Material and methods

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Inoculum was sampled from two continuous anaerobic reactors at steady state (see section 2.1. below). After collection, the inoculums were placed in batch mesophilic anaerobic reactors in which the methane production rate (MPR) was continuously monitored (see section 2.2). To address the issue of initial biomass concentration, inoculum reactors were simulated using the ADM1 to pre-determine the initial conditions required for simulation of batch reactors. The batch reactors were pulsed with specific substrates corresponding to key intermediate steps (see section 2.3.) and the induced MPR was monitored until substrate degradation ended. The batch systems were simulated using the ADM1 with the default parameter set. Finally, the parameters were modified to achieve optimal fit with the measured data (2.4.).

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2.1. Anaerobic sludge sampling and associated CSRT digesters

WAS acclimatised inoculum was sampled from a CSTR fed with WAS and run with a hydraulic retention time (HRT) of 24 days. The organic loading rate (OLR) was 3.8 kgCOD.m-3.d-1 (feed COD concentration of 75.7gO2.kg-1) and, considering the total COD balance of the reactor, the calculated OLR on a biodegradable COD basis was 1.3 kgCODbiodegradable.m-3.d-1 (pH=7.4 ; [N-NH4+]=1.4gN.L-1). The working volume of the reactor was 200 L and the temperature was maintained at 36 °C. PS acclimatised inoculum was taken from a CSTR fed with a mixture of pig slurry (44% of the total COD) and horse feed (56% of the total COD) mainly consisting of a mixture of wheat gluten feed, oats, straw and sunflower cake. HRT was 27 days and OLR was 3.9 kgCOD.m-3.d-1 (feed COD concentration of 106.3gO2.kg-1). On a biodegradable COD basis, the OLR was 2.0 kgCODbiodegradable.m-3.d-1 (pH=7.8; [N-NH4+

]=3.2gN.L-1). The temperature of this reactor was 38 °C and its working volume was 87 L. Both reactors were at steady-state conditions during this study and were operated at least for three times their HRT before the first samples were taken.

2.2. Experiments

2.2.1. Description of the batch device

For the batch experiments, 10 identical reactors with a 1 L working volume were used.

They were continuously mixed by a magnetic stirrer (1200 rpm) and maintained at 37 °C using a specific chamber (Aqualytic, ET637-6, Germany). Each reactor was equipped with a manometer (Vegabar 14, Vega, Germany) and a solenoid valve which allowed continuous monitoring of biogas production. Gas production was calculated by correcting for temperature, the headspace volume of the reactor (between 515 and 550 ml), and pressure measurements. Pressure was automatically released at an overpressure of 50 mbar into a Tedlar Bag® (SKC, 232-01, USA) with 1 L capacity. The biogas composition was regularly determined in terms of CH4 and CO2 contents with a gas chromatograph equipped with a flame ionization detector (Agilent Technologies 6890N, USA) according to the method described in Lucas et al. (2007).

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2.2.2. Batch Experiments

The viscosity of the WAS acclimated inoculum was relatively high, which made stirring difficult. WAS acclimatised inoculum was consequently diluted (1/1) with the centrifuge supernatant. No dilution was required for PS acclimated inoculum. After one day of stabilisation in the batch reactors, substrate pulsing was started. The amount of substrate added was adjusted according to the biodegradable OLR of the CSTRs from which the inoculums were sampled. The dilution factor was taken into account meaning differences in the biomass concentration of the two inoculums were also taken into account. Assessing these factors, taking into account the significant minimal substrate addition and considering references in the literature (Batstone et al, 2003; Pind et al., 2003; Girault et al., 2011), the amounts of substrate added were 1.5gCOD.Linoculum-1

for WAS acclimated inoculum and 3gCOD.Linoculum-1

for PS acclimated inoculum.

Immediately after reactor was opened, the headspace was purged with a gas mixture of N2 and CO2 (70/30). Control experiments were conducted without the substrate pulse. MPRs in the reactors were monitored until MPR receded to the background level observed in the control reactor.

Details on substrate additions are as given in section 2.3. Repeatability had previously been tested between the 10 batch reactors in parallel with an acetate pulse (results not shown).

The relative standard deviation for MPR was below 5% except in the first and the last hours of the pulse, when a slight time lag was observed. That is consistent with those of Raposo et al. (2011) who reported satisfactory repeatability of measurement of the anaerobic biodegradability of standard substrates. Consequently, substrate additions were generally not done in replicate.

