Identification and Quantification of Polycyclic Aromatic Hydrocarbons in Polyhydroxyalkanoates Produced from Mixed Microbial Cultures and Municipal Organic Wastes at Pilot Scale.

Polyhydroxyalkanoates (PHAs) are well-known biodegradable plastics produced by various bacterial strains, whose major drawback is constituted by the high cost of their synthesis. Producing PHAs from mixed microbial cultures and employing organic wastes as a carbon source allows us to both reduce cost and valorize available renewable resources, such as food waste and sewage sludge. However, different types of pollutants, originally contained in organic matrices, could persist into the final product, thus compromising their safety. In this work, the exploitation of municipal wastes for PHA production is evaluated from the environmental and health safety aspect by determining the presence of polycyclic aromatic hydrocarbons (PAHs) in both commercial and waste-based PHA samples. Quantification of PAHs by gas chromatography-mass spectrometry on 24 PHA samples obtained in different conditions showed very low contamination levels, in the range of ppb to a few ppm. Moreover, the contaminant content seems to be dependent on the type of PHA stabilization and extraction, but independent from the type of feedstock. Commercial PHA derived from crops, selected for comparison, showed PAH content comparable to that detected in PHAs derived from organic fraction of municipal solid waste. Although there is no specific regulation on PAH maximum levels in PHAs, detected concentrations were consistently lower than threshold limit values set by regulation and guidelines for similar materials and/or applications. This suggests that the use of organic waste as substrate for PHA production is safe for both the human health and the environment.

Ammonium Recovery and Biogas Upgrading in a Tubular Micro-Pilot Microbial Electrolysis Cell (MEC).

Here, a 12-liter tubular microbial electrolysis cell (MEC) was developed as a post treatment unit for simultaneous biogas upgrading and ammonium recovery from the liquid effluent of an anaerobic digestion process. The MEC configuration adopted a cation exchange membrane to separate the inner anodic chamber and the external cathodic chamber, which were filled with graphite granules. The cathodic chamber performed the CO2 removal through the bioelectromethanogenesis reaction and alkalinity generation while the anodic oxidation of a synthetic fermentate partially sustained the energy demand of the process. Three different nitrogen load rates (73, 365, and 2229 mg N/Ld) were applied to the inner anodic chamber to test the performances of the whole process in terms of COD (Chemical Oxygen Demand) removal, CO2 removal, and nitrogen recovery. By maintaining the organic load rate at 2.55 g COD/Ld and the anodic chamber polarization at +0.2 V vs. SHE (Standard Hydrogen Electrode), the increase of the nitrogen load rate promoted the ammonium migration and recovery, i.e., the percentage of current counterbalanced by the ammonium migration increased from 1% to 100% by increasing the nitrogen load rate by 30-fold. The CO2 removal slightly increased during the three periods, and permitted the removal of 65% of the influent CO2, which corresponded to an average removal of 2.2 g CO2/Ld. During the operation with the higher nitrogen load rate, the MEC energy consumption, which was simultaneously used for the different operations, was lower than the selected benchmark technologies, i.e., 0.47 kW/N·m3 for CO2 removal and 0.88 kW·h/kg COD for COD oxidation were consumed by the MEC while the ammonium nitrogen recovery consumed 2.3 kW·h/kg N.

Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation.

Interspecies electron transfer mechanisms between Bacteria and Archaea play a pivotal role during methanogenic degradation of organic matter in natural and engineered anaerobic ecosystems. Growing evidence suggests that in syntrophic communities electron transfer does not rely exclusively on the exchange of diffusible molecules and energy carriers such as hydrogen or formate, rather microorganisms have the capability to exchange metabolic electrons in a more direct manner. Here, we show that supplementation of micrometer-size magnetite (Fe3O4) particles to a methanogenic sludge enhanced (up to 33%) the methane production rate from propionate, a key intermediate in the anaerobic digestion of organic matter and a model substrate to study energy-limited syntrophic communities. The stimulatory effect most probably resulted from the establishment of a direct interspecies electron transfer (DIET), based on magnetite particles serving as electron conduits between propionate-oxidizing acetogens and carbon dioxide-reducing methanogens. Theoretical calculations revealed that DIET allows electrons to be transferred among syntrophic partners at rates which are substantially higher than those attainable via interspecies H2 transfer. Besides the remarkable potential for improving anaerobic digestion, which is a proven biological strategy for renewable energy production, the herein described conduction-based DIET could also have a role in natural methane emissions from magnetite-rich soils and sediments.

