Biofertigation of Forage With Effluents of Green Line of a Cattle Slaughterhouse : Microbial Diversity and Leaf Dry Mass Productivity

The wastewater has been an environmental problem, but your used as fertilizers could reduce or eliminate the application of commercial fertilizers in soil. Arbuscular mycorrhizal fungi (AMF) and nitrogen fixing bacteria (NFB) are a good parameter to analyze the impacts of this fertigationon soil. We aimed to evaluate the distribution and diversity of AMF and NFB before and after applications of wastewater or manure from green line of a cattle slaughterhouse in the irrigation of B. brizantha cv Marandu in Cerrado soil and leaf biomass productivity. The experimental design was performed in completely randomized blocks with ten biofertigation managements. The seeds of the forage were distributed in grooves with spacing of 5 cm. This seeds were covered with a soil layer. NFB and AMF diversity was performed by denaturing gradient gel electrophoresis (DGGE). The leaf biomass productivity in the biofertigation managements was higher than in the managements without the use wastewater/manure. After biofertigation managements, changes in the DGGE profile of the NFB and AMF communities were observed. These changes may be due to the difference in the sample collection period and in the soil humidification. Thus, these DGGE profiles was a good parameter to diagnose the efficacy of wastewater/manure as an alternative biotechnological irrigation.


Introduction
Cerrado in territorial extension is the second Brazilian biome with 204 million of hectares.This biome plays a fundamental role in the flows of the main hydrographic basins of south American (Lima & Silva, 2007).Cerrado soils present edaphic conditions (e.g.texture, depth and relief) ideal for agropastoral activities.About 55% of Brazilian meat production is made in this biome (Embrapa, 2006).These activities have caused an increase in the deforestation of forest areas, in the water consumption and in the use of synthetic pesticides and fertilizers (da Silva et al., 2017).
Brazil has one of the largest cattle herds in the world, with about 215.2 million animals (IBGE, 2015).Eight million of these cattle are in the Cerrado of Tocantins state.Furthermore, the number of cattle slaughtered in this state is uncertain, due to clandestine slaughter and tax evasion.According to the IBGE (2015), this amount may ranges from 1 to 2 million animals.
water consumption in the green line that corresponds to the process of cleaning stool, urine and vomit is about 1,000 liters per animal (Pacheco, 2006).Thus, in Tocantins/Brazil state the annual volume of wastewater of the green line is about 1 to 2 gigaliters.
Wastewater is an environmental problem if discarded untreated in soil or waterbodies (Hespanhol, 2002;Azevedo, 2007).However, they may be used as agricultural fertilizers, due to the nitrogen, phosphorus, potassium and organic matter contents (Hespanhol, 2002;Azevedo, 2007;Da Silva et al., 2017).In addition, the fertigation also provides the organic matter addition in the soil that is a nutrients source for plants, microorganisms and fauna.
The reduction of water consumption of natural waterbodies, of the wastewater disposal in environmental and of the application of synthetic fertilizers for pasture production is the main advantages of fertigation (Silva et al., 2016).This technology increases in plant productivity and in nutritional quality of biomass.According to Christofidis (2006), about 10 million hectares of Cerrado has potential for fertigation; but less than 10% was used, due to the conflicts of interest between the native human population, farmers and cattle ranchers (Lima et al., 2007).
The presence of fecal microorganisms (coliform bacteria, protozoa and helminths) in wastewater has also been a limiting factor for fertigation (Sousa Neto et al., 2012;Alderson et al., 2015;Ibekwe et al., 2018).However, water treatment and soil management can reduce the risk of contaminations of soil and of agronomic varieties by fecal microorganisms (Rocha et al., 2013;Silva et al., 2016).Fecal coliforms and helminth eggs were not identified, in the soil, after 60 days of the application of biosolid from the wastewater treatment (Rocha et al., 2003).Our study, it was done using the suggestion of Silva et al. (2016).These authors recommend the fertigation for the plantation of agronomic varieties that do not have direct use as human food.In addition, these pathogenic microorganisms from wastewater can also be a source of nutrients for soil microorganisms.According to Ibekwe et al. (2018), bacterial of treated wastewater are very active in soil functions.Pyrosequencing detected sequences of nitrifying bacteria, nitrogen-fixing bacteria, denitrifying bacteria, potential pathogens, and fecal indicator bacteria in treated wastewater (Ibekwe et al., 2018).These authors also show that microbial diversity was not significantly different between soils with treated wastewater and fresh water by Shannon diversity index.
The soil microorganism has several biological activities, such as, organic matter decomposition, atmospheric nitrogen fixation, nitrification and solubilization, and minerals availability to plants (Moreira & Siqueira, 2006;Madigan et al., 2010).In addition, Silva et al. (2016) showed that arbuscular mycorrhizal fungi (AMF) and nitrogen fixing bacteria (NFB) are a good parameter to analyze the impacts of fertigation with domestic wastewater in the Brachiaria brizantha planting in Cerrado soil.
The occurrence and distribution of AMF are influenced by soil use and edaphic factors (Silva et al., 2015).These fungi provide the plant with an increase in rate of nutrient absorption and tolerance to heavy metals, water stress and pathogenic microorganisms (Guo et al., 2013).The genus Glomus is the most abundant in the areas of intensive and extensive pasture and in no-tillage and conventional.In addition, no-tillage provides greatest abundance of AMF spores (Silva et al., 2015).
The soil nitrogen is obtained from the degradation and mineralization of organic matter, biological nitrogen fixation (BNF) and fertilizers (Bloom, 2015).The highest nitrogen content in the rhizosphere comes from the symbiosis between the plants and the diazotrophic bacteria (Vance, 1998;Wartiainen et al., 2008;Silveira et al., 2013).
Thus, the aims of this study were to evaluate the distribution and diversity of AMF and NFB before and after applications of wastewater or manure from green line of a cattle slaughterhouse in the irrigation of Brachiaria brizantha cv Marandu in Cerrado soil and leaf biomass productivity.