The impact of the amount of substrate added on the calibrated parameter values was investigated on PS acclimated inoculum during acetotrophic methanogenesis. Hence, one additional acetate addition of 1.5gCOD.Linoculum-1 was made in this inoculum

Temporal variability was also investigated on WAS and PS acclimated inoculums for acetotrophic methanogenesis. For each inoculum, acetate additions in batch experiments were replicated several months after the first ones. CSTRs in which the inoculums were sampled were kept in the same steady state conditions between each inoculum sampling. Nevertheless, temporal variations were observed especially due to slight variations in the influent and to biological fluctuations.

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2.2.3. Specific substrates added

Tested substrates were chosen as characteristic of the groups of biomass defined in the ADM1 as well as in other mechanistic anaerobic digestion models (Siegrist et al., 2002) and degradation pathway concepts (Pavlostathis and Giraldo Gomez; 1991). The substrates we used were consistent with those proposed by Angelidaki et al. (2009) to test inoculums for their biomethane potential. As such, the substrates used for this study were amorphous cellulose (for polysaccharide hydrolysis), casein (for protein hydrolysis) and triolein (for lipid hydrolysis, purity >60% completed with other triglycerides), glucose (for sugar acidogenesis), oleate (for LCFA acidogenesis, purity between 65 and 88% completed with other long chain fatty acids), a mixture of amino acids resulting from casein hydrolysis (for amino-acid acidogenesis), propionate (for acetogenesis), acetate (specific substrate for acetotrophic methanogenesis). Hydrogenotrophic methanogenesis was not investigated because of difficulties in managing the gas phase (and uncertainties concerning the actual substrate concentration).

2.3. Modelling of batch experiments

The model used to simulate the major biochemical and physicochemical processes occurring during anaerobic digestion was ADM1 (Batstone et al., 2002). The ADM1 was implemented in Scilab® and solved with the ordinary differential equation solver “ode”

(package ODEPACK, solver Isoda). Default parameters were used like for mesophilic systems (Batstone et al., 2002) and decay rate values were thus fixed at 0.02 day-1.

Concerning the concentration of each biomass, the initial states for batch experiments were based on ADM1 simulations of the CSTRs running at steady state. Inputs and effluents for the continuous reactors were fractionated into lipids (Soxhlet extraction of hexane extractable materials), proteins (organic nitrogen converted by 6.25 gCOD gN-1), and saccharides (total COD the balance). The inert fraction was estimated on CSTR results to accurately simulate COD degradation. Biodegradable COD was split into proteins, polysaccharides and lipids according to the biochemical fractionation of the COD of the influent. The steady state resulting from this simulation was used as the initial condition for the inoculum. Specific substrates were added to the corresponding state variable to simulate addition of the substrate.

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Before modelling batch experiments with substrate addition, it was necessary to accurately simulate the MPR in the control experiments without substrate addition. Indeed, the MPR due to residual gas production of biomass was significant in the batch tests (between 25 and 50% of the maximum MPR observed with substrate pulses). As a consequence, at the beginning of the tests, the MPR was strongly dependent on total COD degradation resulting from degradation of the added substrate and residual degradation of the anaerobic sludge.

Hence, the MPR measured in control batch experiments was simulated by optimising the initial concentration of composite materials (Xc) in the inoculum and the related disintegration constant (kdis). The obtained Xc and kdis values were kept when modelling the batch experiments with substrate addition. In the results, MPR of the control test was subtracted to remove residual MPR due to degradation of the inoculum.

2.4. Parameter estimation

For each stage, the maximum growth rate (km) and half-saturation constant (Ks) were optimised simultaneously. To this end, automatic parameter optimisation was done using the Simplex method (Nelder and Mead, 1965), to minimise the objective function (J= sum of squared errors). Parameter uncertainty and correlation were assessed as in Batstone et al.

(2003) using a confidence interval of 95%, and applying an F distribution to the parameter space. The parameter surface is defined by a critical objective function (Jcrit) which is related to the optimal J (Jmin) as found by the Simplex optimisation as follows:





 ×

+ −

= pN −p

data

crit F data

p N

J p

J min 1 0.05, ,

where p is the number of calibrated parameters, Ndata is the number of data points and

p N p data

F0.05, , is the F distribution value for a confidence of 95%, p parameters, and Ndata-p degrees of freedom.

The parameter space that describes the experiment with the defined confidence interval is described within the region where J<Jcrit.

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Parameter estimation was performed from acetotrophic methanogenesis to acidogenesis. In each case, previously calibrated parameters related to downstream stages were used for simulations.

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