Oxygen limitation in aerobic polyhydroxyalkanoates production from sewage sludge anaerobic fermentation liquids under low and medium organic loading rate.

The present study describes the microbial production of polyhydroxyalkanoates (PHA) from thermally pre-treated sewage sludge at pilot scale level, investigating for the first time the effect of the organic loading rate (OLR) under oxygen limitation on biomass storage properties and kinetics. Polymer characteristics have been also evaluated. The selection/enrichment of PHA-storing biomass was successfully achieved in a Sequencing Batch Reactor (SBR) under short hydraulic retention time (HRT; 2 days). Low OLR (2.05 g COD/L d) was ideal for the selection of an efficient PHA-producing consortium cultivated under limited oxygen availability. In the fed-batch accumulation conducted under high DO regime, such biomass was characterized by 51% of PHA content on cell dry weight, with a related storage yield (YP/Sbatch) of 0.61 CODPHA/CODS. On the contrary, medium OLR (4.56 g COD/L d) was not technically feasible to sustain the required consortium's selection under low DO regime. The PHA produced by biomass cultivated under low DO regime was characterized higher thermal stability and crystalline domain compared to PHA traditionally produced under high DO regime. The mass balance assessment highlighted a global yield of 51 g PHA/kg VS (volatile solids of thickened sludge), which was 9% lower than yield obtained under high DO regime, in the face of a realistic reduction of the energy cost of the process.

A Polyhydroxybutyrate (PHB)-Biochar Reactor for the Adsorption and Biodegradation of Trichloroethylene: Design and Startup Phase.

In this work, polyhydroxy butyrate (PHB) and biochar from pine wood (PWB) are used in a mini-pilot scale biological reactor (11.3 L of geometric volume) for trichloroethylene (TCE) removal (80 mgTCE/day and 6 L/day of flow rate). The PHB-biochar reactor was realized with two sequential reactive areas to simulate a multi-reactive permeable barrier. The PHB acts as an electron donor source in the first "fermentative" area. First, the thermogravimetric (TGA) and differential scanning calorimetry (DSC) analyses were performed. The PHB-powder and pellets have different purity (96% and 93% w/w) and thermal properties. These characteristics may affect the biodegradability of the biopolymer. In the second reactive zone, the PWB works as a Dehalococcoides support and adsorption material since its affinity for chlorinated compounds and the positive effect of the "coupled adsorption and biodegradation" process has been already verified. A specific dechlorinating enriched culture has been inoculated in the PWB zone to realize a coupled adsorption and biodegradation process. Organic acids were revealed since the beginning of the test, and during the monitoring period the reductive dichlorination anaerobic pathway was observed in the first zone; no chlorinated compounds were detected in the effluent thanks to the PWB adsorption capacity.

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.

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.

Combined Strategies to Prompt the Biological Reduction of Chlorinated Aliphatic Hydrocarbons: New Sustainable Options for Bioremediation Application.

Groundwater remediation is one of the main objectives to minimize environmental impacts and health risks. Chlorinated aliphatic hydrocarbons contamination is prevalent and presents particularly challenging scenarios to manage with a single strategy. Different technologies can manage contamination sources and plumes, although they are usually energy-intensive processes. Interesting alternatives involve in-situ bioremediation strategies, which allow the chlorinated contaminant to be converted into non-toxic compounds by indigenous microbial activity. Despite several advantages offered by the bioremediation approaches, some limitations, like the relatively low reaction rates and the difficulty in the management and control of the microbial activity, can affect the effectiveness of a bioremediation approach. However, those issues can be addressed through coupling different strategies to increase the efficiency of the bioremediation strategy. This mini review describes different strategies to induce the reduction dechlorination reaction by the utilization of innovative strategies, which include the increase or the reduction of contaminant mobility as well as the use of innovative strategies of the reductive power supply. Subsequently, three future approaches for a greener and more sustainable intervention are proposed. In particular, two bio-based materials from renewable resources are intended as alternative, long-lasting electron-donor sources (e.g., polyhydroxyalkanoates from mixed microbial cultures) and a low-cost adsorbent (e.g., biochar from bio-waste). Finally, attention is drawn to novel bio-electrochemical systems that use electric current to stimulate biological reactions.

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 .

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.

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.

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.

Polychlorinated Biphenyl Profile in Polyhydroxy-alkanoates Synthetized from Urban Organic Wastes.

The microbial synthesis of polyhydroxyalkanoates (PHA) from organic wastes is a valuable process to valorize available renewable resources, such as food wastes and biological sludge. Bioplastics find many applications in various sectors, from medical field to food industry. However, persistent organic pollutants could be transferred from wastes to the final product. The present paper demonstrates that the use of municipal wastes in PHA production is safe for the environment and human health and provides a polychlorinated biphenyl (PCB) profile in both commercial and waste-based PHA samples. PCB analysis in several PHA samples showed very low concentrations of the target analytes. Commercial PHA samples showed a similar PCB level with respect to PHA samples from municipal waste/sludge and higher than PHA samples from fruit waste. For all analyzed PCBs, detected concentrations were consistently lower than the ones reported in regulatory framework or guidelines.

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.

Valorization of Municipal Biowaste into Electrospun Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Biopapers for Food Packaging Applications.

The present study reports on the production and characterization of a new biopackaging material made of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from municipal biowaste (MBW) and produced by the mixed bacterial culture technology. After purification and extraction, the MBW-derived PHBV was processed by electrospinning to yield defect-free ultrathin fibers, which were thermally post-treated. Annealing at 130 °C, well below the biopolymer's melting temperature (Tm), successfully yielded a continuous film resulting from coalescence of the electrospun fibrillar morphology, the so-called biopaper, exhibiting enhanced optical and color properties compared to traditional melt compounding routes. The crystallinity and crystalline morphology were comprehensively studied as a function of temperature by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and combined time-resolved synchrotron small- and wide-angle X-ray scattering (SAXS and WAXS) experiments, which clearly indicated that the molecular order within the copolyester was improved up to a maximum at 130 °C, and then it decreased at the biopolymer's Tm. It was hypothesized that by annealing at the temperature at which the thermally induced molecular order is maximized, the fibers generated sufficient mobility to align alongside, hence reducing surface energy and porosity. The data suggest that this material shows a good balance between enhanced mechanical and improved barrier properties to vapors and gases in comparison to traditional paper and other currently used petroleum-derived polymers, thus presenting significant potential to be part of innovative food biopackaging designs for the protection and preservation of foods in a circular bioeconomy scenario.