2.1. Biomass Sources: Animal Manure and Inoculum
Fresh pig manure (FM), selected as an example of animal slurries, was collected in an intensively rearing pig farm (Girona, Spain) five times for the experiment. FM samples were characterized and used as the influent of four PFR digesters by applying different dilutions with tap water to adjust the inlet total solid content, depending on the experimental conditions (see next section). Both fresh and diluted manure were periodically characterized;
Table 1 shows the characterization of FM samples. The net methane yield (L
CH4 kgVS
−1), at 273 K and 1013 hPa, and the biodegradability (% COD) were calculated through biochemical methane potential assays at 35 °C on FM samples [
35,
36]. Inoculum (5 gVS L
−1), bicarbonate (1g gCOD
−1), deionized water (to accomplish a 0.5 L of medium per vial), and FM (5 gCOD L
−1) were added to 1.2 L glass vials for 30 days at 35 °C. In parallel, controls were prepared to determine the residual biogas production of the inoculum. In order to calculate the methane production rate I and the lag phase (
λ) of the fresh manure (see
Table 1), the modified Gompertz Equation (1) was used [
37]:
The inoculum used for the start-up of the digesters and for the biochemical methane potential assays was sampled in a mesophilic CSTR digester of a municipal wastewater treatment plant (WWTP) in Barcelona (Spain).
2.2. Pilot-Scale Biogas Plant
A pilot-scale plant comprised of four horizontal PFRs (R1, R2, R3, and R4), a feeding system, a heating system, a biogas flow meter, and a programmable logic controller (PLC) to automate the plant equipment (timing and data acquisition system). All recorded data (temperature profiles of each reactor, biogas flows, inlet flows, etc.) were downloaded periodically as Excel files directly from the panel.
Figure 1 shows a scheme of the pilot plant.
Once collected, FM samples were stored at ambient temperature (10–40 °C) in a 1 m3 tank that was diluted with tap water daily just before the feeding of each digester. The weight of tap water and FM added to the dilution tank were registered by a weight cell BL-7 (Sensocar S.A., Terrassa, Spain). The diluted FM was stored in another 1 m3 tank, also at ambient temperature, which was periodically stirred with a waterproof pump GR BluePRO (Zenit Europe, Bascharage, Luxembourg) or a vertical rotor (dilute manure tank) during the storage.
Each PFR (2830 mm length, 646 mm width) consisted of a horizontally oriented U-shaped container of stainless steel and a methacrylate cover, which allowed periodic visual revisions of the inner material, with a working volume of 160 L (total volume of 235 L). Each PFR had its own external gas holder (flexible balloon of 100 L) located on the digester´s cover that, along with the gas headspace of the stainless tank, led to a total volume of 175 L per PFR for gas storage. A flowmeter (TG5, Ritter, Bochum, Germany) recorded the biogas flow, once the biogas passed through a silica filter. The biogas composition was measured off-line daily with a gas analyzer, equipped with electrochemical (H2S, O2) and dual-beam infrared (CH4, CO2) sensors BIOGAS5000 (Geotech Ltd., Conventry, UK). Each digester was heated with individual electric blankets For-Flex Super (Electricfor S.A., Rubí, Spain) and insulated with polyurethane boards. The working temperature was set up at 34 °C and monitored with three temperature probes Pt-100 PR-24-3-100-A-G1/4-6-150 (OMEG, Connecticut, USA) that were distributed regularly along the length of each reactor, controlled by PLC.
The biogas reinjection system was used between the feeding and effluent withdrawal operations. This system includes for each PFR, a silica filter, a gasholder, a compressor V-DTN16 (Elmo Rietschle, Gardner Denver Iberia S.L., Madrid, Spain), and inner 45 polyethylene gas diffusers of 8 mm diameter Tee quick connection (Ningmao Hydraulic Pneumatic Components Factory, Zhejiang, China), which were distributed along the digester floor, near the dividing wall. The gas pipes were made of polyamide. The diffuser distribution fitted well with recommended configurations reported in the literature [
38]. The reinjected biogas, introduced perpendicularly to the PFRs, generated turbulence to mix the reactor’s content. The stored biogas was compressed to attain a specific flow range of 0.270–0.336 m
3 m
−3 h
−1 and intermittently released (2 min h
−1 and 8 times d
−1), with the gas flow range being 4–5 m
3 h
−1. Safety valves were located along the cover of each digester in order to keep the internal pressure of each PFR ≤20 mbar (differential pressure).
