Next Article in Journal
A Symmetric Projection Space and Adversarial Training Framework for Privacy-Preserving Machine Learning with Improved Computational Efficiency
Next Article in Special Issue
Macroissues with Microplastics: A Review on Distribution, Environmental Impacts, Pollutant Interactions, Toxicity, Analytical Methodology and Mitigation Strategies
Previous Article in Journal
The Incorporation of Recycled Aggregate Concrete as a Strategy to Enhance the Circular Performance of Residential Building Structures in Spain
Previous Article in Special Issue
Upcycling Alum Sludge as a Reinforcement in PBAT Composites: A Sustainable Approach to Waste Valorisation
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Sciences
Volume 15
Issue 6
10.3390/app15063272
applsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste

by
Irene Izarra
*,
Irene Álvarez
,
F. Javier Pinar
and
Javier Mena
CLAMBER R&D Biorefinery, Pol. Aragonesas. Ctra. de Calzada (CR-503), 13500 Puertollano, Spain
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(6), 3272; https://doi.org/10.3390/app15063272
Submission received: 4 February 2025 / Revised: 28 February 2025 / Accepted: 13 March 2025 / Published: 17 March 2025
(This article belongs to the Special Issue Waste Recycling Materials and Technologies: Latest Advances and Prospects)
Browse Figures
Versions Notes

Abstract

:
The production of short-chain-length polyhydroxyalkanoates (scl-PHAs) from municipal solid waste-derived volatile fatty acids (VFAs) has been demonstrated. The objective of the study was to evaluate the technical feasibility of the process under real operational conditions. Moreover, the process operation was conducted without pH and temperature control to reduce potential industrial implementation barriers, i.e., by simplifying the process control and minimizing the auxiliary services available for the process. A two-step bioprocess was developed, consisting of an enrichment phase in a 20 m3 fermenter operated for 214 days and an accumulation phase carried out in a 3 m3 batch fermenter across 39 accumulation cycles. In the enrichment phase, steady-state conditions were achieved once the feast/famine ratio was lower than 0.2 h/h. Thus, the impact of environmental conditions was analyzed. It was found that the system’s response was a destabilization of the culture under sharp variations at environmental temperature, followed by an adaptation period and final recovery of the system. During the accumulation phase, the impact of chemical oxygen demand (COD) feeding rates was assessed, with a maximum scl-PHA accumulation of 59 wt.% (2.87 g/L) recorded. The extraction process was also performed at demonstrative scale using dimethyl carbonate (DMC) as the solvent, yielding a scl-PHA recovery of 92% with a purity of 90%. These results confirm the technical feasibility of producing scl-PHAs from municipal organic waste at demonstrative scale, supporting the circular bioeconomy model.

1. Introduction

With the growth of the global population, the consumption of materials, foods, and energy is drastically increasing [1]. This generates a significant increase in the amount of waste generated by the society [2,3,4]. Currently, 2.01 billion tonnes of municipal solid waste (MSW) are generated in the world every year [5]. Specifically, in Europe, each inhabitant generates, on average, 0.5 tonnes of MSW per year, increasing by an annual rate of 10% [6,7]. Around 40–50% of this corresponds to organic waste, highly rich in lipids, proteins, and carbohydrates [7]. The accumulation of MSW poses serious environmental and economic challenges, particularly regarding waste management, greenhouse gas emissions, and the depletion of natural resources [8]. Conventional waste disposal methods, such as landfilling and incineration, not only contribute to environmental pollution but also represent an inefficient use of valuable organic materials. Consequently, there is a growing interest in the development of sustainable waste valorization strategies that align with circular economy principles, aiming to convert waste into valuable bioproducts [8,9]. The valorization of these waste or by-products may decrease environmental pollution, oil dependence, and contribute to the implementation of a renewable circular economy scenario [10,11]. The present work aligns with a circular economy model as it focuses on demonstrating the valorization of the Organic Fraction of Municipal Solid Waste (OFMSW) in a real environment into a biodegradable bioplastic, such as polyhydroxyalkanoate or PHA, on a large scale with the aim of studying its technical viability [6].
PHAs are natural thermoplastic polyesters with interesting environmentally friendly applications [12]. More than 160 types of PHAs, produced using different monomers, have been identified [12,13]. Individual monomers of PHAs can be sorted as short-chain-length (scl) (4 to 5 carbons), medium-chain-length (mcl) (6 to 12 carbons), and long-chain-length (lcl) (more than 14 carbons) [12,14,15].
PHAs can exist as homopolymers or copolymers, consisting of both mcl- and scl-PHAs [16]. Scl-PHAs include poly(3-hydroxybutyrate) (P3HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(4-hydroxybutyrate) (P4HB), copolymers of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (PHB-4HB), copolymers of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), and poly(3-hydroxyoctanoate) (PHO) [12,17]. Nevertheless, poly-(3-hydroxybutyrate) (PHB), a homopolymer with four carbon subunits of 3-hydroxybutyrate (3HB), is the most common and extensively characterized member of the PHAs family [12,18,19].
In this regard, the presented research shows the development of short-chain-length PHAs (scl-PHA) using pretreated OFMSW as a carbon source through a mixed microbial culture (MMC) on a semi-industrial scale. The OFMSW pretreatment consisted of anaerobic digestion to produce a volatile fatty acid (VFA) rich digestate coming from a hydrolytic reactor. The current study contributes to addressing a critical gap in the field of larger-scale biopolymer production than at laboratory or pilot scale by demonstrating under real conditions the feasibility of utilizing the OFMSW as a carbon source for short-chain-length polyhydroxyalkanoate (scl-PHA) biosynthesis on a demonstrative scale.
Two of the main challenges of PHA industrial production are the technical and economic viability of the process, particularly due to the high cost of conventional carbon sources. The origin of the carbon source is crucial for PHA production and represents a significant cost [12,20,21]. The carbon source is metabolized to PHAs, and the chain length of the carbon source has a huge influence on the type of PHA synthesized. The resulting process consists of the following: (1) Transformation of the substrate into VFA; (2) enrichment of PHA accumulating bacteria; (3) PHA accumulation; and (4) extraction [22,23,24]. The existing literature provides extensive insights into PHA production and extraction at laboratory and pilot scales of 5, 10, or 200 L [25,26,27]. So far, there is a significant lack of literature focusing on PHA demonstrative scale production, especially in large fermenters, i.e., the technology has yet to be proven in a real environment, and it has not reached a sufficient readiness level. The transition from small-scale to industrial-scale fermenters introduces various challenges, including process stability, microbial community management, and optimization of operation conditions such as temperature, aeration, and pH control. Specifically, this study evaluates the production of scl-PHAs using MMC and OFMSW-derived VFAs as the primary carbon source in a 20 m3 fermenter and the accumulation fermentation in 3 m3. This research aims to optimize critical operational parameters to improve microbial performance and overall process efficiency.
The microorganism’s ability to store PHA intracellularly can be selected and enriched under aerobic dynamic feeding conditions, where the MMC are exposed to cyclic feast and famine conditions (usually represented as ratio F/F) [28,29,30]. This F/F cycle is carried out under aerobic conditions where during short periods of time, the carbon source is easily accessible for MMC (feast phase) followed by long periods of time in the absence of a carbon source (famine phase) [28,30]. During the feast phase, the MMC is able to metabolize PHA from the carbon source, storing it intercellularly in the form of granules. Once the carbon source has been consumed, the PHA-accumulating microorganisms use the previously stored PHA as an energy source to survive during famine phases. This accumulation capacity of the MMC is crucial for conferring them a significant competitive advantage over other non-PHA-accumulating MMC during the starvation phase [28,31]. Indeed, to achieve an adequate balance between the time lasted for the feast phase and the time lasted for the famine phase, i.e., the F/F ratio, is very important to assure a large number of PHA-storing MMC [32].
The employment of dynamic conditions (discontinuous feeding) together with an adequate balance between the F/F ratio during the enrichment phase lies in the presence of PHA-storing microorganisms in the activated sludge of wastewater treatment plants (WWTT) [33,34,35]. The feast phase must be sufficient to ensure complete substrate removal, while the famine phase must be prolonged enough to allow substantial consumption of the previously accumulated PHA [28,36,37] and to minimize the presence of MMC incapable of accumulating PHAs.
Once the enrichment step of PHA production is completed and the fermentation broth is rich in PHA-accumulated microorganisms, the accumulation reaction takes place [38]. Its objective is to increase as much as possible the intracellular accumulation of the PHA in the MMC cells [28]. Some authors [28,39,40,41] have reported that the fed-batch strategy with MMC reaches a high scl-PHA productivity but, after the feeding cycles take place, the accumulated scl-PHA is largely consumed before the addition of a new pulse with a carbon source and, finally, the scl-PHA productivity decreases.
On the one hand, the feast/famine (F/F) regime is effective in selecting PHA-accumulating microorganisms, but the implementation of this strategy at industrial scale presents several challenges. Thus, maintaining stable F/F cycles under fluctuating environmental and operational conditions can significantly impact microbial selection and PHA accumulation [28,30]. Additionally, optimizing the organic loading rate (OLR) and aeration parameters is crucial to prevent process inhibition while ensuring efficient substrate removal and biomass enrichment. The lack of precise control over temperature and pH can lead to process destabilization [42,43,44] (if the pH is outside the optimal range of 6.5 to 8), particularly during colder seasons. Furthermore, achieving a balance between high PHA productivity and microbial growth remains complex. Excessive substrate availability may lead to biomass proliferation rather than polymer accumulation. Finally, downstream processing, particularly PHA extraction and purification, remains a bottleneck, as conventional solvent-based methods are costly and present environmental concerns [45]. Overcoming these outlined challenges requires fine-tuning operational strategies, and developing cost-effective and scalable recovery techniques to make PHA production viable for its industrialization.
Burniol-Figols et al. [46] successfully converted crude glycerol into VFAs for PHA production. They achieved 76 g scl-PHA/100 g TSS in a 1.7 L sequential batch reactor (SBR) under feast/famine (F/F) conditions, with a PHB:PHV ratio of 84:16. Similarly, Freches et al. [47] used glycerol in a 1.5 L SBR, applying 24 h cycles with three 50 mM carbon source pulses per cycle, obtaining a biomass with 59 wt.% scl-PHA. Bengtsson et al. [48] studied the production of scl-PHAs using paper mill wastewater at laboratory scale with a 24 h cycle length, reaching a maximum PHA content of 48 wt.%.
Few studies have reported PHA production beyond the pilot scale. The RES URBIS H2020 project [25,26,27] investigated PHA production from OFMSW at a pilot scale, using a 380 L anaerobic reactor to produce VFAs, followed by a 100 L SBR for activated sludge cultivation over 8.5 months. The process maintained a 1-day HRT, a cycle length of 6–12 h, and controlled temperature above 24 °C in colder months. The pH was not regulated, and the C:N:P ratio was 100:7:1. Accumulation occurred using a fed-batch strategy, achieving up to 0.4 g scl-PHA/g TSS with a PHV content between 22–40 wt.%.
PHA extraction is also challenging for industry nowadays due to solvent cost, solvent selectivity, environmental concerns of the solvent, or degradation of the polymer during extraction. Since bacterial PHAs are more costly than fossil-based plastics, cost-effective recovery methods are crucial [45]. Factors such as biopolymer type, purity, PHA properties, cost, and environmental impact must be considered [49,50]. PHA extraction typically follows two main approaches: biomass dissolution using oxidants or solvent-based extraction, with a growing emphasis on eco-friendly alternatives such as dimethyl carbonate (DMC) [51,52,53]. The efficiency and feasibility of these methods are highly dependent on process scale, as solvent costs, selectivity, and polymer stability become more critical at larger scales. This study contributes to process scalation by exploring a sustainable DMC-based extraction method, advancing environmentally friendly and industrially viable PHA recovery.
Bio-based scl-PHA obtained after extraction with DMC showed better mechanical properties due to higher plastic deformation capability than that obtained from the same biomass using chloroform and dichloromethane [54]. Samorì et al. [55] extracted PHA at lab scale and employed DMC and a combination of DMC and sodium hypochlorite as solvents for 1 h and 90 °C. The experiment resulted in a PHA recovery of 49 ± 2 wt.% with a purity above 94%. Samori et al. [52] also reported in another study a recovery yield of 88 ± 6% at the same temperature but increasing the contact time of up to 4 h. On the other hand and to the best of our knowledge, studies have not been reported on DMC-base or not PHA solvent extraction processes at demo or pilot scale. This is because of the harmful and flammable characteristics of the most used solvents that can create hazards for the environment and the operators [28]. Therefore, PHA extraction technology requires further maturity. Another study was found that employs supercritical fluid extraction but this methodology requires high investment in equipment and high energy consumption [56].
All in all, the novelty of the presented study lies in the production of scl-PHAs from VFAs derived from municipal solid waste at a demonstrative scale, a bioprocess extensively studied on laboratory scale. Consequently, this study aimed to assess the feasibility of the process under real operational conditions. Furthermore, the pH and temperature control of the process is simplified, aiming to minimize potential industrial implementation barriers. A two-stage bioprocess was implemented, including an enrichment phase in a 20 m3 fermenter operated for 214 days, followed by an accumulation phase conducted in a 3 m3 batch fermenter over 39 cycles. During the enrichment phase, stable conditions were achieved with a feast/famine ratio below 0.2 h/h. On the other hand, a continuous feeding strategy was implemented to maximize scl-PHA production during the accumulation phase [28] with a specific focus on evaluating the influence of the carbon source feeding rate on the ability of MMC cells to generate scl-PHA. Finally, for scl-PHA extraction, a solvent-based method using DMC and ethanol was chosen as a sustainable alternative to traditional solvents like chloroform or methanol. These findings validate the technical viability of the presented scl-PHA production process, reinforcing its potential within the circular bioeconomy framework.