Site Location and Characterization
The experiment was carried out on the campus of CEULP/ULBRA, Palmas-Tocantins, Brazil, located at an altitude of 254 m and the following geographic coordinates: 10º16′34.16″S and 48º20′05.03″W (Figure 1).

The clima evapotrans
The soil o 5%.Thus,  The wing water used ntents of these elements in the depth of 0 to 20 cm and commercial fertilizer containing superphosphate (with 18% P 2 O 5 ), potassium chloride (with 60% K 2 O) and urea (with 45% N) was used.Nitrogen and others elements were determined, respectively, the Kjedahl method and spectrophotometry (APHA, 2005;Embrapa, 2006).

Forage Water Demand
In the pre-planting, a day before the beginning of sowing of the weed on June 22, 2015, a water layer in the experimental plots was applied to raise the water level in the soil to the field capacity.This water layer was calculated from the estimate of the water depth in the soil between the wilting point and the field capacity.After this first addition of water in the soil, the other additions were estimated as a function of the maximum crop evapotranspiration, the irrigation shift (3 and 4 days) and the precipitation occurred between biofertigation.
In each biofertigation and in the experimental plots, the water layer that was applied, with or without wastewater/manure was multiplied by the plot area.This volume of water was divided by the capacity of the sprinkler (10 L) to determine the number of sprinklers to be applied in each plot.

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 48.24%.In the planting of this forage are used 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 done (June 24, 2015) with sowing in equidistant lines (1.00 m).In each plot three planting lines were made 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 made only 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 management (M1 to M10) (Table 1).This table also contains the quantity of inputs for each management.
The wastewater used in this experiment were collected in a cattle slaughterhouse located in Paraíso do Tocantins/TO/Brazil.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.
For the composition of the biofertigation management, the wastewater of slaughterhouse from the 3 rd stabilization pond (M3 a M5), from reception box (M6 a M8) and of solid part (M9 e M10) were used (Table 1).The managements M1 and M2 did not contain wastewater of slaughterhouse (Table 1).
Samples of each of these wastewater of slaughterhouse were collected for determination of the physical-chemical indicators (Table 2).These analyses were made according to Standard Methods (APHA, 2005).Note.N: nitrogen, P: phosphorus, K: potassium.
The inputs amount of each parcels was determined by the availability of NPK in the soil and in the wastewater of slaughterhouse (Table 2).In addition, the N content was used to determine the wastewater of slaughterhouse amount to be applied in the biofertigation management (Tables 1 and 2).
The commercial and wastewater of slaughterhouse inputs were applied together with the water layer of artesian well.Note.* The minerals were determined after a nitroperchloric digestion of the samples.

