Step 1. Inoculum preparation
The experiment was carried out using two different inoculants, one noncommercial (NCI) and one commercial (CI - biological product made up of a mix of microorganisms, being basically lactic acid bacteria, with species not defined by the manufacturer, used for accelerating composting of organic solid waste from agricultural and household waste. According to the manufacturer, it promotes exponential increase in microbial activity, improving the fermentation and composting process by converting organic matter into nutrients such as acids and nitrogen compounds quickly and safely).
The noncommercial inoculum was prepared using 700 g of unsalted rice cooked - not to change the composition of rice, in distilled water (Fig. 1). After cooking, the rice was placed in a plastic tray covered with canvas, which was placed in the native forest on the Federal University of Lavras, during the spring, with mild temperatures. Soil litter was placed over the tray to collect the natural microbiota of the environment. The collection period was of 15 days.
Afterwards, rice with microbial growth was deposited into a 20 liters container, homogenized with 1 liter of sterile sugar cane juice, and then made to a total volume of 20 liters with distilled water. For 20 days the container was stored in a cool, ventilated and closed space .
The commercial inoculum was prepared according to the manufacturer's instructions in which the recommended amount of distilled water and sugar was added to the concentrated product, and after fermentation for 20 days at room temperature, the product was ready for use. Compost without the addition of inoculants was used as the control treatment.
Step 2. Preparation Of Compost Mixture
Compost mixture was performed by mixing organic waste from the Lavras University Restaurant (RU) and garden waste. For the calculation of the proportion of organic residues and residues from the landscaping that were added to the composters, so that if the ideal C/N ratio was reached for the start of composting, the initial characterization of the waste components was performed, described in the Table 1.
Initial characterization of residues.
Organic waste (RU)
|*RU = University restaurant|
The present work used a new type of bioreactor built with concrete blocks and an air injection system. The composters had 1 m3 (1 m x 1 m x 1 m), with monitored temperature and aeration to ensure a minimum oxygen concentration of 10% . Temperature sensors were placed inside each compost (PT10 digital thermometer model MPT2, Lexitron-Guemisa, Madrid, Spain). Forced air (6.3 m³ / min) was supplied from the bottom of the pile through 5 cm diameter PVC tubing at the base of the composting cell, traversing the cell horizontally 10 cm from the rear edge, with 1 cm holes in diameter, in 3 points of the tube (25 cm, 50 cm and 75 cm), connected to a Ventibras air injection pump, with 5 horse axial fan, for 30 minutes every 3 days (Supplementary material, Fig. S1).
The mixture was characterized physicochemical before the addition of the inoculum and the beginning of the fermentation process with a C:N ratio (30:1) and a moisture content of approximately 50 to 60% of the field capacity. The height of the compost piles was monitored during sampling with a tape measure to monitor the compaction of the material and the decomposition of the particles.
All waste was crushed into small particles with the help of a crusher. The organic wastes from the UR were added directly to the garden waste at a ratio of 1:2.5 in each of the composters and mixed with the help of a tractor, resulting in a homogeneous mixture. Immediately after, the starter cultures were inoculated with direct applications of the reactivated inoculum on the mixture of residues. In the initial time, 5 liters of inoculum (both NCI and CI) plus 15 liters of water were used in each compost, except the compost with the control treatment.
Moisture content was controlled by the hand test consisting of “moistening and rubbing a little of the compost between the palms”, if the compost is ready, it will not get dirty, loosening easily, and kept between 50% and 60%, and irrigations are performed when necessary according to this standard throughout the composting process to maintain the ideal moisture content . Irrigations were performed on average every 3 days, with an average amount of 10 liters of water per compost each time .