Effect of feeding regime and the sludge age on the fate of acetate and the microbial composition in sequencing batch reactor.

The study investigated the effects of the feeding regime on the substrate metabolism under aerobic conditions. Throughout the study, two parallel sequencing batch reactors were operated using either short-term (pulse) or long-term (almost continuous) feeding of acetate at two different sludge ages of two days and eight days. The microbial characterization studies showed that the feeding regime did not change the microbial composition as determined by the fluorescent in situ hybridization (FISH) analysis, however did strongly affect the substrate utilization mechanism. Additionally, the same microbial culture was able to utilize acetate with or without poly-ß-hydroxybutyrate (PHB) storage under pulse or continuous feeding, respectively. Conversely, the selected sludge ages induced significant changes in the microbial composition and floc structure, however, the well settling and bulking biomass selected at the sludge ages of eight days and two days, respectively, did not significantly affect the substrate utilization mechanism, where storage or growth prevailed as a result of either pulse or continuous feeding.

Effect of dual substrate environment on the formation of intracellular storage biopolymers.

We investigated the effect of a starch/acetate mixture on the formation of intracellular storage biopolymers compared with system behaviour where these compounds served as a single organic substrate. Three laboratory-scale sequencing batch reactors (SBRs) were operated at steady state with a sludge age of 8 days, one fed with acetate, another with starch and the third with a starch/acetate mixture. The SBR operation involved six cycles per day and continuous feeding during each cycle. Both acetate and starch generated storage biopolymers under continuous feeding. A poly-hydroxybutyrate (PHB) or glycogen pool was formed depending on the selected single substrate. In addition, around 18% of the acetate fed within each cycle was converted to PHB, while the remaining 82% was directly utilized for microbial growth. A higher glycogen formation of 44% was observed for starch. Substrate storage as PHB and glycogen continued with the feeding of the acetate/starch mixture. This observation, supported by microbiological analyses, indicated that the acclimated biomass in the corresponding SBR system sustained microbial fractions capable of performing metabolic functions associated with the formation of the two storage biopolymers. PHB accumulation was reduced as acetate could be more readily used for direct microbial growth in the presence of starch.

Autotrophic Acetate Production under Hydrogenophilic and Bioelectrochemical Conditions with a Thermally Treated Mixed Culture.

Bioelectrochemical systems are emerging technologies for the reduction in CO2 in fuels and chemicals, in which anaerobic chemoautotrophic microorganisms such as methanogens and acetogens are typically used as biocatalysts. The anaerobic digestion digestate represents an abundant source of methanogens and acetogens microorganisms. In a mixed culture environment, methanogen's inhibition is necessary to avoid acetate consumption by the presence of acetoclastic methanogens. In this study, a methanogenesis inhibition approach based on the thermal treatment of mixed cultures was adopted and evaluated in terms of acetate production under different tests consisting of hydrogenophilic and bioelectrochemical experiments. Batch experiments were carried out under hydrogenophilic and bioelectrochemical conditions, demonstrating the effectiveness of the thermal treatment and showing a 30 times higher acetate production with respect to the raw anaerobic digestate. Moreover, a continuous flow bioelectrochemical reactor equipped with an anion exchange membrane (AEM) successfully overcomes the methanogens reactivation, allowing for a continuous acetate production. The AEM membrane guaranteed the migration of the acetate from the biological compartment and its concentration in the abiotic chamber avoiding its consumption by acetoclastic methanogenesis. The system allowed an acetate concentration of 1745 ± 30 mg/L in the abiotic chamber, nearly five times the concentration measured in the cathodic chamber.

Corrigendum: Metagenomic Analysis Reveals Microbial Interactions at the Biocathode of a Bioelectrochemical System Capable of Simultaneous Trichloroethylene and Cr(VI) Reduction.

