Enrichment of a mixed microbial culture for PHA production with used cooking oil as substrate

Enrichment of a mixed microbial culture for PHA production with used cooking oil as substrate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enrichment of a mixed microbial culture for PHA production with used cooking oil as substrate Carlota Ucha, David Correa-Galeote, Ángeles Val del Río, Alba Pedrouso, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8617392/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract The synthesis of polyhydroxyalkanoates (PHA) by a mixed microbial culture (MMC) employing used cooking oil (UCO) as substrate represent a promising bioprocess for organic wastes valorisation. In the present research work, the effect of sequencing batch reactor (SBR) operational strategy on MMC enrichment and PHA accumulation was investigated using UCO without pretreatment as the sole carbon source. Two enrichment SBRs (SBR1 and SBR2) were operated under a feast-famine regime with uncoupled carbon and nitrogen supply, differing in the timing of the biomass withdrawal and nutrient filling. In SBR1, with effluent withdrawal at the end of the feast phase, better poly(3-R-hydroxybutyrate-co-3-R-hydroxyvalerate) (PHBV) accumulation was achieved (average values of 4.2 ± 1.6 wt. %, maximum of 19.9 wt. %) compared to SBR2, with withdrawal at the end of the famine phase (average values of 2.7 ± 1.1 wt. %, maximum of 5.2 wt. %). The highest PHBV accumulation in SBR1 was obtained at a COD/N ratio of 46 g/g, with an active biomass concentration of approximately 1 g VSS/L, highlighting the importance of feeding composition as a key operational control parameter. Additionally, a single UCO pulse resulted in more efficient substrate hydrolysis and consumption than multiple pulses in both systems. Microbial community analysis revealed the enrichment of bacterial OTUs related to Azospirillum , Acinetobacter , Gordonia , Burkholderia , and Bacillus , as well as fungal OTUs such as Geotrichum , Meyerozyma , and Pascua . Multivariate statistical analysis indicated that the synergistic interaction of Burkholderia and Chitinophaga played a crucial role in achieving the highest PHA accumulation levels. COD/N ratio fatty acids pulsed feeding waste valorisation withdrawal strategy. Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights - Mixed culture was enriched in PHA-storing population with raw UCO as substrate. - A COD/N ratio of 46 g/g and a single pulse of raw UCO were determined as the optimal. - SBR setup with withdrawal at the feast end outperformed withdrawal at the famine end. - Up to 19.9 wt.% PHA achieved in enrichment reactor with feast phase withdrawal. - The synergistic action of Burkholderia and Chitinophaga was key for high PHA yields. 1. Introduction Bio-based materials production plays a crucial role in reducing environmental impact and supporting the concept of the circular economy, which has gained interest in the last decades. Among these materials polyhydroxyalkanoates (PHA) have the potential to replace widely used petrochemical plastics. PHAs are polymers biosynthesised by different microorganisms in response to an excess of carbon combined with the limitation of a nutrient in the reaction medium. In the biological process, this polymer serves as a reservoir of carbon and energy that the microorganisms use to grow once the limited nutrient becomes available again in the medium [ 1 – 3 ]. Among the most common PHA monomers, poly(3-R-hydroxybutyrate) (3HB) and poly(3-R-hydroxyvalerate) (3HV) are the main homopolymers typically produced by microorganisms. The copolymer poly(3-R-hydroxybutyrate-co-3-R-hydroxyvalerate) (PHBV) exhibits physicochemical properties that are strongly influenced by the relative proportion of these two monomers. PHBV, the one studied in the present work, is a non-toxic, biodegradable material (in soil, water, and compost) and is biocompatible, with a lower degree of crystallinity and enhanced flexibility and mechanical strength compared to 3HB [ 4 ]. These characteristics make PHBV a particularly attractive biopolymer for biomedical and other high-value applications [ 5 ]. The biological synthesis of PHA could be carried out using either a pure or a mixed microbial culture (MMC). Pure cultures were typically selected for these studies due to the ease of selecting operating conditions and their better performance, achieving high concentrations of cell dry weight (4–50 g/L) [ 6 – 8 ]. However, they require sterile conditions to avoid contamination and are more sensitive to environmental changes [ 9 ]. On the other hand, an MMC, a group of different bacteria with similar growth conditions, offers enhanced adaptability and resilience to environmental changes. For this reason, PHA production by MMC has gained attention in recent years [ 10 – 13 ] In addition to the use of MMCs, a sustainable and cost-effective approach for PHA production also involves the use of waste substrates. Among the possible waste streams that can be utilized, used cooking oil (UCO) stands out. This is a lipidic waste globally produced and selectively collected in various countries like Brazil, Spain, Canada, and Germany [ 14 ]. In Spain alone, an average of 460 million litres of UCO were produced per year between 2022 and 2024; however, only 5 % o the household UCO is recovered for further valorisation [ 15 ]. The valorisation of UCO for bioproduct synthesis remains relatively underexplored. Most existing research works focus on the use of pure cultures or genetically modified bacteria [ 16 , 17 ]. Specifically, PHA production from UCO has been demonstrated using pure cultures cultivated mainly in flasks with a small volume [ 6 , 7 , 16 , 17 ]. In other research works, the UCO is first converted, via hydrolysis and/or acidogenic fermentation, into short-chain organic acids that are subsequently fed to an MMC enriched under a feast-famine regime for PHA storage [ 18 , 19 ]. Other studies include applying a pretreatment step, such as saponification, to favour UCO hydrolysis in the liquid media [ 20 , 21 ] and to obtain adequate MMC enrichment and PHA accumulation. By contrast, actual direct utilization of raw UCO by open MMCs, without prior hydrolysis, emulsification, or conversion to free fatty acids, has scarcely been demonstrated. One of the few targeted studies reported that direct MMC enrichment on PHA-storing bacteria using raw UCO was hindered by filamentous overgrowth, and that it was necessary to perform a previous selection of the MMC using a synthetic substrate different from the raw UCO [ 22 ]. Overall, the direct MMC enrichment with raw UCO is challenging, even though microorganisms are indeed capable of directly utilizing the fatty acids contained in UCO for PHA production [ 29 , 30 ]. The objective of this research work is to study the enrichment of an MMC derived from activated sludge for PHA production using UCO as the carbon source, with the novelty that the UCO was supplied without any type of pretreatment. In addition, the effects of the COD/N ratio and the biomass withdrawal strategy were evaluated. The dominant microbial populations (bacteria and fungi) in the MMC were also analysed to understand how changes in reactor operational conditions influence them. 2. Materials and methods 2.1. Experimental conditions for the enrichment reactors To enrich the MMC with accumulating microorganisms, two lab-scale sequencing batch reactors (SBR1, 10 L, and SBR2, 2 L) were operated. In each reactor, two selection strategies were applied, the aerobic dynamic feeding (ADF) and the double growth limitation (DGL), to promote the enrichment. The ADF strategy was designed to impose a feast-famine regime with 12-hour cycles, comprising 6 hours of feast phase followed by 6 hours of famine phase. The DGL strategy consisted of supplying the carbon source (raw UCO) either in single or multiple pulses to impose a feast phase. In contrast, the nitrogen source (NH 4 Cl) was added 6 hours later, assuming that UCO had already been consumed to begin the famine phase. The difference in cycle configuration between the two SBRs was that in SBR1, effluent withdrawal and filling with nutrient solution occurred at the end of the feast phase, whereas in SBR2, they occurred at the end of the famine phase. The volume exchange ratio was 50% in both SBRs, resulting in hydraulic and solids retention times of 1 day. Details of the operational cycle distribution for each SBR is available in Table S.1. Aeration was continuously supplied to both reactors using an air pump (Laboport N 86 KTP, KNF Neuberger, USA), connected to diffusers placed at the bottom of the reactor to provide dissolved oxygen for the biological reactions and mixing of the biomass inside the reactor. The temperature was maintained at 30°C using a thermostatic bath (Tectron Bio-100, JP Selecta, Spain) connected to a thermal jacket. The pH inside the SBRs was not actively controlled; however, the nutrient solution was adjusted to 7.0 ± 0.5 using KH 2 PO 4 and NaHCO 3 . From day 240 onwards, SBR1 was equipped with a pH controller (42 Series, Chemitec, Italy) connected to two peristaltic pumps, which supplied HCl (0.5 M) or NaOH (0.5 M) to maintain the reactor at pH values around 7.0 ± 0.3. 2.2. Inoculation and operational stages Activated sludge from the municipal wastewater treatment plant of Santiago de Compostela (Northwest Spain) was used as the inoculum for both SBRs, with a concentration of solids in the reactors of 2.5 ± 0.3 g VSS/L (ratio VSS/TSS of 0.73 ± 0.01 g/g). SBR1 (withdrawal at the end of the feast phase) was operated for 500 days, divided into three stages (Table 1 ). The first stage (S-I, days 0-203) was operated with the addition of three UCO pulses, an increasing organic loading rate (OLR) in terms of chemical oxygen demand (COD) from 1.2 to 1.8 g tCOD/(L·d) and a fixed nitrogen loading rate (NLR) of 78.5 mg N/(L·d). In the second stage (S-II, days 204–253), the OLR and NLR were maintained (1.8 g tCOD/(L·d) and 78.5 mg N/(L·d), respectively), but the UCO was added in a single pulse instead of three, that continued till the end of the operation. Finally, in S-III (days 254–500), the OLR remained almost constant at 1.8–1.9 g tCOD/(L·d) while the NLR was decreased and slightly modified between 36–46 mg N/(L·d) to adjust its value and avoid the excess of nitrogen at the end of the famine phase. Table 1 Summary of operational parameters and feeding strategies applied in SBR1 and SBR2 throughout the enrichment process. SBR1 SBR2 Stage S-I S-II 2 S-III Operational days 0–203 204–253 254–500 0–188 UCO pulses/cycle 3 1 1 1 UCO added (mL/pulse) 0.9–1.3 4.0 4.0–4.2 0.7 OLR (g tCOD/(L·d)) 1.2–1.8 1.8 1.8–1.9 1.5 tCOD (mg/L) 1 590–900 900 900–934 736 NLR (mg N/(L·d)) 78.5 78.5 36–46 32.6 NH 4 + -N (mg N/L) 1 40 40 18–23 16 COD/N (g/g) 15–23 23 45–52 46 1 Concentrations of UCO (as tCOD, feast phase) or nitrogen (famine phase) in the reactor just after feeding in each cycle. 2 A control of pH was implemented in SBR1 at day 240. COD: chemical oxygen demand; COD/N: COD fed at the feast phase divided by the nitrogen fed at the famine phase; NLR: nitrogen loading rate; OLR: organic loading rate; UCO: used cooking oil. ), as well as the tCOD () and N () concentrations fed each cycle; (c) percentages of accumulated PHA () and fatty acids () at the end of the feast phase and COD/N ratio () fed through the operation. The different stages are defined with a pointed vertical line ( ⁞ ). SBR2 (withdrawal at the end of the famine phase) was operated for 188 days under conditions like S-III of SBR1 (Table 1 ): feeding with one pulse, OLR of 1.5 g tCOD/(L·d), and NLR of 32.6 mg N/(L·d). 2.3. Feeding composition Both SBRs were fed with: i) raw UCO as the carbon source at the beginning of the feast phase; ii) NH 4 Cl solution as the nitrogen source at the beginning of the famine phase; iii) nutrient solution at the beginning of the famine phase (together with the nitrogen source) in SBR1 and at the beginning of the feast phase in SBR2. The raw UCO was collected from the university's hostelry service and used as the organic substrate without pretreatment. It was characterized by its content in fatty acids (93.84 ± 2.34%), organic matter (2.63 ± 0.29 g tCOD/g UCO) and elemental composition (CH 1.93 O 0.11 N 0.002 ), see more details in Table S.2. Furthermore, an oxygen uptake rate test was performed with activated sludge to confirm the suitability of the raw UCO as the substrate for biological processes, obtaining a biodegradability value of 58.47 ± 2.2%. The nutrient solution (pH 7.0 ± 0.5) comprised KH 2 PO 4 (0.3 g/L), MgSO 4 (0.0099 g/L), KCl (0.066 g/L), NaHCO 3 (0.30–0.45 g/L), allylthiourea (0.0044 g/L) and a micronutrient solution (1 mL/L) described by Vishniac and Santer [ 23 ]. Additionally, in SBR1, the nutrient solution contained NH 4 Cl (0.3 g/L in S-I and S-II and 0.17 g/L in S-III). In SBR2, the NH 4 Cl solution was added separately at the beginning of the famine phase and consisted of 100 mL of a solution of 1.25 g NH 4 Cl/L. 2.4. Sampling and analytical methods In both SBRs, two samples per cycle were analysed (three times per week), collected at the end of the feast phase and at the end of the famine phases. All the samples were analysed in duplicate. To characterise the liquid fraction, the samples were centrifuged and then filtered using a 0.45 µm pore size, mixed cellulose ester membrane (Advantec, Japan). The soluble chemical oxygen demand (sCOD) [ 24 ] and total nitrogen (TN) concentrations (TOC-L analyser with the TNM-module, TOC-5000 Shimadzu, Japan) were determined in the liquid phase. Concentrations of total COD (tCOD) [ 24 ], total suspended solids (TSS) and volatile suspended solids (VSS) [ 25 ], as well as PHBV and fatty acids content were measured in the solid phase. The pH was measured with a pH & Ion-Meter model GLP 22 (Crison, Spain). The PHBV and fatty acids quantification was performed by gas chromatography with a flame ionization detector (GC-FID) equipped with a HP-INNOVAX column (Agilent, USA), using the method described by Fra-Vázquez et al. [ 26 ] slightly modified, drying the biomass at 50°C in an oven (Memmert BE300, Memmert, Germany) instead of freezing and lyophilizing it. Commercial standards of PHBV copolymer (3HB, 90.82 %, nd 3HV, 9.18 %),fatty acids (Palmitic, Stearic, Oleic, and Linoleic), and benzoic acid as an internal standard were used. The dissolved oxygen concentration was continuously measured inside the reactors every 5 min using a portable multimeter (HQ40d, Hach-Lange, USA). 