The effect of different operational strategies on polyhydroxyalkanoates (PHAs) production at short-term biomass enrichment

Journal Pre-proof The effect of different operational strategies on polyhydroxyalkanoates (PHAs) production at short-term biomass enrichment Farinaz Ahmadi, Ali Akbar Zinatizadeh, Azar Asadi

PII:

S2213-3437(19)30654-2

DOI:

https://doi.org/10.1016/j.jece.2019.103531

Reference:

JECE 103531

To appear in:

Journal of Environmental Chemical Engineering

Received Date:

21 July 2019

Revised Date:

4 October 2019

Accepted Date:

9 November 2019

Please cite this article as: Ahmadi F, Zinatizadeh AA, Asadi A, The effect of different operational strategies on polyhydroxyalkanoates (PHAs) production at short-term biomass enrichment, Journal of Environmental Chemical Engineering (2019), doi: https://doi.org/10.1016/j.jece.2019.103531

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The effect of different operational strategies on polyhydroxyalkanoates (PHAs) production at short-term biomass enrichment Farinaz Ahmadi1, Ali Akbar Zinatizadeh1, 2,*, Azar Asadi3 1

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Environmental Research Center (ERC), Department of Applied Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran. 2 Visiting Researcher, Department of Environmental Sciences, University of South Africa, Pretoria, South Africa. 3 Department of Gas and Petroleum, Yasouj University, Gachsaran 75918-74831, Iran. *Corresponding author: [email protected], [email protected]

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Highlights

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 Polyhydroxyalkonate (PHA) production in mixed microbial culture was investigated.  The possibility of PHA production under short-term biomass enrichment was assessed.

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 The potential of different strategies to produce PHA was assessed.

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Abstract: In this study, polyhydroxyalkanoates (PHA) production under different short- time biomass enrichment strategies were investigated. Three strategies were applied in a sequencing batch reactor (SBR) including complete aerobic condition under the feast-famine regime, uncoupled carbon and nitrogen feeding regime and alternating anaerobic-aerobic conditions.

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Also, the effect of two different carbon sources comprising acetate and soft drink wastewaters on PHA production over the accumulation stage was evaluated. From the results, uncoupled carbon and nitrogen feeding regime showed a better performance to accumulate PHA rather than the other strategies. Besides, the accumulated PHA with acetate as a carbon source was significantly higher than soft drink wastewater. PHA production was obtained to be 79% and 25% mg-

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PHA/mg-TSS under uncoupled carbon and nitrogen feeding regime fed by acetate and soft drink wastewater, respectively. However, PHA production was less than 50% and 20% for other strategies with acetate and soft drink wastewater, respectively.

Keywords: Polyhydroxyalkanoate (PHA), culture selection strategy, short-term enrichment

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(STE), acetate; soft drink wastewater. 1. Introduction

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In recent years, wastewater is considered as a valuable source of raw material like organic

matters and main nutrients [1]. Resource recovery besides wastewater treatment processes has

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been highlighted as a promising strategy to develop sustainable wastewater treatment systems. In

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this mean, polyhydroxyalkanoate (PHA) could be synthesized as a biopolymer from carbon source of different wastewaters which has been considered as an alternative material to

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traditional polymers made of fossil fuels [2].

Industrial production of PHAs is usually based on pure microbial cultures [3], requiring high

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operational cost (4-9 times higher than that of conventional plastics) [4]. The maximum polyhydroxybutyrate (PHB) production by mixed microbial culture was reported by K. Johnson and his colleagues [5]. The biomass was enriched in an acetate-fed SBR operated by feast-famine strategy and the maximum PHB content reached up to 89% dry cell weight.

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It should be noted that, PHA presented a better performance rather than PHB in terms of thermal and mechanical properties [6]. PHA has shown a broad potential applications as agriculture, biocontrol agents, biofuels, horticulture, packaging and biomedical, biodegradable implants, drugs carriers, medical devices, memory enhancer and tissue engineering [7].

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In another study, 50% PHA content based on COD was achieved in SBR fed-by acetic and propionic acid as carbon sources and operated by aerobic dynamic feeding strategy (feastfamine) [8]. From the literature, aerobic dynamic feeding (ADF) strategy to produce PHAs in mixed cultures have been investigated with several fermented waste streams including: fermented molasses [9], olive oil mill effluent [10], wastes from pulp industry [8], fermented palm oil mill effluent [11],

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municipal wastewater [12], and wood mill effluents [13]. Liu and his colleagues [14] evaluated

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the potential of tomato cannery wastewater as a substrate for producing PHA in mixed microbial culture SBR. A maximum of 20% PHA content on a cell-weight basis was obtained when

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carbon, ammonia, and phosphorus removal efficiencies were 84%, 100%, and 76%, respectively.

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Another strategy to accumulate PHA in mixed culture systems is to apply anaerobic-aerobic conditions [15]. As a fact, anaerobic-aerobic strategy is used for biologically phosphorus

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removal. In anaerobic condition, polyphosphate-accumulating organisms (PAOs) are capable to utilize the energy stored as poly-P and glycogen when there is no electron acceptor (oxygen or

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nitrate) available for energy generation. During this condition, phosphorus accumulated in PAOs can be released to the liquid phase, therefore the concentration of phosphorus is increased in the liquid phase. In the following aerobic condition, biomass consumes the stored compounds and simultaneously accumulates phosphorus. Therefore, applying these conditions caused to grow

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special microorganisms which are capable to accumulate PHA. Bengtsson [16] assessed PHA production of a mixed microbial culture enriched by glycogen-accumulating organisms (GAOs) under alternating anaerobic-aerobic conditions. The maximum PHA content was 60% of dry cell weight using acetate as a substrate. Many researches have investigated PHA production in mixed cultures fed by various fermented

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waste streams using anaerobic-aerobic strategy including: methanol-enriched pulp-andpaper mill foul condensate, fermented municipal primary solids and biodiesel wastewater [17], fermented sugar cane molasses [18], brewery wastewater [19], and municipal wastewater [20]. Also, it has been proved that an internal growth limitation caused an efficient selection of PHAaccumulating organisms. For this purpose, some reseachers offered uncoupled carbon and

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nitrogen strategy along with a feast-famine strategy to upset the growth process significantly [13,

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21]. In this strategy, the bioreactor is fed by carbon source without any nitrogen content and after reaching famine phase, the nitrogen source was added. Consequently, over feast phase,

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microbial growth is prevented due to lacking of nitrogen, so storing microorganisms can grow

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under the absence of carbon source in the following famine phase. The results showed that uncoupled carbon and nitrogen strategy selected different PHA-producing organisms with higher

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volumetric productivity rather than the conventional feast-famine strategy. In the present study, the efficiency of different operational strategies in SBR for the selection of

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PHA-producing microorganisms was evaluated. For this purpose, an aerobic dynamic feeding strategy, an uncoupled carbon and nitrogen feeding strategy, and an anaerobic-aerobic strategy were used in SBRs fed by soft drink wastewater. Most of the reports in the literature focused on long-term culture selection, requiring plenty of energy and cost. In order to reduce the cost of

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PHA production, this research was aimed to evaluate how rapidly an activated sludge could acclimate PHA producing species under short-term biomass enrichment. Also, the effect of different carbon source on PHA production under accumulating phase was investigated. From the literature, acetate is a favorite carbon source to accumulate PHA,

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therefore, in this study the potential of soft drink wastewater was compared with acetate in terms of PHA production efficiency.