[This corrects the article DOI: 10.3389/fmicb.2021.747670.].

Modelling Mixed Microbial Culture Polyhydroxyalkanoate Accumulation Bioprocess towards Novel Methods for Polymer Production Using Dilute Volatile Fatty Acid Rich Feedstocks.

Volatile fatty acid (VFA) rich streams from fermentation of organic residuals and wastewater are suitable feedstocks for mixed microbial culture (MMC) Polyhydroxyalkanoate (PHA) production. However, many such streams have low total VFA concentration (1-10 gCOD/L). PHA accumulation requires a flow-through bioprocess if the VFAs are not concentrated. A flow through bioprocess must balance goals of productivity (highest possible influent flow rates) with goals of substrate utilization efficiency (lowest possible effluent VFA concentration). Towards these goals, dynamics of upshift and downshift respiration kinetics for laboratory and pilot scale MMCs were evaluated. Monod kinetics described a hysteresis between the upshift and downshift responses. Substrate concentrations necessary to stimulate a given substrate uptake rate were significantly higher than the concentrations necessary to sustain the attained substrate uptake rate. A benefit of this hysteresis was explored in Monte Carlo based PHA accumulation bioprocess numerical simulations. Simulations illustrated for a potential to establish continuous flow-through PHA production bioprocesses even at a low (1 gCOD/L) influent total VFA concentration. Process biomass recirculation into an engineered higher substrate concentration mixing zone, due to the constant influent substrate flow, enabled to drive the process to maximal possible PHA production rates without sacrificing substrate utilization efficiency.

Producing volatile fatty acids and polyhydroxyalkanoates from foods by-products and waste: A review.

Dairy products, extra virgin olive oil, red and white wines are excellent food products, appreciated all around the world. Their productions generate large amounts of by-products which urge for recycling and valorization. Moreover, another abundant waste stream produced in urban context is the Organic Fraction of Municipal Solid Wastes (OFMSW), whose global annual capita production is estimated at 85 kg. The recent environmental policies encourage their exploitation in a biorefinery loop to produce Volatile Fatty Acids (VFAs) and polyhydroxyalkanoates (PHAs). Typically, VFAs yields are high from cheese whey and OFMSW (0.55-0.90 gCOD_VFAs/gCOD), lower for Olive Mill and Winery Wastewaters. The VFAs conversion into PHAs can achieve values in the range 0.4-0.5 gPHA/gVSS for cheese whey and OFMSW, 0.6-0.7 gPHA/gVSS for winery wastewater, and 0.2-0.3 gPHA/gVSS for olive mill wastewaters. These conversion yields allowed to estimate a huge potential annual PHAs production of about 260 M tons.

Microbiome Composition and Dynamics of a Reductive/Oxidative Bioelectrochemical System for Perchloroethylene Removal: Effect of the Feeding Composition.

Chlorinated solvents still represent an environmental concern that requires sustainable and innovative bioremediation strategies. This study describes the microbiome composition of a novel bioelectrochemical system (BES) based on sequential reductive/oxidative dechlorination for complete perchloroethylene (PCE) removal occurring in two separate but sequential chambers. The BES has been tested under various feeding compositions [i.e., anaerobic mineral medium (MM), synthetic groundwater (SG), and real groundwater (RG)] differing in presence of sulfate, nitrate, and iron (III). In addition, the main biomarkers of the dechlorination process have been monitored in the system under various conditions. Among them, Dehalococcoides mccartyi 16S rRNA and reductive dehalogenase genes (tceA, bvcA, and vcrA) involved in anaerobic dechlorination have been quantified. The etnE and etnC genes involved in aerobic dechlorination have also been quantified. The feeding composition affected the microbiome, in particular when the BES was fed with RG. Sulfuricurvum, enriched in the reductive compartment, operated with MM and SG, suggesting complex interactions in the sulfur cycle mostly including sulfur oxidation occurring at the anodic counter electrode (MM) or coupled to nitrate reduction (SG). Moreover, the known Mycobacterium responsible for natural attenuation of VC by aerobic degradation was found abundant in the oxidative compartment fed with RG, which was in line with the high VC removal observed (92 ± 2%). D. mccartyi was observed in all the tested conditions ranging from 8.78E + 06 (with RG) to 2.35E + 07 (with MM) 16S rRNA gene copies/L. tceA was found as the most abundant reductive dehalogenase gene in all the conditions explored (up to 2.46 E + 07 gene copies/L in MM). The microbiome dynamics and the occurrence of biomarkers of dechlorination, along with the kinetic performance of the system under various feeding conditions, suggested promising implications for the scale-up of the BES, which couples reductive with oxidative dechlorination to ensure the complete removal of highly chlorinated ethylene and mobile low-chlorinated by-products.