2.5. Calculations The OLR (as g tCOD/(L·d)) and the NLR (as mg N/(L·d)) were calculated as the amount of UCO added, in terms of tCOD, or the amount of nitrogen added, as mg N, to the reactor per cycle, considering the number of cycles per day (2) and the volume of the reactor (Vr). $$\:OLR=\frac{UCO\:\left(g\:tCOD/cycle\right)\bullet\:2\:(cycles/day)}{Vr\:\left(L\right)}$$ $$\:NLR=\frac{Nitrogen\:\left(mg\:N/cycle\right)\bullet\:2\:(cycles/day)}{Vr\:\left(L\right)}$$ The COD/N ratio was determined as the tCOD fed at the feast phase divided by the nitrogen fed at the famine phase. The active biomass (X) was determined at the end of the famine phase as the solid concentration (g VSS/L) minus the remaining concentration of bioproducts in the microbial cells (PHBV and fatty acids in g/L) [ 27 ]. 2.6. Microbiological analysis DNA was extracted from three independent replicated biomass samples taken from SBR1 using the FastDNA-2 mL SPIN Kit for Soil method, which was briefly modified, and the FastPrep24 instrument (MP-BIO, USA). The diversity of bacterial and fungal communities was characterized using Illumina sequencing with the primers Pro341F/Pro805R [ 28 ] for bacteria and FungiQuantF/FungiQuantR [ 29 ] for fungi. The operational taxonomic units (OTUs) were defined at a 97 % similrity threshold from the raw sequence data using the MothurMiSeq pipeline and Mothur software version 1.44.1 [ 30 ]. Taxonomic classification of Bacteria and Fungi was conducted utilizing the custom BLAST tool in Geneious version 2025.1.2 (Biomatters, New Zealand), comparing sequences against the NCBI bacterial 16S rRNA and fungal 18S rRNA databases [ 31 ]. A non-metric multidimensional scaling (NMS) analysis was used to link the structure of the bacterial and fungal communities to the operational parameters by means of the PC-ORD software (Wild Blueberry Media, Corvallis, OR, USA). 3. Results and Discussion The operation of SBR1 for 500 days, modifying the concentration of COD and N fed, as well as the number of raw UCO pulses added, allowed for determining the adequate operational conditions to increase the concentration of active biomass in a system with an MMC and raw UCO as substrate (without pretreatment). Furthermore, it was evaluated whether the withdrawal and filling with nutrients at the end of the feast phase (SBR1) or at the end of the famine phase (SBR2) influenced the degree of enrichment. 3.1 Dynamics of biomass concentration during the enrichment of the MMC In SBR1, the VSS/TSS ratio consistently remained around 0.94 ± 0.03 (Fig. 1 a), which indicates that most of the solids were organic, although a minor inert fraction may still have been present. The active biomass concentration varied throughout the operation, being low during the first 125 days (S-I) with values ranging from 0.1 to 0.4 g VSS/L, while the bioproducts were only 1.55 ± 0.52 wt. % for PHBV and 10.41 ± 4.05 wt. % for fatty acids (Fig. 1 ). Then, when the fed COD concentration was increased from 590 to 900 mg tCOD/L, the active biomass concentration increased to 0.4–0.6 g VSS/L between days 150 and 203 (S-I), and bioproducts accumulated, with fatty acids values up to 20 wt. % and PHBV peaks of 9 wt. % (day 182) and 20 wt. % (day 203). The increase in PHBV content after day 150 was likely associated with the progressive decrease in nitrogen concentration during the feast phase, as it was consumed for biomass growth during the famine phase [ 32 ]. During stages S-I and S-II, nitrogen was added at the beginning of the famine phase to have a concentration of 40 mg N/L (inside the reactor). However, it was not entirely consumed during the famine phase, and variable concentrations remained available for growth in the following feast phase (Fig. 1 b). Under these conditions, PHBV accumulation was not successful, as limiting conditions of this essential nutrient are necessary at the beginning of the feast phase to stimulate the accumulation of the carbon added as PHBV, while biomass growth is avoided [ 33 ]. Avoiding the presence of an excess of nitrogen during the bioproduct’s accumulation processes becomes very important [ 34 – 37 ]. For this reason, the nitrogen concentration fed was halved in stage S-III (Table 1 ), to support active biomass growth while limiting it during PHBV accumulation. The increase in active biomass, as well as in bioproduct accumulation, with a notable peak in PHBV and fatty acids, was observed during this period (18.5 wt. % of PHBV on day 288 and 31 wt. % of fatty acids on day 268). The highest concentrations of active biomass were achieved between days 330 and 400 (S-III), reaching a maximum of 1 g VSS/L. This period corresponds to a COD feed of 900 mg tCOD/L and a nitrogen addition of 20 mg N/L that was highly consumed during the famine phase (Fig. 1 b). In this stage the remaining nitrogen at the end of the famine phase was too low to affect PHBV synthesis in the subsequent feast phase. This could be a contributing factor to the overall increase in bioproducts accumulation, reaffirming the need for nutrient limitation to achieve the accumulation [ 32 , 37 ]. From day 420 onwards, several minor adjustments to the nitrogen concentration were tested (Fig. 1 b) to enhance PHBV accumulation; however, these changes were unsuccessful, and nitrogen was not fully consumed during the famine phase, remaining available at the beginning of the feast phase. Thus, lower active biomass concentrations were reached compared to the previous days. Therefore, towards the end of the operational period (days 400–500, S-III), the active biomass decreases to approximately 0.32 ± 0.09 g VSS/L (Fig. 1 a). This fact highlights the sensitivity of the enrichment with raw UCO as well as the importance of proper nitrogen concentration feeding. Thus, in SBR1, the increase in active biomass concentration was achieved first by increasing the COD concentration fed during the feast phase, and then by decreasing the nitrogen concentration to restrict its availability during the famine phase. The results showed that among the fed COD/N ratios assayed, in the range from 15 to 52 g/g, the most adequate to promote the increase of active biomass (up to 1 g VSS/L) was approximately 46 g tCOD/g N (Fig. 1 ), equivalent, according to the UCO elemental composition, to approximately 13 g C/g N. This value falls within the range of other reported values obtained from similar enrichment processes (aerobic dynamic feeding) using no pre-fermented substrates such as crude glycerol (15.9 g C/g N) [ 38 ] or UCO as the substrate (10 g C/g N) [ 22 ]. In addition, Zeng et al. [ 39 ], using a mixture of volatile fatty acids (VFAs) as a carbon source, observed that C/N ratios in the range of 40–80 g C/g N favored PHA accumulation at the expense of biomass growth, while lower ratios (5–20 g C/g N) enhanced biomass proliferation but restricted PHA synthesis. Thus, the selection of the C/N ratio of the feeding will help to develop the MMC. However, imposing the desired COD/N ratio requires a successful hydrolysis of the feed raw UCO, which may become a critical bottleneck. Inadequate hydrolysis restricts the availability of soluble substrates for microbial uptake, thereby limiting both biomass growth and PHBV accumulation. For this reason, the soluble COD in the liquid phase was monitored at the end of the feast phase (Fig. 1 b). During the first 100 days, values remained relatively high (200–400 mg sCOD/L), but as the active biomass concentration increased, they decreased to approximately 100 mg sCOD/L and then stabilized for the remainder of the operation. These results indicate that the MMC required approximately 100 days to establish a stable UCO hydrolysis that enables its consumption. 3.2 Feeding strategy: multiple or single pulses Pulsed feeding strategies are reported to reduce substrate inhibition by the carbon source [ 40 , 41 ] and promote high storage of PHA [ 27 ]. Additionally, considering the low solubility of UCO, this feeding strategy was imposed to facilitate the development of the hydrolysis process. Therefore, to avoid a possible inhibition by free fatty acids the operation of SBR1 was started by adding the raw UCO distributed in three pulses during the feast phase. During this stage (S-I) the dissolved oxygen (DO) concentration profile showed a marked decrease after each UCO feeding pulse (first pulse from 7 to 4 mg O 2 /L; second pulse from 6 to 2 mg O 2 /L; third pulse from 5 to 1 mg O 2 /L at day 126) followed by a subsequent recovery once the substrate was consumed (Fig. S.1a), indicating efficient substrate uptake and suggesting that the MMC maintained a good metabolic activity despite the potential inhibitory effects of free fatty acids contained in the raw UCO. Later in S-II, to enhance the UCO consumption and favour more stable PHBV accumulation, a single pulse feeding strategy was implemented from day 204 onwards (Table 1 ), maintaining the same total raw UCO added (900 mg tCOD/L). This change was made to allow more time during the feast phase to promote the UCO hydrolysis and assimilation of the substrate. As shown in Fig. S.1b for day 225, the DO concentration decreased from almost 5 to 2 mg O 2 /L during the first hour after the addition of raw UCO. It rose again to 5 mg O 2 /L before the effluent withdrawal phase, confirming that the MMC was able to metabolize the raw UCO, even using a single pulse. The use of a single pulse seemed to be significant for PHBV accumulation, as the average percentage values achieved with 3 pulses were below 2 wt. %, while with a single pulse, the average accumulation increased progressively from 2 wt. % (day 231) to 12 wt. % (day 476), with a peak of 18.5 wt. % (day 288). Fatty acids content in the biomass (average of 15.6 wt. ± 6.9%), generally higher than those of PHBV (average of 4.1 ± 1.2 wt. %), was also promoted by the shift in feeding strategy, achieving values of 31.6 wt. % on day 268. In contrast to other research works [ 27 , 32 ], which utilized waste fish oil, the present study obtained better results in terms of PHBV accumulation and biomass concentration using a single pulse feeding, and, in the studied operational conditions, confirmed the preference to accumulate fatty acids over PHBV when an oil is used as substrate (Fig. 1 c). 3.3 Uncoupled C and N: the challenge of pH control with oily substrates It is known that uncoupled carbon and nitrogen feeding strategies favour the accumulation of bioproducts like PHA [ 42 ]. However, it was observed that with oily substrates, the uncoupling strategy destabilizes the pH, and acidic conditions can be achieved inside the reactor [ 32 ]. In these conditions, pH control inside the biological reactor is essential. However, the addition of alkaline compounds to increase the pH can cause the saponification of the oil-based substrate, which can also lead to process failure. To manage these situations is therefore vital for the process. In the present study, SBR1 was initially operated without pH control. During the first 170 operational days, no problems of acidification were observed (Fig. 1 a). The pH was likely maintained thanks to the buffering capacity of the nutrient solution (pH fixed at 7.0 ± 0.5), which contained KH 2 PO 4 and NaHCO 3 . Non-consumed ammonium in the initial period might also help maintain a pH stable. However, as nitrogen consumption improved, the pH inside the reactor at the end of the famine phase decreased to values of 3–4 (days 170–250), resulting in an acidic medium and subsequently causing microbial stress. Acidic conditions have been reported to cause different effects on PHA accumulation. Some authors have reported that low pH values (< 4) can inhibit active biomass, ultimately leading to process failure [ 43 ]. Nevertheless, other studies suggest that stress conditions in short time periods, such as low pH, may promote the accumulation of PHA, as microorganisms may utilize this polymer for stress resistance [ 44 – 46 ]. This factor may contribute to the observed higher PHBV accumulations on days 182 and 203 (8.9 and 19.9 wt. %, respectively). However, although this pH stress may be beneficial for short-term PHA accumulation [ 27 , 32 , 47 ], previous studies have reported that pH values of 7.0 or even 8.5 are optimal for PHA synthesis [ 48 , 49 ]. Prolonged exposure to low pH levels, around 3–4, can reduce or even inhibit the growth of MMC as bacteria typically thrive in a more neutral pH environment. Extended periods of stress can reduce or even inhibit the growth of the culture accumulating PHA, leading to a decrease of the PHA produced [ 45 ], as evidenced by the decrease in biomass concentration due to the inability of many microorganisms to survive under such conditions. To mitigate this issue, continuous pH control (7.0 ± 0.3) was implemented in SBR1 from day 240 onwards. After this implementation, the active biomass concentration increased again (Fig. 1 a). The pH value was set at 7.0 to provide suitable conditions for both oil hydrolysis and bacterial growth [ 50 ], as well as for the enrichment of MMCs for PHA production [ 48 ]. This value was also selected to minimize NaOH consumption during pH control and to prevent UCO saponification under alkaline conditions. Although enrichment and PHA accumulation with readily biodegradable substrates such as VFAs are typically carried out under neutral to slightly alkaline conditions [ 51 ], the use of lipid-based wastes like UCO requires an additional hydrolysis step to convert triglycerides into free fatty acids prior to microbial uptake. Since lipase activity is generally favored under mildly acidic to neutral conditions [ 50 ], maintaining a pH around 7.0 represented an appropriate compromise between promoting UCO hydrolysis and ensuring stable microbial activity for PHA accumulation. 