2. Materials and methods 2.1 Culture selection bioreactor Three lab-scale SBRs with 4.5 L working volume were used in this study as shown in Fig. 1. All

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three SBRs were inoculated with activated sludge from the aeration tank of an industrial

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wastewater treatment plant (Kermanshah, Iran) to provide MLSS concentration of 5000–6000 mg TSS/L. SBR1, SBR2, and SBR3 were operated under aerobic dynamic feeding (ADF) strategy

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which is complete aerobic condition under the feast-famine regime, uncoupled carbon and

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nitrogen feeding regime (FF&unCN) and alternating anaerobic-aerobic (A/O) conditions, respectively. The systems were operated in batch mode and each SBR cycle was consisted of

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10 cm Clear zone The operational details for all the bioreactors are presented several phases split up over 12 hours. (25 cm)

in Fig. 2. All the SBRs were fed by soft drink wastewater for 30 minutes. In SBR1 and SBR3, Control valve

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nutrient solution was added to soft drink wastewater, at the beginning of each cycle. In SBR2, Mixer

after 160 min of starting, settling phase (13 min) was performed and the supernatant was discharged to remove residual carbon Sludge zone source, then nitrogen solution (2 L) was added. The (30 cm)

characteristics of the used wastewaters are presented in Table 1. Air was supplied by a Peristaltic pump

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compressed air pump through ceramic diffusers. Effluent tank

Sparger Air pump Feed tank

Timer

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Fig. 1. The experimental set-up.

Fig. 2. Fed-batch reactor for PHA accumulation.

Table 1. The composition of soft drink wastewater used in this study. Selection reactor SBR1

Total sugars (g/L) 1.88

COD Conc. (mg/L)

NH4-N (mg/L)*

PO4-P (mg/L)*

COD/N/P

pH

3000

119.5

29.02

100/4/1

7–8

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SBR2 SBR3

1.88 1.88

3000 3000

45.01 149.99

7.27 58.05

100/1.5/0.25 100/5/2

7–8 7–8

*

N and P were supplied by adding NH4Cl and KH2PO4, respectively.

In order to evaluate the performance of the selected biomass, the accumulating SBR was filled by the selected biomass (2000 mg/L) at the end of 32nd cycle. For this purpose, SBR with an initial working volume of 2 L under aerobic conditions at 20–25 oC was used. Dissolved oxygen

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(DO) level was maintained around 1–2 mg/l. Air was supplied by an air pump through a ceramic diffuser and a proper mixing was provided by magnetic stirring. Two additional carbon sources,

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acetate, and soft drink wastewaters with 5000 mg COD/L were used separately. Growth was

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limited in these experiments as no nitrogen source was supplied and only a small amount of remaining nitrogen source from the previous SBR cycle was available. The accumulation assays

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were operated for at least 48 h. The progress of the experiments was monitored due to online

2.3 Analytical techniques

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(DO, pH) and offline (COD, TSS, PHA, ammonia, and SVI) measurements.

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The concentrations of chemical oxygen demand (COD), total nitrogen (TN), nitrate and nitrite, NH4-N, total phosphorus (TP), and mixed liquor suspended solids (MLSS) were determined by using standard methods [23]. The concentration of dissolved oxygen (DO) in the bioreactor was measured with a DO electrode (WTW DO Cell OX 330) as a percentage of air saturation and pH

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was monitored with a pH electrode (Metrohm model 827, Switzerland). For COD, A colorimetric method with a closed reflux method was developed. Spectrophotometer (DR 5000, Hach, Jenway, USA) at 600 nm was used to measure the absorbance of COD samples. N-NH4 were determined by total Kjeldahl nitrogen (TKN) meter, Gerhardt model (Vapodest 10, Germany). UV–Vis spectrophotometer model JENWAY 6320D was employed.

