Responses of Soil Microbial Activity to Swine Manure Applications

The allocation of the large amount of swine waste from farms is an international concern. An efficient way of managing such waste is its use in farming. It is already known that the incorporation of organic waste into the soil significantly increases the microbial population. Therefore, the objective was to evaluate the impact of the use of swine manure on the soil microbiota in a Eutrophic Oxisol. The experiment was set up in a completely randomized design in a 6 × 4 factorial scheme (sixconcentrations of swine manure and four evaluation periods) with four replications. We evaluate the following characteristics: microbial respiration (C-CO2), microbial biomass (μC g soil) and pH.: microbial respiration (C-CO2), microbial biomass (μC g soil) and pH. A significant effect was found in the interaction between concentrations and time of incubation (p < 0.05) of swine manure on microbial activity in the soil. The amount of microbial carbon increased as a function of increased levels of liquid swine manure. No interaction was observed between concentrations and time of incubation for the pH. The evaluation of the isolated factors allowed to observe that the pH decreased as the doses of manure were incremented. Higher and lower pH values were found after 5 and 30 days of incubation. The application of liquid swine manure up to 6000 L ha increases the release of CO2 and carbon in the microbial biomass. The applications of liquid swine manure cause a gradual reduction in soil pH.


Introduction
The demand for animal husbandry is directly proportional to the growth of the world's population (Safavi & Unnthorsson, 2017). The accelerated growth of swine rearing has caused an environmental issue due to the generation of large quantities of manure with high polluting potential (Veronese et al., 2019). To minimize this problem, one of the most rational forms of final disposal of these wastes is to use them as organic fertilizer (Miyazawa & Barbosa, 2015) since they are rich in nutrients necessary for plant growth. The incorporation of these wastes improves the physical, chemical and biological qualities of the soil as the diet currently supplied to pigs is highly concentrated in elements that are not completely utilized by the metabolism of the animals, therefore causing the excretion of more chemically concentrated waste (Rosov et al., 2020).
The search for farming practices that result in high productivity, but that also consider the various aspects related to environmental quality is a complex equation whose resolution cannot neglect the biotic components of the soil (El-Ramady et al., 2014), which has a close interrelation with the physical and chemical components (Delgado & Gomes, 2016). Thus, all factors that negatively affect microorganisms also cause the deterioration of the physical and chemical properties of the soil (Pereira et al., 2013). Among the parameters used to characterize the adequate microbiological functioning of the soil, the determination of microbial activity through basal respiration, microbial biomass and pH stand out (Novak et al., 2017).
The amount of CO 2 released by the respiration of microorganisms is one of the most traditional and used methods in the assessment of the metabolic activity of the soil microbial population (Sabino et al., 2015). The estimation of microbial biomass can provide useful data on the changes that occur in the biological properties of soils, resulting from the types of the applied management (Semenov et al., 2018). Soils fertilized with organic waste tend to reduce the pH, due to the characteristic processes of organic matter degradation, releasing ions into the medium, therefore, acidifying it (Carmo et al., 2016). Thus, the authors of this paper aimed at evaluating the impact of the use of swine manure on the soil microbial activity in a Eutrophic Oxisol.

