Optimization of an enriched mixed culture to increase PHA accumulation using industrial saline complex wastewater as a substrate

Optimization of an enriched mixed culture to increase PHA accumulation using industrial saline complex wastewater as a substrate Lucía Argiz, Andrea Fra-Vázquez, Ángeles Val del Río, Anuska Mosquera-Corral Accepted Manuscript How to cite: Chemosphere, Volume 247, May 2020, 125873 Doi: 10.1016/j.chemosphere.2020.125873 Copyright information: © 2020 Elsevier Ltd. This manuscript version is made available under the CC-BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by-nc-nd/4.0) Optimization of an enriched mixed culture to increase PHA accumulation using industrial saline complex wastewater as a substrate Lucia Argiz*, Andrea Fra-Vazquez, Angeles Val del Rio and Anuska Mosquera-Corral School of Engineering, Department of Chemical Engineering, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain. * Corresponding author. Tel.: +34 881816784. E-mail address: luciaargiz.m[email protected] Effluent Settling Reaction Feeding Reaction Influent Cooked mussel processing WW NOVELTY Polyhydroxyalkanoates (PHA) production –Enrichment stage CONSEQUENCES OF SETTLING ▪↑YPHA from 0.48 to 0.72 Cmmol PHA/Cmmol VFA ▪↑% máx. PHA accum.from 40 to 60 wt% ▪≠MMC composition ▪≠Biopolymer properties Proteins, carbohydrates Refilling Supernatant Discharge Non-storing bacteria ↓Comammonas ↑Paraccocus 0.8 –1.5 gCOD/L·d 15 min 165 min 20 min 495 min 15 min 5 min 5 min Withdrawal Highlights - Complex saline industrial wastewater as a substrate for PHA production. - A settling stage was included in the enrichment cycle after VFA consumption. - Proteins and carbohydrates promoting growth instead of PHA production were removed. - The PHA production yield increased from 0.48 to 0.72 CmmolPHA / CmmolVFA. - The maximum PHA storage capacity of the MMC improved from 40 to 60 wt%. *Highlights (3 to 5 bullet points (maximum 85 characters including spaces per bullet point) 1 Optimization of an enriched mixed culture to increase PHA 1 accumulation using industrial saline complex wastewater as a 2 substrate 3 Lucia Argiz*, Andrea Fra-Vazquez, Angeles Val del Rio and Anuska Mosquera-Corral 4 School of Engineering, Department of Chemical Engineering, Universidade de Santiago de 5 Compostela, 15782 Santiago de Compostela, Galicia, Spain. 6 * Corresponding author. Tel.: +34 881816784. E-mail address: luciaargiz.m[email protected]7 8 ABSTRACT 9 Polyhydroxyalkanoates (PHA) appear as good candidates to substitute conventional petroleum-based 10 plastics since they have similar properties but with the advantage of being biodegradable. Wastewater 11 streams with high organic content are feasible substrates for PHA production resulting in an opportunity 12 for waste recovery. One of the main challenges is the optimization of the selection of microorganisms 13 with high PHA storage capacity. This microbial selection is performed in sequencing batch reactors 14 (SBR) operated under an aerobic feast/famine (F/F) regime. In the present study, a settling stage was 15 added at the end of the feast phase of the enrichment cycle of a SBR fed with pre-acidified cooked mussel 16 processing wastewater (containing up to 12 g NaCl/L). Settling and subsequent supernatant discharge 17 favoured the wash-out of non-accumulating microorganisms as well as the removal of substances that 18 enhanced their undesired development (proteins and carbohydrates). Microbial analysis performed by 19 fluorescence in situ hybridization (FISH) technique showed shifts in the microbial community; the 20 presence of genus Paracoccus increased whereas genera Comamonas decreased. Moreover, the process 21 efficiency was improved with the increase of the PHA production yield (YPHA) and the maximum PHA 22 storage capacity (max. PHA) from 0.48 to 0.72 CmmolPHA / CmmolVFA and from 40 to 60 wt%, 23 respectively. The polymer composition also changed, its HB:HV ratio varied from 83:17 to 70:30. Results 24 obtained in the present study showed that settling promoted the removal of carbon sources that did not 25 contribute to PHA production and the washout of non-storing bacteria, which favoured the culture 26 enrichment. 