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The percentage of PHA in biomass was measured by using gas chromatography (GC). To prepare samples, 15 ml biomass samples taken at special times and centrifuged firstly at 3600 rpm (1500 ×g) for 20 minutes and the thickened biomass was transferred to 5 ml vials. In the following, 2 ml methanol acidified with H2SO4 (3% v/v) and 2 ml chloroform was added to the thickened biomass. The sample tubes were sealed off and heated at 100 oC for 3.5 h and cooled to room temperature [24]. After digestion, 1 ml of deionized water was added to the sample tubes and shaken well for

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10 minutes. After separation of the three phases (60 minutes), 1 ml of the chloroform phase and 3

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µL methyl benzoate as an internal standard was transferred to a GC vial. 1 µL sample was split injected into an Agilent Technologies Model 7890B GC-FID (Agilent Technologies, Santa Clara,

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CA) equipped with a programmable autosampler. The injection temperature was 210 oC. The

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temperature program was 40 oC for 1 minutes, followed by a ramp of 5 oC/min to 80 oC for 1 minutes, a ramp of 30 oC/min to 160 oC for 1 minutes and a final ramp of 30 oC/min to 210 oC for

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1 minutes. Also, it should be mentioned that standard samples of methyl-3-hydroxybutyrate (Fluka), methyl-3-hydroxy valerite (Fluka) and methyl-3-hydroxyhexanoate (Aldrich) were used

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as external standards.

2.4 Process parameters studied

The PHA content of the biomass (% PHA, mg-PHA/mg-TSS), specific substrate uptake rate (-qs,

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mg COD-S/mg COD-X.h), specific PHA production rate (qp, mg COD-PHA/mg COD-X.h), activated biomass yield (YX/S, mg-VSS/mg COD-S.h), and PHA storage yield (YPHA/S, mg CODPHA/mg COD-Sremoved) were studied in this work as process responses. Also, some calculated parameters were obtained by the following equations. Xa is average activated biomass concentration (it is calculated by the difference between volatile suspended solids (VSS) and

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PHAs storage); t represents the length of feast phase in SBR. Substrate consumption and PHA formation data were obtained by converting the analytical results into COD units by oxidation stoichiometry: 1.067 mg COD/mg acetate, 1.067 mg COD /mg glucose, 1.42 mg COD/mg X (active biomass), 1.67 mg COD/mg (3-hydroxybutyric acid), 1.92 mg COD/mg (3-hydroxyvaleric

× 100

PHAe−PHA0

S0−Se

YX/S =

(3)

Xa.t MLSSe−MLSS0 S0−Se (PHAe−PHA0)×1.67 S0−Se

(4) (5)

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YPHA/S =

(2)

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-qs =

Xa.t

(1)

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MLSS

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qp =

PHAe−PHA0

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%PHA =

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acid), and 2.35 mgCOD/mg (3-hydroxy hexanoic acid).

3. Results and discussion

3.1 SBRs performance in the selection stage In this research, ADF strategy was carried out with COD:N:P of 100:4:1. For the enhancement of

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PHA production in uncoupled carbon and nitrogen feeding strategy, the supernatant was withdrawn to remove carbon content at the end of feast phase. A feed with COD:N:P of 100:1.5:0.25 was added during a cycle. Also, SBR was fed by COD:N:P of 100:5:2 over anaerobic/aerobic strategy. According to the literatures [25, 26], a nitrogen-limited stream in the culture enrichment stage (ADF) positively affected the polymer storage ability of biomass. Therefore, activated sludge was 9

selected in SBR1 operated under nitrogen limitation. The selector system was unstable in the first two cycles of the operation resulted from microbial adaptation with new conditions applied. At the end of feast phase, the measured ammonia, PHA and COD concentrations were 52.1 mg N/L, 113.1 mg PHA/L and 275.5 mg COD/L, respectively. SVI value was less than 100 mg/L during operation time, which indicates that activated sludge enriched under dynamic conditions had good settling properties. Overall COD removal efficiency was about 90.3 % after 32 cycles.

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Fig. 3 shows COD, DO, NH4-N, MLSS and PHA concentration profiles at 32nd cycle of SBR1. As

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can be seen in Fig. 3, initial DO concentration in SBR1 was 2.4 mg/L. After 115 min cultivation (end of feast phase), it sharply increased to 4.9 mg/L during the famine phase. Average values of

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the measured parameters during a cycle of SBR1 with ADF strategy are reported in Table 2. As

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shown in the Table, YX/S in the famine phase was higher than 1, probably due to the presence of other carbon compounds besides PHA. Results indicate that applying dynamic feeding conditions

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and suppressing growth by limiting nitrogen led to a more elevated PHA level in the biomass.

