Biofertigation of Forage With Effluents From a Cattle Slaughterhouse Green Line : Impacts on Physical-Chemical Indicators of Soil Quality and on Production Biomass

Cattle slaughterhouses are potential causes the environmental impacts, as it require a large volume of water in meat processing, generate large effluents amount, and promote the Cerrado deforestation for animal husbandry. Therefore, we aim was carried out to assess the effects of the soil application of a green line wastewater from a cattle slaughterhouse in the Brachiaria brizantha growth. The M1 and M2 managements did not contain wastewater of slaughterhouse. The wastewater from the 3 stabilization pond (M3 to M5), from reception box (M6 to M8), and manure (M9 and M10) were used in the biofertigation managements. The physical-chemical indicators levels did not show significant differences (p < 0.05) before soil preparation and after managements. However, biofertigation in the Cerrado soil can provide a mitigation of the leaching of fine soil particles and cations. In addition, maximum nitrogen dose of wastewater provided a higher leaf biomass productivity than commercial nitrogen. Thus, the fertigation with wastewater can reduce the use of water bodies to crops irrigation and the incorporation of new areas with native vegetation to the agricultural production systems.


Introduction
New forest areas have been incorporated into agricultural production systems, due to the food demand of growing human population (FAO, 2005;ONU, 2013).In Brazil, this agricultural expansion is seen the Cerrado with the replacement of native vegetation by crops and livestock (Andrade, 2002;Cardoso et al., 2011;Costa et al., 2007).The excessive use of agrochemicals, fertilizers, and correctives, the uncontrolled irrigation, soil compaction by trampling of animals, monoculture, and intensive mechanization have contributed to soil and groundwater contamination, and the natural resources degradation (Cunha et al., 2008).To neutralize or mitigate these impacts is necessary to development biotechnologies that can promote the sustainable use of natural resources.
Meat is one of the animal protein sources most consumed by the human population.Brazil has one of the largest cattle herds of the world (IBGE, 2015).However, the meat production is associated with the high water consumption and environmental impacts due to the effluents.In the green line of a cattle slaughterhouse, which assembled effluent from the process of cleaning feces, urine, and vomit, water consumption can be approximately 1,000 liters per animal (Pacheco & Yamanaka, 2008).Furthermore, the highest water consumption is in the washing of the animals and equipment.In this process, law require the use of fresh and potable water, with minimum contents of residual free chlorine (Pacheco, 2006).The water blade with or without wastewater/manure was multiplied by plot area and total volume was divided by the capacity of the sprinkler (10 l).

Fodder Planting
B. brizantha cv Marandu seed was purchased in Palmas/TO/Brazil with, respectively, 60.3% and 80.0% purity and germination rate.Thus, the cultural value was of 48.24%.In the planting of this forage are use of 1.5 to 2.0 kg / ha of viable seeds (Embrapa, 1984).In this study, 1.75 kg/ha of viable seeds were used.
The planting was carried out (June 24, 2015) with sowing in equidistant lines (1.00 m).In each plot, we did 3 planting lines in the form of triangular grooves with depth of 4 cm and 1.62 m in length.The seeds were distributed in these grooves with spacing of 5 cm and were covered with a lightly pressed soil layer.This cover was only made to provide wet soil contact with the seeds.

Biofertigation Management in the Field Experiment
The experimental design was performed in completely randomized blocks (B1 to B4) and 10 biofertigation managements (M1 to M10) (Table 1).This table also contains the inputs quantity for each management.
The wastewater used in this experiment were collected in a cattle slaughterhouse of the Paraíso do Tocantins/TO/Brazil city.In this slaughterhouse, the effluents of the green line are channeled to a reception box.The wastewater of this box are separated into two portions through a pumping system.The liquid part is deposited in three stabilization ponds and solid part (manure) is used as fuel in the boiler heating system.Two liters of these wastewaters were collected for physical-chemical indicators determination (Table 2).These analyses were performed according to Standard Methods (APHA, 2005).
The wastewater from the 3 rd stabilization pond (M3 to M5) and from reception box (M6 to M8) and the manure (M9 and M10) were used in the biofertigation managements (Table 1).The managements, M1 and M2, did not contain wastewater/manure.
The inputs amount of each parcels was determined by NPK availability in the soil and in the wastewater/manure (Table 2).In addition, the N content was used to determine the wastewater amount of the biofertigation managements (Tables 1 and 2).
The commercial and wastewater/manure inputs were applied together with the layer of water of artesian well.

