Substrates and inoculum

The organic fraction of municipal solid waste (OFMSW) was obtained from food processing establishments in Iztapalapa, in Mexico City (Mexico). The stable compost of the mixed municipal solid waste composting plant of the Bordo Poniente of Mexico City (CPBP) was used as a source of microbial consortia (Mc) as an inoculum. The taking of the compost of the CPBP was carried out in accordance with the standard ^{ASTM D-34 (2016)}.

Experimental strategy

The degradation of OFMSW was carried out in four tubular bioreactors (TB) with capacities of 0.98, 1.91 and 3.84 L. The TBs were packed with a wet-based mixture of: OFMSW 83%, microbial consortium 8%, sawdust 4%, paper 3% and pruning waste 2% to achieve an initial C/N ratio of 30 (^{Martínez-Valdez et al., 2015}). The initial moisture was 70% (^{Liang et al., 2003}). During aerobic degradation, aeration rates increased by a proportion of 0.5 in the different experiments (0.032, 0.064, 0.125, 0.251 and 0.392 vkgm). The respirometry system was operated according to ^{Saucedo-Castañeda et al. (2013}); ^{Torres-Mancera et al. (2018}). The calibration of the respirometry equipment was done with standard mixtures of CO₂ y O₂.

Identification of microorganisms

All TBs were inoculated (CPBP compost) and tests were performed at 35 °C due to the increased microbial activity (shown by respirometry) at that temperature (^{Martínez-Valdez et al., 2015}). Once the process was completed, the TBs were unpacked, the material was weighed on an analytical balance (Ohaus Galaxy^® 200) and the moisture, pH and enzymatic activity were determined. All samples were obtained at the time of maximum CO₂ generation rate, as this indicated the highest microbial activity.

The analysis of the microbial population was performed using the technique of denaturing gradient gel electrophoresis (DGGE): samples were taken under all conditions and centrifuged at 8 049.6 g. The precipitate (1 g) was taken, and the DNA extraction and purification was carried out using the PowerSoil^® DNA isolation kit (MoBio). Once the extraction and purification were done a 1% agarose gel was run to corroborate the success of the DNA extraction. Subsequently, a polymerase chain reaction (PCR) was performed to obtain in vitro copies of a fragment of the sequence of the 16S rRNA gene from each of the samples, which was carried out in a thermocycler (Multigene, Labnet International, Inc.).

The reaction mixture, with a final volume of 50 μl, contains 10 X of buffer, 1.5 mmol of MgCl₂, 200 μm of each dNTP (Promega Corporation, USA), 10 pM of each primer, 1.5 U of GoTaq^® Flexi DNA Polymerase (Promega Corporation, USA) and 1-20 ng μl^-1 of DNA template. The amplification conditions were as follows: 2 min at 95 ºC of initial denaturation, 25 cycles consisting of 30 s at 95 ºC, 30 seconds at 50 ºC and 1 min at 72 ºC, followed by a final extension of 5 min at 72 ºC. The amplicons obtained were stored at 4 ºC after running by electrophoresis (100 V, 30 min) in a 1% agarose gel. After amplification of the 16S rRNA gene sequence, a new nested amplification was made using the previously obtained amplicon as a DNA template.

The primers 1070F (5’ ATG GCT GTC GTC AGC T 3’) and 1392R+GC (5’ CGC CCG CCG CGC CGC CCC GCC GCC GCC GCC GCC CCG CCC CCC ACG GGC GGR GRG TAC 3’) were used using the same amplification conditions by changing the alignment temperature to 55°C. The samples were purified with a commercial kit (Wizard^® SV Gel and PCR Clean-up System, Promega Corporation) following the protocol described by the supplier. The quantification of DNA nucleic acids was measured with the NanoDrop ND1000 spectrophotometer (Thermo Scientific). There was 1 μl with 500 ng of DNA from each sample added to 5 μl of loading buffer (0.25% bromophenol blue; 0.25% xylene cyanol; 30% glycerol, in water).

