Effect of Slurry Dilution, Structural Carbohydrates, and Exogenous Archaea Supply on In Vitro Anaerobe Fermentation and Methanogens Population of Swine Slurry

Artificial slurry (4% dry matter) was prepared using fresh feces and urine obtained from four pregnant sows fed with commercial diet. Freeze-dried dairy cattle feces were used (external archaea) as co-inoculum (Co-i) and structural carbohydrates (CHO: apple pulp; sugar beet pulp; and wheat straw) as substrates. Bottles were incubated (39 6 1oC for 56 days), and the gas production was measured (mbar) and converted to the volume. A sample of produced gas was taken and analyzed for methane concentration using a gas chromatography. Bottles were opened at days 0, 25, and 56 to determine total bacteria, total, and hydrogenotrophic methanogens archaea (HMA) concentrations using quantitative polymerase chain reaction and population biodiversity using denaturing gradient gel electrophoresis (DGGE). Incubation time reduced the titers of total bacteria and archaea (P < 0.01) but did not modify HMA population. Doses of Co-i showed a positive correlation with HMA titers, although interacted with an incubation period (P < 0.001); at 5% Coinoculation, total bacteria decreased significantly (0–25 days) but remained steady until day 56 (P > 0.05), whereas at 10% Co inoculation, titers decreased constantly. Most of the archaeal DGGE bands were observed in all samples, suggesting a common microbial population origin but Co-i supply altered the DGGE structure of archaea populations.


INTRODUCTION
air volumes at the same incubation temperature [12]. Gas volume at each incubation time was expressed per gram of incubated OM. After gas pressure measurements, a sample (0.1 mL) from head space gas was collected manually using Hamilton syringe, Gastight (1001SL1.0 mL SYR 22/2"/2 L, Hamilton Company, Reno, Nevada, USA) and immediately analyzed for CH4 concentration. Incubation bottles were opened at days 0, 25, and the two left bottles at day 56, pH was immediately determined (pH meter 2000 Crucible, Crucible Instruments. Barcelona, Spain), and 12 mL of the liquid media were weighed, frozen in liquid nitrogen, and stored at 280 o C for molecular analyses. The remained contents were filtered through a metal sieve (1 mm Ø) and two samples were taken for NH3-N (2 mL were acidified with 0.8 mL HCl 0.5 N) and VFAs determination (4 mL were mixed with preservative solution (1 mL of 2 g/L mercuric chloride, 20 mL/L of orthophosphoric acid, and 2 g/L of 4-methylvaleric acid in distilled water). Samples were immediately frozen (220 o C) until further analyses.

Chemical Analyses
DM content was determined using air oven at 105 o C until a constant sample weight. Ash content was determined by incineration of the samples using muffle furnace (550 o C for 4 h).
CP, crude fiber, and ether extracts were analyzed according to [14]. The NH3-N concentration of the media was measured by distillation-titration based on the Kjeldahl methodology [14]. Neutral detergent fiber and acid detergent fiber were analyzed according to [15] procedures. VFA concentrations were determined by gas chromatography (GC) based on the technique proposed by [16] using a gas chro-matograph (Agilent Technologies 7890A, Net Work GC System, Beijing Elmer, Boston, USA), equipped with a flame ionization detector and capillary column (BP21 30m 3 0.25 mm ID 3 0.25 lm).
CH4 was analyzed using the same GC equipment, equipped with different column (113 4332, GS-Gaspro capillary 30m 3 0.32 mm ID), operating at 70 o C for the column, 150 o C at the injector, and 200 o C at the detector.
The carrier gas was helium (99.999% purity [C50], Carburos Metalicos, Spain) and the total injection time was 2 min. CH4 concentration was calculated from the peak concentration: area ratio using as reference peak area generated from stand-ard gas (CH4; 99.995% purity [C45], Carburos Metalicos, Spain). Then different head space volumes of the standard mixture (0.1, 0.3, 0.5, 0.7, and 0.9 mL) were manually injected into the gas chromatograph to obtain the standard curve.

DNA extraction, DGGE, and Real-Time PCR Analyses
The DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen Ltd., West Sussex, UK) following the manufac-turer's instructions. For denaturing gradient gel electrophoresis (DGGE) analysis, specific Archaea region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the primers described by [17]. PCR amplification condi-tions were as follows: 1 cycle (94 o C for 4 min); 30 cycles (94 o C for 1 min, 55 o C for 1 min, 72 o C for 1 min); 1 cycle (72 o C for 7 min).

Calculations and Statistical Analysis
Pressure values (mbar) were transformed to the volume, by making standard curve using generated pressure against its known air volume, final equation was (y 5 11.46 21 3 20.289; R 2 5 0.9984) being (x, mbar) and (y, mL) pressure and gas volume, respectively.
Hence, there was no replication for VFA, NH3-N, and quantitative PCR analyses and then the replication was not considered as fixed effect in their statistical analyses model.
The copy numbers of total bacteria, total archaea, and hydro-genotrophic methanogens were transformed to their loga-rithm [log10] to perform the statistical analyses. The relative quantification of methanogens was carried out using DCt (DCt 5 Ct total bacteria 2 Ct MA). The mean separation between treatments was performed using the Tukey's test, and the differences were considered significant at P < 0.05.