27 28 Keywords: bioplastics; enrichment; industrial wastewater, mixed microbial culture; 29 polyhydroxyalkanoates; valorization. 30 31 32 33 2 1. INTRODUCTION34 Plastics are polymers that are present in nearly all aspects of modern life. The great 35 majority are petroleum-based products, durable in use but no biodegradable (Zhu et al., 36 2013). This fact, in addition to petroleum depletion, has derived in a growing interest in 37 the development of more sustainable alternatives. Polyhydroxyalkanoates (PHA) are a 38 family of biobased, biodegradable and biocompatible linear polyesters (Keshavarz and 39 Roy, 2010) that have been recognized as good candidates to substitute conventional 40 plastics due to their similar properties (Dias et al., 2006). These polymers are produced 41 by certain microorganisms and accumulated as intracellular carbon sources under stress 42 conditions (Reddy et al., 2003). Nowadays, large scale PHA production is based on the 43 use of pure cultures or genetically modified microorganisms that require sterile 44 conditions and highly costly specific substrates (Jiang et al., 2016). Consequently, PHA 45 are not yet competitive in bulk materials markets (Albuquerque et al., 2010). Its current 46 price ranges from 2.2 – 5.0 €/kg, which is more than one-third of the cost reported from 47 the beginning of the last decade but still high in comparison to petroleum-based 48 polymers, which typically cost less than 1.0 €/kg (Valentino et al., 2017). In recent 49 years, research has been focused on the development of cost-effective processes, which 50 involve the use of low-value substrates and mixed microbial cultures that do not need 51 sterile conditions (Albuquerque et al., 2011) and allow the production of high quality 52 biopolymers with a purity (> 98 %) comparable to those obtained using pure cultures 53 (Samorì et al., 2015). 54 Wastewater streams produced in the agro-alimentary sector are among the most suitable 55 substrates for PHA production due to their high organic matter content (Nikodinovic-56 Runic et al., 2013). This is the case of fish-canning industries, which generate large 57 volumes of effluents with high pollutant load. In this way, wastewater valorization for 58 3 PHA production contributes to the transition to a circular economy model: on one hand, 59 organic matter is recovered and converted into resources and on the other hand, 60 pollutants are removed and treated wastewater can be discharged to natural water 61 bodies. In this way, the concept of wastewater treatment plant (WWTP) moves to a 62 water resource recovery facility (WRRF). 63 The PHA production system with a mixed microbial culture (MMC) using industrial 64 wastewater as substrate generally consists on a three-stage process: (1) substrate pre-65 acidification (if necessary) to obtain a suitable carbon source for PHA production, 66 primarily volatile fatty acids (VFA); (2) selection and enrichment of the MMC in PHA-67 accumulating organisms; and (3) maximization of the PHA storage before biopolymer 68 extraction and purification. The main challenge of this PHA production system is the 69 selection of a MMC with high PHA-storage capacity (Albuquerque et al., 2010). One of 70 the most common strategies to achieve the microbial enrichment is the application of 71 cycles of presence/absence of substrate known as feast/famine regime (F/F) or aerobic 72 dynamic feeding (ADF), operated in sequencing batch reactors (SBR) (Albuquerque et 73 al., 2007). In the last decade, research has been focused on the development of new 74 strategies for the optimization of the culture enrichment. It has been implemented 75 variations such as nitrogen limitation in the feast phase and/or the integration of an 76 intermediate settling phase in the SBR cycle (Kourmentza et al., 2017). 