The samples were collected in triplicate throughout the composting process. In the initial time were collected 3 samples of 1 to 10 cm, 3 samples of 45 cm and 3 samples of 90 cm, respectively. The sampling depth has changed throughout the process due to the reduction in the height of the piles, always maintaining the collection on the surface, interior and bottom of the pile. Surface, interior and bottom composting samples were homogeneously mixed for analysis and after 0, 60 and 120 days for the analysis of the physical and chemical dynamics. Samples were taken after 0, 5, 10, 20, 40, 60 and 120 days for the quantification of the microorganisms.
Step 3. Physicochemical Analyses
The physicochemical analyzes were performed during times 0; 60 and 120 days of composting. The moisture content was evaluated by drying at 105 °C until having the same weight with 3 readings followed. The lowering of the piles was measured using tape measure to follow the lodging of the material and the decomposition of the particles. The fertility of the composting were determined according to the Manual of Soil Analysis . The following parameters were evaluated: C; N; C:N; H + Al (potential acidity); pH in H2O; P-rem (remaining phosphorus); P available; K; SO4; Na; Ca; Mg; S; B; Cu; Fe; Mn; Zn; organic matter (OM); organic carbon (OC); V (base saturation); m (aluminum saturation); SB (sum of bases); total CEC (cations exchange capacity) and effective CTC (Embrapa, 2011). The micronutrients were quantified using the Mehlich method .
Step 4. Microbiological Analyses
Microbial counting of cultivable microorganisms was carried out from inoculum and samples at intervals, 0; 5; 10; 20; 40; 60 and 120 days in triplicate. 25 g of each sample was added to 225 mL of sterile peptone water in a shaker at 120 revolutions per minute (rpm) for 30 min at room temperature . The samples were mixed in a stomacher at normal speed for 60 s, and 10-fold dilutions were prepared.
Seven different types of culture media were used to study the microbial communities. Nutrient Agar (NA, Merck) was used as a general medium for the viable mesophilic bacteria population . GYC (50 g glucose, 10 g yeast extract, 5 g CaCO3 and 20 g agar) for acetic acid bacteria according . MRS (De Man Rogosa Sharpe, Merck) agar containing 0.1% cysteine - HCl was used for Lactic acid bacteria growth under anaerobic conditions according . MRS plates were incubated in acrylic anaerobic jars. After spreading, the plates were incubated at 28 for 48 h. The counting and isolation of actinobacteria were performed using Aaronson medium (2 g KNO3, 0.8 g casein, 2 g NaCl, 2 g K2HPO4, 50 mg MgSO4.7H2O, 20 mg CaCO3, 40 mg FeSO4.7H2O, 15 g agar), incubated at 45 °C for 72 to 120 h . The counting and isolation of yeasts were performed using YEPG (10 g yeast extract, 10 g bacteriological peptone, 20 g glucose, 20 g agar with pH 3.5) and incubated at 28 °C for 48 h . The filamentous fungi population was counting using PDA (200 g raw potatoes, 20 g dextrose, 20 g agar, 1 L distilled water) and incubated at 25 °C for 7 days .
The morphological characteristics of the colonies (cell size, cell shape, edge, color, and brightness) were recorded and the square root of the number of colonies counted for each morphotype was purified by streaking on new agar plates .
The phenotypic characterization of the bacterial colonies was performed using Gram staining, catalase and oxidase activities and motility tests . The pure cultures were stored in an ultra-freezer at -80 °C in the same broth culture media used for plating, containing 20% glycerol (w/w). Yeast colonies were characterized for morphology and biochemical assessments as described by . Filamentous fungi were observed with an optical microscope for preliminary identification. This was done by morphotype analysis of the colony, especially color and appearance using the proposals of Pitt and Hocking, 1997.
Step 5. Experimental Design
The composting process consisted of three treatments: control (without inoculation), noncommercial inoculum (NCI) and commercial inoculum (CI), with 6 replicates, totaling 18 composters. The composters were distributed in a completely randomized design (CRD) in the parcel scheme subdivided in time. The data obtained during the composting process (chemical and microbiological parameters) were analyzed using the statistical software Sisvar and the principal components analysis using the STATISTICA 7.0 software.