Percolates production from OFMSW
Organic fraction of municipal solid waste collection
The source-separated OFMSW coming from street bin containers was collected at a full-scale composting plant located in northern Italy. Collection was done by following the quartering method: in brief, the OFMSW was sampled at different points of the whole mass (500 kg wet weight − w.w. − ×3 times); then the collected waste was mixed, quartered and sub-sampled until a final sample of about 300 kg w.w. material was reached. OFMSW was stored at 4 °C before the trials were set up. A representative sample of approximately 30 kg, obtained by mixing sub-samples of about 5 kg each taken randomly from sampled material, was dried and crushed to 2 mm and then used to perform analytical analyses.
The bulking agent (shredded wood) from the same plant was also collected following a similar approach.
Liquid digestate, used to irrigate OFMSW during dry anaerobic fermentation (see later), was collected directly from a continuously stirred tank reactor anaerobic digester (CSTR-AD) plant fed with OFMSW, located in northern Italy and previously described [19]. Digestate was collected from this plant in large quantities from the discharge pipe system of the post-digester after 30 days of retention time (HRT).
Experimental apparatus
The acidogenesis (SSAD) trials were carried out by developing a pilot-scale anaerobic percolation biocell reactor (APBR) that was made up of an insulated vertical cylinder (100 L of volume) of PVC material with a hermetic cap. Inside the reactor, there was a stainless steel basket with holes at the bottom, into which OFMSW was introduced from the APBR cap, as well as the digestate used to irrigate the organic wastes (Fig. 1). From the bottom of the APBR, the percolate generated daily due to digestate irrigation was extracted and weighed. Digestate irrigation was used to buffer the acidity produced during OFMSW fermentation and to remove OA produced in the APBR. This encouraged high hydrolysis performance from the organic waste, which is the limiting step in producing large amounts of OA from organic substrates [20, 21]. Moreover, large-scale OA production becomes toxic for methanogenic bacteria, limiting methane production during the trials.
Experimental setup
Three different percolation trials were carried out. For all of them, fresh OFMSW (85% w.w.) and bulking agent (chopped green waste material) (15% w.w.) were mixed just before reactor filling to avoid clogging problems in the APBR, allowing the percolation. For each trial, the APBR was filled with about 50 kg of mixture (OFMSWmix-in).
The OFMSWmix-in in the APBR was irrigated continuously by using the liquid digestate obtained from the full-scale CSTR-AD plant in order to optimize OFMSW hydrolysis and subsequent acidogenesis fermentation to produce OA. Anaerobic hydrolysis and acidic fermentation lasted for 21 days for each trial performed. During this time, irrigation with the liquid digestate (previously heated to 55 °C) was done continuously by using a peristaltic pump, adopting an OFMSWmix-in/digestate ratio of 1:0.45 kg kg−1 (flow rate of 0.02 L h−1 kg−1 OFMSWw.w.) for the first trial (Trial 1), 1:0.9 kg kg−1 (flow rate of 0.04 L h−1 kg−1 OFMSWw.w.) for the second trial (Trial 2), and of 1:1.8 kg kg−1 (flow rate of 0.08 L h−1 kg−1 OFMSWw.w.) (day 1–8) and of 1:0.9 kg kg−1 (flow rate of 0.04 L h−1 kg−1 OFMSWw.w.) (day 9–21) for the third trial (Trial 3), respectively. The daily-generated percolates were collected from the bottom of the APBR, quantified and mixed together to get a final mixed percolate sample for each trial.
Percolates treatment before PHA production
The three percolates obtained from anaerobic acidic fermentation trials were submitted to two pre-treatments before their use for polyhydroxyalkanoate (PHA) production. The first pre-treatment was to centrifuge the percolates at 20,000g for 15 min, reducing suspended organic carbon from percolates. Supernatants obtained underwent a second pre-treatment adopted to remove ammonia by stripping. This step was performed by bringing supernatant-pH to 11 by adding 6-mol L−1 KOH and stirring it (magnetic stirring at 200 rpm) under the hood until a final C:N close to 10 was reached [11]. Then ammonia stripping was stopped and the pH was brought again to the initial value of 8 by using 3 mol L−1 H2SO4. Ammonia stripped was not trapped at lab scale and it was not considered in this work. Obviously, this step needs to be considered and further studied on a larger scale.