Dechlorination of trichloroethene in a continuous-flow bioelectrochemical reactor: effect of cathode potential on rate, selectivity, and electron transfer mechanisms.

The exciting discovery that dechlorinating bacteria can use polarized graphite cathodes as direct electron donors in the reductive dechlorination has prompted investigations on the development of novel bioelectrochemical remediation approaches. In this work, we investigated the performance of a bioelectrochemical reactor for the treatment of trichloroethene (TCE). The reactor was continuously operated for about 570 days, at different potentiostatically controlled cathode potentials, ranging from -250 mV to -750 mV vs standard hydrogen electrode. The rate and extent of TCE dechlorination, as well as the competition for the available electrons, were highly dependent on the set cathode potential. When the cathode was controlled at -250 mV, no abiotic hydrogen production occurred and TCE dechlorination (predominantly to cis-DCE and VC), most probably sustained via direct extracellular electron transfer, proceeded at an average rate of 15.5 ± 1.2 µmol e(-)/L d. At this cathode, potential methanogenesis was almost completely suppressed and dechlorination accounted for 94.7 ± 0.1% of the electric current (15.0 ± 0.8 µA) flowing in the system. A higher rate of TCE dechlorination (up to 64 ± 2 µmol e(-)/L d) was achieved at cathode potentials lower than -450 mV, though in the presence of a very active methanogenesis which accounted for over 60% of the electric current. Remarkably, the bioelectrochemical reactor displayed a stable and reproducible performance even without the supply of organic carbon sources with the feed, confirming long-term viability.

Metagenomic Analysis Reveals Microbial Interactions at the Biocathode of a Bioelectrochemical System Capable of Simultaneous Trichloroethylene and Cr(VI) Reduction.

Bioelectrochemical systems (BES) are attractive and versatile options for the bioremediation of organic or inorganic pollutants, including trichloroethylene (TCE) and Cr(VI), often found as co-contaminants in the environment. The elucidation of the microbial players' role in the bioelectroremediation processes for treating multicontaminated groundwater is still a research need that attracts scientific interest. In this study, 16S rRNA gene amplicon sequencing and whole shotgun metagenomics revealed the leading microbial players and the primary metabolic interactions occurring in the biofilm growing at the biocathode where TCE reductive dechlorination (RD), hydrogenotrophic methanogenesis, and Cr(VI) reduction occurred. The presence of Cr(VI) did not negatively affect the TCE degradation, as evidenced by the RD rates estimated during the reactor operation with TCE (111±2 µeq/Ld) and TCE/Cr(VI) (146±2 µeq/Ld). Accordingly, Dehalococcoides mccartyi, the primary biomarker of the RD process, was found on the biocathode treating both TCE (7.82E+04±2.9E+04 16S rRNA gene copies g-1 graphite) and TCE/Cr(VI) (3.2E+07±2.37E+0716S rRNA gene copies g-1 graphite) contamination. The metagenomic analysis revealed a selected microbial consortium on the TCE/Cr(VI) biocathode. D. mccartyi was the sole dechlorinating microbe with H2 uptake as the only electron supply mechanism, suggesting that electroactivity is not a property of this microorganism. Methanobrevibacter arboriphilus and Methanobacterium formicicum also colonized the biocathode as H2 consumers for the CH4 production and cofactor suppliers for D. mccartyi cobalamin biosynthesis. Interestingly, M. formicicum also harbors gene complexes involved in the Cr(VI) reduction through extracellular and intracellular mechanisms.