77 Nitrogen deficiency during the feast phase restricts the growth of non-PHA 78 accumulating bacteria while providing nitrogen during the famine enables their growth, 79 which allows faster selection of a more efficient PHA-storing culture (Ahmadi et al., 80 2018; Oliveira et al., 2017). For example, Marang et al. (2014) observed that the 81 presence of methanol and acetate promoted the development of non-accumulating 82 microorganisms in the MMC. To solve this problem, Korkakaki et al. (2016) proposed 83 4 the addition of a settling phase after the acetate depletion in order to eliminate the 84 methanol with the supernatant and promote the enrichment of the MMC by limiting the 85 growth of non-accumulating bacteria. As a result, they observed a considerable increase 86 in the maximum PHA storage capacity of the biomass from 48 wt% to 70 wt%. 87 Supernatant discharge just after acetate depletion avoided the consumption of the 88 remaining organic matter (methanol) and the consequent growth of side-populations 89 increasing the proportion of PHA-producers in the mixed culture (Kourmentza et al., 90 2017). Therefore, this modification of the SBR enrichment cycle can be considered as a 91 good solution to improve the microbial selection when there is more than one carbon 92 source in the substrate. However, until now few studies have explored this alternative 93 with industrial complex wastewater. 94 The objective of the present study was to improve the selection of PHA-accumulating 95 bacteria in a MMC using pre-acidified cooked mussel processing wastewater (with 4 - 96 12 g NaCl/L and high VFA content) as feedstock. To achieve this goal, a settling stage 97 was implemented in the operational cycle of an enrichment SBR working under F/F 98 regime with the aim of removing undesired substances (mainly proteins and 99 carbohydrates) that promoted the growth of non-accumulating bacteria. The enrichment 100 of the system was evaluated in terms of maximum PHA production and shifts in the 101 microbial community comprising the mixed culture. 102 2. MATERIALS AND METHODS 103 2.1 Experimental set-up 104 For the optimization of the enrichment unit and the consequent evaluation of maximum 105 PHA accumulation of the system, two lab-scale reactors were operated: an enrichment 106 reactor (SBR type) and an accumulation fed-batch reactor (FBR), respectively. 107 5 2.1.1 Enrichment SBR for culture selection 108 A tubular glass SBR with a working volume of 2 L (SBR-S) was inoculated with PHA-109 accumulating enriched biomass (0.80 g VSS/L) adapted to saline conditions from other 110 SBR (SBR-I). The SBR-I was operated under the F/F regime treating pre-acidified 111 wastewater collected from a cooked mussel processing industry. This reactor operated 112 in cycles of 12 hours consisting of 15 min of feeding, 690 min of aerobic reaction and 113 15 min of effluent withdrawal and was completely aerated (Figure S1.A of 114 Supplementary Material). The SBR-S operation was modified with respect to SBR-I by 115 means of including a settling stage at the end of the feast phase. Therefore, the 116 operational cycles of the SBR-S comprised the following stages: 1) 0.8 L of feeding (15 117 min), 2) aerobic reaction (165 min), 3) settling (20 min), 4) 1 L of supernatant 118 discharge (5 min), 5) reactor refilling with 0.8 L of the previous cycle effluent 119 (recirculation) and 0.2 L of tap water (5 min), 6) aerobic reaction, (495 min) and 7) 0.8 120 L of effluent withdrawal, (15 min). Specific details about the cycle distribution can be 121 consulted in the Figure S1. B. 122 Aeration was supplied during all stages, except for settling and supernatant discharge, 123 through a diffuser located at the bottom of the reactor that granted the complete mixture 124 of the system. The temperature was controlled at 30 ˚C by a thermostatic bath (Techne 125 Inc., USA) and the pH was not controlled but periodically measured at the end of the 126 famine phase. A detailed scheme of the SBR-S can be seen in Figure S2 of 127 Supplementary Material. 