M1
Application of dolomitic limestone and layer of water of artesian well 840 Note.* The minerals were determined after a nitroperchloric digestion of the samples.** Values below the limit of detection.

Characterization of Leaf Biomass
The bromatological composition of the forage is indispensable in the evaluation of the impact of the residuary materials on the agronomic performance of the crop and on the changes of the physical-chemical indicators of the soil fertilization.
The leaf biomass cutting was performed in the center of the parcels (1.00 × 0.82 m) to avoid the border effect, with a pruning shears.
The green mass was determined on analytical balance.
The samples in 65 °C forced-ventilation greenhouses during 72 hours to moisture loss were conditioned.
After cooling to room temperature (25±5 °C), the air dry mass (ADM) was determined using the analytical balance.This ADM was crushed with the aid of a Willey mill.Five grams of this powder were used to bromatological analyzes and the other quantity was placed in the oven at 105 °C for greenhouse dry mass (GDM) determination.
The moisture, crude protein, crude fiber, etheric extract, mineral residue and phosphorus of leaf biomass were performed according to methodologies of the Association of Official Analytical Chemists (1990).
The N content was performed by the Kjeldahl method.Phosphorus, potassium and sodium contents by spectrophotometry and flame emission photometry were obtained.Others minerals contents were measured by plasma emission spectrophotometry (EMBRAPA, 1999).These elements were analyzes after the nitric-boric digestion (Tedesco et al., 1985) using 1 g of ADM powder and 5 mL of nitric acid and perchloric acid solution (3:1 v:v).This mixture was incubated for 2 hours at 200 °C.

Statistical Analysis of the Indicators of Soil Quality and of Leaf Biomass
The experiment was in completely randomized block design with factorial unfolding (10 biofertigation managements and three leaf biomass cuttings).
The analysis of variance and Tukey's test at 5% significance were used to compare the soil physical-chemical indicators and bromatological composition of leaf biomass.The estimate of these parameters were performed at 95% confidence level, based on the coefficient of variation (CV) limits (Pimentel- Gomes, 2000).This author classifies the experimental variations in low variation (CV < 10%), medium (10 < CV < 20%), high (20 < CV < at 30%), and very high (CV > 30%).
The mathematical models, two-dimensional and multidimensional, were obtained from the adjustment of the points derived from the correlations of the variables, with higher coefficient of explanation (R 2 ) and significance.
The graphs to present the relationships between the variables were performed from the spreadsheets/software: Excel, Surfer, SigmaPLOT12.0 and Minitab 17.

Wastewater of Slaughterhouse Composition
The physical-chemical indicators contents of wastewater of the green line are presented in Table 2.These indicators contents were similar to those obtained in effluents of cattle slaughterhouse (Gomes, 2010;Louvet et al., 2013;Maldaner, 2008;Masse et al., 2000;Pacheco & Yamanaka, 2008).
The wastewater of slaughterhouse had a diversified composition of primary macronutrients (Nitrogen, Phosphorus and Potassium) and secondary macronutrient (e.g.calcium).Thus, the crude wastewater, despite representing an environmental problem, is used as fertilizer (Azevedo, 2007).This use can reduce or eliminate the addition commercial fertilizers in soil (Hespanhol, 2002).
The wastewater of cattle slaughterhouse had high physical-chemical indicators concentrations (Table 2).Therefore, these wastewaters need a treatment before discarding in the soil and/or in water bodies (Scarassati et al., 2003).The treatment of these effluents, after the removal of the solids and fats, was carried out in stabilization ponds (Silveira, 1999).
After treatment, the effluents of the 3 rd stabilization pond can be discarded in the water body (Conama, 2005).However, the effluents of our study had a nitrogen content seven times higher than the maximum limit allowed by Conama (Table 2).This high N content may contribute to eutrophication of water bodies (Conley et al., 2009).Thus, the biofertigation with the wastewater of slaughterhouse in the B. brizantha cv.Marandu planting may be an alternative for reducing the nitrogen content and the risk of eutrophication.
Forage grasses, due to accelerated leaf growth rates, require a large nutrient amount (Barbero et al., 2013;Costa et al., 2016).Thus, the use of wastewater of the green line in meeting the demand of these plants is a viable alternative (Silva et al., 2012(Silva et al., , 2016)).In soils with adequate aeration capacity, because microbial activity, the nitrogen is in the chemical form of nitrate (De Bona, 2008;Rodrigues et al., 2013;Wu et al., 2015).However, in soils with medium to high drainage capacity (e.g.latosol and neosol) the nitrate leaching occurs, leaving the soil poor in nitrogen (De Bona, 2008).These two types of soil is predominant in the Cerrado biome.
In the wastewater of slaughterhouse, the nitrogen is in different chemical forms that increase of retention in the soil and of absorption by the plant (Table 2).Therefore, the amount and chemical form of this macronutrient changers the plant growth and biomass (Rahayu et al., 2005;Reich et al., 2003).Furthermore, a forage crop with high nitrogen availability has a more vigorous root system than a crop deficient in this nutrient, because of elaborated sap accumulation (Brouwer, 1962).
The wastewater of green line had nitrate and nitrite levels (Table 2) that can be nitrogen source for plant and for the microorganisms of the soil.The roots has the potential to absorb nitrate contained in soil with adequate moisture content (Tinker & Nye, 2000).
The sodium content was lower (Table 2) than at the limit level (40 mg/L), recommended to avoid salinization or sodification of soil and groundwater (Gloaguen et al., 2010;Von Sperling, 2005).Furthermore, this wastewater presented electrical conductivity with low level of soil salinization (Bernardo, 2006).Thus, the use of these wastewaters of green line in biofertigation has little or no potential to cause such environmental damage.
The wastewater pH was higher than those values obtained in effluents of green line of a cattle slaughterhouse in the south region of Brazil (Klank, 2011).According to these authors, the pH of these effluents may vary according to the collection period of the samples.Moreover, the basic pH of wastewater of green line (Table 2) can contribute to the increase of the cation exchange capacity and together with the limestone increase the pH of the soil solution.These soil changes may favor the development of forage crops and shows the potential of biofertigation with wastewater of green line.