2. Materials and Methods

2.1. Enrichment Fermentation

The MMC enrichment in scl-PHA accumulating bacteria was carried out in a 20 m3 fermenter (Figure 1a, FS-20000 series, Bionet, Murcia, Spain) with a working volume of 15 m3. The fermenter consists of the following main parts: fermenter reactor, stirring system able to reach up to 50 rpm (shaft with Rushton-type blades), vent condenser, instrumentation (transmitters for temperature, pressure, pH, and dissolved oxygen, with an air flow meter and upper-level switch for foams) and control system by the control unit. Process air was used as the auxiliary service with a capacity to reach up to 3600 Nm3/h. The auxiliary service is also equipped with a flowmeter, proportional control valve, and automatic on/off valve).
Table 1 shows the operational conditions employed to grow PHA-accumulating MMC at the enrichment stage.
The inoculum (50% v/v) was a mixture of aerobic sludge from the wastewater treatment plant of the municipality of Puertollano, Spain. The MMC was monitored at the end of each F/F cycle by means of total suspended solids (TSS), volatile suspended solids (VSS), pH, and chemical oxygen demand (COD). Oxygen saturation (pO2) was also monitored but during the whole F/F cycle. Steady conditions were considered to be reached at the enrichment fermenter once the TSS and VSS were stabilized, and the feast/famine (F/F) ratio was lower than 0.2 h/h [39].
The carbon source employed was a solution of VFAs. This solution, rich in VFAs, is the result of the pretreatment of the Organic Fraction of Municipal Solid Waste (OFMSW) selectively collected by URBASER in the municipality of Zaragoza, Spain in anaerobic digestion (AD) [57]. Table 2 summarizes the average analytical results of the VFA-rich solution used as the carbon source.
Prior to use, the VFA-rich digestate was clarified in a Alfa Laval Clara 80 centrifugation system (Alfa Laval AB, Lund, Sweden) to eliminate any solid material that could interfere with the subsequent PHA transformation process. Thus, TSS and VSS were reduced after clarification to a final concentration of 1.34 ± 0.10 and 1.1 ± 0.10, respectively. Nevertheless, the average daily VFA feeding composition to the MMC was as follows; acetic acid (5.60 g/L), propionic acid (1.37 g/L), isobutyric acid (0.33 g/L), butyric acid (11 g/L), isovaleric acid (1.12 g/L), valeric acid (1.60 g/L), hexanoic acid (10 g/L), and heptanoic acid (0.70 g/L). The soluble COD of the VFA solution was 47 ± 7.61 g/L. Water dilution was needed to reduce the high VFA concentration of the original VFA-rich digestate solution. Thus, OLR was set to be 1 g COD per L of fermenter and day.
During the commissioning of the bioprocess, OLR was initially set at 0.5 COD/L.d (cycles 1 to 13) and gradually increased. Therefore, OLR was set at 0.7 for cycles 14 to 21), at 0.9 for cycles 22 to 50, and finally it was set at 1 g COD/L.d for cycles 51 to 214. The C:N:P molar ratio in the medium was fixed at 1:0.15:0.05 adding NH4Cl and K2HPO4. Allylthiourea (10–15 mg/L) [58] was also supplied during the first 3 HRTs to avoid nitrifier bacteria growth and Antifoam 204 (Sigma, Saint Louis, MO, USA) was employed when foam formation was observed.

2.2. Scl-PHA Accumulation

Achieving steady conditions in the enrichment fermenter is crucial for the accumulation of the scl-PHA. To accumulate scl-PHA, MMC requires the limitation of an essential nutrient, typically nitrogen (N) or phosphorus (P). In this case, phosphorus was set as the limiting nutrient and it was removed via NaOH precipitation from the clarified VFA-rich digestate stream. The resulting C:N:P ratio was 1:0.08:0 in the accumulation stage diet. Under these conditions, the culture is metabolically driven to store carbon sources as intracellular scl-PHA [59].
Scl-PHA accumulation was run in a 3 m3 fermenter (Figure 1b, F-3000 series, Bionet, Murcia, Spain), geometrically similar to the 20 m3 fermenter but with stirring capabilities up to 190 rpm. The fermenter operated in batch mode, starting with an initial working volume of 1 m3 and filled with continuous feeding of pretreated VFA-rich digestate. The feeding flow rate was adjusted to each trial, based on the maximum VFA consumption rate at steady conditions and the soluble COD of the fermentation broth in the enrichment fermenter. The accumulation process was performed in 39 batches, with continuous feeding of phosphorus-free VFA-rich digestate from the daily discharge of the enrichment fermenter at steady conditions. Each accumulation lasted 8 h, with agitation and aeration control set to maintain dissolved oxygen (DO) above 20%, i.e., ensuring no oxygen limitations. Temperature and pH were monitored but not controlled.

2.3. Scl-PHA Extraction

Once every accumulation cycle was ended, the biomass rich in scl-PHA from the fermenter was centrifugated, thermally treated, and washed with ethanol to prevent PHA content reduction and degradation. Cell debris was separated by batch centrifugation and decantation. The PHA biopolymer was then extracted with dimethyl carbonate (DMC). Finally, a solvent recovery system was used to concentrate scl-PHA in the DMC phase.
Demo-scale extraction was carried out at the Solvent Extraction Unit. This unit includes two 500 L agitated percolators (Model DP1, UTID, Barcelona, Spain) for scl-PHA extraction and a 160 L rotary extractor (Formeco, model Di60 Ax LCD, Noventa Padovana, Italy). The unit also features two 1000 L solvent tanks, a mesh filter, two heat exchangers, four buffer tanks, two condensers, and solvent recovery tanks. The agitated percolators and heating systems ensured efficient mixing and extraction of the biopolymer.
The scl-PHA extraction process is summarized as follows: First, after each accumulation reaction in the fermenter, the biomass of the fermentation broth was separated from the liquid phase using a continuous disk stack centrifugation system (Alfa Laval CLARA 80, Lund, Sweden) at 8000 rpm with a flow rate of 1.5 m3/h in a loop for 4 h. Then, the biomass was heat-treated at 80 °C for 20 min in an oven (JP Selecta, model 2001253, Barcelona, Spain). Next, the biomass was washed with ethanol 96% vol. (CAS number 64-17-5, VWR) at a biomass/ethanol ratio of 1:3 (w/w) at 100 rpm for 4 h to eliminate bacterial debris in the ethanol phase. Subsequently, the biomass was separated from the liquid phase through a 1 µm mesh and the PHA was extracted with DMC (CAS number 616-38-6, GERDISA, Guadalajara, Spain) in percolators at a biomass/DMC ratio of 1:10, at 60 °C for 8 h. After PHA extraction, solid waste biomass was again removed using a 1 µm mesh. The biomass was then subjected to a second and third consecutive extraction step with DMC and solid–liquid separation at the same previously described conditions. The DMC containing scl-PHA from the three extraction steps was concentrated (ratio 1:5) in the rotary evaporator (Formeco, model Di60 Ax LCD, Noventa Padovana, Italy) and stored at 4 °C for scl-PHA precipitation. The scl-PHA precipitated was separated by decantation from the supernatant DMC phase. The solvent was recovered through evaporation for the next extraction process. The remaining DMC was removed from the precipitated scl-PHA by evaporation in a lab fume hood system. Finally, scl-PHA was ground into a powder using a Bosch kitchen chopper. No overheating or melting was observed during grinding, and no additional cooling measures were necessary.