3.4 Reactor configuration: effluent withdrawal and nutrient filling SBR2 was operated similarly to SBR1 in stage S-III, with the feeding added as a single pulse and a COD/N ratio of 46 g/g, but with a modification in the cycle configuration and reactor volume (from 10 to 2 L). The withdrawal and refilling with a nutrient solution in SBR1 occurred during the transition from feast to famine phase, whereas in SBR2, it took place in the transition from famine to feast phase. The SBR1 configuration enables the reactor to act as an enrichment and accumulation unit, as withdrawal occurs immediately after accumulation is complete. In contrast, the SBR2 configuration corresponds to an enrichment reactor, and a subsequent accumulation unit will be necessary because effluent withdrawal occurs at the end of the famine phase, when the bioproducts inside the biomass have already been consumed. Furthermore, in SBR2, the feast/famine lengths were slightly different, 5/7 h/h between days 0 and 94; thereafter, the same values as SBR1 (6/6 h/h) were used until the end of the operation (days 95–188). Regarding the active biomass in SBR2 (Fig. 2a), the higher values were obtained with a feast/famine length of 5/7 h/h, achieving average values of 0.6–0.8 g VSS/L at the end of the famine phase. Increasing the feast phase to 6 hours (6/6 h/h), without modifying the amount of raw UCO added, decreased the active biomass concentration to values of 0.4 g VSS/L. On the other hand, extending the length of the feast phase was beneficial to maintaining the pH value inside the reactor above neutral values. Regarding the soluble COD values, those at the end of the feast phase in SBR2 were consistently below 100 mg sCOD/L after the first 30 days (Fig. 2b), while it took about 100 days in SBR1 to reach similar values. This could indicate that starting the SBR operation with an optimised COD/N ratio (46 g/g in SBR2) can help to balance the hydrolysis and the consumption of the raw UCO in a shorter time. The nitrogen concentration at the end of the famine phase was below 10 mg N/L only on isolated days (Fig. 2b), indicating limited nitrogen consumption for biomass growth, likely due to the low bioproduct synthesis during the feast phase, which is used as the carbon source (Fig. 2c). Similar to SBR1, the SBR2 configuration resulted in a preferential accumulation of fatty acids over PHBV (Fig. 2c). But, if the maximum percentages of accumulation in SBR1 and SBR2 are compared for fatty acids (31.6 and 37.4 wt. %, respectively) and PHBV (19.9 and 5.2 wt. %, respectively), it can be concluded that the SBR1 configuration (withdrawal at the end of the feast phase) is preferable to promote PHBV as a bioproduct. Moreover, in both SBRs, the PHBV copolymer showed variations in the 3HB and 3HV proportions associated with the operational changes, suggesting that the monomeric composition was not determined by the withdrawal configuration (Fig. S.2). The presence of 3HV content is advantageous, as it yields a copolymer with lower crystallinity and melting temperature, improving flexibility and ease of processing [ 4 ]. 3.5 Reactor populations: microbiology analysis The dominant operational taxonomic units (OTUs) for bacteria were OtuB00001 ( Burkholderia , 15.96%), OtuB00002 ( Curvibacter , 5.65%), and OtuB00003 ( Kryptousia , 3.03%) (Fig. 3 a). However, there were significant shifts in the dominant OTUs throughout the operational periods, reflecting a high degree of dynamics within the MMC. Therefore, the imposed operational conditions had a strong capacity to modulate the bacterial community structure over time. Nevertheless, despite the instability of the bacterial population structure throughout the operation of SBR1, the dominant OTUs observed for the majority of the sampling times were described as PHA-accumulating bacteria, mainly OtuB00008 ( Azospirillum) [ 52 ] at day 35, OtuB00004 ( Acinetobacter ) [ 53 ] at days 112 and 154, OtuB00007 ( Gordonia ) [ 54 ] at day 154, OtuB00001 ( Burkholderia ) [ 55 ] at days 182 and 204, OtuB00009 ( Bacillus ) [ 56 ] at day 457. This broad dominance of PHA-accumulating bacteria underscores the importance of high functional redundancy within an MMC, enabling it to withstand the various stresses encountered during operation. Interestingly, the bacterial community was dominated by the OTUB001 taxonomically affiliated with the well-known PHA-accumulating Burkholderia , with a maximum relative abundance of 87% at day 182. In relation to this, the maximum PHBV accumulated percentage in the biomass was only achieved when the relative abundance of this genus was coupled with that of the highly hydrolytic genus Chitinophaga (maximum relative abundances of 16% at day 204). Thus, since both genera seemed to act synergistically in the accumulation of PHBV, it could be hypothesised that Chitinophaga transformed the fats in this residue into free fatty acids through the action of its lipases, and Burkholderia finally metabolised these fats into PHBV by the PhaC enzyme, the key enzyme in the PHA biosynthesis. Regarding the decrease in pH in SBR1, it does not appear to hinder the development of PHA-accumulating bacteria, as a selective increase in the community was observed on days 182 and 204, resulting in the great abundance of the OTU taxonomically classified as Burkholderia . It should be noted that some strains of Burkholderia are acidophiles, capable of surviving under pH values lower than 7 [ 57 ], as is the case for those found at days 170–250, resulting in a significant promotion of the growth of this bacterial genus. Nevertheless, as hypothesised, the establishment of a pH control on day 240 onwards did not result in the stabilisation of bacterial populations within the MMC, as a high degree of dynamics was also observed during this period. Regarding the fungal populations (Fig. 3 b), they were predominantly dominated by OtuF00001 ( Geotrichum , 45.31%), followed by OtuF00002 ( Meyerozyma , 7.66%) and OtuF00003 ( Pascua , 7.57%). Generally considered, the structures of the fungal communities were more stable than those of the bacterial communities, especially after day 182, when the community was predominantly dominated by OtuF00001 ( Geotrichum ), achieving a maximal relative abundance of 99.06%. This dominance pattern can be attributed to the acid tolerance of this genus [ 58 ], which allows its growth once the medium becomes more acidic due to improved nitrogen consumption (days 170–250). This fungus exhibits important antifungal activity, mediated by the production of volatile organic compounds, particularly phenylethyl alcohol, which inhibits fungal growth through various pathways, including disruption of the cell membrane, intracellular leakage, and impairment of energy metabolism and defence systems [ 59 ]. Finally, it should be noted that Geotrichum is an oleaginous yeast [ 60 , 61 ] which, after becoming dominant, could have been responsible for the significant content of fatty acids in the biomass of SBR1. The multivariate statistical analysis carried out revealed that the main operational parameter promoting PHBV accumulation was the tCOD fed (raw UCO), suggesting that a higher organic loading rate, and subsequently a rise in carbon availability, increased the PHBV accumulation capacity (Fig. 4 ). The COD/N ratio fed in each cycle was related to higher PHBV accumulations, and, on the other hand, higher N in the reaction medium at the end of the famine phase was inversely associated to larger PHA yields, suggesting that an increase in the carbon supply and an effective reduction in the nitrogen availability is mandatory to enhance the PHBV accumulation, confirming the previously defined conditions for an effective PHA accumulation from lipidic wastes by MMCs [ 62 ]. In addition, the NMS confirmed that lower pH values in the reaction medium at the end of the famine phase for a short period of time stimulated PHBV biosynthesis, as it was experimentally observed on days 170–250, according to the protective role of PHA in bacterial fitness against various environmental stressors [ 63 ]. The NMS also shows that higher PHBV accumulation yields were correlated with higher relative abundances of Burkholderia and Chitinophaga , endorsing that a synergistic action of both genera is essential for the successful valorisation of raw UCO into bioplastic precursors. To confirm this close relationship, the Spearman rank analysis was also made (Table S.3). In this regard, higher relative abundances of Burkholderia were strongly positively related to a rise of Chitinophaga (ρ = 0.834), which are also correlated with higher PHBV accumulation capacities (ρ = 0.624 for Burkholderia and ρ = 0.803 for Chitinophaga ), reinforcing the synergistic role of both bacteria in the PHBV accumulation. Considering that this synergetic association is first described here, the role of both bacteria should be explored to address the potential biotechnological use of these two genera as a promising alternative to enhance PHBV accumulation for the valorisation of raw UCO. In this regard, co-culture selection and growth of both genera could be based on the phthalic acid resistance of Chitinophaga , as described by Lee et al. [ 64 ] and the tolerance of Burkholderia to this compound, as previously reported by Zhang et al. [ 65 ]. The vectors representing PHBV and fatty acids contents in the biomass are spatially close. In this regard, Geotrichum (OtuF00001 and OtuF00004) has been reported to be capable of biosynthesizing enzymes, such as cellulases, α-amylases, proteases, lipases, β-glucanases, xylanases, and phytases [ 58 ], which could enhance the metabolization of raw UCO, allowing a simultaneous increase in the PHBV and fatty acids contents in the biomass. Based on the high hydrolytic capacities of Geotrichum , the Spearman correlation analysis (Table S.3) suggests a cooperative role in PHBV accumulation, due to the strong correlation between PHBV accumulation and the relative abundances of this fungus (ρ = 0.574). Hence, the operational factor for the simultaneous enrichment of Geotrichum , Burkholderia , and Chitinophaga needs to be thoroughly explored as an alternative for selecting a microbial community of Bacteria and Fungi that synergistically interact in the valorisation of raw UCO into PHBV. 3.6 Challenges of the PHA production process based on MMC and raw UCO Despite the growing interest in replacing conventional fossil-based plastics with PHAs, their large-scale commercialization remains constrained by high production costs, which still limit their competitiveness. Several techno-economic analyses [ 13 , 66 – 69 ] have shown that the use of MMCs can substantially reduce both capital and operational costs by (i) eliminating sterilization requirements, (ii) enabling the use of low-cost waste substrates, and (iii) integrating PHA production into waste treatment and management systems. Using UCO and MMCs accomplishes these requirements. However, the use of MMCs for PHA production with UCO presents several challenges, including substrate complexity, conversion efficiency, and process stability. The poor solubility of UCO requires an effective hydrolysis step to release free fatty acids, which can inhibit microbial activity and compromise reactor performance. Consequently, the enrichment of PHA-storing microorganisms in MMCs fed with lipid-based substrates is typically slower and less stable than with readily biodegradable carbon sources such as VFAs. Nevertheless, the present study demonstrated that this enrichment is achievable using raw UCO without any type of pretreatment. Starting from conventional activated sludge and applying strategies like ADF and DGL, the operational conditions of the enrichment reactor (SBR1) were progressively optimized, achieving notable PHBV contents at the end of the feast phase (9 wt. % on day 182; 20 wt. % on day 203; 18.5 wt. % on day 288; and 12 wt. % on day 476), with 3HB identified as the main constituent, prevailing over 3HV in these days (Fig. S.2). The results obtained in this study, after 500 days of SBR1 operation, confirm that the enrichment of PHA-accumulating MMC with raw UCO is feasible, with key microbial populations identified as Burkholderia and Chitinophaga among bacteria, and Geotrichum among fungi. In contrast, Tamang and Nogueira [ 22 ] reported that direct enrichment using raw UCO was not feasible due to the proliferation of filamentous bacteria, leading to foaming and bulking. In their study, an MMC previously enriched with nonanoic acid achieved a maximum PHA content of 38.2 wt. % from raw UCO at 40°C, but only in a dedicated accumulation reactor. Even with pure cultures, literature reviews [ 70 , 71 ] report a wide range of PHA contents (36–75 wt. %) when using oil-rich wastes, indicating that achieving high intracellular accumulation is not always straightforward, even with specific strains and optimized conditions. In the present research work, the PHBV content reached in SBR1, although significant for an open culture directly fed with raw UCO, was neither stable nor sufficiently high to enable efficient extraction, which typically requires intracellular contents above 30–40 wt. % in MMCs [ 72 ]. This suggests that, although enrichment was achieved, further optimization of the accumulation process is still necessary, either by improving the operational conditions within the same reactor (for example, by extending the feast phase) or by coupling the enrichment stage with a dedicated accumulation unit. Regarding substrate variability, although the composition of raw UCO may differ depending on the type and frequency of use, the UCO used in a potential PHA production process would be supplied by companies specialized in its collection and management. As a result, the UCO composition within a given region tends to be relatively consistent, resulting in reduced variability compared with other complex wastes that require prior fermentation to VFAs. Furthermore, the existing collection and standardization systems established for biodiesel production represent a clear advantage. Additionally, the UCO used for PHA synthesis does not require the extensive pretreatment steps typically applied for biodiesel feedstock preparation (e.g., water or solids removal). Therefore, as in the present study, pretreatment can be omitted or limited to the simple removal of coarse solids. 