Effect of the organic loading rate on the PHA-storing microbiome in sequencing batch reactors operated with uncoupled carbon and nitrogen feeding.

Over the last years, in a search for sustainable and biodegradable alternatives to petrol-based plastics, biotechnological applications turned to the potentialities of mixed microbial cultures (MMC) for producing polyhydroxyalkanoates (PHAs). Under a feast and famine regime, an uncoupled carbon (C) and nitrogen (N)-feeding strategy may be adopted by dosing the C-source at the beginning of the feast and the N-source at the beginning of the famine in order to stimulate a PHA storage response and microbial growth. Even though this strategy has been already successfully applied for the PHA production, very few information is to date available regarding the MMC operating in these systems and the influence of Organic Loading Rate (OLR) on their selection and enrichment. To fill the gap, this study investigated the effect of the OLR on the selection of PHA-accumulating microorganisms in a sequencing batch reactor (SBR) operated with an uncoupled C and N feeding strategy. The SBR cycle length was set at 12 h and four OLRs values (4.25, 8.50, 12.75 and 18 gCOD L-1 d-1) were tested by changing the concentration of the feeding solution, made of a synthetic mixture of acetic (85% of the overall COD) and propionic (15%) acids. The PHA-storage yield increased by increasing the OLR (up to 0.69 COD/COD at 12.75 gCOD L-1 d-1) but significantly decreased (0.27 COD/COD) at 18 gCOD L-1 d-1 concomitantly with a longer feast phase and a lower PHA content in the biomass at the end of the feast phase. The selective pressure induced by the applied OLRs strongly influenced the microbiome composition revealing a high content of putative PHA-storing bacteria, such as Rhodobacter, Thauera and Paracoccus, in the SBR operated at OLRs 4.25, 8.50 and 12.75 g COD L-1 d-1 (up to 97.4% of total reads) and a low content (5.4%) in the SBR at 18 g COD L-1 d-1where the predominance of genus Nitrinicola was instead observed.

Valorization of lipid-rich wastewaters: A theoretical analysis to tackle the competition between polyhydroxyalkanoate and triacylglyceride-storing populations.

The lipid fraction of the effluents generated in several food-processing activities can be transformed into polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs), through open culture biotechnologies. Although competition between storing and non-storing populations in mixed microbial cultures (MMCs) has been widely studied, the right selective environment allowing for the robust enrichment of a community when different types of accumulators coexist is still not clear. In this research, comprehensive metabolic analyses of PHA and TAG synthesis and degradation, and concomitant respiration of external carbon, were used to understand and explain the changes observed in a laboratory-scale bioreactor fed with the lipid-rich fraction (mainly oleic acid) of a wastewater stream produced in the fish-canning industry. It was concluded that the mode of oxygen, carbon, and nitrogen supply determines the enrichment of the culture in specific populations, and hence the type of intracellular compounds preferentially accumulated. Coupled carbon and nitrogen feeding regime mainly selects for TAG producers whereas uncoupled feeding leads to PHA or TAG production function of the rate of carbon supply under specific aeration rates and feast and famine phases lengths.

Subcritical Water as a Pre-Treatment of Mixed Microbial Biomass for the Extraction of Polyhydroxyalkanoates.

Polyhydroxyalkanoate (PHA) recovery from microbial cells relies on either solvent extraction (usually using halogenated solvents) and/or digestion of the non-PHA cell mass (NPCM) by the action of chemicals (e.g., hypochlorite) that raise environmental and health hazards. A greener alternative for PHA recovery, subcritical water (SBW), was evaluated as a method for the dissolution of the NPCM of a mixed microbial culture (MMC) biomass. A temperature of 150 °C was found as a compromise to reach NPCM solubilization while mostly preventing the degradation of the biopolymer during the procedure. Such conditions yielded a polymer with a purity of 77%. PHA purity was further improved by combining the SBW treatment with hypochlorite digestion, in which a significantly lower hypochlorite concentration (0.1%, v/v) was sufficient to achieve an overall polymer purity of 80%. During the procedure, the biopolymer suffered some depolymerization, as evidenced by the lower molecular weight (Mw) and higher polydispersity of the extracted samples. Although such changes in the biopolymer's molecular mass distribution impact its mechanical properties, impairing its utilization in most conventional plastic uses, the obtained PHA can find use in several applications, for example as additives or for the preparation of graft or block co-polymers, in which low-Mw oligomers are sought.

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 .

Carbon Source Applied in Enrichment Stage of Mixed Microbial Cultures Limits the Substrate Adaptability for PHA Fermentation Using the Renewable Carbon.

