Analytical methods

Sampling

The collection and the analysis of gas composition during anaerobic processes was made directly from the Tedlar sampling bags, evaluating the general composition of biogas emissions. While gas circulation is active (during methane production phases and aeration), results of this methodology are consistent with a direct sampling from interstitial pores. On the contrary, if gas flow is negligible (during semi-aerobic phases and anaerobic aftercare), analysis on Tedlar bag gas can be slightly affected by possible intrusion of oxygen from the connection pipes. In this case, for avoiding uncertainty, sampling was made directly form the headspace of reactor.

Leachate sampling was always made from the collection tanks (Figure 1.3.8, C). If the reactor management comprises a daily leachate recirculation, sampling was made immediately after the recirculation and prior to the new fresh distilled water injection.

The solid waste sampling was a tricky operation, affecting consistently the analytical results obtained. Take samples only from the upper part of reactors is never suitable because the extracted material will not be representative for the whole waste. Moreover, the small dimensions of the lab scale equipment (Figure 1.3.8, B) will probably complicate the operation. Opening the reactor with ongoing processes can consistently disturb the processes itself so this operation must be avoided, if possible. For the experiments carried out in the following chapters, solid waste samples was taken at the beginning and at the end of the test and managed with the same methodology.

The initial solid waste prior to be loaded into reactors and the treated waste, fully extracted at the end of the test, was disposed in a tank and manually mixed (Figure 1.3.8, A).

After that, for obtaining a homogeneous sample with dimensions consistent with lab-scale analytical methods, 2-3 kg of the material were milled at 10 mm and mixed again. The milling procedure at 10 mm does not change chemical characteristics of the sample or its humidity.

The produced material constituted the final matrix to be used for the analysis.

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Figure 1.3.8: Waste samples extracted from a lab-scale reactor, after methane production end (A). Internal view of a columnar reactor at the end of the test (B). Leachate samples collected (C).

Analytical methods

International standard methods were used for the analysis of all samples (solid, liquid and gaseous) in order to allowing a precise comparison with other scientific tests in literature. In particular, chemical analysis followed Italian official procedures, derived from international certified standards (CEN, 2002).

A portable analyser (Eco-Control LFG20) were used for measure carbon dioxide, methane and oxygen concentrations in the biogas. The LFG20 was calibrated with normal air before every use, with a standard gas mixture (50% CH4, 50% CO2) once a week and the results were periodically compared with an LFG 2000 analyser and with a micro Gas Chromatography.

Stripped N-NH3(g) could be measured through an acid scrubber placed immediately after the off-gas valve of each reactor. A solution of boric acid (0.5 M) trapped the gaseous ammonia, retaining it in solution as ammonium ion. Periodically, the scrubbing solution was titrated with sulfuric acid (0.01 M) to evaluate the amount of N-NH3(g) exiting from the reactor. This measurement system for the gaseous ammonia emissions was periodically checked with the portable gas analyser (Analitica Strumenti LFG 2000).

Chemical and biochemical analysis on liquid samples were made in a laboratory without the support of ready to use kits. The routine analysis performed on leachate were pH, COD, TOC, BOD5, TKN, N-NH4+, N-NO3-, SO42-, Cl^- and Heavy Metals (Cr, Cu, Fe, Mn, Ni, Pb, Zn). In particular, routine procedures were the following ones:

 pH and conductibility are measured directly with specific probes.

 Chemical Oxygen Demand (COD) analysis starts with an acid digestion at 150°C with a strong chemical oxidizer (potassium dichromate). The oxygen chemically consumed is stoichiometric calculated by means of a titration with Mohr salt.

A C

B

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 Total Organic Carbon (TOC) is measured directly with the “TOC-V CSN” analytical equipment.

 Biological Oxygen Demand (BOD5) is a batch test quantifying the oxygen biologically consumed by the sample in 5 days.

 Total Kjeldahl Nitrogen (TKN) is evaluated with the standard Kjeldahl methodology.

The sample is digested with acid at 350°C for destroying all the organic bonds. After that, soda is added to increase pH over 10 and stripping of gaseous ammonia is enhanced by distillation. The extracted N-NH3(g) is caught in boric acid and the solution is titrated with sulfuric acid (0.01 M) to evaluate the amount of nitrogen present in the initial solution.

 Ammonia ion (N-NH4+) is analysed as TKN, without the initial digestion.

 Nitrates (N-NO3) and Sulphates (SO42-) are evaluated by means of a spectrophotometer.

 Chloride (Cl^-) concentration is quantified after a titration with argent nitrate 0,1 M.

 Heavy Metals (HM) are analysed by means of an Inductively Coupled Plasma (ICP) after an acid digestion of the sample.