4. Conclusions The increase of the active biomass concentration in the enrichment reactors was successfully achieved through a progressive rise in the organic loading rate, fed as raw UCO, followed by a nitrogen limitation. A COD/N ratio around 46 g/g allowed to maintain an active biomass concentration up to 1 g VSS/L. Furthermore, when a single UCO pulse was provided, the biomass exhibits better performance in synthetizing PHBV (12 wt. %) compared to the three-pulse strategy (< 2 wt. %), which was attributed to better substrate hydrolysis and assimilation. The SBR configuration with the withdrawal and filling with nutrients at the end of the feast phase (SBR1) and 10 L of working volume, consistently reached better PHBV accumulation results (average values of 4.2 ± 1.6 wt. %, maximum of 19.9 wt. %) than the configuration with the withdrawal and filling with nutrients at the end of the famine phase and 2 L of working volume (SBR2, average values of 2.7 ± 1.1 wt. %, maximum of 5.2 wt. %). However, although a maximum accumulation of 19.9 wt. % for PHBV and 31.6 wt. % for fatty acids were reached in SBR1, process operation under these conditions was unstable, and high storage levels could not be maintained over time. Process pH was demonstrated to be a key parameter for system operation. A transient pH decrease enhanced PHBV production due to its stress resistance function, whereas prolonged exposure to acidic conditions negatively affected microbial activity and bioproducts synthesis. Microbial community analysis revealed a strong successional dynamic of PHA-accumulating bacterial populations, primarily including Azospirillum , Acinetobacter , Gordonia , Burkholderia , and Bacillus. The high degree of functional redundancy within the MMC enabled resilience to operational and environmental changes, while multivariate analysis confirmed that synergistic interactions between Burkholderia and Chitinophaga were essential for the successful valorisation of UCO into PHBV. Overall, the present study demonstrates the feasibility of producing PHBV from raw UCO using MMC, while underling the role of the operational strategy, feeding mode, and microbial interactions. Future work should focus on optimize this process stability, targeting larger active biomass concentrations and higher percentages of accumulated PHBV to support the scale-up and implementation of this method for lipid-rich waste valorisation. Declarations Author Contribution C.U. made the writing of the original draft; C.U. and D.C.G. made the investigation and formal analysis; C.U., D.C.G and A.V. made the visualization of the data; C.U., D.C.G, A.V. and A.M.C made the methodology; A. V., A.P and A.M.C made the conceptualization; A. V. and A.M.C made the supervision and validation; A.M.C made the project administration and funding acquisition; All authors made the review and editing of the manuscript. Acknowledgement This research was supported by the Spanish Government (AEI) through the ECOPOLYVER project [MACROPOLYVER PID2020-112550RB-C21/ AEI / 10.13039/501100011033 & MICROPOLYVER PID2020-112550RB-C22/ AEI / 10.13039/501100011033] and the project POLYGO1 [TED2021-130164B-I00 / AEI / 10.13039 / 501100011033 / Unión Europea NextGenerationEU / PRTR]. Carlota Ucha, Ángeles Val del Río, Alba Pedrouso and Anuska Mosquera-Corral belong to a Galician Competitive Research Group (GRC ED431C 2025/19) and CRETUS Research Centre (ED431G 2023/12). Data Availability The data that support the findings of this study are not openly available but are available from the corresponding author upon reasonable request. References Valentin HE, Broyles DL, Casagrande LA et al (1999) PHA production, from bacteria to plants Satoh H, Mino T, Matsuo T (1999) PHA production by activated sludge Wen Q, Chen Z, Tian T, Chen W (2010) Effects of phosphorus and nitrogen limitation on PHA production in activated sludge. J Environ Sci 22:1602–1607. https://doi.org/10.1016/S1001-0742(09)60295-3 García-Chumillas S, Guerrero-Murcia T, Nicolás-Liza M et al (2024) PHBV cycle of life using waste as a starting point: from production to recyclability. 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Front Bioeng Biotechnol 9:624021. https://doi.org/10.3389/FBIOE.2021.624021/FULL Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx floatimage1.png Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 Mar, 2026 Reviews received at journal 04 Mar, 2026 Reviews received at journal 04 Mar, 2026 Reviewers agreed at journal 09 Feb, 2026 Reviewers agreed at journal 08 Feb, 2026 Reviewers agreed at journal 07 Feb, 2026 Reviewers agreed at journal 07 Feb, 2026 Reviews received at journal 06 Feb, 2026 Reviews received at journal 06 Feb, 2026 Reviewers agreed at journal 06 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviewers invited by journal 05 Feb, 2026 Editor assigned by journal 19 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 16 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Department","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Correa-Galeote","suffix":""},{"id":588004450,"identity":"a84abf20-f325-43e7-9b8e-61362dfb0ae9","order_by":2,"name":"Ángeles Val del Río","email":"","orcid":"","institution":"CRETUS","correspondingAuthor":false,"prefix":"","firstName":"Ángeles","middleName":"Val del","lastName":"Río","suffix":""},{"id":588004452,"identity":"8c588508-7bc5-499c-bd7f-2f1ec853f5e3","order_by":3,"name":"Alba Pedrouso","email":"","orcid":"","institution":"CRETUS","correspondingAuthor":false,"prefix":"","firstName":"Alba","middleName":"","lastName":"Pedrouso","suffix":""},{"id":588004453,"identity":"2d34fd11-0e1c-4b5f-a16d-27c90d842367","order_by":4,"name":"Anuska Mosquera-Corral","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYJACxgYkjhzpWoxJ15LYgEMVHPDP7jH+OKOGIZqf//A2iY977NI3nD/8gOHDH9xaJO6cMZPccIwhd+aMtDLJGc+SczfcSDNgnNmGx5obOWaMD9gYgCp5zKR5DjCDGAzMvHicJ38jx/jjg39ALefPmEn/OVCfbnD+DAPzHzwOM7iRYyC5sQ2o5UCOmTTDgcMJBgdyGJgZ2HBrMbwB9MLMPgmQX4otew4cN5wJ9MvBXjx+kbuRvPljzzeb3H7+wxtv/DhQLc93/vDDBz/wOAwKJMCOhHMPENQA8xexCkfBKBgFo2CEAQCg4FbudyXUZgAAAABJRU5ErkJggg==","orcid":"","institution":"CRETUS","correspondingAuthor":true,"prefix":"","firstName":"Anuska","middleName":"","lastName":"Mosquera-Corral","suffix":""}],"badges":[],"createdAt":"2026-01-16 11:04:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8617392/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8617392/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102354131,"identity":"b53b7e45-4c20-4af7-ba45-ad83ceb9adf2","added_by":"auto","created_at":"2026-02-10 20:05:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":832849,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"f1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8617392/v1/46e970ee9e858c3f39ecaf76.jpg"},{"id":102354133,"identity":"ef422271-faa7-44aa-b0a3-51f057080714","added_by":"auto","created_at":"2026-02-10 20:05:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":773519,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"f2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8617392/v1/2c30535d06e2952df2349e80.jpg"},{"id":102397916,"identity":"103fd99b-78ac-4c69-801f-96efbbe5f474","added_by":"auto","created_at":"2026-02-11 10:20:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":681379,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"f3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8617392/v1/2850908f04c098d2f7255c52.jpg"},{"id":102397835,"identity":"deb46c56-4327-459a-afe4-ba0ad6b42702","added_by":"auto","created_at":"2026-02-11 10:19:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":593244,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"f4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8617392/v1/f98119ca88c503e4a113602d.jpg"},{"id":102399111,"identity":"bc1b46b0-a390-4dd0-8a16-2a8e2ee8a2ea","added_by":"auto","created_at":"2026-02-11 10:33:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3764205,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8617392/v1/b4fe7acb-0f7e-42bf-b687-75f3269f8c1e.pdf"},{"id":102354136,"identity":"b4ad759e-0ae5-4dfe-b9ce-1311c171bb0c","added_by":"auto","created_at":"2026-02-10 20:05:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":128033,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8617392/v1/9907c3c4f271401e8a426a60.docx"},{"id":102398318,"identity":"2fb435db-1a95-467e-b256-7a6dc20add2f","added_by":"auto","created_at":"2026-02-11 10:22:07","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":135872,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8617392/v1/21cfe6511b017923aa7352ba.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enrichment of a mixed microbial culture for PHA production with used cooking oil as substrate","fulltext":[{"header":"Highlights","content":"\u003cp\u003e- Mixed culture was enriched in PHA-storing population with raw UCO as substrate.\u003c/p\u003e\u003cp\u003e- A COD/N ratio of 46 g/g and a single pulse of raw UCO were determined as the optimal.\u003c/p\u003e\u003cp\u003e- SBR setup with withdrawal at the feast end outperformed withdrawal at the famine end.\u003c/p\u003e\u003cp\u003e- Up to 19.9 wt.% PHA achieved in enrichment reactor with feast phase withdrawal.\u003c/p\u003e\u003cp\u003e- The synergistic action of Burkholderia and Chitinophaga was key for high PHA yields.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eBio-based materials production plays a crucial role in reducing environmental impact and supporting the concept of the circular economy, which has gained interest in the last decades. Among these materials polyhydroxyalkanoates (PHA) have the potential to replace widely used petrochemical plastics. PHAs are polymers biosynthesised by different microorganisms in response to an excess of carbon combined with the limitation of a nutrient in the reaction medium. In the biological process, this polymer serves as a reservoir of carbon and energy that the microorganisms use to grow once the limited nutrient becomes available again in the medium [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among the most common PHA monomers, poly(3-R-hydroxybutyrate) (3HB) and poly(3-R-hydroxyvalerate) (3HV) are the main homopolymers typically produced by microorganisms. The copolymer poly(3-R-hydroxybutyrate-co-3-R-hydroxyvalerate) (PHBV) exhibits physicochemical properties that are strongly influenced by the relative proportion of these two monomers. PHBV, the one studied in the present work, is a non-toxic, biodegradable material (in soil, water, and compost) and is biocompatible, with a lower degree of crystallinity and enhanced flexibility and mechanical strength compared to 3HB [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These characteristics make PHBV a particularly attractive biopolymer for biomedical and other high-value applications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe biological synthesis of PHA could be carried out using either a pure or a mixed microbial culture (MMC). Pure cultures were typically selected for these studies due to the ease of selecting operating conditions and their better performance, achieving high concentrations of cell dry weight (4\u0026ndash;50 g/L) [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, they require sterile conditions to avoid contamination and are more sensitive to environmental changes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. On the other hand, an MMC, a group of different bacteria with similar growth conditions, offers enhanced adaptability and resilience to environmental changes. For this reason, PHA production by MMC has gained attention in recent years [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn addition to the use of MMCs, a sustainable and cost-effective approach for PHA production also involves the use of waste substrates. Among the possible waste streams that can be utilized, used cooking oil (UCO) stands out. This is a lipidic waste globally produced and selectively collected in various countries like Brazil, Spain, Canada, and Germany [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In Spain alone, an average of 460\u0026nbsp;million litres of UCO were produced per year between 2022 and 2024; however, only 5 % o the household UCO is recovered for further valorisation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The valorisation of UCO for bioproduct synthesis remains relatively underexplored. Most existing research works focus on the use of pure cultures or genetically modified bacteria [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Specifically, PHA production from UCO has been demonstrated using pure cultures cultivated mainly in flasks with a small volume [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In other research works, the UCO is first converted, via hydrolysis and/or acidogenic fermentation, into short-chain organic acids that are subsequently fed to an MMC enriched under a feast-famine regime for PHA storage [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Other studies include applying a pretreatment step, such as saponification, to favour UCO hydrolysis in the liquid media [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and to obtain adequate MMC enrichment and PHA accumulation.