After pre-treatments, the three supernatants were diluted with deionized water, to produce a final COD content close to 1300 mg L−1 [11]. These OFMSW-derived supernatants (OFMSW-supernatantsin) were used for subsequent inoculum selection.
Substrates (OFMSW-supernatantsacc.) used for PHA accumulation tests were derived from percolates that had undergone the same treatments shown for those destined for the inoculum selection. Three OFMSW-supernatantsacc. were obtained but, in this case, the ammonia content was lower than those reported for OFMSW-supernatantsin, since it has been reported that N starvation can determine a greater conversion of carbon into PHA because of cell growth limitation [22]. Moreover, OFMSW-supernatantsacc. were less diluted, giving a final COD concentration of about 7500 mg L−1, in order to avoid an excessive dilution of the biomass in the accumulation reactor [10].
Inoculum production and PHA-producing bacteria enrichment
The enrichment of PHA-producing bacteria was performed by using an inoculum constituted by an activated sludge (5 g total suspended solids L−1) collected from the secondary sedimentation tank of a wastewater treatment plant (5.2 × 105 equivalent inhabitants) located at Peschiera Borromeo (Milan, Italy).
The enrichment in PHA-producing bacteria was carried out in a Sequencing Batch Reactor (SBR) with a working volume of 800 mL, applying an aerobic dynamic feeding (ADF) strategy [11]. In brief, the SBR cycle length was of 12 h, consisting of four discrete phases: (1) influent filling (4 min), (2) aeration (675 min), (3) settling (40 min) and (4) withdrawal of the exhausted effluent (5 min), with 1 day of hydraulic retention time (HRT) and 5 days of Sludge Retention Time (SRT), keeping the temperature at 25 °C and the pH at 8.8, this latter controlled automatically by adding 1 mol L−1 HCl. Aeration and agitation were provided by supplying air at 6 L min−1 and stirring set at 110 rpm. Pumping, aeration and stirring were automatically controlled.
The selection process lasted for 3 months; for each month, a different pre-treated supernatant was used as substrate: OFMSW-supernatantin 1, OFMSW-supernatantin 2 and OFMSW-supernatantin 3, respectively, for the first, second and third month of selection.
The selection trend was monitored by determining the duration of the feast phase achievable by using the dissolved oxygen (DO) concentration in the selection media [10], measured by an optical probe (FDO 925, WTW, Germany).
In particular, the feast (h)-to-famine (h) ratio (F/F ratio) was calculated as the ratio between the lengths in hours of the two phases. For a correct selection of PHA-storing bacteria, the F/F ratio had to be equal or less than 0.33 [23]. To carry out the selection of PHA-accumulating bacteria, 400 mL of activated sludge was used as the inoculum feed for each cycle, with 400 mL of OFMSW-supernatantin. Organic Loading Rate (OLR) was kept close to 1300 mg COD L−1 day−1 and the C:N:P ratio was of about 100:13:0.4 mol C: mmole N: mmole P. Every month, two cycles were monitored in order to evaluate the performance of the selected culture. In particular, monitoring was performed between cycles 31 and cycle 45 since it was reported that the cultures reach stability after three SRTs from the beginning of the trial (cycle 31) [24].
PHA accumulation
The ability of the MMC to accumulate PHA was assessed by fed-batch assays carried out in a 300-mL working volume glass reactor, with continuous aeration and stirring. These assays consisted of feeding the substrate to 160 mL of enriched culture (at least 3 SRTs from the beginning of the selection) [24] adopting a pulse-wise feeding method. The assays were monitored continuously by measuring the concentration of the dissolved oxygen in the accumulation media [10]. In particular, substrate (OFMSW-supernatantacc.) was fed to the reactor when DO showed a strong increase [10]. Total C dosed was calculated taking into account that the ratio of the carbon to the microorganisms had to be the same as that inside the selection reactor. The assays were stopped when no DO variation followed the substrate feeding.
For the accumulation tests, the operating conditions used were those adopted in the selection reactor, i.e. temperature of 25 °C, aeration of 6 L min−1 and stirring at 110 rpm.