128 The length of the aerobic reaction before the settling phase was established by the 129 duration of the feast phase, and stopped when the VFA had just been depleted 130 (Korkakaki et al., 2016). Since during the operation of the SBR-I, reactor where the 131 inoculum was obtained, the feast phase lasted from 2.5 to 3.0 h, the settling stage in the 132 12 During the first operational days, the VSS concentration inside the SBR-S and end of 261 the cycle (min 705) decreased while its concentration increased in the supernatant 262 discharge (min 200) (Figure 1.B). Then, when the feast length was stabilized, the VSS 263 concentrations were maintained at values of 0.66 ± 0.05 g/L and 0.23 ± 0.04 g/L in the 264 reactor (measurements performed at the end of the cycle) and in the supernatant, 265 respectively. Therefore, not only non-accumulating microorganisms were removed from 266 the system at the end of the cycle at min 705 (wash-out at the end of the famine phase), 267 but also part of them were discharged with the supernatant at min 200 (wash-out at the 268 end of the feast phase). 269 Consequently, apart from the removal of non-desired substances in the supernatant, 270 which favoured the development of non-accumulating microorganisms (proteins and 271 carbohydrates) during the famine phase, the implementation of the settling stage at the 272 end of the feast phase favoured the growth of PHA-accumulating microorganisms in the 273 MMC that could use the accumulated PHA as carbon source for growth (Korkakaki et 274 al., 2016; Kourmentza et al., 2017). 275 The settling stage was implemented after the feast phase, when accumulating bacteria 276 had more intracellular PHA. It is known that the cell weight of these microorganisms is 277 proportional to their cell size and PHA content, which increases the cell density and 278 therefore their settling velocity (Chen et al., 2016). Thus, non-accumulating bacteria 279 presented a lower settleability and they were washed-out with the discharged 280 supernatant after settling. This was in accordance with the results obtained by Chen et 281 al., (2015), who only used acetate as a carbon source (there was no residual COD 282 present in the experiments) in such a way that the improvement of the selection capacity 283 could only be attributed to differences in the cell density that led to physical selection. 284 285 13 3.2.2 Enrichment cycle profile and kinetics with (SBR-S) and without (SBR-I) 286 settling phase 287 The differences in the cycle profile between SBR-I and SBR-S can be seen in Figure 2. 288 In the case of SBR-I, the DO concentration after feeding decreased, corresponding to 289 VFA depletion during the feast phase, and then increased during the famine phase due 290 to exhaustion of external carbon source (Figure 2.A). 291 When the settling stage was implemented in the SBR-S, the aeration was stopped to 292 settle the biomass and then, the supernatant was discharged in the absence of oxygen. 293 For this reason, the DO concentration profile showed a strong decrease between the 294 feast and the famine phases (Figure 2.B). Furthermore, it can be observed that the 295 period of VFA consumption was reduced from 3 h in the SBR-I (Figure 2.A) to less 296 than 1 h at day 62 of operation in the SBR-S (Figure 2B), which implied a faster PHA 297 accumulation. 298 To quantify this effect Table 1.A compares the kinetics of SBR-I and SBR-S at day 62. 299 Despite both MMC showed similar specific substrate uptake rates (qVFA = 0.26-0.27 300 CmmolVFA/(CmmolX·h)), an increase on the qPHA from 0.07 to 0.22 301 CmmolVFA/(CmmolX·h) was observed. Consequently, a higher PHA production yield 302 (YPHA = 0.80 CmmolPHA/h) and a higher quantity of PHA accumulated (18.32 wt%) 303 were obtained. 