FACTORES DE LA INDUSTRIA

3.2.1 Materials

Eighty-three substrates were sampled and assessed. Criteria for selection included the most abundant and available substrates in Ireland. The substrates included first generation substrates (food crops) such as: beets, maize, cereal crops, oil seed rape, potatoes and turnips. Second generation substrates included grass silage, agri-food waste streams, and agricultural and municipal wastes. Third generation substrates included marine biomass such as green seaweed (U. lactuca) and brown seaweeds (Saccharina latissima, Laminaria digitata and Ascophylum nodosum). Various pretreatments were assessed such as drying, ensiling and macerating to highlight differences in potential methane yields.

Specific substrates such as dairy slurry, food waste and grass silage were examined in closer detail due to their abundance and the variety of sub-streams. Nine different slurries were sampled as well as poultry manure and farmyard manure. Ten variations of grass silage were sampled. These represent the various methods Irish farmers used to preserve and cut silage and include baling, ensiling, hay and 1st (early and late stage cuts) and 2nd cuts of silage. Eleven variants of food wastes were sampled. Milk processing wastes (MPW) and abattoir wastes were also sampled as various wastes are produced from these processes. Appendix A and B in supplementary data includes for a full list of substrates.

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3.2.2.1 Proximate and analytical methods

Each sample was analysed initially for total solids (TS) content and volatile solid (VS) content by drying to 105oC for 24 hours and again placing samples in a furnace heated to 550oC for 6 hours as described by APHA standards [9]. To determine a theoretical yield for each sample the Carbon, Nitrogen, Hydrogen and Oxygen percentage of each sample (<1mm particle size) was obtained from a dried sample of each substrate by a CE 440 elemental analyser in triplicate. The C:N ratio was also observed from the elemental analysis. To facilitate in the completion of successful BMP assays and to ensure homogeneity amongst each substrate, all samples were macerated to 5mm particle size by a lab scale macerator. Liquid samples were mixed by a lab scale blender prior to all analysis tests. Liquid samples were tested for soluble chemical oxygen demand (SCOD) using Hach Lange CLK 914 cuvettes. pH was obtained using a Jenway 3510 pH meter.

The biodegradability index (ratio of BMP assay yield to theoretical yield) was calculated for all substrates. This indicates the level of VS destruction of the substrate over 30 days.

3.2.2.2 BMP procedure

The same BMP apparatus was used on all of the substrates to ensure repeatability of results. Two Bioprocess AMPST® II units were used in tandem: all samples were run in triplicate. Inoculum and cellulose controls were conducted for each run; this allowed for 8 substrates to be analysed for each run of the BMP system. An inoculum to substrate ratio (I:S) on a VS basis, of 2:1 was chosen for these trials [10]. Nitrogen was used to flush the head space of the 400 ml reactors prior to commencing each BMP assay. The mixing system alternated between on and off for 60 seconds at 30 rpm. Reactor vessels were placed in a water bath and heated to 37oC for the duration of the experiment. The biogas produced was then passed through a solution of 3M NaOH to remove CO2,

H2S and other gaseous impurities. An electronic gas-tipping device recorded the volume of

biomethane, which was produced from each reactor vessel. The total biomethane produced from the inoculum was averaged and subtracted from the volume of biomethane produced by the individual substrate, to determine the specific methane yield of the substrate. Automatic adjustment

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was completed for standard temperature and pressure for all results. Overestimation in the flush gas was corrected for also by the AMPTS II system. In order to allow for the numerous substrates the inoculum was chosen from two different sources. One half was from a continuous lab scale reactor, which was fed grass silage, dairy slurry and macro algae. The other half of the inoculum came from the previous BMP assay run. Both inoculum sources were mixed to a homogeneous state and analysed for solids composition.

3.2.2.3 Statistical analysis

To compare a run of 8 BMP substrates to another run of 8 other substrates, the method of analysis of variance (ANOVA) was used to determine the influence of the substrate on biochemical yield. The BMP run was regarded as the block effect and the substrate as the main effect for the ANOVA test. The test procedure SIMULATE was used from the statistical analysis programme SAS 9.3. A significance of differences in methane yields between substrates was determined by multiple comparisons, with a significance level, α (set at p < 0.05).

3.2.2.4 Kinetic analysis

Kinetic analysis was performed on the cumulative production curves produced from each BMP assay. Kinetic modelling was used to create an insight into the biodegradability of each substrate. It is also used as a method of physical comparison for the cellulose BMP assays from each run. This can help to illustrate any differences between BMP runs and highlight any outlying results if kinetic values deviate from average results. A first order differential equation was used to determine the decay constant value, k (Eqn. 3.1). The modified Gompertz formula (Eqn. 3.2) was used to determine the remaining kinetic biodegradability values associated with BMP assays.

= . 1 − exp Eqn. 3.1

= ∙ exp{ − exp[ ∙ ∆ − ] + 1} Eqn. 3.2

Y(t) is the cumulative biomethane yield (L CH4 kg-1 VS) at a digestion time t (days). Ym is the maximum

biomethane potential (L CH4 kg-1 VS) of the substrate added. k the decay constant (days-1). k is a

measure of the rate that the substrate has been degraded. M(t) is the cumulative biomethane yield (L CH4 kg-1 VS) at a given time t (days). P is the maximum biomethane potential (L CH4 kg-1 VS) of the

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substrate from the BMP test. Rmax is the maximum biomethane production rate (L CH4 kg-1 VSd-1). Δ

the lag phase is a measure of how long it takes (days) before biochemical methane production starts to occur. t is the time (days). T50 is the half-life (days) and is a measure of how long it takes to

produce half of the maximum cumulative yield of biomethane. R2 _{is a measure of how the kinetic}

equation model fits the curve of biomethane production (%).

3.2.2.5 Theoretical yields

Data for elemental compositions collected on each substrate was used to create theoretical methane yields using the Buswell equation (Eqn. 3.3). The Buswell equation is a method to determine the maximum BMP yield of substrates by converting all available VS to methane and carbon dioxide. This stoichiometric equation however assumes all donated electrons are used entirely for metabolic energy, which does not allow for the development of the anaerobic bacteria or losses within the system. This lends itself to an overestimation in the theoretical yield. A ceiling value however is established for the chosen substrates and a biodegradability index (BMPassay/

BMPBuswell) can be established which can indicate how well a substrate can be degraded.

"# $%&'+ () − %_*−'₊, $+& → (#₊+ %_.−'_*, "$* + (#₊− %_.+'_*, "&+ Eqn. 3.3