The biomass from the selection process was submitted to accumulation tests using the same substrate as the carbon source; every month, two accumulation trials were performed in duplicate, between the cycles 31 and 45.
PHA extraction
The biomass collected after PHA accumulation tests was centrifuged at 8000g for 15 min, washed with 0.9% sodium chloride solution and centrifuged again at 8000g for 20 min. The pellet obtained was lyophilized and then suspended in chloroform (in a ratio of ca. 40 mL CHCl3 g−1 dried cells) and left to dissolve for a period of 3 days at 37 °C [10]. The solution was then filtered to remove all undissolved material. The extracted PHA in chloroform was then precipitated by the addition of 5 volumes of methanol and allowed to settle down for 30 min [25]. The white precipitate formed was then filtered, suspended in chloroform and used to fill glass Petri dishes. Finally, chloroform was evaporated allowing polymer recovery in the form of a thin film.
Analytical procedures
OFMSW, digestate and percolates characterization
Total solids (TS), volatile solids (VS), total nitrogen (TKN), pH and ammonia (N-NH4
+) (detected on fresh material) contents were determined according to the standard procedures [26]. Total OA expressed as acetic acid, were detected on fresh material and determined according to the acid titration method [27].
Substrate and biomass characterization during PHA production
The substrates (OFMSW-supernatants) fed during the selection and accumulation processes were characterized in terms of pH, TS, VS, Chemical Oxygen Demand (COD), OA content (acetate, butyrate, lactate, propionate and valerate), TKN, N-NH4
+ and phosphorus (P) content.
During the selection trials, samples were taken during the cycle once in each SRT; every sample was characterized in terms of total suspended solids (TSS), volatile suspended solids (VSS), soluble COD, OA content, N-NH4
+ content and PHA content. During accumulation trials, samples were taken continuously in order to measure TSS, VSS, soluble COD, OA content and PHA content. Biomass concentration was calculated as VSS according to the standard methods [10].
TSS and VSS were determined as reported by Valentino et al. in 2015 [28]. OA concentrations measured on filtered samples (filter diameter of 0.45 µm) were determined by high-performance liquid chromatography (HPLC) using a chromatograph equipped with a UV detector and Aminex HPX-87H column (column temperature 20 °C, 0.0025 M H2SO4 eluent, flow rate 0.6 mL min−1). The OA concentrations were calculated through a standard calibration curve (20–1000 mg L−1 of each organic acid). The COD and the N-NH4
+ content (filtered at 0.45 µm) were determined using cuvette test kits (Macherey-Nagel, Germany).
PHAs were determined by GC MS using a method adapted from Serafim et al. in 2004 [22]. Lyophilized biomass was incubated for methanolysis in a 20% v/v H2SO4 in MeOH solution (1 mL) and extracted with chloroform (1 mL). The mixture was digested at 100 °C for 3.5 h. After the digestion step, the organic phase (methylated monomers dissolved in chloroform) was extracted and injected (1 mL) into a gas chromatograph equipped with a detector (7980, Agilent Technologies, USA) and a ZB-Wax column (30 m, 0.25 mm internal diameter, 0.25 µm film thickness, Zebron, Phenomenex, USA), using helium as a carrier gas at 1.0 mL min−1. Samples were analysed under a temperature regime starting at 40 °C, increasing to 100 °C at a rate of 20 °C min−1, to 175 °C at a rate of 38 °C min−1 and reaching a final temperature of 220 °C at a rate of 20 °C min−1 for ensuring cleaning of the column after each injection. Injector and detector temperatures were at 280 and 230 °C, respectively. Hydroxybutyrate (HB) and hydroxyvalerate (HV) concentrations were determined through the use of two calibration curves, one for HB and another for HV, using standards (0.1–8 g L−1) of a commercial P (HB–HV) (88/12%) (Sigma-Aldrich, Germany), and corrected using heptadecane as internal standard (concentration of approximately 1 g L−1) (Sigma Aldrich, Germany).