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COD conc.

MLSS conc.

PHA conc.

DO conc.

Famine

Feast

1400

20

1300

18

1200 1100

16

1000

14

900 12

700

10

600

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800

8

500

6

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400 300

100 0 30

130

230

330

430

4 2 0

530

630

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Time, min

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200

PHA Conc. (mg PHA/L), DO Conc. (mg/L)

COD Conc. (mg/L), MLSS Conc. (g TSS/L), N-NH4 Conc. (mg N/L )

N-NH4 conc.

Fig. 3. DO, COD, NH4-N, MLSS and PHA concentration profiles in SBR1 after 32 cycles.

Parameter

Unit

mg COD/L mg COD/L mg COD/L.h mg-VSS/mg COD-S mg COD-PHA/mg COD-S mg-PHA/mg-TSS mg COD-PHA/mg CODSpecific PHA production rate (qp) X.h Specific substrate uptake rate (-qS) mg COD-S/mg COD-X.h

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Substrate concentration Polymer concentration Substrate consumption rate Biomass yield (YX/S ) PHA storage yield (YPHA/S) Max. PHA content (%)

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Aerobic dynamic feeding strategy (ADF)

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Table 2. Average values of the measured parameters during a cycle of SBR1, SBR2, and SBR3

Substrate concentration Uncoupled carbon Polymer concentration and nitrogen Substrate consumption rate feeding strategy. Biomass yield (YX/S ) PHA storage yield (YPHA/S) Max. PHA content (%) Specific PHA production rate (qp)

mg COD/L mg COD/L mg COD/L.h mg-VSS/mg COD-S mg COD-PHA/mg COD-S mg-PHA/mg-TSS mg COD-PHA/mg CODX.h 11

275.5 336.34 125.08 0.34 0.23 9.4

End of the famine phase 130 84.3 3.57 1.08 — 2.35

0.022

—

0.063

0.008

176 337.5 108.7 0.029 0.26 9.4

42.5 78.5 3.11 2.29 — 2.3

0.016

—

End of the feast phase

Specific substrate uptake rate (-qS) mg COD-S/mg COD-X.h mg COD/L mg COD/L mg COD/L.h mg-VSS/mg COD-S mg COD-PHA/mg COD-S mg-PHA/mg-TSS mg COD-PHA/mg CODSpecific PHA production rate (qp) X.h Specific substrate uptake rate (-qS) mg COD-S/mg COD-X.h

0.001

165.66 474.65 43.86 0.22 0.275 12.55

71 153.09 3.5 0.1 — 4.05

0.0062

—

0.022

0.0017

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Substrate concentration Polymer concentration Substrate consumption rate Anaerobic/aerobic Biomass yield (YX/S ) strategy PHA storage yield (YPHA/S) Max. PHA content (%)

0.058

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According to the results obtained from previous experiments [27] and some studies [10], in the

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uncoupled carbon and nitrogen feeding strategy, to provide a real famine, the supernatant at the end of feast phase is discharged after a settling period. Fig. 4 reports COD, PHA, MLSS,

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phosphorus, and ammonia nitrogen concentration profiles obtained in 32nd cycle during the selection stage for SBR2. As shown in the Fig., DO concentration was relatively low at the

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beginning of the cycle due to an increase in metabolic activity as a result of the presence of rbCOD. At the end of the feast phase, DO concentration was sharply increased to 3.4 mg/L. The

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settling phase was started at 145 minutes after starting of a cycle. After settling phase, DO concentration was slowly increased (almost 3.7 mg/L) due to very low level of COD concentration despite the presence of nutrients.