2.4. Analytical Methods

Fresh samples of the fermenters were taken before and after feeding in the enrichment and accumulation fermenters. TSS and VSS were measured according to standard protocols [60].
The samples were filtered (0.45 μm) prior to analysis of soluble nitrogen N-NH4, COD, and VFA content. Soluble nitrogen N-NH4 was measured by spectrophotometry using the Ammonium Cell Test (Merck, Darmstadt, Germany, ref 1.14558.0001). COD was also measured by spectrophotometry using the COD Cell Test (Merck, ref 1.14541.0001). The employed spectrophotometer was a Spectroquant 100 (Merck). VFA composition was analyzed by High Pressure Liquid Chromatography in a Chromaster HPLC system (Hitachi, Tokyo, Japan) equipped with an RI detector and Agilent MetaCarb 67H 300 × 6.5 mm chromatographic column.

2.4.1. Gas Chromatography Analysis of scl-PHA

Analysis of the methyl-3-hydroxy acids was performed on a gas chromatograph (GC) (Shimadzu, Nexis GC-2030, Kyoto, Japan) equipped with SH-Stabilwax-DA, 0.25 mm ID, 0.25 umdfM 30 m UN column. A total of 1 µL of the sample was injected by split injection. Temperature programming for scl-PHAs analysis was 2 min at 60 °C, the first temperature ramp was set at 10 °C/min up to 160 °C, and the second temperature ramp was set at 20 °C/min up to 230 °C. Hydrogen was used as the gas carrier, the injector temperature was 260 °C, and the detector temperature was 275 °C. The GC was previously calibrated to measure PHB and PHV by means of a calibration line and using poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) as a pattern [25]. Methyl-3-hydroxy acids concentrations were calculated from peak areas as determined by an integrator [61].

2.4.2. Scl-PHA Determination in the Whole Cells

After biomass accumulation, the samples were centrifuged at 4000× g for 10 min using a laboratory centrifuge (Ultra Centrifugal Retsch ZM 200, Haan, Germany). The resulting pellets were freeze-dried using a freeze dryer (Noxair, model LYOBENCH 55, Barcelona, Spain). The lyophilized cell pellets containing PHA were methanolized in duplicate. Each sample consisted of 20 mg of freeze-dried cells combined with a mixture of 2 mL of methanol containing sulfuric acid and 2 mL of chloroform containing methyl benzoate as an internal standard. The mixture was vigorously agitated in a screw cap tube and incubated at 100 °C for two hours. After cooling for 5 min, 4 mL of distilled water was added and vortexed for 1 min. Following phase separation, the organic phase was dried with Na2SO4 and analyzed by gas chromatography (GC) [61].
The purity of scl-PHA and the contents of PHB and PHV were calculated as the percentage of TSS on a mass basis with respect to the concentration of PHB and PHV detected by GC.
The scl-PHA storage yield (based on VFA) during the accumulation stage was calculated as the ratio between the amount of stored PHA and the amount of the removed soluble VFA. The scl-PHA content in the biomass was calculated at the end of each feast/famine cycle and at the end of each accumulation stage and expressed as the ratio of scl-PHA concentration in the biomass and TSS.

3. Results

3.1. Enrichment of scl-PHA-Accumulating Microorganisms

The enrichment process was carried out in a 20 m3 fermenter over 214 days to develop a microbial culture capable of accumulating scl-PHA.
Due to the slow growth of MMC observed during the first cycles, and to favor the growth of accumulating bacteria, allylthiourea (ATU) was added (2 g/L) to the daily feeding from cycle 28 during three TRH, i.e., up to cycle 43, to reach a faster stabilization of culture broth rich in accumulating bacteria. ATU was employed to remove the nitrifying bacteria that can subsist in the famine phase due to its capacity to obtain energy from ammonium oxidation without a carbon source [46]. The addition of ATU to inhibit nitrifying bacteria in PHA-producing fermenters is a common practice to avoid the competition of these bacteria with PHA-accumulating bacteria for ammonium and oxygen [58,62]. Figure 2 shows the evolution of TSS, VSS, pH, and COD over all the enrichment cycles performed.
An example of a F/F cycle at steady-state conditions and COD consumption during the cycle can be observed in Figure 3.
Feast and famine phases were monitored through the variation in DO concentration over the cycle. When an external carbon source was available (VFAs), DO decreased due to substrate consumption and the shift between feast and famine was characterized by a sudden increase in DO as a result of carbon source depletion [47]. The VFA profile during the whole cycle was not monitored but it was indirectly measured through the soluble COD (CODs) as samples were taken at different times during an enrichment cycle. The CODs reached values lower than 100 mg/L from approximately the third hour of the cycle until its end. The CODs monitorization started right once the VFA daily feeding of the enrichment fermenter was completed. Hence, the initial COD measure was 850 mg/L instead of 1000 mg/L.
Thus, when the OLR was set at 1 g COD/L.d (cycle 51), the COD consumption rate was monitored at different cycles during operation (Table 3).
The scl-PHA average percentage in the enriched biomass (before accumulation stage), assessed over 214 days of operation, was 0.39 ± 0.12 wt.%.
Figure 4 shows the temperature profile of the MMC during the whole enrichment operation.

3.2. Accumulation of scl-PHA in Microorganisms

Once the fermenter reached steady conditions, MMC in the enrichment fermenter was ready to start with the accumulation stage. Therefore, a total amount of 39 accumulation batches were performed. This accumulation stage was performed in the 3 m3 fermenter to increase the amount of PHA stored in the MCC. Every batch performed in the scl-PHA accumulation stage consisted of a fed-batch mode at a constant flow rate of the free phosphorus VFA-rich solution into a fixed volume of the daily discharge of the enrichment fermenter. Initially, the applied COD feeding rate was similar than the COD consumption rate obtained in the feast phase of the enrichment fermenter.
In this study, different COD feeding rates in the accumulation fermenter were analyzed. First, ten accumulation batches were performed using the biomass obtained in selected enrichment cycles between 111 and 131. The COD consumption rate was 13.91 mg COD/L.min (Table 3). Thus, the COD feeding rate was varied from 9.86 to 24.44 mg COD/L.min to study the scl-PHA storage capacity influenced by the COD feeding rate. Moreover, the aeration and agitation were automatically controlled in a cascade control by varying the aeration and agitation from 0 to 700 m3/h, and 20 to 190 rpm, respectively, assuring a DO concentration higher than 20%. Table 4 summarizes the data of the first ten accumulation batches.
The influence of the COD feeding rate as function of the percentage of scl-PHA in the biomass from accumulation batches 1 to 10 can be seen in Figure 5.
To deeply study the influence of DO, a second set of 29 accumulation batches was run. In this case, the discharge of the enrichment fermenter from cycle 136 up to cycle 204 was employed. The COD consumption rate during those cycles was 19.56 mg COD/L.min (Table 3).
Aeration and agitation were set to the maximum values allowed by the 3 m3 fermenter system (700 m3/h and 190 rpm, respectively). The high aeration and agitation rates ensured a dissolved oxygen concentration exceeding 70%, thereby deactivating the cascade control.
Table 5 summarizes the data of this second set of accumulation batches.
The influence of the COD feeding rate as function of the percentage of scl-PHA in the biomass from accumulation batches 11 to 39 can be seen in Figure 6.
The average composition of the scl-PHA, in terms of PHB and PHV percentage ratio can be observed in Figure 7 for all the accumulation batches performed.
Accumulation batches from 11 to 39 have been performed under the same aeration and agitation conditions, while accumulation batches from 1 to 10 suffered important variations in those parameters due to cascade control as explained above.
To sum up, a total amount of 83 kg of scl-PHA was generated by using 75 t of digestate rich in VFAs during the 214 days of operation during enrichment stager. In the case of the accumulation stage, 23.50 t of VFA-rich digestate was used for all scl-PHA accumulation batches. Considering an average soluble COD of 47 g/L in the VFA-rich digestate, it was necessary in the accumulation reaction an amount of 13.5 kg CODs/kg scl-PHA.

3.3. Extraction of scl-PHA from the Produced Biomass at Demo Plant

The biomass obtained from the accumulation batches 11 to 39 was also extracted at Demo scale following the procedure described in Section 2.3. The scl-PHA obtained in accumulation batches 1 to 10 was not able to be extracted due to the low scl-PHA content observed in the cells. The biomass obtained from each accumulation batch was separated from the liquid phase, thermally treated, and subsequently stored at 4 °C along with the other batches.
Figure 8 shows the appearance of one of the fermentation broths before and after its centrifugation. The total amount of wet biomass recovered (concentrated fraction) after solid–liquid separation was 543 kg with a relative humidity of 80% and scl-PHA weight content of 22.3%.
Table 6 summarizes the results of the scl-PHA extraction process.
The appearance of wet extracted scl-PHA in DMC, the wet scl-PHA after DMC removal, dry scl-PHA, and grinded scl-PHA with a purity of 90% is shown in Figure 9.