\u003c/p\u003e \u003cp\u003eBy contrast, actual direct utilization of raw UCO by open MMCs, without prior hydrolysis, emulsification, or conversion to free fatty acids, has scarcely been demonstrated. One of the few targeted studies reported that direct MMC enrichment on PHA-storing bacteria using raw UCO was hindered by filamentous overgrowth, and that it was necessary to perform a previous selection of the MMC using a synthetic substrate different from the raw UCO [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Overall, the direct MMC enrichment with raw UCO is challenging, even though microorganisms are indeed capable of directly utilizing the fatty acids contained in UCO for PHA production [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe objective of this research work is to study the enrichment of an MMC derived from activated sludge for PHA production using UCO as the carbon source, with the novelty that the UCO was supplied without any type of pretreatment. In addition, the effects of the COD/N ratio and the biomass withdrawal strategy were evaluated. The dominant microbial populations (bacteria and fungi) in the MMC were also analysed to understand how changes in reactor operational conditions influence them.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental conditions for the enrichment reactors\u003c/h2\u003e \u003cp\u003eTo enrich the MMC with accumulating microorganisms, two lab-scale sequencing batch reactors (SBR1, 10 L, and SBR2, 2 L) were operated. In each reactor, two selection strategies were applied, the aerobic dynamic feeding (ADF) and the double growth limitation (DGL), to promote the enrichment. The ADF strategy was designed to impose a feast-famine regime with 12-hour cycles, comprising 6 hours of feast phase followed by 6 hours of famine phase. The DGL strategy consisted of supplying the carbon source (raw UCO) either in single or multiple pulses to impose a feast phase. In contrast, the nitrogen source (NH\u003csub\u003e4\u003c/sub\u003eCl) was added 6 hours later, assuming that UCO had already been consumed to begin the famine phase. The difference in cycle configuration between the two SBRs was that in SBR1, effluent withdrawal and filling with nutrient solution occurred at the end of the feast phase, whereas in SBR2, they occurred at the end of the famine phase. The volume exchange ratio was 50% in both SBRs, resulting in hydraulic and solids retention times of 1 day. Details of the operational cycle distribution for each SBR is available in Table S.1.\u003c/p\u003e \u003cp\u003eAeration was continuously supplied to both reactors using an air pump (Laboport N 86 KTP, KNF Neuberger, USA), connected to diffusers placed at the bottom of the reactor to provide dissolved oxygen for the biological reactions and mixing of the biomass inside the reactor. The temperature was maintained at 30\u0026deg;C using a thermostatic bath (Tectron Bio-100, JP Selecta, Spain) connected to a thermal jacket. The pH inside the SBRs was not actively controlled; however, the nutrient solution was adjusted to 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 using KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and NaHCO\u003csub\u003e3\u003c/sub\u003e. From day 240 onwards, SBR1 was equipped with a pH controller (42 Series, Chemitec, Italy) connected to two peristaltic pumps, which supplied HCl (0.5 M) or NaOH (0.5 M) to maintain the reactor at pH values around 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Inoculation and operational stages\u003c/h2\u003e \u003cp\u003eActivated sludge from the municipal wastewater treatment plant of Santiago de Compostela (Northwest Spain) was used as the inoculum for both SBRs, with a concentration of solids in the reactors of 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 g VSS/L (ratio VSS/TSS of 0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g/g).\u003c/p\u003e \u003cp\u003eSBR1 (withdrawal at the end of the feast phase) was operated for 500 days, divided into three stages (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The first stage (S-I, days 0-203) was operated with the addition of three UCO pulses, an increasing organic loading rate (OLR) in terms of chemical oxygen demand (COD) from 1.2 to 1.8 g tCOD/(L\u0026middot;d) and a fixed nitrogen loading rate (NLR) of 78.5 mg N/(L\u0026middot;d). In the second stage (S-II, days 204\u0026ndash;253), the OLR and NLR were maintained (1.8 g tCOD/(L\u0026middot;d) and 78.5 mg N/(L\u0026middot;d), respectively), but the UCO was added in a single pulse instead of three, that continued till the end of the operation. Finally, in S-III (days 254\u0026ndash;500), the OLR remained almost constant at 1.8\u0026ndash;1.9 g tCOD/(L\u0026middot;d) while the NLR was decreased and slightly modified between 36\u0026ndash;46 mg N/(L\u0026middot;d) to adjust its value and avoid the excess of nitrogen at the end of the famine phase.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of operational parameters and feeding strategies applied in SBR1 and SBR2 throughout the enrichment process.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eSBR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSBR2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS-I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS-II\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS-III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOperational days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026ndash;203\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e204\u0026ndash;253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e254\u0026ndash;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u0026ndash;188\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUCO pulses/cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUCO added (mL/pulse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026ndash;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.0\u0026ndash;4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOLR (g tCOD/(L\u0026middot;d))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2\u0026ndash;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.8\u0026ndash;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etCOD (mg/L) \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e590\u0026ndash;900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e900\u0026ndash;934\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e736\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLR (mg N/(L\u0026middot;d))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u0026ndash;46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (mg N/L) \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18\u0026ndash;23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOD/N (g/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u0026ndash;23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45\u0026ndash;52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Concentrations of UCO (as tCOD, feast phase) or nitrogen (famine phase) in the reactor just after feeding in each cycle.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e A control of pH was implemented in SBR1 at day 240.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eCOD: chemical oxygen demand; COD/N: COD fed at the feast phase divided by the nitrogen fed at the famine phase; NLR: nitrogen loading rate; OLR: organic loading rate; UCO: used cooking oil.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e), as well as the tCOD () and N () concentrations fed each cycle; (c) percentages of accumulated PHA () and fatty acids () at the end of the feast phase and COD/N ratio () fed through the operation. The different stages are defined with a pointed vertical line (\u003cb\u003e⁞\u003c/b\u003e).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSBR2 (withdrawal at the end of the famine phase) was operated for 188 days under conditions like S-III of SBR1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e): feeding with one pulse, OLR of 1.5 g tCOD/(L\u0026middot;d), and NLR of 32.6 mg N/(L\u0026middot;d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Feeding composition\u003c/h2\u003e \u003cp\u003eBoth SBRs were fed with: i) raw UCO as the carbon source at the beginning of the feast phase; ii) NH\u003csub\u003e4\u003c/sub\u003eCl solution as the nitrogen source at the beginning of the famine phase; iii) nutrient solution at the beginning of the famine phase (together with the nitrogen source) in SBR1 and at the beginning of the feast phase in SBR2.\u003c/p\u003e \u003cp\u003eThe raw UCO was collected from the university's hostelry service and used as the organic substrate without pretreatment. It was characterized by its content in fatty acids (93.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34%), organic matter (2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 g tCOD/g UCO) and elemental composition (CH\u003csub\u003e1.93\u003c/sub\u003eO\u003csub\u003e0.11\u003c/sub\u003eN\u003csub\u003e0.002\u003c/sub\u003e), see more details in Table S.2. Furthermore, an oxygen uptake rate test was performed with activated sludge to confirm the suitability of the raw UCO as the substrate for biological processes, obtaining a biodegradability value of 58.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%.\u003c/p\u003e \u003cp\u003eThe nutrient solution (pH 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5) comprised KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (0.3 g/L), MgSO\u003csub\u003e4\u003c/sub\u003e (0.0099 g/L), KCl (0.066 g/L), NaHCO\u003csub\u003e3\u003c/sub\u003e (0.30\u0026ndash;0.45 g/L), allylthiourea (0.0044 g/L) and a micronutrient solution (1 mL/L) described by Vishniac and Santer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, in SBR1, the nutrient solution contained NH\u003csub\u003e4\u003c/sub\u003eCl (0.3 g/L in S-I and S-II and 0.17 g/L in S-III). In SBR2, the NH\u003csub\u003e4\u003c/sub\u003eCl solution was added separately at the beginning of the famine phase and consisted of 100 mL of a solution of 1.25 g NH\u003csub\u003e4\u003c/sub\u003eCl/L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Sampling and analytical methods\u003c/h2\u003e \u003cp\u003eIn both SBRs, two samples per cycle were analysed (three times per week), collected at the end of the feast phase and at the end of the famine phases. All the samples were analysed in duplicate. To characterise the liquid fraction, the samples were centrifuged and then filtered using a 0.45 \u0026micro;m pore size, mixed cellulose ester membrane (Advantec, Japan). The soluble chemical oxygen demand (sCOD) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and total nitrogen (TN) concentrations (TOC-L analyser with the TNM-module, TOC-5000 Shimadzu, Japan) were determined in the liquid phase. Concentrations of total COD (tCOD) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], total suspended solids (TSS) and volatile suspended solids (VSS) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], as well as PHBV and fatty acids content were measured in the solid phase. The pH was measured with a pH \u0026amp; Ion-Meter model GLP 22 (Crison, Spain).\u003c/p\u003e \u003cp\u003eThe PHBV and fatty acids quantification was performed by gas chromatography with a flame ionization detector (GC-FID) equipped with a HP-INNOVAX column (Agilent, USA), using the method described by Fra-V\u0026aacute;zquez et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] slightly modified, drying the biomass at 50\u0026deg;C in an oven (Memmert BE300, Memmert, Germany) instead of freezing and lyophilizing it. Commercial standards of PHBV copolymer (3HB, 90.82 %, nd 3HV, 9.18 %),fatty acids (Palmitic, Stearic, Oleic, and Linoleic), and benzoic acid as an internal standard were used.\u003c/p\u003e \u003cp\u003eThe dissolved oxygen concentration was continuously measured inside the reactors every 5 min using a portable multimeter (HQ40d, Hach-Lange, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Calculations\u003c/h2\u003e \u003cp\u003eThe OLR (as g tCOD/(L\u0026middot;d)) and the NLR (as mg N/(L\u0026middot;d)) were calculated as the amount of UCO added, in terms of tCOD, or the amount of nitrogen added, as mg N, to the reactor per cycle, considering the number of cycles per day (2) and the volume of the reactor (Vr).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:OLR=\\frac{UCO\\:\\left(g\\:tCOD/cycle\\right)\\bullet\\:2\\:(cycles/day)}{Vr\\:\\left(L\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:NLR=\\frac{Nitrogen\\:\\left(mg\\:N/cycle\\right)\\bullet\\:2\\:(cycles/day)}{Vr\\:\\left(L\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe COD/N ratio was determined as the tCOD fed at the feast phase divided by the nitrogen fed at the famine phase. The active biomass (X) was determined at the end of the famine phase as the solid concentration (g VSS/L) minus the remaining concentration of bioproducts in the microbial cells (PHBV and fatty acids in g/L) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Microbiological analysis\u003c/h2\u003e \u003cp\u003eDNA was extracted from three independent replicated biomass samples taken from SBR1 using the FastDNA-2 mL SPIN Kit for Soil method, which was briefly modified, and the FastPrep24 instrument (MP-BIO, USA). The diversity of bacterial and fungal communities was characterized using Illumina sequencing with the primers Pro341F/Pro805R [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] for bacteria and FungiQuantF/FungiQuantR [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] for fungi. The operational taxonomic units (OTUs) were defined at a 97 % similrity threshold from the raw sequence data using the MothurMiSeq pipeline and Mothur software version 1.44.1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Taxonomic classification of \u003cem\u003eBacteria\u003c/em\u003e and \u003cem\u003eFungi\u003c/em\u003e was conducted utilizing the custom BLAST tool in Geneious version 2025.1.2 (Biomatters, New Zealand), comparing sequences against the NCBI bacterial 16S rRNA and fungal 18S rRNA databases [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA non-metric multidimensional scaling (NMS) analysis was used to link the structure of the bacterial and fungal communities to the operational parameters by means of the PC-ORD software (Wild Blueberry Media, Corvallis, OR, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe operation of SBR1 for 500 days, modifying the concentration of COD and N fed, as well as the number of raw UCO pulses added, allowed for determining the adequate operational conditions to increase the concentration of active biomass in a system with an MMC and raw UCO as substrate (without pretreatment). Furthermore, it was evaluated whether the withdrawal and filling with nutrients at the end of the feast phase (SBR1) or at the end of the famine phase (SBR2) influenced the degree of enrichment.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Dynamics of biomass concentration during the enrichment of the MMC\u003c/h2\u003e \u003cp\u003eIn SBR1, the VSS/TSS ratio consistently remained around 0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), which indicates that most of the solids were organic, although a minor inert fraction may still have been present. The active biomass concentration varied throughout the operation, being low during the first 125 days (S-I) with values ranging from 0.1 to 0.4 g VSS/L, while the bioproducts were only 1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 wt. % for PHBV and 10.41\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05 wt. % for fatty acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Then, when the fed COD concentration was increased from 590 to 900 mg tCOD/L, the active biomass concentration increased to 0.4\u0026ndash;0.6 g VSS/L between days 150 and 203 (S-I), and bioproducts accumulated, with fatty acids values up to 20 wt. % and PHBV peaks of 9 wt. % (day 182) and 20 wt. % (day 203). The increase in PHBV content after day 150 was likely associated with the progressive decrease in nitrogen concentration during the feast phase, as it was consumed for biomass growth during the famine phase [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring stages S-I and S-II, nitrogen was added at the beginning of the famine phase to have a concentration of 40 mg N/L (inside the reactor). However, it was not entirely consumed during the famine phase, and variable concentrations remained available for growth in the following feast phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Under these conditions, PHBV accumulation was not successful, as limiting conditions of this essential nutrient are necessary at the beginning of the feast phase to stimulate the accumulation of the carbon added as PHBV, while biomass growth is avoided [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAvoiding the presence of an excess of nitrogen during the bioproduct\u0026rsquo;s accumulation processes becomes very important [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For this reason, the nitrogen concentration fed was halved in stage S-III (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), to support active biomass growth while limiting it during PHBV accumulation. The increase in active biomass, as well as in bioproduct accumulation, with a notable peak in PHBV and fatty acids, was observed during this period (18.5 wt. % of PHBV on day 288 and 31 wt. % of fatty acids on day 268). The highest concentrations of active biomass were achieved between days 330 and 400 (S-III), reaching a maximum of 1 g VSS/L. This period corresponds to a COD feed of 900 mg tCOD/L and a nitrogen addition of 20 mg N/L that was highly consumed during the famine phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In this stage the remaining nitrogen at the end of the famine phase was too low to affect PHBV synthesis in the subsequent feast phase. This could be a contributing factor to the overall increase in bioproducts accumulation, reaffirming the need for nutrient limitation to achieve the accumulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom day 420 onwards, several minor adjustments to the nitrogen concentration were tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) to enhance PHBV accumulation; however, these changes were unsuccessful, and nitrogen was not fully consumed during the famine phase, remaining available at the beginning of the feast phase. Thus, lower active biomass concentrations were reached compared to the previous days. Therefore, towards the end of the operational period (days 400\u0026ndash;500, S-III), the active biomass decreases to approximately 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 g VSS/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This fact highlights the sensitivity of the enrichment with raw UCO as well as the importance of proper nitrogen concentration feeding.\u003c/p\u003e \u003cp\u003eThus, in SBR1, the increase in active biomass concentration was achieved first by increasing the COD concentration fed during the feast phase, and then by decreasing the nitrogen concentration to restrict its availability during the famine phase. The results showed that among the fed COD/N ratios assayed, in the range from 15 to 52 g/g, the most adequate to promote the increase of active biomass (up to 1 g VSS/L) was approximately 46 g tCOD/g N (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), equivalent, according to the UCO elemental composition, to approximately 13 g C/g N. This value falls within the range of other reported values obtained from similar enrichment processes (aerobic dynamic feeding) using no pre-fermented substrates such as crude glycerol (15.9 g C/g N) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] or UCO as the substrate (10 g C/g N) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, Zeng et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], using a mixture of volatile fatty acids (VFAs) as a carbon source, observed that C/N ratios in the range of 40\u0026ndash;80 g C/g N favored PHA accumulation at the expense of biomass growth, while lower ratios (5\u0026ndash;20 g C/g N) enhanced biomass proliferation but restricted PHA synthesis. Thus, the selection of the C/N ratio of the feeding will help to develop the MMC.\u003c/p\u003e \u003cp\u003eHowever, imposing the desired COD/N ratio requires a successful hydrolysis of the feed raw UCO, which may become a critical bottleneck. Inadequate hydrolysis restricts the availability of soluble substrates for microbial uptake, thereby limiting both biomass growth and PHBV accumulation. For this reason, the soluble COD in the liquid phase was monitored at the end of the feast phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). During the first 100 days, values remained relatively high (200\u0026ndash;400 mg sCOD/L), but as the active biomass concentration increased, they decreased to approximately 100 mg sCOD/L and then stabilized for the remainder of the operation. These results indicate that the MMC required approximately 100 days to establish a stable UCO hydrolysis that enables its consumption.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Feeding strategy: multiple or single pulses\u003c/h2\u003e \u003cp\u003ePulsed feeding strategies are reported to reduce substrate inhibition by the carbon source [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and promote high storage of PHA [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, considering the low solubility of UCO, this feeding strategy was imposed to facilitate the development of the hydrolysis process. Therefore, to avoid a possible inhibition by free fatty acids the operation of SBR1 was started by adding the raw UCO distributed in three pulses during the feast phase. During this stage (S-I) the dissolved oxygen (DO) concentration profile showed a marked decrease after each UCO feeding pulse (first pulse from 7 to 4 mg O\u003csub\u003e2\u003c/sub\u003e/L; second pulse from 6 to 2 mg O\u003csub\u003e2\u003c/sub\u003e/L; third pulse from 5 to 1 mg O\u003csub\u003e2\u003c/sub\u003e/L at day 126) followed by a subsequent recovery once the substrate was consumed (Fig. S.1a), indicating efficient substrate uptake and suggesting that the MMC maintained a good metabolic activity despite the potential inhibitory effects of free fatty acids contained in the raw UCO. Later in S-II, to enhance the UCO consumption and favour more stable PHBV accumulation, a single pulse feeding strategy was implemented from day 204 onwards (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), maintaining the same total raw UCO added (900 mg tCOD/L). This change was made to allow more time during the feast phase to promote the UCO hydrolysis and assimilation of the substrate. As shown in Fig. S.1b for day 225, the DO concentration decreased from almost 5 to 2 mg O\u003csub\u003e2\u003c/sub\u003e/L during the first hour after the addition of raw UCO. It rose again to 5 mg O\u003csub\u003e2\u003c/sub\u003e/L before the effluent withdrawal phase, confirming that the MMC was able to metabolize the raw UCO, even using a single pulse.\u003c/p\u003e \u003cp\u003eThe use of a single pulse seemed to be significant for PHBV accumulation, as the average percentage values achieved with 3 pulses were below 2 wt. %, while with a single pulse, the average accumulation increased progressively from 2 wt. % (day 231) to 12 wt. % (day 476), with a peak of 18.5 wt. % (day 288). Fatty acids content in the biomass (average of 15.6 wt. \u0026plusmn; 6.9%), generally higher than those of PHBV (average of 4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 wt. %), was also promoted by the shift in feeding strategy, achieving values of 31.6 wt. % on day 268.\u003c/p\u003e \u003cp\u003eIn contrast to other research works [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], which utilized waste fish oil, the present study obtained better results in terms of PHBV accumulation and biomass concentration using a single pulse feeding, and, in the studied operational conditions, confirmed the preference to accumulate fatty acids over PHBV when an oil is used as substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Uncoupled C and N: the challenge of pH control with oily substrates\u003c/h2\u003e \u003cp\u003eIt is known that uncoupled carbon and nitrogen feeding strategies favour the accumulation of bioproducts like PHA [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, it was observed that with oily substrates, the uncoupling strategy destabilizes the pH, and acidic conditions can be achieved inside the reactor [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In these conditions, pH control inside the biological reactor is essential. However, the addition of alkaline compounds to increase the pH can cause the saponification of the oil-based substrate, which can also lead to process failure. To manage these situations is therefore vital for the process.\u003c/p\u003e \u003cp\u003eIn the present study, SBR1 was initially operated without pH control. During the first 170 operational days, no problems of acidification were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The pH was likely maintained thanks to the buffering capacity of the nutrient solution (pH fixed at 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5), which contained KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and NaHCO\u003csub\u003e3\u003c/sub\u003e. Non-consumed ammonium in the initial period might also help maintain a pH stable. However, as nitrogen consumption improved, the pH inside the reactor at the end of the famine phase decreased to values of 3\u0026ndash;4 (days 170\u0026ndash;250), resulting in an acidic medium and subsequently causing microbial stress.\u003c/p\u003e \u003cp\u003eAcidic conditions have been reported to cause different effects on PHA accumulation. Some authors have reported that low pH values (\u0026lt;\u0026thinsp;4) can inhibit active biomass, ultimately leading to process failure [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Nevertheless, other studies suggest that stress conditions in short time periods, such as low pH, may promote the accumulation of PHA, as microorganisms may utilize this polymer for stress resistance [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This factor may contribute to the observed higher PHBV accumulations on days 182 and 203 (8.9 and 19.9 wt. %, respectively). However, although this pH stress may be beneficial for short-term PHA accumulation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], previous studies have reported that pH values of 7.0 or even 8.5 are optimal for PHA synthesis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Prolonged exposure to low pH levels, around 3\u0026ndash;4, can reduce or even inhibit the growth of MMC as bacteria typically thrive in a more neutral pH environment. Extended periods of stress can reduce or even inhibit the growth of the culture accumulating PHA, leading to a decrease of the PHA produced [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], as evidenced by the decrease in biomass concentration due to the inability of many microorganisms to survive under such conditions.\u003c/p\u003e \u003cp\u003eTo mitigate this issue, continuous pH control (7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3) was implemented in SBR1 from day 240 onwards. After this implementation, the active biomass concentration increased again (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe pH value was set at 7.0 to provide suitable conditions for both oil hydrolysis and bacterial growth [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], as well as for the enrichment of MMCs for PHA production [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This value was also selected to minimize NaOH consumption during pH control and to prevent UCO saponification under alkaline conditions. Although enrichment and PHA accumulation with readily biodegradable substrates such as VFAs are typically carried out under neutral to slightly alkaline conditions [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], the use of lipid-based wastes like UCO requires an additional hydrolysis step to convert triglycerides into free fatty acids prior to microbial uptake. Since lipase activity is generally favored under mildly acidic to neutral conditions [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], maintaining a pH around 7.0 represented an appropriate compromise between promoting UCO hydrolysis and ensuring stable microbial activity for PHA accumulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Reactor configuration: effluent withdrawal and nutrient filling\u003c/h2\u003e \u003cp\u003eSBR2 was operated similarly to SBR1 in stage S-III, with the feeding added as a single pulse and a COD/N ratio of 46 g/g, but with a modification in the cycle configuration and reactor volume (from 10 to 2 L). The withdrawal and refilling with a nutrient solution in SBR1 occurred during the transition from feast to famine phase, whereas in SBR2, it took place in the transition from famine to feast phase. The SBR1 configuration enables the reactor to act as an enrichment and accumulation unit, as withdrawal occurs immediately after accumulation is complete. In contrast, the SBR2 configuration corresponds to an enrichment reactor, and a subsequent accumulation unit will be necessary because effluent withdrawal occurs at the end of the famine phase, when the bioproducts inside the biomass have already been consumed. Furthermore, in SBR2, the feast/famine lengths were slightly different, 5/7 h/h between days 0 and 94; thereafter, the same values as SBR1 (6/6 h/h) were used until the end of the operation (days 95\u0026ndash;188).\u003c/p\u003e \u003cp\u003eRegarding the active biomass in SBR2 (Fig.\u0026nbsp;2a), the higher values were obtained with a feast/famine length of 5/7 h/h, achieving average values of 0.6\u0026ndash;0.8 g VSS/L at the end of the famine phase. Increasing the feast phase to 6 hours (6/6 h/h), without modifying the amount of raw UCO added, decreased the active biomass concentration to values of 0.4 g VSS/L. On the other hand, extending the length of the feast phase was beneficial to maintaining the pH value inside the reactor above neutral values.\u003c/p\u003e \u003cp\u003eRegarding the soluble COD values, those at the end of the feast phase in SBR2 were consistently below 100 mg sCOD/L after the first 30 days (Fig.\u0026nbsp;2b), while it took about 100 days in SBR1 to reach similar values. This could indicate that starting the SBR operation with an optimised COD/N ratio (46 g/g in SBR2) can help to balance the hydrolysis and the consumption of the raw UCO in a shorter time.\u003c/p\u003e \u003cp\u003eThe nitrogen concentration at the end of the famine phase was below 10 mg N/L only on isolated days (Fig.\u0026nbsp;2b), indicating limited nitrogen consumption for biomass growth, likely due to the low bioproduct synthesis during the feast phase, which is used as the carbon source (Fig.\u0026nbsp;2c). Similar to SBR1, the SBR2 configuration resulted in a preferential accumulation of fatty acids over PHBV (Fig.\u0026nbsp;2c). But, if the maximum percentages of accumulation in SBR1 and SBR2 are compared for fatty acids (31.6 and 37.4 wt. %, respectively) and PHBV (19.9 and 5.2 wt. %, respectively), it can be concluded that the SBR1 configuration (withdrawal at the end of the feast phase) is preferable to promote PHBV as a bioproduct. Moreover, in both SBRs, the PHBV copolymer showed variations in the 3HB and 3HV proportions associated with the operational changes, suggesting that the monomeric composition was not determined by the withdrawal configuration (Fig. S.2). The presence of 3HV content is advantageous, as it yields a copolymer with lower crystallinity and melting temperature, improving flexibility and ease of processing [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Reactor populations: microbiology analysis\u003c/h2\u003e \u003cp\u003eThe dominant operational taxonomic units (OTUs) for bacteria were OtuB00001 (\u003cem\u003eBurkholderia\u003c/em\u003e, 15.96%), OtuB00002 (\u003cem\u003eCurvibacter\u003c/em\u003e, 5.65%), and OtuB00003 (\u003cem\u003eKryptousia\u003c/em\u003e, 3.03%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, there were significant shifts in the dominant OTUs throughout the operational periods, reflecting a high degree of dynamics within the MMC. Therefore, the imposed operational conditions had a strong capacity to modulate the bacterial community structure over time. Nevertheless, despite the instability of the bacterial population structure throughout the operation of SBR1, the dominant OTUs observed for the majority of the sampling times were described as PHA-accumulating bacteria, mainly OtuB00008 (\u003cem\u003eAzospirillum)\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] at day 35, OtuB00004 (\u003cem\u003eAcinetobacter\u003c/em\u003e) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] at days 112 and 154, OtuB00007 (\u003cem\u003eGordonia\u003c/em\u003e) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] at day 154, OtuB00001 (\u003cem\u003eBurkholderia\u003c/em\u003e) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] at days 182 and 204, OtuB00009 (\u003cem\u003eBacillus\u003c/em\u003e) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] at day 457. This broad dominance of PHA-accumulating bacteria underscores the importance of high functional redundancy within an MMC, enabling it to withstand the various stresses encountered during operation. Interestingly, the bacterial community was dominated by the OTUB001 taxonomically affiliated with the well-known PHA-accumulating \u003cem\u003eBurkholderia\u003c/em\u003e, with a maximum relative abundance of 87% at day 182. In relation to this, the maximum PHBV accumulated percentage in the biomass was only achieved when the relative abundance of this genus was coupled with that of the highly hydrolytic genus \u003cem\u003eChitinophaga\u003c/em\u003e (maximum relative abundances of 16% at day 204). Thus, since both genera seemed to act synergistically in the accumulation of PHBV, it could be hypothesised that \u003cem\u003eChitinophaga\u003c/em\u003e transformed the fats in this residue into free fatty acids through the action of its lipases, and \u003cem\u003eBurkholderia\u003c/em\u003e finally metabolised these fats into PHBV by the PhaC enzyme, the key enzyme in the PHA biosynthesis. Regarding the decrease in pH in SBR1, it does not appear to hinder the development of PHA-accumulating bacteria, as a selective increase in the community was observed on days 182 and 204, resulting in the great abundance of the OTU taxonomically classified as \u003cem\u003eBurkholderia\u003c/em\u003e. It should be noted that some strains of \u003cem\u003eBurkholderia\u003c/em\u003e are acidophiles, capable of surviving under pH values lower than 7 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], as is the case for those found at days 170\u0026ndash;250, resulting in a significant promotion of the growth of this bacterial genus. Nevertheless, as hypothesised, the establishment of a pH control on day 240 onwards did not result in the stabilisation of bacterial populations within the MMC, as a high degree of dynamics was also observed during this period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the fungal populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), they were predominantly dominated by OtuF00001 (\u003cem\u003eGeotrichum\u003c/em\u003e, 45.31%), followed by OtuF00002 (\u003cem\u003eMeyerozyma\u003c/em\u003e, 7.66%) and OtuF00003 (\u003cem\u003ePascua\u003c/em\u003e, 7.57%). Generally considered, the structures of the fungal communities were more stable than those of the bacterial communities, especially after day 182, when the community was predominantly dominated by OtuF00001 (\u003cem\u003eGeotrichum\u003c/em\u003e), achieving a maximal relative abundance of 99.06%. This dominance pattern can be attributed to the acid tolerance of this genus [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], which allows its growth once the medium becomes more acidic due to improved nitrogen consumption (days 170\u0026ndash;250). This fungus exhibits important antifungal activity, mediated by the production of volatile organic compounds, particularly phenylethyl alcohol, which inhibits fungal growth through various pathways, including disruption of the cell membrane, intracellular leakage, and impairment of energy metabolism and defence systems [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Finally, it should be noted that \u003cem\u003eGeotrichum\u003c/em\u003e is an oleaginous yeast [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] which, after becoming dominant, could have been responsible for the significant content of fatty acids in the biomass of SBR1.\u003c/p\u003e \u003cp\u003eThe multivariate statistical analysis carried out revealed that the main operational parameter promoting PHBV accumulation was the tCOD fed (raw UCO), suggesting that a higher organic loading rate, and subsequently a rise in carbon availability, increased the PHBV accumulation capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The COD/N ratio fed in each cycle was related to higher PHBV accumulations, and, on the other hand, higher N in the reaction medium at the end of the famine phase was inversely associated to larger PHA yields, suggesting that an increase in the carbon supply and an effective reduction in the nitrogen availability is mandatory to enhance the PHBV accumulation, confirming the previously defined conditions for an effective PHA accumulation from lipidic wastes by MMCs [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In addition, the NMS confirmed that lower pH values in the reaction medium at the end of the famine phase for a short period of time stimulated PHBV biosynthesis, as it was experimentally observed on days 170\u0026ndash;250, according to the protective role of PHA in bacterial fitness against various environmental stressors [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe NMS also shows that higher PHBV accumulation yields were correlated with higher relative abundances of \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eChitinophaga\u003c/em\u003e, endorsing that a synergistic action of both genera is essential for the successful valorisation of raw UCO into bioplastic precursors. To confirm this close relationship, the Spearman rank analysis was also made (Table S.3). In this regard, higher relative abundances of \u003cem\u003eBurkholderia\u003c/em\u003e were strongly positively related to a rise of \u003cem\u003eChitinophaga\u003c/em\u003e (ρ\u0026thinsp;=\u0026thinsp;0.834), which are also correlated with higher PHBV accumulation capacities (ρ\u0026thinsp;=\u0026thinsp;0.624 for \u003cem\u003eBurkholderia\u003c/em\u003e and ρ\u0026thinsp;=\u0026thinsp;0.803 for \u003cem\u003eChitinophaga\u003c/em\u003e), reinforcing the synergistic role of both bacteria in the PHBV accumulation. Considering that this synergetic association is first described here, the role of both bacteria should be explored to address the potential biotechnological use of these two genera as a promising alternative to enhance PHBV accumulation for the valorisation of raw UCO. In this regard, co-culture selection and growth of both genera could be based on the phthalic acid resistance of \u003cem\u003eChitinophaga\u003c/em\u003e, as described by Lee et al. [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] and the tolerance of \u003cem\u003eBurkholderia\u003c/em\u003e to this compound, as previously reported by Zhang et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe vectors representing PHBV and fatty acids contents in the biomass are spatially close. In this regard, \u003cem\u003eGeotrichum\u003c/em\u003e (OtuF00001 and OtuF00004) has been reported to be capable of biosynthesizing enzymes, such as cellulases, α-amylases, proteases, lipases, β-glucanases, xylanases, and phytases [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], which could enhance the metabolization of raw UCO, allowing a simultaneous increase in the PHBV and fatty acids contents in the biomass. Based on the high hydrolytic capacities of \u003cem\u003eGeotrichum\u003c/em\u003e, the Spearman correlation analysis (Table S.3) suggests a cooperative role in PHBV accumulation, due to the strong correlation between PHBV accumulation and the relative abundances of this fungus (ρ\u0026thinsp;=\u0026thinsp;0.574). Hence, the operational factor for the simultaneous enrichment of \u003cem\u003eGeotrichum\u003c/em\u003e, \u003cem\u003eBurkholderia\u003c/em\u003e, and \u003cem\u003eChitinophaga\u003c/em\u003e needs to be thoroughly explored as an alternative for selecting a microbial community of Bacteria and Fungi that synergistically interact in the valorisation of raw UCO into PHBV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Challenges of the PHA production process based on MMC and raw UCO\u003c/h2\u003e \u003cp\u003eDespite the growing interest in replacing conventional fossil-based plastics with PHAs, their large-scale commercialization remains constrained by high production costs, which still limit their competitiveness. Several techno-economic analyses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR67 CR68\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] have shown that the use of MMCs can substantially reduce both capital and operational costs by (i) eliminating sterilization requirements, (ii) enabling the use of low-cost waste substrates, and (iii) integrating PHA production into waste treatment and management systems.