Material and Method
The experiment was carried out in the Microbiology and Chemistry laboratories of the State University of Montes Claros. The samples of Eutrophic Oxisol were collected in the city of Jaíba, Minas Gerais (MG). The samples were sieved in a 2 mm mesh and maintained at 60% of the soil's total water retention capacity. The swine manure used in the experiment was collected from a biodigester in the Jaíba Project region, in the municipality of Jaíba-MG. This was digested anaerobically at 56 °C for 30 days. The chemical composition of swine manure is described in table 1. The experiment was conducted in a 6 × 4 factorial design, with six different concentrations of swine manure: 0, 2000, 3000, 4000, 5000 and 6000 L ha -1 and 4 evaluation periods (5, 10, 20 and 30 days), with four replications. Swine manure concentrations were determined according to the amount already applied to the soil by rural producers in the Jaíba region. It is thus adjusted, two concentrations upwards (5000 and 6000 L ha -1 ) and two concentrations downwards (2000 and 3000 L ha -1 ). The following characters were evaluated: microbial respiration (C-CO 2 ), microbial biomass and pH. Note. Average of four repetitions, ±standard deviation.
Five levels of manure were applied to each soil sample and homogenized using a glass stick. The soil microbial respiration was estimated by the amount of CO 2 released from the soil during 5, 10, 20 and 30 days of incubation, according to the methodology proposed by Mendonça and Matos (2005). The 50 g samples of Eutrophic Oxisol were placed in plastic containers of 2 dm 3 at 28 ºC and incubated aerobically in order to stimulate microbial activity. The moisture of the soil samples was adjusted to 60% of its total water retention capacity. The CO 2 produced was captured by 30 mL of a 0.5 mol L -1 NaOH solution and quantified by titration with 0.25 mol L -1 HCl, 10 mL of 0.05 mol L -1 BaCl 2 was previously added and phenolphthalein at 1% was used as an indicator. Four titrations were performed after the beginning of the samples incubation in which the first was performed at 5 days, the second at 10 days, the third at 20 days and the fourth at 30 days. The microbial respiration data were expressed in mg C-CO 2 100 cm -3 and calculated using the formula: where, B = HCl volume in the blank (mL); V = Volume of HCl used in the sample (mL); M = Real HCl concentration (mol L -1 ); 6 = Carbon atomic mass (12) divided by the number of moles of CO 2 that react with NaOH (2); V1 = Total volume of NaOH used to capture CO 2 (mL); V2 = Volume of NaOH used in the titration (mL).
The carbon in the microbial biomass was determined through irradiation-extraction according to the methodology proposed by Ferreira et al. (1999). Therefore, 40 g of soil were weighed in duplicates, one was irradiated in a microwave oven for 120 seconds to eliminate microorganisms and the other was not. After that, the duplicates were placed in 500 mL glass flasks for the carbon extraction process, with the addition of 50 mL of 0.5 mol L -1 K 2 SO 4 to each flask. The set was kept under stirring at 220 rpm for 30 minutes in a Tecnal-TE 420-Incubator orbital shaker. After this period, the samples were filtered on filter paper. In 500 mL glass flasks, a 25 mL aliquot of the filtered extract and 10 mL of K 2 Cr 2 O 7 extracting solution 0.167 mol L -1 and 20 mL of H 2 SO 4 MW were added. The set was then manually homogenized for one minute and allowed to cool for 30 minutes. Next, 200 mL of H 2 O and 10 mL of H 3 PO 4 MW were added. After, three drops of the Diphenylamine indicator were added, and the excess of Potassium Dichromate was titrated with Fe (NH 4 ) 2 (SO 4 ) 26 H 2 O 0.4 mol L -1 . The microbial carbon values were expressed in µg g -1 of soil and calculated using the formula: Microbial carbon (µg g -1 C in the soil) = (IC -NIC)/Kc (2) where, IC = irradiated carbon sample; NIC = not-irradiated carbon sample; Kc = 0.33 (correction factor proposed by Sparling and West (1988).
The pH was determined according to the methodology described by Donagema et al. (2011) and performed in Tecnal® pH-meter. In 250 mL containers, 20 grams of soil and 25 mL of distilled water were placed. The containers were stirred at 220 rpm for 30 minutes on a Tecnal-TE 420-Incubator® orbital shaker, then subjected to rest for 60 minutes. Soon after, the electrodes were soaked in the suspension solution and the pH was read. The pH analysis was performed after 5, 10, 20 and 30 days of soil incubation. The data obtained in the evaluation of the experiment were subjected to tests of assumption of analysis of variance (Bartlett and Shapiro Wilk). The data were considered normal and were subjected to analysis of variance at 5% probability by the t test.
When the interaction was significant (p < 0.05), it unfolded. The swine manure concentrations were subjected to regression analysis at 5% probability by the t test and the incubation days at the Tukey test at 5% probability. R Core Team software version 3.5 (2015) was performed.

Results
A significant effect was observed on the interaction between concentrations and incubation time (p < 0.05) of swine manure on microbial activity in the soil. The unfolding of the interaction between the dose level and the incubation periods allowed to observe that at doses 0, 2000, 4000 and 6000 L ha -1 , the highest values of basal respiration were observed on days 5, 10 and 20 of incubation (Table 2). At the doses of 3000 and 5000 L ha -1 , the highest value of basal respiration was found at 10 days of incubation. Regardless of the dose used, the lowest basal respiration values were found at 30 days of incubation (Table2). All variables of the regressions in the four evaluation periods were significant at 5% probability by the T-test. The determination of the microbial activity of the soil by basal respiration allowed verifying the effect of the application of different concentrations of swine manure in four incubation periods (Figure 1).    Hydrogen pote same letter in bon (µC g -1 so significant an Incubation n was observe olated factors as found at 600 B).

