LANZAMIENTO DE LA REPRESENTACION DE DEMEYERE EN ECUADOR

The Bioprocess™ automatic methane potential test system (AMPTS) was used to carry out BMP assays in triplicate on the selected substrates, as well as a cellulose standard (Sigma Aldrich, CAS Number: 9004-34-6) and an inoculum control. Each bottle had a 400 mL working volume with 250 mL of headspace. The bottle contents were individually mixed by stirrers at 30 rpm and operated every other minute. Temperature for all bottles was held constant at 37°C by means of a large heated water bath. A calculated quantity of each substrate and inoculum was initially added to the bottles corresponding to a 2:1 inoculum-to-substrate ratio which is

recommended to overcome any problems with process inhibition (Chynoweth et al., 1993). Distilled water was added to bring the content level in the bottle to 400 mL and the headspace was flushed with nitrogen prior to start-up to ensure anaerobic conditions. A 3 M sodium hydroxide (NaOH) solution was used to remove carbon dioxide and other trace gases from the biogas produced. The resultant methane was

sent to a flow measurement device which measured gas through water displacement. Pressure and temperature were recorded continuously and gas yields were logged on a bespoke software package. Seven BMP assays (in triplicate) ran for a period of 30 days and included for: (1) mono-digestion of >3 cm grass silage (25 g wwt.); (2) mono-digestion of <1 cm grass silage (25 g wwt.); (3) inoculum; (4) mono-digestion of >3 cm grass silage (25 g wwt.) with rumen fluid addition (1.25 mL); (5) mono- digestion of <1 cm grass silage (25 g wwt.) with rumen fluid addition (1.25 mL); (6) Inoculum with rumen fluid (1.25 mL) (corresponding to 50 mL per kg silage as in 4 and 5); (7) cellulose.

6.2.4 Semi-continuous trials

Two identical continuously stirred tank reactors (CSTRs) were run for a period of 30 weeks. The cylindrical PVC pipe reactors operated with a 4 L working volume and a 1 L headspace. Temperature was controlled at mesophilic range (37 ± 1°C) by a heated water circulator which pumped water through brass coils around both

reactors. Mixing was provided by vertically mounted stirrers attached to 24 V direct- current motors. Feeding was performed through an inlet port at the top of the reactor that was otherwise sealed by a rubber bung. The removal of digestate was achieved through an outlet port at the bottom of the reactor. The production of biogas was measured by wet-tip gas meters that were individually calibrated (50-55 mL/tip). The counting of gas tips was recorded by a data logger. All gas data were corrected for standard pressure and temperature.

The CSTRs operated with four permutations in total. These permutations included for both particle sizes and rumen fluid addition. Reactor R1 was used in mono- digestion of >3 cm grass silage (Week 1–18) and mono-digestion of <1 cm grass

silage (Week 19–30). Reactor R2 was used in mono-digestion of >3 cm grass silage with rumen fluid addition (Week 1–18) and mono-digestion of <1 cm grass silage with rumen fluid addition (Week 19–30). The organic loading rate (OLR) was held constant at 2.5 kg VS m-3 d-1 for the duration of the trial (30 weeks) for both R1 and R2. This equated to an effective HRT of 31 days allowing for daily recirculation of effluent liquor (< 25 g DS kg-1) separated from the digestate. The addition of rumen fluid was executed by adding ca. 3 mL daily to the grass silage input feed, which corresponded to the daily input equivalent of 50 mL per kg silage.

The inocula for the BMP and CSTR trials were sourced from laboratory scale digesters operating on grass silage co-digested with dairy slurry. The inoculum was sieved through a 1 mm mesh to remove any larger particles and acclimatised by heating at 39°C for the week prior to start-up.

6.2.5 Analytical methods

For weekly calculations of FOS/TAC (volatile organic acids/total inorganic carbon), the Nordmann-method was used with 0.1 N sulphuric acid (Nordmann, 1977). A Titronic Universal Automatic titrator was used to perform a two-point titration (endpoints at pH 5.0 and pH 4.4). FOS/TAC values of below 0.300 indicate stable fermentation (Drosg, 2013). pH was measured daily using a Jenway 3510 pH meter. DS and VS analyses were determined according to Standard Methods 2540 G (APHA, 2005). Chemical oxygen demand (COD) and total ammonical nitrogen (TAN) were determined weekly using Hach Lange cuvette tests (LCK 914 and LCK 313, respectively) and evaluated by a DR3900 Hach Lange Spectrophotometer. Biogas samples from the continuous trials were collected three times per week from the outlet of the wet-tip gas meters using 1 L Tedlar gas bags. Analysis of the biogas

composition (% CH4 v/v) was measured by an Agilent 6890 GC with thermal conductivity detector. For evaluation of DSD and NDF of the grass silage, samples were dried at 40°C for 48 h and subsequently ground using a Wiley hammer mill with a 1 mm pore screen. DSD was evaluated by the Tilley and Terry (1963) method but with final residue isolated by filtration (Whatman GF/A 55 mm, pore size 1.6 µm, Whatman International) instead of centrifugation. NDF, assayed with a heat stable amylase and sodium sulphite, concentrations were evaluated using the

ANKOM filter bag technique (ANKOM, 2006a; ANKOM, 2006b) with reference to the analytical method of Van Soest et al. (1991), and expressed on an ash-free basis. VFA concentrations were measured using an Agilent 6890 GC with flame ionisation detector as described in Allen et al. (2014). VFA analysis was performed on an almost weekly basis for the CSTR trials, with samples prepared by centrifuging for 10 minutes at 15,000 rpm with 0.2 mL HPO3 added to remove any particulate matter.