Amplicon profiles were analyzed with the universal mutation detection system DCode (Bio-Rad Laboratories, Hercules, CA) using acrylamide gels of 16 cm x 16 cm, using a 7% acrylamide gel with a denaturation gradient of 30-60%, with a ratio of (37.5:1), which were filtered at 0.45 μm. To do this, two solutions were prepared for the elaboration of the degradation gel from 30 to 60%. The 30% solution was prepared using 12.5 ml of 40% acrylamide/Bis, 2 ml of TAE 50X, 12 ml of formamide and 12.6 g of urea Molecular Biology grade (Sigma-Aldrich) and 60.9 ml of milli Q water per 100 ml. The 60% solution contained 12.5 ml 40% acrylamide/Bis, 2 ml of TAE 50X, 24 ml of formamide, 25.2 g of urea and 36.3 ml of milli Q water per 100 ml.

Both solutions were filtered through a 0.45 μm filter and degassed. The formation of the gel was carried out by the BioRad 475 gradient former, which was loaded with 20 ml of denaturing solution, 70 μl of 10% persulfate and 10 μl of TEMED. Gel electrophoresis was performed for 16 hours in TAE buffer (0.5 X, pH 8) at 60 °C using a PowerPac universal power supply (Bio-Rad), with an initial voltage of 200 V for 10 min, then the voltage was reduced at 85 V for 16 h (^{Neilson et al., 2013}). The gel was stained according to the technique described by ^{Radojkovic and Kušic (2000}). The staining procedure consisted of an initial pre-stain fixation for 3 min in 10% ethanol, 0.5% acetic acid.

Staining for 5 min in fixation solution plus 0.2% silver nitrate, the washing of the gel in water for 2 min, and development for about 5 min in 30% NaOH and 0.1% formaldehyde in Milli-Q water. Once the gel was run, the identified bands were cut, and a 5 min extraction was performed. With water and stir. A 30% agarose gel (80 V, 45 min) was applied to corroborate the presence of DNA and then sequencing was performed by Macrogen.

Evolutionary history was deduced using the maximum likelihood method based on the ^{Kimura 2-parameters model (Kimura, 1980}), previously selected 8 by a Bayesian information criterion. The tree with the highest probability of trunk (-1499) is shown. The percentage of trees in which the associated taxa were grouped is shown next to the branches.

The initial trees for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with the superior log likelihood value. The tree is drawn to scale, with the lengths of the branches measured in the number of substitutions per site. The analysis used 32 nucleotide sequences. All positions containing gaps and missing data were removed. There were 239 positions in the final dataset. Evolutionary analyses were performed in MEGA7 (^{Kumar et al., 2016}).

Measurement of enzymatic activity

The activities of the extracellular enzymes, pectinase, cellulase and xylanase, were quantified by the release of reducing sugars. Reactions were carried out in a citrate buffer (50 mmol, pH 5.5). Pectinase activity was measured using 1% birch pectin incubated at 37 °C for 30 minutes (^{Zhang et al., 2000}). Cellulase activity was quantified using 2% carboxymethylcellulose incubated at 50°C for 30 minutes (^{Ghose, 1987}).

Xylanase activity was measured using 1% xylan from birch wood incubated at 50 °C for 15 minutes (^{Loera and Córdova, 2003}). All reaction volumes were: 750 ml of substrate with 250 ml of enzymatic extract, mixed in an orbital stirring of 250 rpm. The solutions were measured at a wavelength of 540 nm with a Beckman^® DU 640 spectrophotometers.

Protease activity was determined using a modified method from ^{Alef and Nannipieri (1995}). An aliquot of 1 ml of enzymatic extract was added to 5 ml of 2% casein solution and incubated at 50 °C under stirring for 2 h. Enzymatic activities were expressed as U per gram of dry matter, where 1 unit (U) is the amount of enzyme that catalyzes the generation of 1 μmol of product per minute under enzymatic reaction conditions.

Statistical analysis

All measurements and tests were performed in triplicate. A multi-variable analysis (α= 0.05) and the principal components analysis were performed using Statgraphics^® Centurion XVII.

Effect of aeration during composting

To analyze the effect of the aeration rate (vkgm), the CO₂ content (v/v) produced under all conditions was evaluated (Figure 1). CO₂ production was 31, 20, 14, 9, and 2% at 0.032, 0.064, 0.125, 0.251, and 0.392 vkgm, respectively. A principal component analysis (PCA) is shown in Figure 2a. The PCA showed that a main group containing cumulative CR, instantaneous CR, pectinase and CO₂ as grouped variables, which are associated with each other, and another main group contained reducing sugars, xylanase and cellulase (Figure 2a). In addition, it was observed that vkgm and protease are negatively correlated with pectinase, CO₂ and CR.