Discussion
The residues organic in the soil activate physiological responses of microorganisms, which are shown on the performance of the microbial community (Grenni et al., 2018). These physiological effects can be assessed using variables such as basal respiration and microbial biomass, since they display the metabolic processes involved in the growth and reproduction of these organisms (Anderson & Domsch, 2010). The increase in the concentration of swine manure resulted in the rise for CO 2 (Figure 1). The linear model equations show an increase for CO 2 due to the increase in the levels of liquid swine manure. In all concentrations of swine manure, the C-CO 2 values were higher than in the control treatment. The increase in theamount of mineralizable carbon is explained by the increase and availability of organic matter in the soil, which improves the environment for the development of microbial flora (Quadro et al., 2011).
Basal respiration is an indicator of soil quality and the available organic carbon for heterotrophic microorganisms. In addition, the greater the amount of CO 2 released per unit of soil weight, the greater the amount of assimilable substrate for the growth of microbial biomass . The concentration of residue organic applied to the soil influences the amount of nutrients and mainly of the organic carbon available to the soil microbiota. Silva et al. (2015) found that the concentration of 200m 3 ha -1 swine liquid manure applied to the soil resulted in higher microbial respiration values.
According to Morales et al., (2016), the high values of basal respiration point to a greater metabolic activity of microorganisms as the CO 2 flow correlates with the intensity of the catabolic processes. Aerobic decomposition of organic residues occurs in biochemical redox reactions (Hullebusch et al., 2019) through which the microorganisms obtain energy using part of the carbon in the substrate in catabolic reactions while CO 2 is released (Podmirseg et al., 2019). The evolution of CO 2 as a measure of respiration, then, represents a measure of total decomposition (Londoño, 2012).
Firstly, microbial biomass degrades the substrates recently added to the soil, or part of them (Ali et al., 2018). In addition, after that, the metabolic activity and consumption of the remaining substrates increases (Bonner et al., 2018). In the final stage, the substrate reaches a status similar to the initial one of the soils, and there is a decline in microbial activity and biomass (Cortes-Tolalpa et al., 2018), therefore, reducing basal respiration, which was observed in this study at 30 days of incubation.
There was an increase in carbon due to the increase in swine liquid manure concentrations in the evaluated period. Microbial biomass is the total mass of microorganisms in a particular volume of soil or even the living component of the organic matter according to Miltner et al. (2012). Biomass is responsible for a large part of the biological activity of the soil, this is the main source of enzymes responsible for the biochemical transformations that occur there (Singh et al., 2014). Balota et al. (2012) when working with the concentrations of 0, 30, 60 and 120 m 3 ha -1 , also saw significant increases for biomass as the volume of swine manure applied to the soil increased. Studies evaluating microbial biomass in soils fertilized with swine manure have shown increases in the carbon content of microbial biomass in relation to soils without fertilization (Morales et al., 2016). These changes occur through the addition of microorganisms by means of manure, which contribute to the increase in carbon in microbial biomass . Moreover, the addition of low molecular-weight compounds and easy transport across the cell membrane increases the number of resources available or partially utilized by the microorganisms, which can increase in quantity due to the favorable condition that the manure creates temporarily (Londoño, 2012).
Microbial biomass has the ability to mineralize and to immobilize nutrients. In addition, the characterization of microbial communities, as well as their cycles, can be useful for understanding and explaining changes of these organisms, of the nutrient cycling and decomposition and mineralization processes . Microbial biomass be as the central compartment in the carbon cycle, where it is associated with the processes of decomposition of plant residues until the cycling of the constituents of the organic matter (Cronan, 2018).
Currently, an immense range of organic compounds it is used to support the demand for nutrients by crops such as sewage sludge, sludge from tanneries, oil residues, animal waste, among others (Zhang et al., 2017). Because microbial biomass uses such organic wastes as a source of nutrients, it directly affected when applied to the soil (Teutscherova et al., 2017). Tavares et al. (2019) when working with swine manure (25, 50, 100 and 200 m 3 ha -1 ) application in cerrado soils in Brazil, concluded that the application of this residue promoted the mineralization of organic matter, resulting in the formation of humic substances and improvement in the degree of soil structure.
The fluctuation of soil pH caused by the metabolic activity of microorganisms is dependent on the type of the substrate metabolized. The addition of carbohydrate reduces the initial pH by producing acidic and CO 2 metabolites (Song et al., 2018). The application of nitrogen fertilizers has an acidic residual effect caused by the nitrification reaction. The decomposition of organic matter can decrease the pH of the soil due to the formation of organic acids in the first stage of the decomposition of the anaerobic phase (Santos et al., 2015), which justifies the pH 6.08 at concentration 0 and more acid in other concentrations (2000, 3000, 4000, 5000 and 6000 L ha -1 ).
The soil microbial components have been being the indicators of the dynamics of organic matter due to their rapid turnover (Cagnarini et al., 2019). In this context, the parameters evaluated in this experiment showed microbial activity, when these are offered a lesser or greater amount of nutrients. As well as the period in which this source of nutrients is available in the soil, affecting its dynamics.

Conclusion
The application of liquid swine manure up to the dose of 6000 L ha -1 increases the release of CO 2 and the carbon in the microbial biomass at days 5, 10, 20 and 30 of incubation. The applications of liquid swine manure cause a gradual reduction in soil pH.