\u003c/p\u003e \u003cp\u003eUsing UCO and MMCs accomplishes these requirements. However, the use of MMCs for PHA production with UCO presents several challenges, including substrate complexity, conversion efficiency, and process stability. The poor solubility of UCO requires an effective hydrolysis step to release free fatty acids, which can inhibit microbial activity and compromise reactor performance. Consequently, the enrichment of PHA-storing microorganisms in MMCs fed with lipid-based substrates is typically slower and less stable than with readily biodegradable carbon sources such as VFAs. Nevertheless, the present study demonstrated that this enrichment is achievable using raw UCO without any type of pretreatment. Starting from conventional activated sludge and applying strategies like ADF and DGL, the operational conditions of the enrichment reactor (SBR1) were progressively optimized, achieving notable PHBV contents at the end of the feast phase (9 wt. % on day 182; 20 wt. % on day 203; 18.5 wt. % on day 288; and 12 wt. % on day 476), with 3HB identified as the main constituent, prevailing over 3HV in these days (Fig. S.2). The results obtained in this study, after 500 days of SBR1 operation, confirm that the enrichment of PHA-accumulating MMC with raw UCO is feasible, with key microbial populations identified as \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eChitinophaga\u003c/em\u003e among bacteria, and \u003cem\u003eGeotrichum\u003c/em\u003e among fungi.\u003c/p\u003e \u003cp\u003eIn contrast, Tamang and Nogueira [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] reported that direct enrichment using raw UCO was not feasible due to the proliferation of filamentous bacteria, leading to foaming and bulking. In their study, an MMC previously enriched with nonanoic acid achieved a maximum PHA content of 38.2 wt. % from raw UCO at 40\u0026deg;C, but only in a dedicated accumulation reactor. Even with pure cultures, literature reviews [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] report a wide range of PHA contents (36\u0026ndash;75 wt. %) when using oil-rich wastes, indicating that achieving high intracellular accumulation is not always straightforward, even with specific strains and optimized conditions.\u003c/p\u003e \u003cp\u003eIn the present research work, the PHBV content reached in SBR1, although significant for an open culture directly fed with raw UCO, was neither stable nor sufficiently high to enable efficient extraction, which typically requires intracellular contents above 30\u0026ndash;40 wt. % in MMCs [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. This suggests that, although enrichment was achieved, further optimization of the accumulation process is still necessary, either by improving the operational conditions within the same reactor (for example, by extending the feast phase) or by coupling the enrichment stage with a dedicated accumulation unit.\u003c/p\u003e \u003cp\u003eRegarding substrate variability, although the composition of raw UCO may differ depending on the type and frequency of use, the UCO used in a potential PHA production process would be supplied by companies specialized in its collection and management. As a result, the UCO composition within a given region tends to be relatively consistent, resulting in reduced variability compared with other complex wastes that require prior fermentation to VFAs. Furthermore, the existing collection and standardization systems established for biodiesel production represent a clear advantage. Additionally, the UCO used for PHA synthesis does not require the extensive pretreatment steps typically applied for biodiesel feedstock preparation (e.g., water or solids removal). Therefore, as in the present study, pretreatment can be omitted or limited to the simple removal of coarse solids.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe increase of the active biomass concentration in the enrichment reactors was successfully achieved through a progressive rise in the organic loading rate, fed as raw UCO, followed by a nitrogen limitation. A COD/N ratio around 46 g/g allowed to maintain an active biomass concentration up to 1 g VSS/L. Furthermore, when a single UCO pulse was provided, the biomass exhibits better performance in synthetizing PHBV (12 wt. %) compared to the three-pulse strategy (\u0026lt;\u0026thinsp;2 wt. %), which was attributed to better substrate hydrolysis and assimilation. The SBR configuration with the withdrawal and filling with nutrients at the end of the feast phase (SBR1) and 10 L of working volume, consistently reached better PHBV accumulation results (average values of 4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 wt. %, maximum of 19.9 wt. %) than the configuration with the withdrawal and filling with nutrients at the end of the famine phase and 2 L of working volume (SBR2, average values of 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 wt. %, maximum of 5.2 wt. %). However, although a maximum accumulation of 19.9 wt. % for PHBV and 31.6 wt. % for fatty acids were reached in SBR1, process operation under these conditions was unstable, and high storage levels could not be maintained over time.\u003c/p\u003e \u003cp\u003eProcess pH was demonstrated to be a key parameter for system operation. A transient pH decrease enhanced PHBV production due to its stress resistance function, whereas prolonged exposure to acidic conditions negatively affected microbial activity and bioproducts synthesis.\u003c/p\u003e \u003cp\u003eMicrobial community analysis revealed a strong successional dynamic of PHA-accumulating bacterial populations, primarily including \u003cem\u003eAzospirillum\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003eGordonia\u003c/em\u003e, \u003cem\u003eBurkholderia\u003c/em\u003e, and \u003cem\u003eBacillus.\u003c/em\u003e The high degree of functional redundancy within the MMC enabled resilience to operational and environmental changes, while multivariate analysis confirmed that synergistic interactions between \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eChitinophaga\u003c/em\u003e were essential for the successful valorisation of UCO into PHBV.\u003c/p\u003e \u003cp\u003eOverall, the present study demonstrates the feasibility of producing PHBV from raw UCO using MMC, while underling the role of the operational strategy, feeding mode, and microbial interactions. Future work should focus on optimize this process stability, targeting larger active biomass concentrations and higher percentages of accumulated PHBV to support the scale-up and implementation of this method for lipid-rich waste valorisation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.U. made the writing of the original draft; C.U. and D.C.G. made the investigation and formal analysis; C.U., D.C.G and A.V. made the visualization of the data; C.U., D.C.G, A.V. and A.M.C made the methodology; A. V., A.P and A.M.C made the conceptualization; A. V. and A.M.C made the supervision and validation; A.M.C made the project administration and funding acquisition; All authors made the review and editing of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by the Spanish Government (AEI) through the ECOPOLYVER project [MACROPOLYVER PID2020-112550RB-C21/ AEI / 10.13039/501100011033 \u0026amp; MICROPOLYVER PID2020-112550RB-C22/ AEI / 10.13039/501100011033] and the project POLYGO1 [TED2021-130164B-I00 / AEI / 10.13039 / 501100011033 / Uni\u0026oacute;n Europea NextGenerationEU / PRTR]. Carlota Ucha, \u0026Aacute;ngeles Val del R\u0026iacute;o, Alba Pedrouso and Anuska Mosquera-Corral belong to a Galician Competitive Research Group (GRC ED431C 2025/19) and CRETUS Research Centre (ED431G 2023/12).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are not openly available but are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eValentin HE, Broyles DL, Casagrande LA et al (1999) PHA production, from bacteria to plants\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSatoh H, Mino T, Matsuo T (1999) PHA production by activated sludge\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen Q, Chen Z, Tian T, Chen W (2010) Effects of phosphorus and nitrogen limitation on PHA production in activated sludge. 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Front Bioeng Biotechnol 9:624021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/FBIOE.2021.624021/FULL\u003c/span\u003e\u003cspan address=\"10.3389/FBIOE.2021.624021/FULL\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"COD/N ratio, fatty acids, pulsed feeding, waste valorisation, withdrawal strategy.","lastPublishedDoi":"10.21203/rs.3.rs-8617392/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8617392/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe synthesis of polyhydroxyalkanoates (PHA) by a mixed microbial culture (MMC) employing used cooking oil (UCO) as substrate represent a promising bioprocess for organic wastes valorisation. In the present research work, the effect of sequencing batch reactor (SBR) operational strategy on MMC enrichment and PHA accumulation was investigated using UCO without pretreatment as the sole carbon source. Two enrichment SBRs (SBR1 and SBR2) were operated under a feast-famine regime with uncoupled carbon and nitrogen supply, differing in the timing of the biomass withdrawal and nutrient filling. In SBR1, with effluent withdrawal at the end of the feast phase, better poly(3-R-hydroxybutyrate-co-3-R-hydroxyvalerate) (PHBV) accumulation was achieved (average values of 4.2 ± 1.6 wt. %, maximum of 19.9 wt. %) compared to SBR2, with withdrawal at the end of the famine phase (average values of 2.7 ± 1.1 wt. %, maximum of 5.2 wt. %). The highest PHBV accumulation in SBR1 was obtained at a COD/N ratio of 46 g/g, with an active biomass concentration of approximately 1 g VSS/L, highlighting the importance of feeding composition as a key operational control parameter. Additionally, a single UCO pulse resulted in more efficient substrate hydrolysis and consumption than multiple pulses in both systems. Microbial community analysis revealed the enrichment of bacterial OTUs related to \u003cem\u003eAzospirillum\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003eGordonia\u003c/em\u003e, \u003cem\u003eBurkholderia\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e, as well as fungal OTUs such as \u003cem\u003eGeotrichum\u003c/em\u003e, \u003cem\u003eMeyerozyma\u003c/em\u003e, and \u003cem\u003ePascua\u003c/em\u003e. Multivariate statistical analysis indicated that the synergistic interaction of \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eChitinophaga\u003c/em\u003e played a crucial role in achieving the highest PHA accumulation levels.\u003c/p\u003e","manuscriptTitle":"Enrichment of a mixed microbial culture for PHA production with used cooking oil as substrate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-10 20:05:10","doi":"10.21203/rs.3.rs-8617392/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-04T16:58:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T16:52:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T09:49:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149218189348571543030875335342653696623","date":"2026-02-09T06:11:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168187989795770130082147981087326305689","date":"2026-02-08T15:46:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142913552276279908344282623586634168231","date":"2026-02-07T09:41:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269853991284246077177995326782987167301","date":"2026-02-07T08:18:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T04:17:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-06T06:27:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165347528794905628605102512041562598682","date":"2026-02-06T05:12:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126892537607771814008488874105373385605","date":"2026-02-05T10:49:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-05T10:25:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T00:30:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T22:33:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioprocess and Biosystems Engineering","date":"2026-01-16T09:08:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9831cc98-d66a-4757-8b3b-116ce7339d36","owner":[],"postedDate":"February 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T08:39:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-10 20:05:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8617392","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8617392","identity":"rs-8617392","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}