4. Discussion

4.1. Enrichment of scl-PHA-Accumulating Microorganisms

The enrichment phase successfully selected an MMC capable of accumulating scl-PHAs under dynamic feast/famine (F/F) conditions. Steady-state conditions were established after approximately 65 cycles (Figure 2), characterized by stable total suspended solids (TSS), volatile suspended solids (VSS), and a consistent F/F ratio below 0.2 h/h. The biomass concentration (VSS) increased until reaching an organic loading rate (OLR) of 1 g COD/L·d (cycle 51), maintaining an average VSS concentration of ~2 g/L.
This stabilization of biomass concentration is particularly relevant in the context of PHA-accumulating microbial culture selection, where the F/F ratio plays a crucial role [47,63,64]. Studies have shown that low F/F ratios (<0.2) confer a competitive advantage to PHA-accumulating microorganisms over non-accumulating bacteria [47,65]. A very low F/F ratio is indicative that the process could tolerate a higher OLR. The different shoulder shapes observed in the DO profile (Figure 3) may be attributed to varying rates of VFA consumption by MMC.
The lack of temperature and pH control is the main limitation of the process studied. On the one hand, this approach simplifies the process control and requires auxiliary services for its further industrialization. On the other hand, it also introduces variability into process stability. A notable destabilization occurred between cycles 150 and 180, where TSS, VSS, and pH dropped significantly. This event coincided with a sharp decrease in the environmental temperature (Figure 4), likely reducing microbial growth rates and metabolic activity. Although the MMC exhibited resilience and was recovered after ~30 cycles, this adaptation period could carriage challenges at larger-scale applications where process stability is critical. Additionally, the inherent heterogeneity of the OFMSW used as the feedstock may have also contributed to fluctuations in substrate composition, further impacting microbial selection and process performance. It has been found that the variability in the composition of agri-food waste significantly influences the efficiency and yield of VFA and subsequent PHA production. According to Arriaga et al. [66], the type and proportion of organic substrates present in the feedstock directly affect the metabolic pathways of microbial communities involved in fermentation. For instance, carbohydrate-rich wastes generally promote the formation of acetic and propionic acids, whereas lipid- and protein-rich residues favor the accumulation of butyric and valeric acids. Additionally, process parameters such as pH, retention time, and the presence of specific microbial consortia further modulate the VFA profile, ultimately impacting the composition of PHAs, including the proportion of HB and HV monomers in PHBV. The study highlights the importance of substrate selection and process optimization to achieve tailored VFA compositions, which can enhance the mechanical and thermal properties of PHBV bioplastics, ensuring their suitability for various industrial applications.
Conversely, the VSS concentration remained stable (Table 3), although an increase in COD consumption was observed. The COD consumption rate progressively increased from 10.10 mg COD/L·min to 19.60 mg COD/L·min by cycle 133, suggesting improved microbial metabolic activity. However, the study did not explore the upper limit of OLR that the MMC could tolerate. Higher OLRs could enhance productivity but may also increase the risk of system overload, requiring further optimization [67].
Valentino et al. [42] studied PHA production with MMC using SBR. They reported COD consumption rates ranging from 21.8 to 33.45 mg COD/L·min. The higher consumption rates were typically achieved with shorter cycle lengths (2 to 6 h) and increased feeding frequencies (4 to 12 times per day), demonstrating the influence of operational strategies on COD removal efficiency. In contrast, the present study that was conducted under real operational conditions, i.e., without synthetic VFA addition, recorded a lower consumption rate (19.56 mg COD/L.min), suggesting that feed composition and cycle duration significantly impact system performance.
Temperature control has been identified as a key factor in optimizing PHA production by several authors [42,44]. The studies have shown that optimal temperatures (25–30 °C) enhance microbial activity, substrate uptake, and PHA accumulation. While previous research demonstrated improved performance under controlled temperature conditions [42,43,44], maintaining such temperature control at an industrial scale is energy-intensive, and as a result, it increases the cost of the final bioproduct. The observed microbial adaptation to environmental fluctuations in this study suggests that implementing modifications in other process variables, rather than strict temperature control, could be a more viable strategy for large-scale applications.
The enhancement of the process’s robustness and industrial scalability must be the center of future research. Thus, prioritizing the mitigation of temperature fluctuations should be examined by developing more efficient process control strategies, such as optimizing organic loading rates (OLR) and/or conducting comprehensive microbial community analyses. The implementation of precise environmental regulations, refining substrate feeding regimes, and leveraging advanced bioprocess monitoring techniques will be crucial for improving system stability and ensuring consistent biopolymer yields in large-scale operations.
While temperature control may not be feasible in large-scale operations with MMC, adaptive strategies such as adjusting HRT or modifying feeding cycles during colder months could also be helpful in mitigating microbial activity loss. Correspondingly, further research should evaluate increasing OLRs beyond 19.6 mg COD/L·min to determine the threshold at which microbial performance is maximized without causing system instability. A more comprehensive study on microbial population dynamics during the enrichment phase may provide feedback on identifying key species responsible for scl-PHA accumulation and environmental stressors. Moreover, rather than implementing comprehensive pH and temperature control, partial regulation methodologies, such as passive insulation for temperature stabilization or intermittent buffering for pH control, could be further investigated to ensure economic and technical feasibility.
Regarding pH control in the process, Figure 2 shows how the pH profile remained relatively stable throughout the enrichment cycles, fluctuating around 8–9 with minor variations. This stability suggests that the lack of pH control did not compromise microbial selection or PHA accumulation. The observed pH range aligns with the literature reports [42,43,44] indicating optimal conditions for PHA-accumulating bacteria (6.5–8.0), preventing the proliferation of non-accumulating species. A slight pH decreased around cycles 150–180 coincided with fluctuations in TSS and VSS, suggesting that environmental factors, such as temperature, may influence process stability. However, COD removal remained consistent, supporting the feasibility of operating without pH control in an industrial scenario.
Despite its limitations, this study demonstrates that PHA-accumulating MMC can be enriched under real operational conditions without strict process control. However, ensuring consistency in biomass concentration and microbial performance remains a challenge.

4.2. Accumulation of scl-PHA in Microorganisms

The primary objective during the accumulation study was to maximize intracellular scl-PHA storage by optimizing the carbon feeding strategy. It can be seen how the scl-PHA concentration (expressed as wt.%) in the biomass exponentially grows with increasing VFA concentration (Figure 5) and once the COD feeding rate is higher than the COD consumption rate. Hence, it is observed in Table 4 that the low values of the scl-PHA storage yield show a slightly ascendant tendency as the COD feeding increases, which means that the total amount of VFAs employed during the accumulation process was not only employed for scl-PHA accumulation but also for MMC growth. A. de Lucas et al. [68] studied the kinetics of stored wastewater substrates by a mixed microbial culture, they concluded that only a certain proportion of the COD present in industrial wastewater can be accumulated inside the cell. We detected that a COD feeding rate lower than 13.91 mg COD/L.min almost did not produce scl-PHA accumulated in the cells (0.05–0.12 g/L). When the COD feeding rate increased above 13.91 mg COD/L.min, a significant increase in accumulated scl-PHA in the biomass is achieved (0.18 g/L, accumulation nº 6). Thus, we detected an increase in scl-PHA percentage storage in the biomass of 13.58 wt.% by increasing the COD feeding rate from (nº 5) 13.80 up to (nº 10) 24.22 mg COD/L.min.
This low polymer accumulation was due to the DO cascade control. During the accumulation reaction the DO was higher than 20%, but in any case, rose up to 70%. With the selected DO cascade control, when the DO rises above 20% the aeration and/or agitation decreased keeping that DO condition. This control system turned out to be unfavorable for this kind of fermentation, causing large oscillations in aeration and agitation without providing a high DO concentration.
The similar exponential tendency for the scl-PHA content in the biomass than the previous accumulation experiments 1 to 10 is observed in Figure 6. When the COD feeding rate applied is lower than the COD consumption rate in the enrichment fermenter (fermentation broth used for accumulation) the scl-PHA accumulated in the cells is low (0.66 g/L). Even though the obtained scl-PHA content in the biomass in accumulation batch nº 11 is higher than the obtained ones for accumulation batches from nº 1 to nº 5. Comparing Figure 5 and Figure 6, the higher the COD consumption rate is in the enrichment fermenter, the higher is the scl-PHA storage yield. Moreover, the average scl-PHA accumulation between batches nº 11 and nº 29 (Table 5) was 20 wt.%, which is higher than the average one obtained between batches nº 1 and nº 10 (Table 4), 5.6 wt.%. Therefore, maintaining adequate aeration and agitation conditions that ensure a dissolved oxygen (DO) concentration above 70%, enhances scl-PHA accumulation by microorganisms.
A precise understanding of aeration conditions in a bioprocess can significantly reduce productive costs by optimizing oxygen transfer efficiency and minimizing energy consumption. Proper aeration control ensures that microorganisms receive the necessary oxygen levels for metabolic activity, avoiding both oxygen limitations that could hinder MMC growth and excessive aeration that would lead to unnecessary energy consumption [69]. Additionally, well-regulated aeration enhances process stability, improves substrate utilization, and maximizes product yield [70]. In large-scale industrial applications, implementing advanced aeration strategies, such as dynamic oxygen supply control or real-time monitoring, can further contribute to sustainability by reducing operational costs and lowering the environmental footprint of the production process [69,70].
On the other hand, in this second sequence of experiments, several accumulations were performed by applying a COD feeding rate of 81.5 mg COD/L.min (accumulation batches from nº 32 to nº 37), obtaining an average scl-PHA in the biomass of 43.95 ± 10.14 wt.%, in which two reached almost 60 wt.% of scl-PHA within the cells. However, higher COD feeding rates by applying 81.5 mg COD/L.min did not improve the scl-PHA storage by the cells (accumulation batches nº 38 and nº 39). These values agree with the ones obtained in other studies at lower scales (40–50 wt.%) [25,48]. Thus, the scl-PHA production process up-scaling has demonstrated to be technically feasible. However, further studies should explore a broader range of feeding rates to define the most effective balance between substrate availability, microbial growth, and PHA storage efficiency. In this context, comparative studies have studied the impact of different operational conditions and carbon sources on PHA production efficiency. Among them, Eriksson E. [71] investigated the production of PHAs using VFAs derived from anaerobic digestion of municipal waste as a carbon source, employing a mixed microbial culture in SBR (5 L), achieving similar results than those obtained in this study. The research focuses on utilizing a mixed microbial culture enriched through a feast/famine strategy to maximize PHA accumulation. The fermentation process was based on a VFA mixture, (primarily rich in hexanoic acid, 29.5 g/L). The maximum biomass concentration achieved during the production phase was 31.4% of VSS, which was lower than the maximum accumulation obtained in the present work. Regarding the PHA composition, a very similar ratio was achieved, 97.1% and 2.9% of PHB and PHV, respectively.
On the one hand, Lorini et al. [72] were able to reach a laboratory scale of 2.4 g/L of scl-PHA in a mixed culture, where synthetic VFAs were used as a carbon source. In addition, a similar scl-PHA accumulation percentage was obtained by Valentino et al. [73] at pilot plant scale (120 L for enrichment fermentation) from a combined treatment of OFMSW and sewage sludge (46 wt.%). On the other hand, Korkakaki et al. [43] reached up to 78 wt.% of scl-PHA in the biomass by using an SBR for the enrichment process of 2 L of OFMSW. Singh et al. [74] explored the use of acidogenic fermentation of food waste to produce VFAs as a cost-effective carbon source for PHA synthesis. In their work, a pure culture was used, Pseudomonas pseudoflava, which was tested under varying carbon concentrations (4–55 g/L), achieving the highest PHA yield (52.5%) at 19 g/L and obtaining an average PHB and PHV composition of 97.1 and 2.9, respectively.
Regarding the PHB/PHV composition of the obtained scl-PHA, a consistent PHB/PHV ratio was observed across most of the accumulation batches (Figure 7). The average PHB and PHV compositions were 85.67 ± 6.09 and 13.16 ± 6.09%, respectively. For instance, Colombo et al. [75] obtained a scl-PHA with a PHB/PHV ratio of 53/47% from OFMSW by using a mixed microbial culture. The enrichment and accumulation processes were carried out in 800 and 300 mL reactors, respectively. Even though, from the accumulation batches from nº 1 to nº 10 a remarkably higher presence of PHV than PHB was observed, in comparison with the accumulation batches from nº 11 to nº 39. This may be due to the influence of aeration in the composition of PHA [76]. Nevertheless, from the accumulation batches from nº 11 to nº 39 the PHA composition was very similar, except for accumulation batch 13 (PHB/PHV = 44/57%).