Ethylic Esters as Green Solvents for the Extraction of Intracellular Polyhydroxyalkanoates Produced by Mixed Microbial Culture.

Volatile fatty acids obtained from the fermentation of the organic fraction of municipal solid waste can be used as raw materials for non-toxic ethyl ester (EE) synthesis as well as feedstock for the production of polyhydroxyalkanoates (PHAs). Taking advantage of the concept of an integrated process of a bio-refinery, in the present paper, a systematic investigation on the extraction of intracellular poly(3-hydroxybutyrate-co-3-hydroxyvalerate), produced by mixed microbial culture by using EEs was reported. Among the tested EEs, ethyl acetate (EA) was the best solvent, dissolving the copolymer at the lowest temperature. Then, extraction experiments were carried out by EA at different temperatures on two biomass samples containing PHAs with different average molecular weights. The parallel characterization of the extracted and non-extracted PHAs evidenced that at the lower temperature (100 °C) EA solubilizes preferentially the polymer fractions richer in 3HV comonomers and with the lower molecular weight. By increasing the extraction temperature from 100 °C to 125 °C, an increase of recovery from about 50 to 80 wt% and a molecular weight reduction from 48% to 65% was observed. The results highlighted that the extracted polymer purity is always above 90 wt% and that it is possible to choose the proper extraction condition to maximize the recovery yield at the expense of polymer fractionation and degradation at high temperatures or use milder conditions to maintain the original properties of a polymer.

Control of Sulfate and Nitrate Reduction by Setting Hydraulic Retention Time and Applied Potential on a Membraneless Microbial Electrolysis Cell for Perchloroethylene Removal.

A membraneless microbial electrolysis cell (MEC) has been developed for perchloroethylene (PCE) removal through the reductive dechlorination reaction. The MEC consists of a tubular reactor of 8.24 L equipped with a graphite-granule working electrode which stimulates dechlorinating microorganisms while a graphite-granule cylindrical envelopment contained in a plastic mesh constituted the counter electrode of the MEC. Synthetic PCE-contaminated groundwater has been used as the feeding solution to test the nitrate and sulfate reduction reactions on the MEC performance at different hydraulic retention times (HRTs) (4.1, 1.8, and 1.2) and different cathodic potentials [-350, -450, and -650 mV vs standard hydrogen electrode (SHE)]. The HRT decrease from 4.1 to 1.8 d promoted a considerable increase in sulfate removal from 38 ± 11 to 113 ± 26 mg/Ld with a consequent current increase, while a shorter HRT of 1.2 d caused a partial inhibition of sulfate reduction with a consequent current decrease from -99 ± 3 to -52 ± 6 mA. Similarly, the cathodic potential investigation showed a direct correlation of current generation and sulfate removal in which the utilization of a cathodic potential of -350 mV versus SHE allowed for an 80% decrease in the sulfate removal rate with a consequent current decrease from -163 ± 7 to 41 ± 5 mA. The study showed the possibility to mitigate the energy consumption of the process by avoiding side reactions and current generation, through the selection of an appropriate HRT and applied cathodic potential.

Chromate fate and effect in bioelectrochemical systems for remediation of chlorinated solvents.

A continuous-flow bioelectrochemical reactor was developed in a previous study to address the bioremediation of groundwater contaminated by trichloroethene (TCE). The present report investigated the applicability of the same system in the presence of Cr(VI) and its possible inhibitory effect on dehalorespiring bacterial populations. Preliminary batch tests were performed at the optimal cathodic reducing potential for the reductive dechlorination (RD) of TCE (-0.65 V vs. the standard hydrogen electrode) with two different dechlorinating microorganism consortia. The results demonstrated that Cr(VI) removal efficacy was increased by microorganisms that had been previously acclimatised to Cr(VI). Specifically, Cr(VI) was completely reduced only in the presence of acclimated microorganisms. The presence of chromate negatively affected RD performance, by either (i) limiting the TCE transformation to cis-dichloroethene at lower concentrations, or (ii) completely inhibiting RD at higher concentrations. In contrast, after the acclimation period, RD was extended down to vinyl chloride, which is the main TCE daughter product. Finally, the continuous flow reactor was fed by synthetic groundwater contaminated with TCE (50 µM) and Cr(VI) (45 µM), and the experimental results showed that Cr(VI) was completely reduced under RD conditions. Moreover, TCE removal was complete, with vinyl chloride and ethene as the main intermediates, thus indicating that chromate inhibition was decreased by Cr(VI) removal.