304 These results correlate with the downward trend of the feast phase length (Figure 1) and 305 indicate a progressive enhancement on PHA-accumulating microorganisms selection 306 with the implementation of the settling phase. 307 308 309 14 310 311 Figure 2. Evolution of the parameters monitored in representative enrichment cycles: DO (-), VFA 312 (●), X (■) and percentage of PHA accumulated (wt%) (▲). A) Reactor operated without settling stage 313 (SBR-I, - - -) at the moment of inoculum collection. B) Reactor operated with settling stage (SBR-S, ──) 314 at day 62 of operation. 315 316 B) A) 15 Table 1. Comparison of experimental kinetic parameters and yields of: A) enrichment reactors 317 operated with (SBR-S) and without settling stage (SBR-I). B) accumulation essays with enriched biomass 318 from the reactors operated with (FBR-S) and without settling stage (FBR-I). 319 A) SBR-I SBR-S Day 24 Day 62 Feast lenght (h) 2.62 ± 0.10 2.00 1.50 qVFA (Cmmol VFA/Cmmol X·h) 0.26 ± 0.06 0.22 0.27 qPHA (Cmmol PHA/Cmmol X·h) 0.07 ± 0.02 0.14 0.22 qHB (Cmmol HB/Cmmol X·h) 0.06 ± 0.01 0.12 0.15 qHV (Cmmol HV/Cmmol X·h) 0.01 ± 0.01 0.02 0.07 YPHA (Cmmol PHA/Cmmol VFA) 0.32 ± 0.21 0.61 0.80 max. PHA (wt%) 12.80 ± 0.76 14.77 18.32 HB:HV 88:12 ± 6:6 88:12 70:30 320 B) FBR-I FBR-S Day 35 Day 64 qVFA (Cmmol VFA/Cmmol X·h) 0.20 0.30 0.26 qPHA (Cmmol PHA/Cmmol X·h) 0.10 0.14 0.19 qHB (Cmmol HB/Cmmol X·h) 0.08 0.12 0.14 qHV (Cmmol HV/Cmmol X·h) 0.02 0.02 0.05 YPHA (Cmmol PHA/Cmmol VFA) 0.48 0.49 0.72 max. PHA (wt%) 40.87 44.28 59.92 HB:HV 83:17 82:18 70:30 qVFA (Cmmol VFA/Cmmol X·h) 0.20 0.30 0.26 3.2.3 Organic matter balance 321 The pre-acidified cooked mussel processing wastewater used as feedstock contained 322 two different carbon sources: (1) preferred substrates that are readily available and can 323 be efficiently converted into PHA by PHA producers (desirable VFA) and (2) 324 compounds that constitute a carbon source that allows non-accumulating 325 microorganisms growth (non-desirable carbohydrates and proteins) (Valentino et al., 326 2017). 327 The carbon balance of these compounds (detailed in Table S2 of Supplementary 328 Material) was calculated (as sCOD) during the operational enrichment cycle of the 329 16 SBR-S: between the beginning of the cycle (Initial), the supernatant discharge after 330 settling (Supernatant) and the end of the cycle (Withdrawal). It was observed an 331 effective removal of non-desired carbon sources with the implementation of the settling 332 stage. In the supernatant were discharged proteins and to a lesser extent carbohydrates 333 (40.16 ± 8.15 % and 34.55 ± 10.13 % of the present at the beginning of the cycle, 334 respectively), which were in a much lower concentration. 335 The concentration of proteins and carbohydrates inside the reactor before settling and 336 after refilling decreased from 120 – 215 mg/L to 60 – 110 mg/L (Figure 3.A) and from 337 20 – 40 mg/L to 15 – 30 mg/L (Figure 3.B) respectively. It should be pointed out that 338 the refilling stream (from the previous cycle effluent) presented low carbohydrates 339 concentrations (14.57 ± 2.58 mg/L) and normally there were no proteins due to their 340 hydrolysis into NH4+, except in isolated cases in which it was measured concentrations 341 of 5 – 10 mg/L. Therefore, the supernatant discharge promoted the removal of 342 substances that could favour the development of non-accumulating microbial 343 populations. 344 345 346 347 348 17 Figure 3. Proteins A), carbohydrates B) and TN C) concentrations before settling (*) and after refilling (■) in the SBR-S reactor. B) A) C) 18 3.2.4 Nitrogen balance 349 Nitrogen balances of enrichment were calculated in the SBR-S (more information is 350 detailed in Table S3 and Table S4 of Supplementary Material) showing that 41 – 65 % 351 of the TN present at the beginning of the cycle was discharged with the supernatant 352 after the settling stage. It was also observed that approximately 15 % of the TN present 353 at the beginning of the cycle was consumed during the feast phase, whereas 50 % of the 354 TN present after the reactor refilling was consumed during the famine phase. 