PHA and active biomass growth yield calculation
The PHA content in cells was referred to VSS on a mass basis [PHA = (g kg−1 VSS)], considering VSS to be constituted by both active biomass (X) and PHA [10]. PHA was converted into COD according to the following oxidation stoichiometry: 1.67 mg COD mg−1 HB monomer and 1.92 mg COD mg−1 HV monomer [23].
Acetate, butyrate and lactate were considered as HB precursors, valerate and propionate as HV precursors [10]. X was calculated on a COD basis considering that 1 g of X contains 1.42 g of COD [23]. For the SBR, PHA storage yield was expressed in COD and referred to both COD consumed (CODcons.) and OA consumed (CODOA-cons.), calculated, respectively, as the ratio between the amount of PHA accumulated during the feast phase (CODPHA) and the amount of COD depleted or OA depleted.
In the accumulation batches, these PHA storage yields were calculated as described before, for each pulse. In order to compare different accumulation tests, the average values of the first three pulses and for each parameter were considered [23].
In accumulation batches, PHA storage yield was also related to COD fed (CODin) and OA fed (CODOAin); moreover, it was also related back to total solids of OFMSW (OFMSWTS) and to OFMSWw.w..
The X growth yield in the SBR was expressed in COD and referred to COD consumed (CODcons.), calculated as the ratio between the new X produced during the feast phase (CODX) and the amount of COD depleted [23].
PHA characterization by solid-state NMR and solution 1H NMR, and molecular weight
Solid-state 13C NMR spectra of lyophilized biomass containing PHA were recorded on a Bruker Avance 300 spectrometer operating at 75.47 MHz, using a 4 × 21 mm cylindrical zirconium rotor spun at 11,000 Hz to avoid the side bands. The 13C cross-polarization magic angle spinning (CPMAS) NMR spectra were acquired using recycle delay of 8 s, 1H 90 pulse length of 3.5 μs, 1 m contact time, acquisition time of 35 ms and from 1K to 4K scans. The 13C single-pulse excitation (SPE) NMR spectra were recorded with delays of 160 s and 1–2K scans.
The chemical shifts were recorded relative to tetramethylsilane via benzene as a secondary reference.
Liquid NMR experiments on extracted PHA were performed on a Bruker 500 MHz AVANCE III NMR spectrometer (Bruker GmbH, Germany) with a 5-mm TCI cryoprobe. Deuterated chloroform (99.96%, Sigma Aldrich) was used as solvent. 1H NMR spectra were recorded at 303 K using recycle delay of 12 s, 64K fid size and 64 scans.
PHA molecular weight was determined by HP-SEC/TDA measurement. The HPLC equipment consisted of a Viscotek system (Malvern Instrument Ltd, Malvern, UK) equipped with a Knauer HPLC pump K501, and a Biotech Degasi GPC degassing device. The detector system was a Viscotek mod. 302 Triple Detector Array (TDA), which is composed by Laser Light Scattering detector (90° and 7°; wavelength 670 nm), Refractive index (RI) detector [cell volume of 12 μL; light-emitting diode (LED) at 660 nm wavelength] and Viscosimeter detector (four capillaries with a differential Wheatstone bridge configuration). A PL GEL 20-um MIXED A column (7.5 × 300 mm) was used. Chloroform was used as the mobile phase at a flow rate of 1 mL min−1. Columns, injector and detectors were maintained at 30 °C. Samples were dissolved in chloroform at concentrations of 2–8 mg mL−1 and filtered on a 0.2-μm membrane before injection. Injection volume was of 100 μL.
The system was calibrated with the PS narrow standard of known Mw, polydispersity and intrinsic viscosity (Malvern PolyCAL PS std 105 k). Using a standard PHA sample at different concentrations (2.3, 4.3, 6.0, 8.5 mg mL−1), the differential refractive index increment (dn/dc) value was found to be equal to 0.024 and used for further calculations.
Statistical analysis
Average and standard deviation values were calculated according to standard procedures and the results were analysed by an ANOVA test. A Tukey’s test was used to compare mean values and to assess the significance of the differences between mean values, adopting a fixed effects model, i.e. the digestate flow adopted modified the chemical composition of both the percolates produced and the supernatants derived.
All statistical analyses were carried out using the SPSS statistical software, version 15.0 (SPSS, Chicago, IL, USA).