Methodological Approach
Pig slurry is a mixture of excreta, including feces and urine together with water and some feed orts [25] stored in the animal's pit and bulked temporally in the pool. Slurry hydration comes mostly from urine but also, at different pro-portions, from water refusals, animal's cleaning or drinking losses. Moreover slurry storage periods are based on farm structure, management, and regulations imposed for their application in the field. Indeed, it results in a wide heterogeneity of slurry compositions and characteristics. As an example, slurry DM content (g DM/100 mL) in the Spanish commercial pig farms range from 1 to 12. Thus, in this work, we restricted the study on evolution of archaea titers during the anaerobic OM conversion to CH4 at specific slurry dilutions varying from 2 to 6% of DM.
Fresh pigs' feces were diluted with fresh urine up to 10% to simulate the normal feces/urine ratio and further diluted up to 6% (2, 4, and 6%) of DM using aforementioned buffer and tap water. To com-pensate the low C/N ratio of the slurry and thus to improve bacterial growth, media were supplemented with crop or agroindustrial by-products characterized by their high pro-portion of different type of structural carbohydrates. It was assumed that starch and other nonstructural sources were previously digested in the pig's intestine. The anaerobic digestion system used originated from the methodology pro-posed by Theodorou et al.
[12] designed to optimize anaero-bic degradation of substrate at 39 o C, by buffering the pH variation and using small amounts of substrate. Under such experimental approach, this article was made and discussed. Figure 1 shows the accumulated CH4 production through-out the incubation period (from 0 to 56 days) at different substrate and slurry dilutions in the incubation media (Figure 1a and 1b, respectively). The CH4 production was modified by slurry concentration (P < 0.001), However, the addition of SBP depressed both total (211 mL CH4/bottles) and production efficiency (88.48 mL CH4/ g OM) compared with blanks. Co-inoculation with cows' feces did not affect CH4 production.

CH4 Production and Incubation Media Characteristics
CH4 production at 56 days (mL/bottle) was directly related to the amount of available OM, but differences among treatments in relation to the production efficiency (mL/g OM fermented) were relevant. CH4 production (mL/g OM) was within the range reported by other authors which studied the similar conditions for both slurry concentration (4.3-13.6% DM) and incubation periods (up to 56 days) [26-28], although higher production has been reported in some literatures (290-490 mL/g OM; [2]). Differences in efficiency of production must be interpreted in terms of variation in the fermentation conditions and initial substrate [29].
During the incubation period, pH showed the pattern showed in Figure 2, which decreased at 25-days and increased at 56-days period (P < 0.001), although the variation was more pronounced in supplemented than in nonsupplemented media (blanks) in which pH reduction at 25-days period was negligible (interaction days 3 substrate: P < 0.01). A differential evolution among the substrates was observed with slurry dilution as pH decreased in SBP-supplemented bottles with 6% slurry concentration (6.9, 6.79, and 6.67). The opposite was observed in nonsupplemented bottles (blanks: 7.07, 7.27, and 7.33 for 2, 4, and 6% slurry DM concentration), whereas no significant variations were detected in those bottles supplemented with WS or AP (inter-action slurry dilution 3 substrate: P < 0.01). Type of sub-strate altered average media acidity (P < 0.01) and pH was lower in the pulps (pH 57.02 and 6.8 for AP and SBP, respectively) than in blanks bottles (7.23; SE: 0.04).
Ammonia-N concentration in the incubation media varied between 1.18 to 3.02 g/L and it was significantly modified by experimental treatment. Ammonia concentration increased throughout the incubation period in the highest slurry con-centration (2.57, 2.72, and 3.02 g/L in 6% DM slurry at 0, 25, and 56 days compared with 1.58, 1.92, and 2.11 g/L at 4% DM slurry, respectively). However, no changes were observed at the lowest concentration level of the slurry (1.56, 1.18, and 1.22 in 2% DM slurry at 0, 25, and 56 days incubation period, respectively; interaction slurry concentration 3 days: P > 0.01). The effect of slurry concentration on ammonia was also modulated by type of substrate; thus, for SBPsupplemented bottles and for blanks bottles, ammonia concentration increased proportionally with DM concentra-tion, but in those bottles supplemented with WS or AP, the increase was only observed when slurry concentration changed from 4 to 6% of DM.
CH4 production did not increase with the highest slurry DM concentration. Thereby, it was found that there was not a direct relationship between the OM concentration in the media and CH4 production efficiency, which agrees with pre-vious reports [30,31]. In this sense, it has been hypothesized that the absence of a direct relationship could be derived either from the presence of toxic substances in the slurry (i.e., lignin and its derivatives: [18,32] or because the incuba-tion media became inappropriate or toxic, for the acidity induced by the excess of VFAs [32] or on the contrarily, alka-linity induced by high ammonia loads [18]. The average of VFAs concentration (mM) in the original media (12.7) was increased (28.8) at day 25 and then decreased (6.1) at day 56 of the incubation period (Tables 3 and 4).
The differences among VFA concentration of different slurries during the incubation course did reach statistical significance (interaction; slurry concentration 3 days: P > 0.05).
The highest production (from day 0 to 25) was registered for the highest DM The high buffer capability of the slurry was able to com-pensate induced acidity by increases in VFA production in the case of blanks and bottles supplemented with WS, as pH level was kept within the appropriate range reported for anaerobic OM digestion (6.6-7.6; [33]). However, pH was below this threshold level and reached a critical value in those media supplemented with pulps, which explains the inhibition of the fermentation processes and the registered depression in CH4 production using pulps [34]. SBP is quickly fermented in the initial digestion process (days 1-10; [35] due to its high proportion of high digestible hemicellulo-ses and low proportion of lignin and cellulose