355 TN removal with supernatant discharge could limit nutrients availability during the 356 famine phase (when PHA accumulating microorganisms used the accumulated carbon 357 source (PHA) for growth) whereas it was still present during the feast phase (when non-358 storing microorganisms could use non-desired carbon sources for growth) negatively 359 affecting the enrichment of the system (Figure 3.C). However, refilling provides the 360 SBR with nutrients that lack in the famine phase. In fact, if comparing the reactors with 361 (SBR-S) and without (SBR-I) settling, there was no evidence of a negative effect on the 362 enrichment of the system due to nitrogen removal with supernatant discharge (Table 1). 363 These results controvert the advantages of the carbon-nitrogen uncoupled systems, in 364 which the development of non-accumulating microorganisms was observed to be 365 restricted by applying nitrogen deficiency during the feast phase, resulting in higher 366 PHA production yields and productivities compared to conventional ADF systems 367 (Kourmentza et al., 2017). It is presumed that the decoupling of the system could further 368 improve the enrichment of the MMC. However, the complex and changeable 369 characteristics of the feedstock would surely make it difficult to implement a system 370 like this with such a substrate. It would be necessary to add a pre-treatment of the 371 substrate to remove the ammonium content and later, if possible, recover the previously 372 removed ammonium and reintroduce it in the system, to have nutrient availability 373 19 during the famine. If not, it would be necessary to add an external nitrogen source 374 which, in addition to the needed pre-treatment, could have an important economic 375 impact in the process. 376 3.2.5 Considerations for the settling stage implementation 377 It is necessary to point out that the supernatant should be treated before discharge 378 because of the COD and TN content. It could also be considered its possible reuse or 379 valorization as a fertilizer due to the high NH4+ concentration. However, this effluent is 380 also characterized by a high salinity because of the nature of the industrial cooked 381 mussel process, which could be an important hindrance. Regarding economic 382 considerations, settling implementation will presumably lead to an increase in the costs 383 due to the need for an extra pump for recirculation. However, 20 % and 25 % increases 384 of PHA accumulation and PHA production yield, respectively, are expected to 385 compensate investment costs. Operational costs mainly concerning energy requirements 386 for pumping, might be compensated with the lack of aeration during the settling stage. 387 The results obtained suggested an enrichment of the MMC in PHA-accumulating 388 bacteria and therefore an optimization of the system due to the implementation of the 389 settling stage. Moreover, it was observed a reduction of the feast phase length from 3.0 390 to 1.5 hours because of the enrichment of the MMC. Therefore, in order to discharge the 391 supernatant just after VFA depletion and avoid the consumption of the remaining 392 carbon sources (proteins and carbohydrates), which are preferably used for growth of 393 non-accumulating bacteria, the pre-settling reaction stage could be reduced from 165 394 min (imposed in the present SBR-S operation) to 90 minutes. It was also observed an 395 improvement of the enriched biomass settleability and therefore a higher settling 396 velocity of the enriched culture, which suggested a possible reduction of the settling 397 stage length. This coincided with the results obtained by (Korkakaki et al., 2016), who 398 20 observed compaction of the flocs due to their selection after settling implementation and 399 removal of suspended cells. 