Physical-Chemical Indicators Characterization of Soil
The pH, base saturation and cation exchange capacity of the first soil samples were used to determine the soil acidity adjustment (Table 3).However, for pH adjustment only the values obtained in the rhizosphere depths (0 to 10 and 10 to 20 cm) were used.About 80% of the mass of the root bulb of B. brizantha cv.Marandu is in the depths of up to 20 cm.
Soil samples, at depths up to 20 cm, had lower base saturation and cation exchange capacity (Table 3) than the recommended values for Marandu (Malavolta, 1989;Primavesi et al., 2008).Thus, in this study, the soil acidity adjustment using 1.728 t/ha of dolomitic limestone was made.For addition of limestone up to 20 cm, the depth factor was 1.0.
The pH and sand had low dispersion in the soil profile (CV < 10%).The others soil physical-chemical indicators had average to very high dispersion (Figure 2, Table 3).This high dispersion may be due to soil morphological characteristics, nutrient availability and microbial activity (Rodrigues et al., 2013;Santos et al., 2009).
The pH in the depth of 0 to 30 cm did not present a significant difference (p < 0.05) in the blocks, but of 90 to 100 cm it presented a significant difference when compared to the depths of 0 to 10 and 10 to 20 cm (Table 3).
The Cerrado soils has a low acidity and it can vary depending on the time, geographical position and soil depth (Ronquim, 2010).
In the depth of 20 to 30 cm (Figure 2C), the physical-chemical indicators concentration was lower than in the others depths (Figures 2A,2B and 2D).However, this fact may be because a very high variability (CV = 201.3%) in the potassium content (Figure 2C, Table 3).
In depth greater than 10-20 cm, a reduction in the ions (H + Al) concentration was observed.While, the base saturation increased with soil depth (Table 3).The soil of the experimental area was sandy-loam, due to, respectively, high percentage (> 60) of sand and medium (< 34) concentration of clay in 100 cm of depth (Embrapa, 2006).Rainfall that it has highest infiltration volume in the effective zone of the roots is high in sandy-loam soil (Ronquim, 2010).Therefore, this volume of infiltration could contributed to leaching of the bases and the increase of the ions (H + and Al +3 ) concentration in the soil (Ronquim, 2010;Roscoe et al., 2006).
The concentration and movement of physical-chemical indicators under natural conditions may limit the use of Cerrado soil for agropastoral activities (Gucker et al., 2009;Malavolta & Kliemann, 1985).In this study, the use dolomitic limestone and fertilizers contributed to the Marandu production in this soil.Furthermore, the biofertigation in Cerrado soil can contribute to reduce the incorporation of new areas with native vegetation to the agropastoral systems.Note.Sd: standard deviation; CV: coefficient of variation; CEC: cation exchange capacity. jas.ccsenet.

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In the biof (Figure 6) determinat In this study, the root system and soil penetration capacity were directly proportional (Table 4).The root system well developed, gives the plant greatest capacity to withstand stress, such as severe winters, dry summers and grazing (Cunha et al., 2010).
The increase of the wastewater of slaughterhouse doses may be limited the roots growth in resistance tension of 2.5 MPa (Figure 7, Table 4).However, soil resistance to root penetration between 1.0 and 3.5 MPa restricts root growth (Benghough & Mullins, 1990).In soils with pasture crops has been indicated a limit of 2.5 MPa (Leão et al., 2004).Furthermore, the plant growth are also limited by morphological characteristics and the water retention capacity of the soil (Lapen et al., 2004;Leão et al., 2004;Tormena et al., 1998).
Mathematical models of the soil resistance determined the relationship between the nitrogen of wastewater and of root depth (Figure 7).These models may aid in the estimation of root depth and of nitrogen dose for be used in planting of B. brizantha.Root depth is fundamental for acidity adjustment, soil fertility and addition of water blade (Doran & Parkin, 1994;Stenberg, 1999).Furthermore, this depth has a positive influence on leaf growth and biomass production (Stenberg, 1999;Cunha et al., 2010).
The root penetration had a positive correlation with the increase of up to 17.5% of nitrogen of wastewater of reception box.In the largest doses of this element, the correlation was zero or negative depending on the applied voltage (Figure 7A).This behavior was due to the high fat content that caused an obstruction of the soil porosity (Table 2).The obstruction reduced the capacity of water infiltration and air circulation in the soil.Meanwhile, the root penetration had a positive correlation with depth at doses up to 50% of nitrogen of wastewater of 3 rd stabilization pond (Figure 7B).The application of nitrogen of the manure also showed a positive correlation up to the dose of 20% and in the largest doses, the vertical root growth showed a negative correlation (Figure 7C).These results show the necessity of the treatment of wastewater of green line for application in the soil.This wastewater when distributed on the soil can form a layer that hinders the penetration of water and compromises the elevation of moisture to the field capacity.-------------------------------------------------------kg/ha ------------------------------------------------------ Note.CV = coefficient of variation (%).
Similar to the 2 nd cutting, the biomass productivity in the 3 rd cutting also showed high to very high variability and did not have significant differences (p < 0.05) in the biofertigation managements (Table 5).
A linear increase in biomass productivity by nitrogen dose was observed in the managements with wastewater of 3 rd stabilization (Table 5).Meanwhile, in managements with wastewater of reception box (untreated) and manure showed an increase in biomass productivity, respectively, in 40% and 20% of nitrogen (Table 5).These results confirm the need to treat the effluents for application in biofertigation and shows the importance of the casualization of the experiment.Silva (2017) also observed the importance of the casualization of managements with domestic wastewater applied to the Marandu grass fertigation.This author showed that the dry mass productivity depends of dose and the cutoff period.Our results also show a proportional increase of the biomass productivity by cutting.* There was no significant difference (p < 0.05) between the biofertigation managements.
The nitrogen, sulfur and iron contents was difference (p < 0.05) between the biofertigation managements (Table 7).In the managements with manure, the nitrogen and iron concentrations were lower than in the controls.The sulfur concentration varied between the managements.
Thus, the wastewater/manure of the green line of a slaughterhouse and did not negatively affect the nutritional quality of the B. brizantha cv.Marandu biomass.

Conclusions
The wastewater of green line from cattle slaughterhouse have potential for use in the irrigation of B. brizantha cv.
Marandu.This biofertigation in the Cerrado soil, of medium to long term, provides a mitigation of the leaching of fine particles of soil and cations.The maximum dose of nitrogen of the wastewater/manure meet the maximum demand of this nutrient.The nitrogen of treated wastewater provided a greatest leaf biomass productivity per unit area and a highest use in volume than the commercial nitrogen.In addition, biofertigation can provide a reduction in water demand of water bodies.

Table 1 .
Biofertigation Management and quantity of inputs applied in the planting of Brachiaria brizantha cv Marandu

Table 2 .
Physical-chemical indicators of the green line wastewater of slaughterhouse used in the experiment

Table 3 .
Concentration of the physical-chemical indicators in the experimental area before of the planting Brachiaria brizantha cv Marandu

Table 4 .
Penetration of the roots of Brachiaria brizantha cv.Marandu in differ management of biofertigation (M1 to M10) with wastewater of slaughterhouse

Table 7 .
Bromatological composition of the leaf biomass of roots of Brachiaria brizantha cv.Marandu after biofertigation managements (see Table1)