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Also, nitrogen concentration was decreased over famine phase, indicating the occurrence of microbial growth during famine phase by using PHA stored as a carbon source. After nitrogen depletion (250 min after beginning of the cycle), PHA consumption was slowed down, which proves that the biomass growth in the overall process was controlled by nitrogen concentration. Based on oxygen profile, the duration ratio of feast phase to famine phase was 0.2. At the end of the feast phase, PHA content was 9.5 % (mg-PHA/mg-TSS). Average results during a cycle (only 12

considering pseudo-steady state) are reported in Table 2. High biomass yield (2.29, mg-VSS/mg COD-S) during the famine phase could imply the growth of PHA accumulating bacteria. As expected, uncoupled carbon and nitrogen feeding strategy could accumulate PHA-storing species quicker than ADF strategy [28].

N-NH4 conc.

MLSS conc.

DO conc.

P-PO4 conc.

Famine

Feast

20

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1400

PHA conc.

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16

1000

14 12

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800

10 8

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600

400

200

0 130

230

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30

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COD conc. (mg/l), N-NH4 conc. (mg/L×10-1)

18

1200

330

6 4 2

MLSS conc. (g TSS/L), P-PO4 conc. (mg/L), PHA conc. (mg/L×10)

COD conc.

0 430

530

630

Time, min

Fig. 4. DO, COD, NH4-N, PO4-P, MLSS and PHA concentration profiles in SBR2 after 32 cycles.

In anaerobic/aerobic strategy, to enrich mixed microbial culture with polyphosphate-accumulating

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organisms (PAOs), more phosphorous and less nitrogen concentrations (COD:N:P of 100:5:2 against to 100:10:1 at the previous study [29]) were supplied. Within 10 days after inoculating with municipal activated sludge, SBR reached a steady state. The profiles of studied parameters are depicted in Fig. 5. As observed in the Fig., PHA and phosphorus concentrations show a reverse trend, an increase in anaerobic phase and a decrease in aerobic phase. The maximum amount of PHA and phosphorus concentration were achieved around 127 mg P-PO4/L and 251.14 13

mg PHA/L, respectively, at the end of anaerobic phase. The function of inoculum exhibits the presence of PAOs in the culture. From the Fig., PHA production was in agreement with COD consumption over anaerobic phase. During the subsequent aerobic phase, PHA was consumed and polyphosphate was accumulated in PAOs which is caused a decrease in P-PO4. Anaerobic phosphorus release over substrate uptake (0.08 mg P-PO4/mg COD-S) was observed in anaerobic phase. High aerobic phosphorus uptake was obtained (1.17 mg P-PO4/mg COD-S removed in the

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aerobic phase), as a result of simultaneous consumption of external carbon source (95 mg

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COD/L), stored carbon source in biomass (PHA), and growth of microbial biomass. Analysis of the performance of the enriched biomass during 32nd cycle is shown in Table 2. COD removed

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during anaerobic phase was 1034 mg/L (corresponding to a COD removal of 94.08%). At the end

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of anaerobic phase, average PHA content was 12.54% (mg-PHA/mg-TSS).

Anaerobic

P-PO4 conc.

PHA conc. Aerobic

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1400

1200

200

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1000

250

800

600

400

150

100

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COD conc. (mg COD/L), MLSS conc. (mg TSS/L×10)

COD conc.

50

200

0

30

0 130

230

330

430

530

630

Time, h

Fig. 5. COD, PHA and phosphorus concentration profiles in SBR after 32 cycles.

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P-PO4 conc. (mg/L), PHA conc. (mg PHA/L)

MLSS conc.

3.2 PHA production in the accumulation stage In order to optimize PHA accumulation process to achieve the maximum PHA biomass content, batch experiments were conducted using two different substrates. Each batch test was carried out under nutrient limiting conditions, with continuous aeration and mixing. The batch test lasted 48 h. 3.2.1 Acetate as a carbon source

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Three batch experiments were carried out with acetate as a sole carbon source. The changing

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trend of PHA concentration during batch experiments is shown in Fig. 6. From the Fig., the

initial PHA concentration in all batch experiments was less than 130 mg PHA/L. The maximum

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PHA concentration was obtained from SBR3 after 24 h cultivation. During the next 24 hours,

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PHA content sharply increased to 3500 mg/L in batch2, however, it was increased gently in batch1 and batch3. It might be due to the strong selective pressure applied to mixed microbial

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culture of batch2 over the culture selection stage. At the end of cultivation, the maximum PHA content was 16, 79, and 44.7% mg-PHA/mg-TSS for batch1, batch2, and batch3, respectively. The

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overall performance of the selected biomass during the accumulation stage after 48 h were reported in Table 5. By comparing the results presented in Table 3, it could be concluded that the culture selection under uncoupled carbon and nitrogen feeding strategy showed a better PHA production performance mainly in terms of storage yield and specific PHA productivity relative

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to the other two strategies. The stored PHAs were identified as copolyesters with three types of monomers in SBR3 and two types of monomers for the other two batches. As reported by Valentino et al. [30], the obtained PHA after 22–40 cycles was very similar to those achieved by fully acclimated biomass under a longer acclimation period. Table 4 compares some studies used acetate as a sole carbon source to produce PHA with different strategies. From the Table,

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uncoupled carbon and nitrogen feeding strategy shows the best results over short term operation, however, short-term enrichment is not efficient for ADF strategy. Also, anaerobic and aerobic strategy presents insignificant differences between 16, 100, and 450 days of operation.

Batch1

Batch2

Batch3

3500

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2500

2000

1500

-p

PHA Conc. (mg/L)

3000

re

1000

500

0

5

10

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0 15

20

25

30

35

40

45

50

Time (h)

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Fig. 6. PHA concentration profile in the accumulation reactor using acetate as a carbon source after 48 hours.

Table 3. The overall performance of selected biomass during accumulation stage after 48 h and the analysis of PHA content (acetate as a carbon source). Polymer conc. (mg PHA/L)

PHA storage yield (mg CODPHA/mg CODSremoved)

Specific PHA production rate (mg COD-PHA/ mg COD-X.h)

PHA content ( mg-PHA/mgTSS×100)

3-HB/PHA (mg 3-HB /mg-PHA)

3-HV/PHA (mg 3-HV /mg-PHA)

3-HHx/PHA (mg 3-HHx /mg-PHA)

Batch1

317.96

0.197

0.0029

16 %

0.85

0.15

—

Batch2

3414.63

0.64

0.019

79 %

0.84

0.16

—

Batch3

893.047

0.468

0.012

44.7 %

0.68

0.23

0.09

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Batch test

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Table 4. Comparison of some researches regarding PHA production using acetate as carbon source Operation time (day)

COD/N/P

Content of PHA (%)

Reference

Aerobic dynamic feeding strategy (ADF)

60 100 8 16

100/5.5/4.5 100/2.8/1.7 100/10/1 100/4/1

52 51 13.2 16

[31] [32] [29] This study

8

100/3/0.5

22.36

[29]

16 100 450 16

100/1.5/0.25 100/4.8/1.2 100/2.9/0.9 100/5/2

79 51 60 44.7

This study [33] [16] This study

Anaerobic/aerobic strategy

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Uncoupled carbon and nitrogen feeding strategy

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Culture selection strategy

3.2.2 Soft drink wastewater as a carbon source

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In order to investigate PHA production ability using soft drink wastewater as a carbon source, three batch tests were performed. The PHA production trends for all three conditions are shown

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in Fig. 7. As shown in the Fig., an increase in PHA concentration was observed after 48 h of

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cultivation for all batch tests, while batch2 displayed sharply increasing trend rather than the others. At the end of the accumulation test, the maximum PHA content was 12, 25, and 17.2 % (mg-PHA/mg-TSS) for batch1, batch2, and batch3, respectively. Table 5 summarizes the results