400 3.3 Effect of MMC selection on the maximum PHA accumulation capacity 401 To determine the effects of settling in the maximum PHA-accumulation capacity of the 402 MMC, discontinuous accumulation assays were carried out with biomass from SBR-S 403 (FBR-S) and compared with previous assays with biomass from SBR-I (FBR-I). 404 Results of the accumulation assays with biomass from both reactors are considered in 405 Figure 4. Table 1.B shows the experimental kinetic parameters and yields obtained. The 406 maximum PHA-accumulation capacity (max. PHA) of the MMC notably increased in 407 the FBR-S assays (60 %) in comparison with the FBR-I ones (41 %), which correlated 408 with the higher production yield obtained (YPHA = 0.72 CmmolPHA/h). For similar 409 values of the specific substrate uptake rate (qVFA), the improvement of the enrichment in 410 SBR-S promoted an increase of the specific PHA production rate (qPHA) in the 411 accumulation FBR-S experiments, which indicated the improvement of the efficiency of 412 the global PHA production process. In addition, results showed a positive effect on the 413 accumulation kinetic parameters of the FBR-S between days 35 and 64, which 414 correlates with the progressive enhancement of the enrichment SBR-S selection 415 capacity (Table 1.B). 416 417 21 418 419 Figure 4. Evolution of the parameters monitored in accumulation essays (FBR) performed with 420 enriched biomass: VFA (●) and percentage of accumulated PHB (■) and PHV (♦). A) Reactor operated 421 without settling stage (FBR-I, - - -) at the moment of inoculum collection. 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PHA (wt%) 12.80 ± 0.76 14.77 18.32 HB:HV 88:12 ± 6:6 88:12 70:30 4 B) FBR-I FBR-S Day 35 Day 64 qVFA (Cmmol VFA/Cmmol X·h) 0.20 0.30 0.26 qPHA (Cmmol PHA/Cmmol X·h) 0.10 0.14 0.19 qHB (Cmmol HB/Cmmol X·h) 0.08 0.12 0.14 qHV (Cmmol HV/Cmmol X·h) 0.02 0.02 0.05 YPHA (Cmmol PHA/Cmmol VFA) 0.48 0.49 0.72 max. PHA (wt%) 40.87 44.28 59.92 HB:HV 83:17 82:18 70:30 qVFA (Cmmol VFA/Cmmol X·h) 0.20 0.30 0.26 5 6 7 2 Table 2. Data from accumulation essays carried out with biomass obtained under different enrichment strategies. 8 MMC origin & Enrichment strategy Feedstock YPHA/S (Cmmol PHA/ Cmmol S) Max. PHA (wt%) qPHA (CmmolPHA/Cmmol X·h) Reference Activated sludge ADF without settling VFA mixture and lactate YPHA/S: 0.05 – 0.45 Dionisi et al., 2007 Activated sludge ADF without settling Sodium acetate Max. PHA: 69.00 – 89.00 Johnson et al., 2009 Water from a river estuary ADF + settling before withdrawal Sodium acetate, glucose or starch YPHA/S: 0.60 sodium acetate / 0.54 glucose / 0.30 starch Max. PHA: 64.70 sodium acetate / 60.50 glucose / 23.70 starch Cui et al., 2016 Activated sludge ADF without and with settling after feast phase Mixture of sodium acetate and methanol Max. PHA: 48.00 without settling / 58.00-70.00 with settling Korkakaki et al., 2016 Activated sludge ADF + settling after feast phase Nutrients rich fermented centrate sludge YPHA/S: 0.25 - 0.27 Max. PHA: 17.00 - 39.00 Morgan-Sagastume et al., 2015 Activated sludge ADF uncoupled C and N feeding strategy + settling after feast phase Industrial soft drink wastewater Max. PHA: 13.80 Ahmadi et al., 2018 Activated sludge ADF coupled and uncoupled carbon and nitrogen feeding strategy + settling before withdrawal Fermented cheese whey YPHA/S: 0.86 coupled / 0.96 uncoupled qPHA: 0.29 coupled / 0.40 uncoupled Oliveira et al., 2017 Activated sludge ADD (aerobic dinamic discharge) + two settling stages (after feast phase and before withdrawal) Mixture of sodium acetate, NH4Cl and KH2PO4 qPHA: 0.47 - 1.89 Chen et al., 2016 Enriched biomass from the C-SBR, which was inoculated with activated sludge ADF + settling stage after feast phase Synthetic VFA mixture mimicking the composition of pre-acidified cooked mussel wastewater YPHA/S: 0.72 Max. PHA: 59.92 qPHA: 0.19 This work 9 1 Figure 1 1 Figure2 3 4 5 6 7 8 B) A) 2 Figure 2 9 10 11 12 B) A) 3 Figure 3 13 14 B) A) C)