Characterization of Leaf Biomass
The cut of leaf biomass were made with a pruning shears.The samples of these biomass were collected in the center of the parcels, with 1.00 m × 0.82 m of dimensions to avoid the border effect.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 for determination of greenhouse dry mass (GDM) at 105 ºC.

Measurement of Viable Microorganisms in Soil
Ten grams of soil and 90 mL of sodium chloride (0.85% w/v) were used to quantify the microorganisms (Sabino, 2007).This mixture was stirred for one hour at 200 rpm, filtered on filter paper and stored at 4 °C.A series of dilutions (10 -1 to 10 -7 ) of 1 mL of the suspension was made.One hundred μl of each dilution was added on solid culture medium and spread with a Drigalski handle.The plates were incubated at 25 °C.This procedure was performed in triplicates.
In the measurement of BFN, the nutrient agar culture medium containing 0.3 ml of nystatin was used (Sabino, 2007).The pH in this medium culture was adjusted to 7. The plates were incubated for 3 days.
Martin medium containing rose bengal (0.1% w/v) was used for counting filamentous fungi (Martin, 1950).In this medium, 1 ml of streptomycin (0.3 mg/ml) was added and the pH was adjusted to 5.8.The plates were incubated for 7 days.
The actinomycete counts in selective medium containing glycerol were made (Rodrigues, 2007).The plates for 7 days were incubated.
The microbial measurements were expressed in log scale of the colony-forming unit (CFU) per gram of soil.

Characterization of Microbial Diversity by DGGE Profile
Diversity of NFB and AMF was performed by denaturing gradient gel electrophoresis (DGGE).These microbial groups were selected for this analysis due to species diversity and their contributions to soil fertility and structuring (Moreira & Siqueira, 2006).
DNA of the soil samples was extracted using a soil DNA Mega Prep Kit (Kit-MO BIO, Ultraclean TM).In this extraction, 0.5 g of soil were added in plastic tubes (Eppendorff type) containing polypropylene beads.After, several steps of adding solutions and centrifugations, according to the manufacturer's protocol, the suspension containing the total DNA was stored at -20 °C.
The nifH and 18S rDNA genes were amplified by polymerase chain reaction (PCR) from the total DNA for analysis of NFB and AMF, respectively.
Bionumerics software (Version 5.10) was used for normalization, conversion and comparison of the images in presence/absence and band intensity matrices.

DGGE Profile of NFB
The PCR of the nifH gene was done with the 19F and 407R primers (Ueda et al., 1995).In this amplification, a 390 base pair (bp) fragment was obtained.This fragment was used in the Nested-PCR with the 19F-GC (with GC clamp) and 278R primers (Direito & Teixeira, 2002).In this new amplification, a 260 bp fragment was obtained.
The program used in the thermal cycler was similar to the described by Direito and Teixeira (2002).In the negative controls, 1 μL of MilliQ water was used instead of the DNA fragments.
The Nested-PCR fragments were analyzed by DGGE (Model DCodeTM Systems, BIO-RAD California).20 μL of these fragments were loaded onto 8% polyacrylamide gel (w/v) in TAE buffer (1×).This gel was prepared with denaturation gradient varying from 45 to 70% using urea (7M) and formamide.The gel was subjected to vertical electrophoresis for 12 h at 60 V and 60 °C.This gel was stained for 40 min with SYBR Gold (1×) (Molecular Probes, Leiden, The Netherlands) and photographed on ultraviolet light on the Molecular Imaging Locococentor (Loccus biotechnological L-Pix Chemi).

DGGE Profile of AMF
PCR was similar to those described in DDGE profile of NFB.
The AM1 and NS31 primers were used to amplify the fragments of the 18S rDNA gene of the first PCR (Simon et al., 1992;Helgason et al., 1998).In this reaction, a 580 bp fragment was obtained which was used in the Nested-PCR with primers NS31-GC (with GC clamp) and Glo1 (Kowalchuk et al., 2002;Cornejo et al., 2004).
The DNA fragments of the Nested-PCR were used to obtain the DGGE profile (DCodeTM Systems Model, BIO-RAD California).Twenty μL of this fragment (150 to 200 ng of DNA) was loaded onto polyacrylamide gel (8%, w/v) in TAE buffer (1×).
The next steps to obtain DDGE prolife of AMF were done similar to DGGE prolife of NFB.