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of main parameters collected in three batch tests. From the Table, the obtained results showed a significant similarity with acetate feeding condition. It has been found that lower PHA content using glucose (soft drink wastewater) can be attributed to accumulate glucose in the form of

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carbohydrate and likely glycogen [21]. Therefore, a lower fraction of carbon source is available for PHA production. The stored PHA contains copolymers with two types of monomers, namely 3-hydroxybutyrate (3-HB) and 3-hydroxyvalerate (3-HV). Table 6 presents PHA production by mixed microbial cultures from different industrial carbon sources. From the Table, PHA production efficiency in this study is lower than other researches which it could be related to short-term biomass enrichment (16 days). In overall, short-term strategy is ineffective when 17

industrial wastewaters are used as carbon sources, since microorganisms should be compatible with industrial wastewaters. Batch1

Batch2

Batch3

500

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300

200

-p

PHA Conc. (mg/L)

400

0 0

5

10

15

20

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100

25

30

35

40

45

50

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Time, h

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Fig. 7. PHA concentration profile in the accumulation reactor using soft drink wastewater as a carbon source after 48 hours.

Table 5. The overall performance of selected biomass during accumulation stage after 48 h and the analysis of PHA content (soft drink wastewater as a carbon source). Polymer conc. (mg PHA/L)

PHA storage yield (mg CODPHA/mg CODSremoved)

Specific PHA production rate (mg COD-PHA/mg CODX.h)

PHA content ( mg-PHA/mgTSS×100)

3-HB/PHA (mg 3-HB /mg-PHA)

3-HV/PHA (mg 3-HV /mg-PHA)

241.85 500.52 342.89

0.068 0.218 0.24

0.0022 0.0058 0.0047

12 % 25 % 17.2 %

0.74 0.7 0.68

0.26 0.3 0.32

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Batch test

Batch1 Batch2 Batch3

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Table 6. Comparison of some researches regarding PHA production using different industrial wastewaters

Anaerobic/aerobic strategy

COD/N/P

100 40

100/6/1 100/4/0.6

Content of PHA (%) 61.26 30

16

100/4/1

12

This study

40 100

100/0.4/0.1 100/2.1/0.8

50 62.16

[28] [10]

16

100/4/1

25

This study

365 50 250

100/4.2/1.1 100/4/1.6 100/ 3.7/ 0.88

37 20 42

[18] [20] [34]

16

100/5/2

17.13

This study

Reference [22] [28]

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Uncoupled carbon and nitrogen feeding strategy

Sugar cane wastewater Fermented cheese whey Soft drink industrial wastewater Fermented cheese whey Olive oil mill wastewater Soft drink industrial wastewater Fermented sugar cane molasses Municipal wastewater Paper mill wastewater Soft drink industrial wastewater

Operation time (day)

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Aerobic dynamic feeding strategy (ADF)

Type of carbon source

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Culture selection strategy

4. Conclusion

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In this study, three different strategies to enrich PHA-accumulating bacteria under short-term

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operation were investigated. The obtained results indicated that operational strategy of culture selection stage was a significant factor for PHA production. Also, two carbon sources were

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applied in accumulation stage to evaluate the capability of selected biomass for PHA production. The maximum storage response when uncoupled carbon and nitrogen feeding strategy was applied as culture selection strategy was 79 and 25% mg-PHA/mg-TSS for acetate and soft drink wastewaters, respectively. Moreover, complete aerobic condition under feast-famine regime

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showed the lowest potential for PHA production (16 and 12% mg-PHA/mg-TSS for acetate and soft drink wastewaters, respectively). As a conclusion, carbon source is an effective factor in PHA production besides operational strategy, so that acetate showed better performance for PHA production respect to soft drink wastewater. Declaration of interests

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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Acknowledgment The authors would like to acknowledge Iran National Science Foundation (INSF) for the full

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financial support provided for this research work. The authors also wish to thank Razi University

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re

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and the Ministry of Science and Technology-Iran for their financial support and lab equipment.

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