Figure 1 Concentration of a) CO₂; and b) O₂ at the gaseous outlet during the aerobic degradation of OFMSW at different aeration rates (vkgm) ■ 0.032, □ 0.064, ▲ 0.125, ∆ 0.201 and ● 0.392). 

Figure 2 a) principal component analysis (PCA); and b) correlation matrix and Pearson’s correlation coefficients between aeration rate, physicochemical, respirometric and enzymatic responses determined. 

The analysis showed that a 3-component model explains 96.34% of the variability and a 2-component model explains 76%. A dispersion matrix and Pearson’s correlation coefficients between each pair of variables are included in Figure 2b. It was observed that the aeration rate (vkgm) has a significant negative correlation with respect to pectinase activity (-0.86) and with respirometric activity responses such as CO₂ content (-0.91), instantaneous CR (-0.82) and cumulative CR (-0.84). on the other hand, vkgm has a significant positive correlation with protease activity (0.97).

In addition, the high activity of proteases indicates that soluble proteins are degraded by proteases, and it could be a consequence of pectinase activity being reduced as a result. Protease activity has a significant positive correlation with cellulase activity (0.41). These observations were also reported by ^{Goyal et al. (2005}); ^{He et al., (2013}), who observed an increase in protease and cellulase activity during the active phase of composting. ^{Kayikçioğlu et al. (2011}) reported that the maximum protease activity coincided with the maximum microbial activity.

The PCA revealed that pectinase has a significant positive correlation with the respirometric indices, namely CO₂ (0.89), instantaneous CR (0.85) and cumulative CR (0.87) and a negative correlation with vkgm (-0.86). Meanwhile, proteases have a significant negative correlation with respirometric indices such as CO₂ (-0.93), instantaneous CR (-0.86) and cumulative CR (-0.85) and a significant positive correlation with vkgm (0.97).

The above confirms that protease is correlated with aerobic conditions, while pectinase is only present in anaerobic conditions. ^{Puyuelo et al. (2011}); ^{Xu et al. (2012}) have pointed out that the increase in enzymatic activity depends on the microbial consortia present and the concentration of microorganisms. Therefore, the change in protease and pectinase activity could indicate changes in microbial consortia during the process.

Microorganisms present in different aeration conditions

The identification of bacteria (Figure 3) Citrobacter freundi, Klebsiella oxytoca can be found. Klebsiella oxytoca has been reported as a high producer of cellulose enzymes (^{Golias et al., 2002}). In low aeration rates (0.032, 0.064 and 0.125 vkgm), the presence of Enterobacteria has been revealed, which is related to low protease and high pectinase activities (i.e., the prevalence of semi-anaerobic environments). These bacteria are chemoorganotrophic with both respiratory and fermentative metabolism. Most are negative oxidases, except for Plesiomonas. Most reduce nitrate to nitrite, except for fermentations of Saccharobacter and some strains of Erwinia and Yersinia (^{Brenner and Farmer, 2005}).

Some strains, such as E. cloacae, E. hafniae or E. aerogenes are producers of lipase. On the other hand, Buttiauxella sp. is known as a phytase producer and a facultative anaerobe of the Enterobacteriacea family (^{Mitra et al., 2010}). In the condition with the highest rate of aeration tested (0.392 and 0.251 vkgm), Acinetobacter bacteria were the most representative, being strictly aerobic with positive catalase and negative oxidase activity. Most strains can grow in a simple minimum medium with a single carbon source and an incubation temperature between 20-30 ºC.

^{Lewin et al. (2016}); ^{Wang et al. (2016}) mentioned that Actinobacter contributes to the global carbon cycle through the decomposition of plant biomass. Its enzymes may contribute to the industrial-scale decomposition of cellulosic plant biomass into simple sugars that could be converted into biofuels. Actinobacter hosts a substantially broad catalog of cellobiohydrolase, β-glucosidase, acetyl xylan esterase, arabinofuranosidase, pectin lyase, and ligninase genes.

Figure 3 Molecular phylogenetic analysis by the maximum probability method. The experimental conditions used were: Inoculation by composting of CPBP, temperature set at 35 °C, initial moisture 70% (wb), pH 6, various aeration rates, namely 0.032, 0.064, 0.125, 0.251 and 0.392 vkgm. All samples were obtained at the time of maximum CO2 generation rate.