Statistical Analysis of the Indicators of Soil Quality and of Leaf Biomass
The experiment was conducted in a completely randomized block design with factorial unfolding (10 biofertigation management and 3 cuts of leaf biomass).
Physical-chemical indicators content and leaf biomass were compared using analysis of variance followed by pos-hocTukey test, both at 5% significance.The estimates of these parameters were made at 95% confidence level, based on the coefficient of variation (CV) limits proposed by 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 DGGE profiles were analyzed in the Bionumerics software (Version 5.1).In this software were made the unweighted pair group method with arithmetic mean (UPGMA) dendograms using the Jaccard similarity index.The similar bands were considered those with probability level of 0.5% by the post-hoc Bonferroni test.
The graphs to present the relationships between the variables were made from the spreadsheets/software: Excel, Surfer, SigmaPLOT12.0 and Minitab 17.

Wastewater/Manure of Slaughterhouse Composition
The wastewater/manure of slaughterhouse had a diversified composition of primary macronutrients (Table 2).Thus, crude wastewater, despite representing an environmental problem may be reduces or eliminates the use of commercial fertilizers (Hespanhol, 2002).
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 in meeting the demand of these plants is a viable alternative (Silva et al., 2016(Silva et al., , 2017)).
A forage crop with high availability in nitrogen has more vigorous roots than a crop deficient in this nutrient, because root growth is related to the accumulation of elaborated sap (Brower, 1962).
The basic pH of wastewater/manure of green line (Table 2) can contribute to the increase of the cation exchange capacity in the soil and together with the limestone increase the pH in soil solution.These soil changes may favor the development of forage crops and shows the potential of biofertigation with wastewater/manure of green line.

Characterization of Physical-Chemical Indicators of Soil
The concentration, movement and distribution of the physical-chemical indicators in natural conditions limit the use of the Cerrado soil for the exploitation of agropastoral activities.The use of adjusts acidity and fertilizeris are alternative to reduce or eliminate this limitation.Thus, in this study the dolomitic limestone was added for soil acidity adjustment before the planting of B. brizantha cv Marandu.
We observed a reduction in the ions (H + Al) concentration and an increase in base saturation and pH in the effective depth (20 to 30 cm) of the roots (Figure 2).These results may be due to the morphological characteristics of the sandy loam soil.In this type of soil has a greatest volume of infiltration of water in the roots depth that favors the leaching of the bases and the increase of the H + and Al 3+ concentration.
The pH in the soil depth of 0 to 30 cm did not significant difference (p < 0.05) in the blocks.However, in the depth of 90 to 100 cm the pH presented a significant difference when compared to the other depths (Figure 2).
Cerrado soils have acidic pH and vary according to the time, geographical location and soil depth (Ronquim, 2010).le 4).ential evapotranspiration (Table 4).The ASWC represented about 50% of the ASW that is the maximum limit of water in the soil to avoid the water stress of the plant.
During the biofertigation management, an excess of water in the soil was observed (Table 4).This fact was due to precipitations with intensity higher than the ASW.This excess water caused the leaching of fine soil particles (silt and clay) and nutrients.Furthermore, the mean water demand of Brachiaria brizantha calculated by the ratio between the maximum crop evapotranspiration and the time of biofertigation management was 4.1 mm/d (Table 4).

Characterization of Physical-Chemical Indicators of Soil
The physical-chemical indicators did not present significant differences in their contents along the soil depth, in all biofertigation management (Table 5).These indicators in the soil solution presented a greater dispersion with the biofertigation than dispersion before the preparation of the soil for the planting.According to Koura et al. (2002), it requires several years of irrigation with wastewater to achieve changes in physical-chemical characteristics.However, we results show o potential of use of wastewater/manure of green line as nutrient source and water by B. brizantha cv Marandu that is important for cattle food.

Characterization of Leaf Biomass
In the first cut, leaf biomass productivity was low.This result may be due to the initial stages of plant growth or interference of wastewater/manure in the formation of foliar mass (Figure 6A).However, leaf biomass productivity did not show a significant difference (p < 0.05) between the treatments without or with the use of wastewater/manure (Figure 6A).Therefore, the low productivity, regardless of the management, was due to the culture not having completed the development, at this stage.
The second cut of the leaf mass was performed at 209 days of planting and at 49 days after the first cut (Figure 6B).The organic and mineral masses were larger in this cut than in the other cuts (Figure 6).Thus, the ideal period for cutting the leaf mass, regardless of the use of biofertigation, was after 200 days of forage planting.
Leaf biomass productivity in the second cut had significant differences (p < 0.05) between the biofertigation managements (Figure 6B).
Similar to that observed in the first cut, the leaf biomass productivity in the second cut was greater in managements with manure than other (Figure 6).In addition, the leaf biomass productivity in the biofertigation managements was higher than in the managements without the use wastewater/manure.These results show that, at this stage of plant growth, the use of the biofertigation has a positive effect on the growth and production of B. brizantha cv.Marandu in the Cerrado soil.

Characterization of Microbial Diversity by DGGE Profile
After the biofertigation managements, changes in the DGGE profile of the NFB and AMF communities were observed (Figures 4,5,7 and 8).We observed a decrease in the amount and an increase in the intensity of the bands which shows a reduction in species richness, but an increase in the in the pre-existing microbial community.These changes may be due to the difference in the sample collection period and the soil humidification.Due to the sensitivity the environmental and anthropogenic interferences, the NFB and AMF communities and the soil microbial activity are good indicators to soil quality (Barros et al., 2010).
Management without addition of fertilizer or wastewater/manure (M1) had greatest similarity in the profile of NFB bands with the M2 and M3 managements (Figure 7).The M7 and M8 with wastewater from the receiving box had the same band profile.These results demonstrate the influence of the chemical composition of the wastewater/manure on the NFB community (Figure 6).In addition, the changes on the NFB were due to the addition of NPK sources and dolomitic limestone.Biotic and abiotic factors, including soil acidity, affect the NFB community and decrease the symbiotic association efficiency between NFB and plants (Rufini et al., 2011).profiles, the A l for use of w s (Figures 4, 5 The sensitivity tion with or w nicellular and est diversity of ell in Amazon  Second factor and perhaps most important is the time of germination and or growth.NFB may be free-living or symbiotic.Thus, they can grow and multiply on the soil without necessarily being associated with another living organism.However, the AFM depend on being associated with the plants roots for growth and reproduction (Moreira & Siqueira, 2006).Moreover, the sexual reproduction of AFM depends on spore germination (Madigan et al., 2010;Moreira & Siqueira, 2006).In this context, to verify the alterations on the AMF community after the management of depends on a longer time than the NFB.However, the seven months of soil management could have been adequate to investigate microbial changes in soil.Several authors have shown that the soil microbial community undergoes rapid changes in relation to environmental conditions (Faleiro & Andrade, 2011;Barros et al., 2010;Moreira & Siqueira, 2006).Furthermore, the AMF communities were most similar among samples from a similar geographical location (Figures 5 and 8).The AMF has also divergence in management types within a given location (Schneider et al., 2015).
Therefore, the DGGE profile of NFB and AMF before and after biofertigation was a good parameter to diagnose the efficacy of wastewater/manure as an alternative biotechnological irrigation, which will provide a reduction in the water demand of the water bodies.

Conclusions
In this study, we had the following conclusions: (1) Biofertigation with wastewater form the green line of a cattle slaughterhouse contributes for development of forage crops in Cerrado soil.
(2) The nutrients of wastewater from the green line do not have a significant infiltration in the soil.
(3) Biofertigation with wastewater or manure from the green line has a positive influence on the increase in the number of viable microbial cells and the amount and intensity of NFB and AMF bands on the DGGE.
(4) The increase of the microbial biomass in the soil causes an increase, directly proportional, in the biomass productivity of Brachiaria brizantha cv.Marandu.
(5) The evaluation of the NFB and AMF communities by DGGE showed to be a good parameter to study the changes caused by biofertigation managements in the Cerrado soil.
(6) The wastewater may be a viable alternative to reduces or eliminates the use of commercial fertilizers for Marandu grass production in the Cerrado soil.

Table 1 .
Biofertigation management and quantity of wastewater/manure applied in the planting of Brachiaria brizantha cv Marandu

Table 3 .
Counts of viable microbial cells of the Cerrado soil before the planting of Brachiaria brizantha cv Marandu and of the use of biofertigation with cattle slaughterhouse wastewater/manure

Table 5 .
Physical-chemical indicators concentration after of the planting Brachiaria brizantha cv Marandu and biofertigation management (M1 at M10) with wastewater/manure of the green line

Table 6 .
Counts of viable microbial cells in different soil depth after in the planting of Brachiaria brizantha cv Marandu and of the use of biofertigation management (M1 a M10) with cattle slaughterhouse wastewater/manure