4.3. Extraction of scl-PHA from the Produced Biomass at Demo Plant

The scl-PHA extraction yield obtained was 92.78%, demonstrating the technical feasibility of the extraction process developed at demonstrative scale. As observed in Table 6, successive solvent extractions increased the total amount of scl-PHA recovered from the biomass; however, the scl-PHA content in each individual extraction progressively decreased. Consequently, while increasing the number of extraction steps may enhance the overall yield, it could also lead to process inefficiencies by increasing solvent and energy consumption.
A major limitation of the extraction process is the presence of impurities in the final scl-PHA product. While pure PHA is naturally off-white, the extracted material exhibited a yellow-brownish appearance, indicating contamination (see Figure 9). These impurities may originate from the raw feedstock (OFMSW-Derived Contaminants), bacterial debris [77], or residual solvents. Given that the OFMSW serves as the primary carbon source, residual organic compounds, pigments, and heavy metals could also remain in the polymer. The presence of proteins, endotoxins, and lipids generated during the microbial fermentation process may not have been completely removed during the ethanol washing steps. While DMC was selected as an alternative to hazardous solvents such as chloroform and dichloromethane, incomplete solvent removal could also impact negatively on polymer purity. Additionally, the need for multiple extraction steps highlights a trade-off between extraction yield and efficiency. Although additional solvent cycles improve overall polymer recovery, they increase the operational costs of the final bio-product and may not be economically viable at an industrial scale. The energy-intensive nature of solvent evaporation and recovery further raises concerns about the scalability of the process.
To sum up, this study demonstrates that scl-PHA can be successfully extracted at demonstrative scale using dimethyl carbonate (DMC) as a green solvent. However, impurity content, solvent consumption, and process scalability remain key challenges to further overtake. Future research on the improvement of solid–liquid separation steps, solvent recovery, and polymer purification strategies will be essential to enhance both the technical and economic viability of scl-PHA production from OFMSW on industrial scale. Additionally, evaluating biodegradable solvents could improve sustainability and cost-effectiveness. Finally, exploring alternative extraction methods such as mechanical disruption, aqueous phase extraction, or biopolymer affinity purification may offer more efficient and eco-friendly solutions [51].

5. Conclusions

This study successfully demonstrates the feasibility of scaling up scl-PHA production from the OFMSW, validating the bioprocess under real operational conditions. The results confirm that the conversion of OFMSW into VFAs and their subsequent utilization as a carbon source for PHA synthesis is not only technically viable but also aligns with the principles of circular economy and sustainable waste valorization.
The industrial-scale implementation of the bioprocess presented shows significant potential to reduce dependency on fossil-based plastics while contributing to waste management strategies. The production of 83 kg of scl-PHA over 29 accumulation batches demonstrates the robustness of the bioprocess, with promising yields comparable to those obtained at earlier stages of the technology readiness level. The scl-PHA has been produced from 23.5 t of VFA-rich digestate. It can be concluded that not controlling pH did not promote a detrimental effect on the production of PHAs, but low environmental temperatures did cause a destabilization in the enrichment fermenter during the winter season, although it was followed by an MMC adaption. Good aeration and agitation strategies are essential to obtain high percentages of PHAs during the accumulation step. Thus, DO concentration must be ensured to be over 70% to reach high rates of scl-PHA accumulated by microorganisms. The COD feeding rate during the accumulation reaction could be increased up to four times the COD consumption rate of the enrichment reaction, by reaching up to 58 wt.% of PHA in the MMC cells. In addition, most of the organic matter was used for MMC growing at the PHA accumulation phase.
Moreover, the high extraction efficiency (92%) and purity (90%) achieved highlight the effectiveness of the solvent-based recovery method at demonstrative scale. However, for full-scale industrial deployment, the economic viability of the process must be further assessed. Key factors such as process optimization, energy consumption, solvent recovery, and market competitiveness must be carefully analyzed to ensure the financial sustainability of PHA production. The cost-effectiveness of aeration, substrate feeding strategies, and downstream processing, particularly in solvent extraction, will be critical in determining the commercial feasibility of this technology.
Additionally, exploring the scalability of different feeding rates and substrate compositions will be essential to maximize PHA storage efficiency while maintaining process stability.
In summary, this work represents a significant step towards the industrialization of scl-PHA production from municipal waste streams. While technical feasibility has been demonstrated, further advancements in process efficiency, cost reduction, and sustainability assessments will be necessary to establish PHA bioplastics as a competitive alternative to conventional plastics.

Author Contributions

Conceptualization, I.I.; Methodology, I.I., I.Á. and F.J.P.; Formal analysis, I.I. and I.Á.; Investigation, I.I., I.Á., F.J.P. and J.M.; Data curation, I.I.; Writing—original draft, I.I.; Writing—review & editing, F.J.P. and J.M.; Supervision, F.J.P. and J.M.; Project administration, J.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Bio-Based Industries Joint Undertaking through the project URBIOFIN (Grant Agreement Number 745785). And The APC was funded by IRIAF (Regional Institute for Agri-Food and Forestry Research and Development of Castilla-La Mancha).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article.

Acknowledgments

The authors would like to acknowledge to URBASER (Zaragoza, Spain) for providing the OFMSW digestate and to AINIA (Valencia, Spain) for the scientific advice. This study has received funding from the European Commission, supporting this work by the Bio-Based Industries Joint Undertaking through the project URBIOFIN (Grant Agreement Number 745785).