Suitability of different substrates for enriched mixed microbial cultures (MMCs) is of importance to the polyhydroxyalkanoate (PHA) fermentation using renewable carbon. In this study, three enriched MMCs were evaluated for their fermentation features and kinetics with different carbon sources (sodium acetate, glucose, or starch). The results showed that the highly specific bacterial community composition was developed depending on the applied carbon source. Correspondence analysis suggested that the genus affiliated in Gammaproteobacteria_unclassified was related to 3-hydroxybutyrate (HB) synthesis in acetate-fed MMC (relative abundance of 38%) and glucose-fed MMC (relative abundance of 76.7%), whereas Vibrio genus was related to 3-hydroxyvalerate (HV) production in glucose-fed MMC (relative abundance of 0.4%) and starch-fed MMC (relative abundance of 94.6%). The acetate-fed MMC could not use glucose and starch as fermentation carbon sources, showing the limitation of microbial species developed with the specific metabolic substrate. Glucose-fed MMC produced the highest PHA cell content of 64.2% cell dry weight when using sodium acetate as the fermentation carbon. Glucose-fed MMC showed wide resilience and adaptation to various carbon sources. When actual landfill leachate was used for fermentation by glucose-fed MMC, maximum PHA cell content of 45.5% cell dry weight and the PHA volumetric productivity of 0.265 g PHA/(L·h) were obtained. This study suggested carbon sources applied in the MMC enrichment stage had a significant influence on utilization of carbon in the fermentation stage.

Exploring the feasibility of nitrous oxide reduction and polyhydroxyalkanoates production simultaneously by mixed microbial cultures.

Nitrous oxide (N2O), as a powerful greenhouse gas, has drawn increasing attention in recent years and different strategies for N2O reduction were explored. In this study, a novel strategy for valuable polyhydroxyalkanoates (PHA) production coupling with N2O reduction by mixed microbial cultures (MMC) using different substrates was evaluated. Results revealed that N2O was an effective electron acceptor for PHA production. The highest PHA yield (0.35 Cmmol PHA/Cmmol S) and PHA synthesis rate (227.47 mg PHA/L/h) were obtained with acetic acid as substrate. Low temperature (15℃) and pH of 8.0 were beneficial for PHA accumulation. Results of the thermogravimetric analysis showed that PHA produced with N2O as electron acceptor has better thermal stability (melting temperature of 99.4℃ and loss 5% weight temperature of 211.4℃). Our work opens up new avenues for simultaneously N2O reduction and valuable bioplastic production, which is conducive to resource recovery and climate protection.

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.

Microbiome dynamics and phaC synthase genes selected in a pilot plant producing polyhydroxyalkanoate from the organic fraction of urban waste.

This study analyses the bacterial population dynamics of a mixed microbial community (MMC) selected in a pilot plant producing polyhydroxyalkanoate (PHA) from the fermentation of the organic fraction of urban waste (OFMSW) and sewage sludge (SS). 16S rRNA gene high-throughput sequencing revealed the occurrence of a variety of PHA accumulating bacteria that ensured a stable PHA production in an open system operating with real substrates and without temperature control. The Volatile Fatty Acids (VFA) changes in the feed and the temperature variation affected the dynamics of the PHA-accumulating bacteria over the plant operation. Remarkably, the higher PHA content was associated to a MMC largely comprising of Hydrogenophaga species during the operation at higher working temperature. The involvement of a heterogeneous PHA-accumulating MMC was associated with a high phaC synthase genes biodiversity confirming the occurrence of a functional redundancy.

Polyhydroxyalkanoates (PHAs) production from fermented cheese whey by using a mixed microbial culture.

Two fermented cheese wheys (FCW), FCW1 composed of lactic, acetic and butyric acids in the proportion of 58/16/26 (% CODOrganic Acid (OA)) and FCW2 composed of acetic, propionic, butyric, lactic and valeric acids in the proportion of 58/19/13/6/4 (% CODOA) were used to produce polyhydroxyalkanoates (PHAs) by using a pre-selected mixed microbial culture (MMC). PHA accumulation gave for fermented FCW1 a PHA yield (Ytot) of 0.24±0.02mgCODPHAmgCODSolubleSubstrate(SS)(-1) and a total PHA production, referred to the substrate used, of 60gPHAkgcheesewheyTotalSolids(TS)(-1). For fermented FCW2 results were: PHA yield (Ytot) of 0.42±0.03mgCODPHAmgCODSS(-1) and PHA from a substrate of 70gPHAkgcheesewheyTS(-1). Qualitatively, PHAs from FCW1 was made up exclusively of 3-hydroxybutyrate (HB), while those obtained from FCW2 were composed of 40% of 3-hydroxyvalerate (HV) and 60% of HB.

A new method for polyhydroxyalkanoate (PHA) accumulating bacteria selection under physical selective pressure.

An aerobic dynamic discharge (ADD) process involving physical selective pressure to favor the accumulation of polyhydroxyalkanoates (PHAs) accumulating bacteria in microbial mixed cultures (MMCs) is proposed and evaluated in this study. Sequencing batch reactor PHA production in the ADD mode was compared to the conventional aerobic dynamic feeding (ADF) mode. Results showed that the cultures selected under the ADD mode showed a better PHA producing potential than ADF mode cultures in terms of both maximum PHA content and selection time, explained by altered physical selective pressure. The maximum PHA content of 74.16 (± 0.03)% and PHA yield of 0.72 (± 0.07) C mol PHA/C mol Ac were achieved after 30 days of the ADD operation, which were much higher than those obtained under the same organic loading rate in the ADF operation. The ADD mode is a promising method for quick selection of a strong-PHA accumulating culture.