Feeding, effluent recirculation and effluent withdrawal were done manually once per day, from Monday to Friday, dividing the loading equally over the 5 days. Daily flows were recorded directly in the PLC.
Sampling ports were located in the cover and in the bottom of the tank, allowing the collection of sludge samples from the initial, intermediate, and final points of the digesters. Influents and effluents of each PFR were characterized once per week by their content of total chemical oxygen demand (COD), total solids (TS), volatile solids (VS), total Kjeldahl nitrogen (TKN), and total ammonium nitrogen (TAN). Effluents were also characterized by pH, total and partial alkalinities (TA, PA), and volatile fatty acid (VFA) concentration once a week. All parameters were determined following standard methods [
39], except for COD, which was determined as per Noguerol-Arias et al. [
40] and VFA profile (acetic, propionic, i-butyric, n-butyric, i-valeric, n-valeric) which was determined by gas chromatography determined as per Rodríguez-Abalde et al. [
35].
2.3. Experimental Conditions
Table 2 shows the recorded experimental conditions, which are also shown in
Figure 2, in order to improve the understanding of the different conditions in each period and the dependence between the different experimental parameters (
inlet-TS,
HRT,
RR and
OLR) shown. The experiment was developed in four periods (P1, P2, P3, and P4), being evaluated in terms of
MY (L
CH4 kgVS
−1, expressed at 273 K and 1013 hPa) and organic matter removal efficiency (
VS removal). The length of each period was at least 2.5 × HRT or the minimum amount of time to achieve a “stable condition”, which was defined as that moment in which the biogas production and COD concentration in the effluent were inside 15% of the average value [
27].
Period P1 consisted in the start-up of all four digesters (R1, R2, R3, and R4), which were inoculated with 150 L inoculum obtained from a CSTR operating at a WWTP in Barcelona (Spain). Conservative conditions were established for 25 days. Different conditions were applied for each reactor in the next periods, P2 to P4, by changing
inlet-TS,
HRT, and
RR values. The
inlet-TS content was in the range of 3.0% and 7.0%, as the representative TS range of FMs. The
HRT was between 10 and 40 days, as these values were reported as the minimum for anaerobic digesters in the literature [
10,
12]. The
RR was fixed between 20% and 50% of the influent flow to ensure a stable process through the anaerobic biomass recirculation into the reactors.
Digesters R1 and R2 were used to evaluate the effect of changing the inlet flow and/or the inlet TS concentration. In this way, in R1 a progressive increase of the inlet flow and inlet-TS content through the periods were done, which increased the OLR and decreased the HRT from period P2 to P4.
In R2, the OLR was promptly doubled by pulse additions in each period. Two additions of dilute FM were done in period P2 reaching 2.5 kgVS m−3 d−1 these days. Three pulses of different VFA (acetic acid 30 g pulse−1, propionic acid 20 g pulse−1, or butyric acid 16 g pulse−1) were done in period P3 reaching 2.5 kgVS m−3 d−1 these days. Finally, three glucose additions (30 g pulse−1) in period P4 were done reaching 2.5 kgVS m−3 d−1 these days. These increases in OLR were performed to evaluate resilience of the different group of microorganisms involved in the anaerobic digestion process: FM pulses help to evaluate reactor response focusing mainly on hydrolytic and acidogenic bacteria, VFA pulses help to evaluate reactor response focusing on methanogenic bacteria, whereas glucose pulses help to evaluate reactor response focusing on acidogenic and acetogenic bacteria.
Digester R3 was a used to evaluate possible problems at full-scale plants, such as pumping failures or feed blockages. In this way, during period P2, both feeding and recirculation of the effluent were stopped for 1 week and RR was stopped for another week at the end of this period.
Finally, digester R4 was used as a control by maintaining stable conditions, with an
OLR of 1.6 kgVS m
−3 d
−1 and a
HRT of 24–36 days. The only parameter changed was the effluent
RR, varying from 50% to 25% depending on the experimental period (see
Table 2).
Periodically, a mass balance calculation was done in terms of total N and COD. For that purpose, an accumulation term was estimated based on total N balance (since total N concentration is conservative in AD processes assuming a constant growth of the involved microorganisms). This accumulation was included in the COD mass balance in order to calculate both methane and biogas production. In this regard, Kinyua et al. [
41] included N related compounds, such as struvite, and the presence of a dead volume inside tubular anaerobic digesters. Similarly, Jagadish et al. [
26] discovered a floating layer that remained during the entire fermentation period inside horizontal PFRs when digesting a chopped blend of herbaceous terrestrial weeds and leaf biomass.