Conflicts of Interest

All authors were employed in CLAMBER R&D Biorefinery. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Khajuria, A.; Atienza, V.A.; Chavanich, S.; Henning, W.; Islam, I.; Kral, U.; Liu, M.; Liu, X.; Murthy, I.K.; Oyedotun, T.D.T.; et al. Accelerating circular economy solutions to achieve the 2030 agenda for sustainable development goals. Circ. Econ. 2022, 1, 100001. [Google Scholar] [CrossRef]
  2. Sengupta, D.; Ilankoon, I.M.S.K.; Dean Kang, K.; Nan Chong, M. Circular economy and household e-waste management in India: Integration of formal and informal sectors. Miner. Eng. 2022, 184, 107661. [Google Scholar] [CrossRef]
  3. Sakthivelmurugan, E.; Senthilkumar, G.; Karthick, K.N. Analysis of the impact of circular economy over linear economy in the paper processing industry. Mater. Today Proc. 2022, 66, 1446–1452. [Google Scholar] [CrossRef]
  4. Bongers, A.; Casas, P. The circular economy and the optimal recycling rate: A macroeconomic approach. Ecol. Econ. 2022, 199, 107504. [Google Scholar] [CrossRef]
  5. Noor, T.; Javid, A.; Hussain, A.; Bukhari, S.M.; Ali, W.; Akmal, M.; Hussain, S.M. Chapter 14—Types, sources and management of urban wastes. In Urban Ecology; Verma, P., Singh, P., Singh, R., Raghubanshi, A.S., Eds.; Elsevier: Amsterdam, The Netherlands, 2020; pp. 239–263. [Google Scholar] [CrossRef]
  6. Pérez, V.; Pascual, A.; Rodrigo, A.; García Torreiro, M.; Latorre-Sánchez, M.; Coll Lozano, C.; David-Moreno, A.; Oliva-Dominguez, J.M.; Serna-Maza, A.; Herrero García, N.; et al. Chapter 2—Integrated innovative biorefinery for the transformation of municipal solid waste into biobased products. In Waste Biorefinery; Bhaskar, T., Pandey, A., Rene, E.R., Tsang, D.C.W., Eds.; Elsevier: Amsterdam, The Netherlands, 2020; pp. 41–80. [Google Scholar] [CrossRef]
  7. URBIOFIN, Urban Biorefinery: From Waste to Bioproducts Through Biorefiney. Available online: https://circulareconomy.europa.eu/platform/en/good-practices/urbiofin-waste-bioproducts-through-biorefinery (accessed on 14 September 2023).
  8. Mor, S.; Ravindra, K. Municipal solid waste landfills in lower- and middle-income countries: Environmental impacts, challenges and sustainable management practices. Process Saf. Environ. Prot. 2023, 174, 510–530. [Google Scholar] [CrossRef]
  9. Najar, I.N.; Sharma, P.; Das, R.; Tamang, S.; Mondal, K.; Thakur, N.; Gandhi, S.G.; Kumar, V. From waste management to circular economy: Leveraging thermophiles for sustainable growth and global resource optimization. J. Environ. Manag. 2024, 360, 121136. [Google Scholar] [CrossRef]
  10. Yaashikaa, P.R.; Kumar, P.S.; Saravanan, A.; Varjani, S.; Ramamurthy, R. Bioconversion of municipal solid waste into bio-based products: A review on valorisation and sustainable approach for circular bioeconomy. Sci. Total Environ. 2020, 748, 141312. [Google Scholar] [CrossRef]
  11. Oliveira, M.; Cocozza, A.; Zucaro, A.; Santagata, R.; Ulgiati, S. Circular economy in the agro-industry: Integrated environmental assessment of dairy products. Renew. Sustain. Energy Rev. 2021, 148, 111314. [Google Scholar] [CrossRef]
  12. Muthuraj, R.; Valerio, O.; Mekonnen, T.H. Recent developments in short- and medium-chain- length Polyhydroxyalkanoates: Production, properties, and applications. Int. J. Biol. Macromol. 2021, 187, 422–440. [Google Scholar] [CrossRef]
  13. Taguchi, S.; Matsumoto, K.i. Evolution of polyhydroxyalkanoate synthesizing systems toward a sustainable plastic industry. Polym. J. 2020, 53, 67–79. [Google Scholar] [CrossRef]
  14. Zhuang, Q.; Wang, Q.; Liang, Q.; Qi, Q. Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid β-oxidation cycle in Escherichia coli. Metab. Eng. 2014, 24, 78–86. [Google Scholar] [CrossRef] [PubMed]
  15. Singh, A.; Mallick, N. Enhanced production of SCL-LCL-PHA co-polymer by sludge-isolated Pseudomonas aeruginosa MTCC 7925. Lett. Appl. Microbiol. 2008, 46, 350–357. [Google Scholar] [CrossRef] [PubMed]
  16. Phan, H.T.; Hosoe, Y.; Guex, M.; Tomoi, M.; Tomita, H.; Zinn, M.; Matsumoto, K.i. Directed Evolution of Sequence-Regulating Polyhydroxyalkanoate Synthase to Synthesize a Medium-Chain-Length–Short-Chain-Length (MCL–SCL) Block Copolymer. Biomacromolecules 2022, 23, 1221–1231. [Google Scholar] [CrossRef]
  17. Israni, N.; Shivakumar, S. Chapter 13—Polyhydroxybutyrate: Development and applications as a biodegradable biotextile. In Materials for Biomedical Engineering; Grumezescu, V., Grumezescu, A.M., Eds.; Elsevier: Amsterdam, The Netherlands, 2019; pp. 405–444. [Google Scholar] [CrossRef]
  18. Sudesh, K.; Abe, H.; Doi, Y. Synthesis, structure and properties of polyhydroxyalkanoates: Biological polyesters. Prog. Polym. Sci. 2000, 25, 1503–1555. [Google Scholar] [CrossRef]
  19. Cammas-Marion, S.; Martínez-Barbosa, M.E. 15—Microbial polyesters: Synthesis and applications. In Fundamentals of Natural Fibres and Textiles; Mondal, M.I.H., Ed.; Woodhead Publishing: Cambridge, UK, 2021; pp. 515–555. [Google Scholar] [CrossRef]
  20. Chavan, S.; Yadav, B.; Tyagi, R.D.; Drogui, P. A review on production of polyhydroxyalkanoate (PHA) biopolyesters by thermophilic microbes using waste feedstocks. Bioresour. Technol. 2021, 341, 125900. [Google Scholar] [CrossRef]
  21. Allegue, L.D.; Ventura, M.; Melero, J.A.; Puyol, D. Unraveling PHA production from urban organic waste with purple phototrophic bacteria via organic overload. Renew. Sustain. Energy Rev. 2022, 166, 112687. [Google Scholar] [CrossRef]
  22. Rajesh Banu, J.; Ginni, G.; Kavitha, S.; Yukesh Kannah, R.; Kumar, V.; Adish Kumar, S.; Gunasekaran, M.; Tyagi, V.K.; Kumar, G. Polyhydroxyalkanoates synthesis using acidogenic fermentative effluents. Int. J. Biol. Macromol. 2021, 193, 2079–2092. [Google Scholar] [CrossRef] [PubMed]
  23. Corrado, I.; Varriale, S.; Pezzella, C. Microbial Processes for Upcycling Food Wastes into Sustainable Bioplastics. In Reference Module in Food Science; Elsevier: Amsterdam, The Netherlands, 2023. [Google Scholar] [CrossRef]
  24. Bellini, S.; Tommasi, T.; Fino, D. Poly(3-hydroxybutyrate) biosynthesis by Cupriavidus necator: A review on waste substrates utilization for a circular economy approach. Bioresour. Technol. Rep. 2022, 17, 100985. [Google Scholar] [CrossRef]
  25. Lorini, L.; Martinelli, A.; Pavan, P.; Majone, M.; Valentino, F. Downstream processing and characterization of polyhydroxyalkanoates (PHAs) produced by mixed microbial culture (MMC) and organic urban waste as substrate. Biomass Convers. Biorefinery 2021, 11, 693–703. [Google Scholar] [CrossRef]
  26. Crognale, S.; Tonanzi, B.; Valentino, F.; Majone, M.; Rossetti, S. Microbiome Dynamics and phaC Shyntase Genes Selected in a Pilot Plant Producing Polyhydroxyalkanoates Production from Organic Fraction of Urban Waste; Elsevier: Amsterdam, The Netherlands, 2019. [Google Scholar]
  27. Francesco Valentino, M.R.; Silva, F.; Majone, M. Report on Optimization of PHA Production at Pilot Scale; European Comision: Brussels, Belgium, 2019. Available online: https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e080165c080162e080107c080106&appId=PPGMS (accessed on 20 June 2022).
  28. Mannina, G.; Presti, D.; Montiel-Jarillo, G.; Carrera, J.; Suárez-Ojeda, M.E. Recovery of polyhydroxyalkanoates (PHAs) from wastewater: A review. Bioresour. Technol. 2020, 297, 122478. [Google Scholar] [CrossRef]
  29. Oliveira, C.; Silva, C.; Carvalho, G.; Reis, M. Strategies for efficiently selecting PHA producing mixed microbial cultures using complex feedstocks: Feast and famine regime and uncoupled carbon and nitrogen availabilities. New Biotechnol. 2016, 37, 69–79. [Google Scholar] [CrossRef]
  30. Di Caprio, F.; Proietti Tocca, G.; Stoller, M.; Pagnanelli, F.; Altimari, P. Control of bacterial contamination in microalgae cultures integrated with wastewater treatment by applying feast and famine conditions. J. Environ. Chem. Eng. 2022, 10, 108262. [Google Scholar] [CrossRef]
  31. Jayakrishnan, U.; Deka, D.; Das, G. Waste as feedstock for polyhydroxyalkanoate production from activated sludge: Implications of aerobic dynamic feeding and acidogenic fermentation. J. Environ. Chem. Eng. 2021, 9, 105550. [Google Scholar] [CrossRef]
  32. Cui, Y.-W.; Zhang, H.-Y.; Lu, P.-F.; Peng, Y.-Z. Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process. Sci. Rep. 2016, 6, 30766. [Google Scholar] [CrossRef] [PubMed]
  33. Moretto, G.; Lorini, L.; Pavan, P.; Crognale, S.; Tonanzi, B.; Rossetti, S.; Majone, M.; Valentino, F. Biopolymers from Urban Organic Waste: Influence of the Solid Retention Time to Cycle Length Ratio in the Enrichment of a Mixed Microbial Culture (MMC). ACS Sustain. Chem. Eng. 2020, 8, 14531–14539. [Google Scholar] [CrossRef]
  34. Valentino, F.; Morgan-Sagastume, F.; Campanari, S.; Villano, M.; Werker, A.; Majone, M. Carbon recovery from wastewater through bioconversion into biodegradable polymers. New Biotechnol. 2017, 37, 9–23. [Google Scholar] [CrossRef]
  35. Majone, M.; Dircks, K.; Beim, J.J. Aerobic storage under dynamic conditions in activated sludge processes. The state of the art. Water Sci. Technol. 1999, 39, 61–73. [Google Scholar] [CrossRef]
  36. Sun, Y.; Angelotti, B.; Brooks, M.; Wang, Z.-W. Feast/famine ratio determined continuous flow aerobic granulation. Sci. Total Environ. 2021, 750, 141467. [Google Scholar] [CrossRef]