Characterization of Polyhydroxyalkanoates Produced at Pilot Scale From Different Organic Wastes.

Polyhydroxyalkanoates (PHAs) production at pilot scale has been recently investigated and carried out exploiting different process configurations and organic wastes. More in detail, three pilot platforms, in Treviso (North-East of Italy), Carbonera (North-East of Italy) and Lisbon, produced PHAs by open mixed microbial cultures (MMCs) and different organic waste streams: organic fraction of municipal solid waste and sewage sludge (OFMSW-WAS), cellulosic primary sludge (CPS), and fruit waste (FW), respectively. In this context, two stabilization methods have been applied, and compared, for preserving the amount of PHA inside the cells: thermal drying and wet acidification of the biomass at the end of PHA accumulation process. Afterward, polymer has been extracted following an optimized method based on aqueous-phase inorganic reagents. Several PHA samples were then characterized to determine PHA purity, chemical composition, molecular weight, and thermal properties. The polymer contained two types of monomers, namely 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) at a relative percentage of 92.6-79.8 and 7.4-20.2 w/w, respectively, for Treviso and Lisbon plants. On the other hand, an opposite range was found for 3HB and 3HV monomers of PHA from Carbonera, which is 44.0-13.0 and 56.0-87.0 w/w, respectively. PHA extracted from wet-acidified biomass had generally higher viscosity average molecular weights (M v ) (on average 424.8 ± 20.6 and 224.9 ± 21.9 KDa, respectively, for Treviso and Lisbon) while PHA recovered from thermally stabilized dried biomass had a three-fold lower M v .

Effect of the temperature in a mixed culture pilot scale aerobic process for food waste and sewage sludge conversion into polyhydroxyalkanoates.

The utilisation of urban organic waste as feedstock for polyhydroxyalkanoates (PHA) production is growing since it allows to solve the main concerns about their disposal and simultaneously to recover added-value products. A pilot scale platform has been designed for this purpose. The VFA-rich fermentation liquid coming from the anaerobic treatment of both source-sorted organic fraction of municipal solid waste (OFMSW) and waste activated sludge (WAS) has been used as substrate for the aerobic process steps: a first sequencing batch reactor (SBR, 100 L) for the selection of a PHA-producing biomass, and a second fed-batch reactor (70 L) for PHA accumulation inside the cells. The SBR was operated at 2.0-4.4 kg COD/(m3 d) as OLR, under dynamic feeding regime (feast-famine) and short hydraulic retention time (HRT; 1 day). The selected biomass was able to accumulate up to 48% g PHA/g VSS. Both steps were performed without temperature (T) control, avoiding additional consumption of energy. In this regard, the applied OLR was tuned based on environmental T and, as a consequence, on biomass kinetic, in order to have a constant selective pressure. The latter was mainly quantified by the PHA storage yield (YP/Sfeast 0.34-0.45 CODP/CODS), which has been recognized as the main parameters affecting the global PHA productivity [1.02-1.82 g PHA/(L d)] of the process.

High rate selection of PHA accumulating mixed cultures in sequencing batch reactors with uncoupled carbon and nitrogen feeding.

The selection and enrichment of a mixed microbial culture (MMC) for polyhydroxyalkanoates (PHA) production is a well-known technology, typically carried out in sequencing batch reactors (SBR) operated under a feast-famine regime. With a nitrogen-deficient carbon source to be used as feedstock for PHA synthesis, a nutrient supply in the SBR is required for efficient microbial growth. In this study, an uncoupled carbon (C) and nitrogen (N) feeding strategy was adopted by dosing the C-source at the beginning of the feast and the N-source at the beginning of the famine, at a fixed C/N ratio of 33.4 g COD/g N and 12 h cycle length. The applied organic loading rate (OLR) was increased from 4.25 to 8.5 and finally to 12.725 g COD/L d. A more efficient selective pressure was maintained at lower and intermediate OLR, where the feast phase length was shorter (around 20 % of the whole cycle length). However, at the higher OLR investigated, the PHA content in the biomass reached a value of 0.53 g PHA/g VSS at the end of the feast phase, as a consequence of the increased C-source loaded per cycle. Moreover, 2nd stage PHA productivity was 2.4 g PHA/L d, 1.5 and 3.0-fold higher than those obtained at lower OLR. The results highlight the possibility of simplifying the process by withdrawing the biomass at the end of the feast phase directly to downstream processing, without a need for the intermediate accumulation step.

An urban biorefinery for food waste and biological sludge conversion into polyhydroxyalkanoates and biogas.

This study focuses on the application of the concept of circular economy, with the creation of added-value marketable products and energy from organic waste while minimizing environmental impacts. Within this purpose, an urban biorefinery technology chain has been developed at pilot scale in the territorial context of the Treviso municipality (northeast Italy) for the production of biopolymers (polyhydroxyalkanoates, PHAs) and biogas from waste of urban origin. The piloting system (100-380 L) comprised the following units: a) acidogenic fermentation of the organic fraction of municipal solid waste (OFMSW) and biological sludge; b) two solid/liquid separation steps consisting of a coaxial centrifuge and a tubular membrane (0.2 µm porosity); c) a Sequencing Batch Reactor (SBR) for aerobic PHA-storing biomass production; d) aerobic fed-batch PHA accumulation reactor and e) Anaerobic co-digestion (ACoD). The thermal pre-treatment (72 °C, 48 h) of the feedstock enhanced the solubilization of the organic matter, which was converted into volatile fatty acids (VFAs) in batch mode under mesophilic fermentation conditions (37 °C). The VFA content increased up to 30 ± 3 g COD/L (overall yield 0.65 ± 0.04 g CODVFA/g VS(0)), with high CODVFA/CODSOL (0.86 ± 0.05). The high CODVFA/CODSOL ratio enhanced the PHA-storing biomass selection in the SBR by limiting the growth of the non-storing microbial population. Under fully aerobic feast-famine regime, the selection reactor was continuously operated for 6 months at an average organic loading rate (OLR) of 4.4 ± 0.6 g COD/L d and hydraulic retention time (HRT) of 1 day (equal to SRT). The ACoD process (HRT 15 days, OLR 3.0-3.5 kg VS/m3 d) allowed to recover the residual solid-rich overflows generated by the two solid/liquid separation units with the production of biogas (SGP 0.44-0.51 m3/kg VS) and digestate. An overall yield of 7.6% wt PHA/VS(0) has been estimated from the mass balance. In addition, a preliminary insight into potential social acceptance and barriers regarding organic waste-derived products was obtained.

Elemental concentration and migratability in bioplastics derived from organic waste.

In line with the Circular Economy approach, the production of polyhydroxyalkanoate (PHA) with organic waste as the feedstock may a biotechnological application to reduce waste and recover high-value materials. The potential contaminants that could transfer from bio-waste to a PHA include inorganic elements, such as heavy metals. Hence, the total content and migratability of certain elements were evaluated in several PHA samples produced from different origins and following different methods. The total content of certain elements in PHA ranged between 0.0001 (Be) and 49,500 mg kg-1 (Na). The concentrations of some alkaline (Na and K) and alkaline earth (Ca and Mg) metals were highest, which are of little environmental concern. The feedstock type and PHA stabilisation and extraction procedures affected the element contents. Several sets of experiments were conducted to evaluate the migration of elements from the PHA samples under different storage times, temperatures, and pH levels. The total contents of some heavy metals (As, Cd, Fe, Hg, Ni, Pb, and Zn) in PHA produced from fruit waste or crops (commercial PHA) were lower than those in the PHA samples produced from the mixture of the organic fraction of municipal waste and sludge from wastewater treatment. Both the PHA obtained by extraction from wet biomass (acid storage) with aqueous phase extraction reagents and commercial PHA were below the migration limits stipulated by the current Toy Safety Directive and by Commission Regulation (EU) October 2011 on plastic materials and articles intended to come into contact with food under frozen and refrigerated conditions.