The analysis on solid samples were performed both on the material as it is, to evaluate the massive compounds concentrations (TS, VS, TOC, TKN, and HM) and on the eluate from a standard leaching test, to evaluate the emission potential of some mobile species (pH, COD, TOC, BOD5, TKN, N-NH4+, N-NO3-, SO42-, Cl^- and HM). To decrease the analytical uncertainty due to waste heterogeneity, all the tests on solid samples were performed at least in duplicate:

 Total Solid (TS) quantifies the dry mass of a sample (in percentage respect to the wet mass), heating the sample at 105 °C for 24 hours, evaporating the interstitial pores water.

 Total Volatile Solids (VS) quantify the mass of the volatile compounds present in a sample (in percentage respect to the dry mass), heating the sample at 550 °C for 4 hours, burning all combustible volatile substances.

 TOC on solid sample is directly measured with the “TOC-V CSN” analytical equipment plus the burning chamber for solid samples.

 TKN and HM are evaluated with the same methodology used for leachate samples.

The leaching test used for evaluating the emission potential of soluble compounds present in solid waste is the international standard methodology (UNI EN 12457-2). The solid sample was milled at 4 mm diameter, the dust was mixed with distilled water for reaching a liquid to solid ratio of 10 L/kgTS, the mix was turned for 24 hours at 20 °C and, finally, filtered at 0.45 µm. The eluate obtained with this procedure can be analysed with the same methods used for liquid samples.

Dealing with degradable substances, some biological tests are particularly useful for understanding potential methane production or residual degradability of a sample:

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 BOD5, as explained before, quantifies the oxygen consumption due to the aerobic degradation processes that happens in a batch liquid test. This index is presented as a concentration and can be evaluated for either leachates and eluates from leaching tests. To evaluate the biodegradability of a sample avoiding dilution effect present in any concentration, BOD5/COD ratio can be estimated (Cossu et al., 2012). This index can assume values ranging between 0.02 and 0.13 for low biodegradability and values greater than 0.4 for high biodegradability (Sekman et al., 2011).

 Respiration Index (RI4) is a German developed tool (AT4) internationally certified as a biological stability index for solid waste samples (ANPA 3/2001 n.12.1.2.3). RI4 is a respiration test in which cumulative oxygen consumption in 4 days is measured under the controlled standard conditions of “SAPROMAT” equipment (H+P Labortechnik, Germany). The aerobic microorganisms in the sample consume oxygen producing carbon dioxide, which is caught by soda, generating a negative pressure in the system.

Sapromat automatically registers this pressure difference and inject new oxygen in the batch system. The oxygen consumption is progressively measured respect to the dry mass of sample.

 Biological Methane Potential (BMP) is an anaerobic batch test lasting until the methane production becomes negligible. Sample is loaded in a bottle at 40°C with sufficient headspace to store the gas produced, which has to be emptied at least daily.

This test is useful to calculate the theoretical maximum production of methane in optimum conditions and some kinetic parameters useful to design other laboratory tests.

During the aftercare phases, more than 10% of the organic carbon initially present in the samples was transformed into not mobile forms, probably due to the formation of humic substances (Brandstätter et al., 2015). The determination of humic and fulvic acids in the eluate form leaching tests can be made following the procedure proposed by Baddi et al.

(2004). Some mL of eluate sample undergo to a double step centrifugation (7000 rpm for 25 min) with subsequent distilled water washing in order to remove any particulate matter in solution. The pH of the supernatant was decreased below 2 with a sulfuric acid solution (2 M) to enhance humic acids precipitation. The mixture is settled (24 h at 4 °C) for a complete coagulation. Subsequently, a second double step centrifugation was performed and the precipitate was dried under vacuum. The obtained residue represented the amount of humic acids. The supernatant coming from centrifugation is collected and dialyze through Spectra/Por® Dyalysis Membranes (3500 Da). The amount of eluate retained is dried to evaluate the fulvic acids content of the sample.

Heavy Metals speciation can be performed on solid samples through the multistep procedure proposed by Krishnamurti et al. (2002). This method allowed to separate eight different forms under which Heavy Metals could be found: ion exchangeable, carbonate-bound (adsorbed), as metal-organic complexes (associated with humic and fulvic acids), as easily reducible metal-oxides, bound to organic matter (other than humic and fulvic acids), in amorphous mineral colloids, as crystalline iron-oxides and bound to aluminum-silicate

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minerals. Every step of the procedure require a precise mixing time, temperature, pH and reagent. After each step, a double centrifugation (at 10500 rpm for 20 min) with distilled water washing is necessary to separate the supernatant which is analysed with ICP.