  37. Iorhemen, O.T.; Zaghloul, M.S.; Hamza, R.A.; Tay, J.H. Long-term aerobic granular sludge stability through anaerobic slow feeding, fixed feast-famine period ratio, and fixed SRT. J. Environ. Chem. Eng. 2020, 8, 103681. [Google Scholar] [CrossRef]
  38. Sousa, B.V.; Silva, F.; Reis, M.A.M.; Lourenço, N.D. Monitoring pilot-scale polyhydroxyalkanoate production from fruit pulp waste using near-infrared spectroscopy. Biochem. Eng. J. 2021, 176, 108210. [Google Scholar] [CrossRef]
  39. Albuquerque, M.; Martino, V.; Pollet, E.; Avérous, L.; Reis, M. Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: Effect of substrate composition and feeding regime on PHA productivity, composition and properties. J. Biotechnol. 2010, 151, 66–76. [Google Scholar] [CrossRef]
  40. Chen, H.; Meng, H.; Nie, Z.; Zhang, M. Polyhydroxyalkanoate production from fermented volatile fatty acids: Effect of pH and feeding regimes. Bioresour. Technol. 2013, 128, 533–538. [Google Scholar] [CrossRef] [PubMed]
  41. Mannina, G.; Presti, D.; Montiel-Jarillo, G.; Suárez-Ojeda, M.E. Bioplastic recovery from wastewater: A new protocol for polyhydroxyalkanoates (PHA) extraction from mixed microbial cultures. Bioresour. Technol. 2019, 282, 361–369. [Google Scholar] [CrossRef]
  42. Valentino, F.; Beccari, M.; Fraraccio, S.; Zanaroli, G.; Majone, M. Feed frequency in a Sequencing Batch Reactor strongly affects the production of polyhydroxyalkanoates (PHAs) from volatile fatty acids. New Biotechnol. 2014, 31, 264–275. [Google Scholar] [CrossRef] [PubMed]
  43. Korkakaki, E.; Mulders, M.; Veeken, A.; Rozendal, R.; van Loosdrecht, M.C.M.; Kleerebezem, R. PHA production from the organic fraction of municipal solid waste (OFMSW): Overcoming the inhibitory matrix. Water Res. 2016, 96, 74–83. [Google Scholar] [CrossRef] [PubMed]
  44. Jiang, Y.; Hebly, M.; Kleerebezem, R.; Muyzer, G.; van Loosdrecht, M.C.M. Metabolic modeling of mixed substrate uptake for polyhydroxyalkanoate (PHA) production. Water Res. 2011, 45, 1309–1321. [Google Scholar] [CrossRef]
  45. Choi, J.-i.; Lee, S.Y. Process analysis and economic evaluation for Poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng. 1997, 17, 335–342. [Google Scholar] [CrossRef]
  46. Burniol-Figols, A.; Varrone, C.; Le, S.B.; Daugaard, A.E.; Skiadas, I.V.; Gavala, H.N. Combined polyhydroxyalkanoates (PHA) and 1,3-propanediol production from crude glycerol: Selective conversion of volatile fatty acids into PHA by mixed microbial consortia. Water Res. 2018, 136, 180–191. [Google Scholar] [CrossRef]
  47. Freches, A.; Lemos, P.C. Microbial selection strategies for polyhydroxyalkanoates production from crude glycerol: Effect of OLR and cycle length. New Biotechnol. 2017, 39, 22–28. [Google Scholar] [CrossRef]
  48. Bengtsson, S.; Werker, A.; Christensson, M.; Welander, T. Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater. Bioresour. Technol. 2008, 99, 509–516. [Google Scholar] [CrossRef]
  49. Jacquel, N.; Lo, C.-W.; Wei, Y.-H.; Wu, H.-S.; Wang, S.S. Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochem. Eng. J. 2008, 39, 15–27. [Google Scholar] [CrossRef]
  50. Koller, M.; Niebelschütz, H.; Braunegg, G. Strategies for recovery and purification of poly[(R)-3-hydroxyalkanoates] (PHA) biopolyesters from surrounding biomass. Eng. Life Sci. 2013, 13, 549–562. [Google Scholar] [CrossRef]
  51. Kosseva, M.R.; Rusbandi, E. Trends in the biomanufacture of polyhydroxyalkanoates with focus on downstream processing. Int. J. Biol. Macromol. 2018, 107, 762–778. [Google Scholar] [CrossRef] [PubMed]
  52. Samorì, C.; Basaglia, M.; Casella, S.; Favaro, L.; Galletti, P.; Giorgini, L.; Marchi, D.; Mazzocchetti, L.; Torri, C.; Tagliavini, E. Dimethyl carbonate and switchable anionic surfactants: Two effective tools for the extraction of polyhydroxyalkanoates from microbial biomass. Green Chem. 2015, 17, 1047–1056. [Google Scholar] [CrossRef]
  53. Mongili, B.; Abdel Azim, A.; Fraterrigo Garofalo, S.; Batuecas, E.; Re, A.; Bocchini, S.; Fino, D. Novel insights in dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB). Biotechnol. Biofuels 2021, 14, 13. [Google Scholar] [CrossRef] [PubMed]
  54. de Souza Reis, G.A.; Michels, M.H.A.; Fajardo, G.L.; Lamot, I.; de Best, J.H. Optimization of Green Extraction and Purification of PHA Produced by Mixed Microbial Cultures from Sludge. Water 2020, 12, 1185. [Google Scholar] [CrossRef]
  55. Samorì, C.; Abbondanzi, F.; Galletti, P.; Giorgini, L.; Mazzocchetti, L.; Torri, C.; Tagliavini, E. Extraction of polyhydroxyalkanoates from mixed microbial cultures: Impact on polymer quality and recovery. Bioresour. Technol. 2015, 189, 195–202. [Google Scholar] [CrossRef]
  56. Khosravi-Darani, K.; Vasheghani-Farahani, E.; Yamini, Y.; Bahramifar, N. Solubility of Poly(β-hydroxybutyrate) in Supercritical Carbon Dioxide. J. Chem. Eng. Data 2003, 48, 860–863. [Google Scholar] [CrossRef]
  57. Urbaser. (2020, Agosto 11). Urbaser Integra la Transformación de Residuos Orgánicos en Bioproductos Para su uso en otros Sectores. Urbaser. Available online: https://www.urbaser.com/2020/08/11/urbaser-integra-la-transformacion-de-residuos-organicos-en-bioproductos-para-su-uso-en-otros-sectores-2/ (accessed on 20 August 2023).
  58. Ospina, C.; Echeverri, S.; Rodriguez-Gonzalez, C.; Wist, J.; Combariza, M.; Sanabria, J. Enhancement of PHA Production by a Mixed Microbial Culture Using VFA Obtained from the Fermentation of Wastewater from Yeast Industry. Fermentation 2022, 8, 180. [Google Scholar] [CrossRef]
  59. Miu, D.M.; Eremia, M.C.; Moscovici, M. Polyhydroxyalkanoates (PHAs) as Biomaterials in Tissue Engineering: Production, Isolation, Characterization. Material 2022, 15, 1410. [Google Scholar] [CrossRef]
  60. Apha, A.; Barron, J. Standard Methods for the Examination of Water & Wastewater, 21st ed.; American Public Health Association (APHA); American Water Works Association (AWWA); Water Environment Federation (WEF): Washington, DC, USA, 2005. [Google Scholar]
  61. Huijberts, G.N.M.; van der Wal, H.; Wilkinson, C.; Eggink, G. Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria. Biotechnol. Tech. 1994, 8, 187–192. [Google Scholar] [CrossRef]
  62. Wang, X.; Bengtsson, S.; Oehmen, A.; Carvalho, G.; Werker, A.; Reis, M.A.M. Application of dissolved oxygen (DO) level control for polyhydroxyalkanoate (PHA) accumulation with concurrent nitrification in surplus municipal activated sludge. New Biotechnol. 2019, 50, 37–43. [Google Scholar] [CrossRef]
  63. Pinto-Ibieta, F.; Serrano, A.; Cea, M.; Ciudad, G.; Fermoso, F.G. Beyond PHA: Stimulating intracellular accumulation of added-value compounds in mixed microbial cultures. Bioresour. Technol. 2021, 337, 125381. [Google Scholar] [CrossRef] [PubMed]
  64. Cruz, R.A.P.; Oehmen, A.; Reis, M.A.M. The impact of biomass withdrawal strategy on the biomass selection and polyhydroxyalkanoates accumulation of mixed microbial cultures. New Biotechnol. 2022, 66, 8–15. [Google Scholar] [CrossRef] [PubMed]
  65. Leong, Y.K.; Chang, J.-S. Chapter 6—Bioprocessing for production and applications of bioplastics from algae. In Algae-Based Biomaterials for Sustainable Development; Ngo, H., Guo, W., Pandey, A., Chang, J.-S., Lee, D.-J., Eds.; Elsevier: Amsterdam, The Netherlands, 2022; pp. 105–132. [Google Scholar] [CrossRef]
  66. Arriaga, M.; Pinar, F.J.; Izarra, I.; Amo, J.D.; Vicente, J.; Fernández-Morales, F.J.; Mena, J. Valorization of Agri-Food Waste into PHA and Bioplastics: From Waste Selection to Transformation. Appl. Sci. 2025, 15, 1008. [Google Scholar] [CrossRef]
  67. Liu, C.; Wang, W.; Anwar, N.; Ma, Z.; Liu, G.; Zhang, R. Effect of Organic Loading Rate on Anaerobic Digestion of Food Waste under Mesophilic and Thermophilic Conditions. Energy Fuels 2017, 31, 2976–2984. [Google Scholar] [CrossRef]
  68. de Lucas, A.; Rodríguez, L.; Villaseñor, J.; Fernández, F.J. Biodegradation kinetics of stored wastewater substrates by a mixed microbial culture. Biochem. Eng. J. 2005, 26, 191–197. [Google Scholar] [CrossRef]
  69. Krzeminski, P.; Leverette, L.; Malamis, S.; Katsou, E. Membrane bioreactors—A review on recent developments in energy reduction, fouling control, novel configurations, LCA and market prospects. J. Membr. Sci. 2017, 527, 207–227. [Google Scholar] [CrossRef]
  70. Jwara, T.; Musonge, P.; Babatunde, F.; Mnguni, M. Biological Nutrient Removal Efficiencies for Hydraulically Overloaded Wastewater Works; WIT Press (Wessex Institute of Technology Press): Southampton, UK, 2019; pp. 223–231. [Google Scholar]
  71. Eriksson, E. Polyhydroxyalkanoate Production from Municipal Waste Streams. In Kth Skolan för Kemi, Bioteknologi Och Hälsa; KTH Royal Institute of Technology: Stockholm, Sweden, 2020. [Google Scholar]
  72. Lorini, L.; Di Re, F.; Majone, M.; Valentino, F. High rate selection of PHA accumulating mixed cultures in Sequencing Batch Reactors with uncoupled carbon and nitrogen feeding. New Biotechnol. 2020, 56, 140–148. [Google Scholar] [CrossRef]
  73. Valentino, F.; Moretto, G.; Lorini, L.; Bolzonella, D.; Pavan, P.; Majone, M. Pilot-Scale Polyhydroxyalkanoate Production from Combined Treatment of Organic Fraction of Municipal Solid Waste and Sewage Sludge. Ind. Eng. Chem. Res. 2019, 58, 12149–12158. [Google Scholar] [CrossRef]
  74. Singh, A.; Srivastava, V. Production of polyhydroxyalkanoates (PHA) by pseudomonas from waste products. Int. J. Health Sci. 2022, 6, 11450–11459. [Google Scholar] [CrossRef]
  75. Colombo, B.; Favini, F.; Scaglia, B.; Sciarria, T.P.; D’Imporzano, G.; Pognani, M.; Alekseeva, A.; Eisele, G.; Cosentino, C.; Adani, F. Enhanced polyhydroxyalkanoate (PHA) production from the organic fraction of municipal solid waste by using mixed microbial culture. Biotechnol. Biofuels 2017, 10, 201. [Google Scholar] [CrossRef] [PubMed]
  76. Nazir, H.; Batool, M.; Bolivar Osorio, F.J.; Isaza-Ruiz, M.; Xu, X.; Vignarooban, K.; Phelan, P.; Inamuddin; Kannan, A.M. Recent developments in phase change materials for energy storage applications: A review. Int. J. Heat Mass Transf. 2019, 129, 491–523. [Google Scholar] [CrossRef]
  77. Pagliano, G.; Galletti, P.; Samorì, C.; Zaghini, A.; Torri, C. Recovery of Polyhydroxyalkanoates From Single and Mixed Microbial Cultures: A Review. Front. Bioeng. Biotechnol. 2021, 9, 624021. [Google Scholar] [CrossRef]
Applsci 15 03272 g001
Figure 1. Fermenters of 20 (a) and 3 (b) m3 employed for enrichment and accumulation, respectively.
Figure 1. Fermenters of 20 (a) and 3 (b) m3 employed for enrichment and accumulation, respectively.
Applsci 15 03272 g001
Applsci 15 03272 g002
Figure 2. Evolution of COD, TSS, VSS, and pH in the enrichment fermenter feeding VFA-rich digestate during 214 days.
Figure 2. Evolution of COD, TSS, VSS, and pH in the enrichment fermenter feeding VFA-rich digestate during 214 days.
Applsci 15 03272 g002
Applsci 15 03272 g003
Figure 3. DO profile and COD consumption of the enrichment fermenter in 20 m3 during cycle 137.
Figure 3. DO profile and COD consumption of the enrichment fermenter in 20 m3 during cycle 137.
Applsci 15 03272 g003
Applsci 15 03272 g004
Figure 4. Temperature evolution in fermenter during the enrichment operation.
Figure 4. Temperature evolution in fermenter during the enrichment operation.
Applsci 15 03272 g004
Applsci 15 03272 g005
Figure 5. Influence of the scl-PHA percentage in the biomass varying the COD feeding rate to the accumulation reaction having the enrichment fermenter a COD consumption rate of 13.91 mg COD/L.min.
Figure 5. Influence of the scl-PHA percentage in the biomass varying the COD feeding rate to the accumulation reaction having the enrichment fermenter a COD consumption rate of 13.91 mg COD/L.min.
Applsci 15 03272 g005
Applsci 15 03272 g006
Figure 6. Effect of COD feeding rate on scl-PHA percentage in the biomass for a COD consumption rate of 19.56 mg COD/L.min. in the enrichment fermenter.
Figure 6. Effect of COD feeding rate on scl-PHA percentage in the biomass for a COD consumption rate of 19.56 mg COD/L.min. in the enrichment fermenter.
Applsci 15 03272 g006
Applsci 15 03272 g007
Figure 7. PHA composition from accumulation batches 1 to 39.
Figure 7. PHA composition from accumulation batches 1 to 39.
Applsci 15 03272 g007
Applsci 15 03272 g008
Figure 8. Fermentation broth before its centrifugation (a) and the biomass after centrifugation (b) (concentrated phase).
Figure 8. Fermentation broth before its centrifugation (a) and the biomass after centrifugation (b) (concentrated phase).
Applsci 15 03272 g008
Applsci 15 03272 g009
Figure 9. Wet extracted scl-PHA concentrated in DMC (a), wet scl-PHA after DMC removal (b), dry scl-PHA (c), and grinded scl-PHA with a purity of 90% (d).
Figure 9. Wet extracted scl-PHA concentrated in DMC (a), wet scl-PHA after DMC removal (b), dry scl-PHA (c), and grinded scl-PHA with a purity of 90% (d).
Applsci 15 03272 g009
Table 1. Operational condition of the enrichment fermenter.
Table 1. Operational condition of the enrichment fermenter.
Enrichment Fermenter
Working volume (L)15,000
HRT (d)5
Cycle length (h)24
Feeding period (min)30
OLR (gCOD/L. d)1
Aeration (m3/h)781
Stirring (rpm)50
pHNo control
TemperatureNo control
Table 2. Average analytical results of the VFA-rich solution generated.
Table 2. Average analytical results of the VFA-rich solution generated.
Total COD (mg/L)62,165.00 ± 8922.16
Soluble COD (mg/L)42,483.13 ± 8374.85
N-NH4 (mg/L)1397.25 ± 161.12
NT (mg/L)6450.00 ± 1990.81
NO3− (mg/L)26.13 ± 14.99
NO2− (mg/L)2.93 ± 1.48
Ca (mg/L)2888.75 ± 620.89
PT (mg/L)84.38 ± 30.53
P-PO4 (mg/L)40.70 ± 19.25
SST (g/L)2.06 ± 0.93
SSV (g/L)1.55 ± 0.83
Fe (mg/L)110.33 ± 60.37
Mn (mg/L)15.31 ± 2.45
Zn (mg/L)4.96 ± 2.87
Cu (mg/L)5.00 ± 1.30
Ca (mg/L)2888.75 ± 620.89
Na (mg/L)1555.00 ± 77.78
K (mg/L)3930.00 ± 49.49
SO4 (mg/L)1778.00 ± 142.02
Table 3. Evolution of the COD consumption rate monitored at state conditions in the enrichment fermenter at the beginning of different cycles.
Table 3. Evolution of the COD consumption rate monitored at state conditions in the enrichment fermenter at the beginning of different cycles.
CycleTSS (g/L)VSS (g/L)COD Consumption Rate in the Enrichment Fermenter
(mg COD/L.Min)		g COD/g SSV·h
29–342.89 ± 0.241.95 ± 0.2710.110.31
35–623.05 ± 0.522.21 ± 0.4510.410.28
63–813.30 ± 0.662.28 ± 0.4811.980.32
82–1322.99 ± 0.482.19 ± 0.6613.910.38
133–2142.91 ± 0.741.92 ± 0.5519.56 0.61
Table 4. Data from accumulation batches 1 to 10 performed with an average COD consumption rate in enrichment fermenter of 13.91 mg COD/L.min.
Table 4. Data from accumulation batches 1 to 10 performed with an average COD consumption rate in enrichment fermenter of 13.91 mg COD/L.min.
NºCycleCOD Feeding Rate
(mg COD/L.min)		Initial
TSS
(g/L)		Final TSS
(g/L)		Scl-PHA
in Final Biomass
(wt.%)		Scl-PHA
(g/L)		Scl-PHA Generated (kg)/100 kg COD
Fed (%)		Final Soluble COD (mg/L)
11119.862.75 ± 0.264.61 ± 0.092.080.100.82<200
212111.542.91 ± 0.085.19 ± 0.122.300.121.89
311912.073.05 ± 0.102.61 ± 0.502.000.050.68
411713.593.67 ± 0.595.01 ± 0.171.720.090.97
511713.803.51 ± 0.473.69 ± 0.572.480.090.88
612717.053.16 ± 0.106.92 ± 0.872.610.182.72
713220.284.12± 0.9014.72 ± 0.156.710.9912.16
812820.613.32 ± 0.186.43 ± 0.217.650.496.35
912924.443.16 ± 0.056.11 ± 0.1112.440.766.11237
1013124.442.84 ± 0.287.44 ± 0.2716.061.1910.48299
Table 5. Summarizes the data of these 29 accumulation batches.
Table 5. Summarizes the data of these 29 accumulation batches.
NºCycleReal COD Feeding Rate Applied in the Accumulation Reaction
(mg COD/L.min)		TSS
Initial
(g/L)		TSS
Final
(g/L)		Scl-PHA
in Biomass
(wt.%)		Scl-PHA
(g/L)		Scl-PHA Generated (kg)/100 kg COD
Fed (%)		Final Soluble COD (mg/L)
1113613.633.18 ± 0.057.74 ± 0.118.550.665.23198
1213823.273.39 ± 0.157.04 ± 0.4719.711.3911.61375
1313333.852.99 ± 0.088.32 ± 0.819.060.754.97222
1414138.413.91 ± 1.099.92 ± 0.307.940.794.83478
1514041.903.81 ± 0.0810.17 ± 1.1216.001.638.22200
1620953.552.29 ± 0.044.52 ± 0.2719.000.866.06405
1721454.112.79 ± 0.794.26 ± 1.4815.490.654.83489
1820855.322.44 ± 0.074.09 ± 0.2124.731.016.39510
1921455.322.79 ± 0.794.35 ± 0.3019.390.846.46456
2020656.032.50 ± 0.154.18 ± 0.1328.301.188.25501
2120756.712.67 ± 0.145.40 ± 0.4029.351.5810.48375
2221357.922.87 ± 0.674.40 ± 0.1619.820.875.02406
2320860.072.44 ± 0.074.35 ± 0.3620.800.905.81375
2420960.072.29 ± 0.043.89 ± 0.2319.090.744.71401
2520562.522.47 ± 0.055.90 ± 0.2627.151.6010.23346
2621162.312.61 ± 0.252.98 ± 0.0813.280.402.40479
2713465.263.00 ± 0.2711.97 ± 0.9730.623.6715.67198
2820167.133.23 ± 0.376.79 ± 0.0129.922.039.55245
2919867.882.74 ± 0.123.58 ± 0.4624.570.883.99605
3020072.383.51 ± 0.513.57 ± 0.0634.221.226.90547
3119773.162.96 ± 0.073.98 ± 0.4329.731.186.43705
3218181.502.22 ± 0.041.99 ± 0.0154.051.083.63458
3318581.502.54 ± 0.121.78 ± 0.1637.770.672.91427
3418781.502.71 ± 0.012.00 ± 0.0334.980.703.25515
3519081.503.16 ± 0.204.94 ± 0.1558.002.8711.68470
3619481.502.61 ± 0.139.5 ± 0.3038.343.6422.32200
3719681.503.00 ± 0.344.53 ± 0.7040.611.848.66347
3820383.503.00 ± 0.275.63 ± 0.0932.541.837.65750
3920483.504.11 ± 1.304.64 ± 0.1429.351.365.69874
Table 6. Scl-PHA extraction data.
Table 6. Scl-PHA extraction data.
Data Before Solvent Extraction
Wet biomass treated (after centrifugation) (kg)543
Scl-PHA content (wt.%)22.3
Humidity (%)80
Scl-PHA content (kg)24.21
Data after solvent extraction
Remaining scl-PHA in the biomass after the first extraction with DMC (wt.%)7.56
Remaining scl-PHA in the biomass after the second extraction with DMC (wt.%)4.17
Remaining scl-PHA in the biomass after the third extraction with DMC (wt.%)1.6
Total extracted scl-PHA (kg)22.4
Extraction yield (%) (respect to the wet biomass treated)92.78
Purity (%)90
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Izarra, I.; Álvarez, I.; Pinar, F.J.; Mena, J. Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste. Appl. Sci. 2025, 15, 3272. https://doi.org/10.3390/app15063272

AMA Style

Izarra I, Álvarez I, Pinar FJ, Mena J. Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste. Applied Sciences. 2025; 15(6):3272. https://doi.org/10.3390/app15063272

Chicago/Turabian Style

Izarra, Irene, Irene Álvarez, F. Javier Pinar, and Javier Mena. 2025. "Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste" Applied Sciences 15, no. 6: 3272. https://doi.org/10.3390/app15063272

APA Style

Izarra, I., Álvarez, I., Pinar, F. J., & Mena, J. (2025). Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste. Applied Sciences, 15(6), 3272. https://doi.org/10.3390/app15063272

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop