Polyhydroxyalkanoate synthesis using different carbon sources by two enhanced biological phosphorus removal microbial communities

Process Biochemistry 44 (2009) 97–105

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Polyhydroxyalkanoate synthesis using different carbon sources by two enhanced biological phosphorus removal microbial communities Maite Pijuan a,b, Carles Casas a, Juan Antonio Baeza a,* a b

Department of Chemical Engineering, ETSE, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain Advanced Water Management Centre, The University of Queensland, St. Lucia 4072, Queensland, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 May 2008 Received in revised form 4 September 2008 Accepted 19 September 2008

Polyhydroxyalkanoate (PHA) is a biodegradable plastic synthesised by bacteria as energy and carbon storage material. PHA production is mostly based on pure cultures operated under sterile conditions, which increase the costs of this biopolymer. The use of inexpensive mixed culture biomass, such as activated sludge, to produce biodegradable plastics from renewable waste streams has been proposed as an alternative. The effect of carbon sources (acetate, propionate, butyrate and glucose) on the type and quantity of PHA synthesis obtained with different enhanced biological phosphorus removal (EBPR) microbial communities enriched with acetate and propionate are reported in this work. Two sequencing batch reactors (SBRs) were seeded with biomass withdrawn from a non-EBPR wastewater treatment plant (WWTP). The same operational conditions were kept, but using acetate or propionate as the sole carbon source of each reactor. These conditions produced two microbial communities with different P-removal capacity. The results presented in this study show the effect of the carbon source on the PHA composition (amount of polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and polyhydroxy-2-methylvalerate (PH2MV)), which differed not just between substrates but also between the two EBPR communities used. In addition, some monomers not always analysed contribute significantly to the total amount of PHA, especially when using butyrate, showing that if they are not considered this can lead to erroneous calculated yields. ß 2008 Elsevier Ltd. All rights reserved.

Keywords: Volatile fatty acid (VFA) Enhanced biological phosphorus removal (EBPR) Polyhydroxyalkanoate (PHA) Polyphosphate accumulating organisms (PAO)

1. Introduction Polyhydroxyalkanoate (PHA) is a biodegradable plastic synthesised by bacteria as energy and carbon storage material. It has typical properties similar to those of thermoplastics [1], such as a polypropylene (PP) and polyethylene (PE) and the large number of copolymer blends that are possible allows for the potential to engineer polymers with the desired properties for a wide range of applications [2]. The prevailing technology for PHA production is mostly based on pure cultures operated under sterile conditions involving the use of pure carbon sources [3]. These conditions increase considerably the costs of this biopolymer, preventing its commercial application because the available synthetic polymers are much cheaper. However, these synthetic polymers are made from non-renewable resources and are characterised by difficulty in disposal where recycle and re-use are not feasible [4]. Moreover,

* Corresponding author. Tel.: +34 93 5811587; fax: +34 93 5812013. E-mail address: [email protected] (J.A. Baeza). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.09.017

the production of PHA is more beneficial in a full cradle-to-gate life cycle assessment study than PP or PE production [4]. The use of inexpensive mixed culture biomass, such as activated sludge, to produce biodegradable plastics from renewable waste streams has been proposed as an alternative [5], due to the reduction in costs of sterility, equipment and control requirements and the utilization of cheaper substrates as carbon sources. However, there is still a definite need for optimisation of the entire mixed culture PHA production process [2]. In addition, to know the composition of PHA is important because it will determine the properties of the bioplastic obtained. For example, PHB, since is made up solely of 3HB monomers, has been found to possess high melting and glass transition temperatures, and being stiffer than synthetic plastics. On the other hand, P(3HB–3HV) copolymer has been found to have better physical and mechanical properties having greater flexibility than PHB [1]. Some researchers have suggested that the introduction of more diverse hydroxyalkanoate monomers into PHA copolymers could likely improve the quality of the plastic produced [5,6]. Further study is required to test the quality of the bioplastic produced with the different PHA

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fractions that could potentially lead to the finding of new and better bioplastics. Several activated sludge cultures have been proposed to produce significant amounts of PHA: aerobic cultures subjected to feast and famine cycles [7,8]; glycogen accumulating organisms (GAO) [5,6] or polyphosphate accumulating organisms (PAO) [9– 12]. PAO are the responsible of the enhanced biological phosphorus removal (EBPR) process for removing phosphorus from wastewater. Under anaerobic conditions, PAO take up organic substrates (preferably volatile fatty acids—VFAs) and store them as PHA, while the reducing equivalents needed are provided by glycolysis of internally stored glycogen [13–18]. The energy for PHA storage is obtained partly from the glycogen utilization but mostly from the hydrolysis of the intracellular stored polyphosphate (polyP), resulting in an orthophosphate release into solution. In the subsequent aerobic phase, PAO take up excessive amounts of orthophosphate to recover the intracellular polyP levels using the oxidation of stored PHA as energy source. Meanwhile, they grow and replenish the glycogen pool using PHA as both carbon and energy sources [14,19]. Net phosphorus removal is achieved by wasting sludge after the aerobic period when the biomass contains a high level of polyP. ‘‘Candidatus Accumulibacter phosphatis’’ has been found in many lab scale studies as the PAO responsible of EBPR occurring in reactors fed with acetate [20,21] or propionate [22–24] and has been observed in abundance in full-scale EBPR treatment plants [25]. Moreover, GAO have been also reported to play a significant role in some of the EBPR systems, causing their deterioration. GAO can compete with PAO for VFA uptake under anaerobic conditions without performing anaerobic P-release or aerobic P-uptake [15,26]. ‘‘Candidatus Competibacter phosphatis’’ [27] was identified to perform this GAO phenotype and was present in significant amounts in some EBPR treatment plants [25,27,28]. Recently, it was demonstrated [22,24] that Accumulibacter have an advantage over Competibacter when propionate is the predominant carbon source, although other groups could be competing with PAO for the propionate as Alphaproteobacterial-GAO [29], which include Defluviicoccus vanus clusters 1 and 2 related bacteria [30]. The effect of carbon source on EBPR cultures has been previously studied [9,10,31,32] and Lemos et al. [9] already focused their research on PHA production. However, only data of some PHA synthesis with acetate and propionate is usually reported for EBPR cultures [12,22]. The present work was focused on the study of the PHA composition (PHB, PHV and PH2MV) and synthesis with several carbon sources (acetate, propionate, butyrate and glucose) using two different EBPR microbial communities. Two SBR were seeded with biomass withdrawn from the same WWTP and were developed using the same operational conditions except for the carbon source: one SBR was fed with acetate and the other one with propionate. The synthesis of different PHA depending on the substrate used (acetate, propionate, butyrate, glucose and its mixture) was tested in different cycle studies in both reactors. The results were compared and related to the different microbial communities.

32 cm  23 cm  23 cm (H  W  D), had a working volume of 10 L and was jacketed for using a heating fluid to have a stable temperature. Both reactors were inoculated with activated sludge from a non-EBPR municipal WWTP (Granollers, Catalonia, Spain), the only difference was the carbon source used: propionate (PrSBR) or acetate (Ac-SBR). They were operated in four cycles of 6 h per day. Each cycle consisted of 2 h anaerobic react, 3 h aerobic react, 55 min of settling and in the last 5 min, extraction of 5 L of supernatant. A volume of 5 L of synthetic wastewater was added during the first 5 min of the subsequent cycle, producing a HRT of 12 h. The SRT was kept at 9 days in both reactors by periodic sludge wastage at the end of the aerobic react phase. To achieve anaerobic and aerobic conditions, nitrogen gas and air were bubbled through the liquid, respectively. The pH was controlled during the aerobic period at 7.0  0.1 with 1 M HCl. The temperature was controlled at 25  1 8C. DO was kept between 3.5 and 4.5 mg/L during the aerobic phase with an on/off control manipulating an aeration valve. The cycle studies were conducted after 90 days of operation, when the SBRs had a stable operation, corroborated with the monitoring of different cycles with very similar profiles of the main compounds being the biomass concentration also very similar. The biomass concentration during the experimental period was around 4300 mg VSS L 1 in the Ac-SBR, with a VSS/TSS ratio of 0.87 at the end of the aerobic period, and 4000 mg VSS L 1 in the Pr-SBR, with a VSS/TSS ratio of 0.65 at the end of the aerobic period. 2.2. Synthetic media The synthetic wastewater used in this study was constituted by mixing two different solutions. The first one, called ‘‘concentrated feed’’, represented 0.25 L of the 5 L of synthetic wastewater prepared. The second one, called ‘‘P-water’’, represented 4.75 L per 5 L of synthetic wastewater prepared. The ‘‘concentrated feed’’ consisted of (g/L distilled water): 0.5 peptone, 0.84 NH4Cl, 1.8 MgSO4
7H2O, 3.2 MgCl2
6H2O, 0.84 CaCl2
2H2O, 0.4 yeast extract, 0.01 allylthiourea (ATU) to inhibit nitrification and 12 mL of nutrient solution [33]. The concentrated feed was adjusted to pH 5.5 with 2 M NaOH and autoclaved to prevent contamination. One SBR was fed using acetate as the only carbon source (Ac-SBR) and the initial concentration in the SBR (after the feeding phase) was increased progressively over a 26 days period from 30 to 200 mg Acet/L. The other SBR was developed using propionate (Pr-SBR) as the sole carbon source and the initial concentration in the SBR (after the feeding phase) was increased progressively over a 26 days period from 20 to 160 mg Prop/L. The P-water consisted initially of (mg/L distilled water): 81.6 KH2PO4 and 62.0 K2HPO4 and the pH was adjusted to 10.0 with 2 M NaOH. Consequently, the synthetic wastewater had a concentration of 28 mg/L of P-PO43 . When the acetate and the propionate were increased to 200 and 160 mg/L, respectively, the phosphorus concentration was doubled in the media (56 mg/L of P-PO43 ) to avoid any limitation. The synthetic media described above was also used in the cycle studies, except for the carbon source that was substituted by the one used in each study. The cycle studies performed with individual substrates in the Pr-SBR were carried out with an initial concentration of propionic acid: 200 mg Prop/L, acetic acid: 250 mg Acet/L, butyric acid: 150 mg Buty/L or glucose: 200 mg Gluc/L, respectively. When these substrates were combined, the concentrations were 60 mg Prop/L, 100 mg Acet/L, 100 mg Buty/L and 70 mg Gluc/L. The cycle studies performed with individual substrates in the Ac-SBR were carried out with an initial concentration of: 250 mg Prop/L, 300 mg Acet/L, 220 mg Buty/L or 150 mg Gluc/L. When these substrates were combined, the concentrations were 80 mg Prop/L, 110 mg Acet/L, 110 Buty/L and 90 mg Gluc/L. 2.3. Cycle studies Five cycle studies were carried out in each SBR, being the carbon source used the only difference between them. 5 L of synthetic feed was added as a pulse at the beginning of the cycle. Liquid and solid-phase samples were taken for chemical analysis with sampling intervals of 10 min during the first hour of the anaerobic or aerobic period and each 30 min afterwards. The liquid samples were immediately filtered through disposable Millipore filter units (0.22 mm pore size) for the analysis of phosphate, VFA and glucose. The solid-phase samples were fixed with formaldehyde immediately to stop reaction, and then washed with phosphorus buffer solution (PBS) and subsequently frozen for further analysis of PHA and glycogen. Samples for mixed liquor total suspended solids (TSS) and volatile suspended solids (VSS) were taken at the end of the anaerobic period and at the end of the aerobic period. Cycle studies were carried out once per day and at least three 6-h SBR cycles with normal VFA concentration elapsed between each study. 2.4. Chemical analysis

2. Materials and methods 2.1. Bench-scale reactors 1

Two SBRs monitored (pH, temperature, DO and ORP sensors, from Knick , Germany) and controlled (pH, temperature and DO) were used to enrich the two EBPR microbial populations. All the sensors and actuators were connected to a programmable logic controller (PLC) (SIMATIC S7-226, Siemens1), which was programmed for controlling all the key parameters of the reactors and the influent and effluent pumps. The PLC was linked to a PC running software developed in Visual Basic1 that was used for monitoring the system. Each reactor was

Analyses of phosphate in filtered samples were performed with an Electrophoresis Capillar System (Quanta 4000E CE – WATERS). The electrolyte used was a commercial solution (Ionselect High Mobility Anion Electrolyte). Ammonium was analysed with a continuous flow analyser (CFA) based on potentiometric determination of ammonia. The VFA (propionate, acetate and butyrate) were measured by gas chromatography (GC). A volume of 0.1 mL of 10% phosphoric acid was added to 0.9 mL of filtered sample (Millipore 0.2 mm pore size) and stored at 4 8C. A Hewlett Packard 5890 GC equipped with a HP-7673 column (30 m  0.53 mm  1.0 mm; length  internal diameter  film thickness) and a flame ionisation detector (FID) was used. The

M. Pijuan et al. / Process Biochemistry 44 (2009) 97–105 injector temperature was 260 8C, and a sample volume of 1 mL was injected. The carrier gas was high purity helium at a flow rate of 49 mL/min. The GC worked with a precolumn and the temperature of the column was 240 8C. The run time was 22 min. The software used was Millennium 3.20 Waters. Glucose filtered samples (Millipore 0.2 mm pore size) were measured using a Yellow Spring Instrument (2700 Select). For the solid-phase analysis, 50 mL of mixed liquor samples were withdrawn from the reactor and centrifuged at 5000 rpm. The supernatant was removed and the sludge was washed with 0.1 M PBS. Then, it was centrifuged again, and the pellet was freeze dried after removing the supernatant. Glycogen was determined by a modification of the method of Smolders et al. [16]. An amount of 30 mg of lyophilised sludge samples were digested in a water bath with 0.6N HCl aqueous solution at 100 8C during 6 h. After cooling and filtering through 0.22 mm filter (Millipore), the concentration of glucose was measured using a Yellow Spring Instrument (2700 Select). Triplicates of each sample were done. PHA was measured according to a modification of the method of Comeau et al. [34]. 30 mg of lyophilised sludge samples were digested and methylated with 4 mL of acidified methanol (3% H2SO4) and 4 mL of chloroform during 6 h at 100 8C. Benzoic acid was used as internal standard. The calibration of the method was performed using as standard 3-hydroxybutyric acid and 3-hydroxyvaleric acid copolymer (7:3) (Fluka) for 3HB and 3HV, while 2-hydroxycaproic acid (Aldrich) was the standard for PH2MV. The analyses were performed in a GC system (Hewlett

99

Packard 5890). Triplicates of each sample were done. Finally, TSS and VSS were done according to Standard Methods [35]. 2.5. Microbial analyses Fluorescence in situ hybridisation (FISH) was performed as in Amann [36] with Cy5-labelled EUBMIX probes (for most bacteria [37]) and either Cy3-labelled PAOMIX probes (for ‘‘Candidatus Accumulibacter phosphatis’’, comprising equal amounts of probes PAO462, PAO651 and PAO846 [20]) or Cy3-labelled GAOMIX (for ‘‘Candidatus Competibacter phosphatis’’, comprising equal amounts of probes GAOQ431 and GAOQ989 [27]). Sludge was fixed by 4% paraformaldehyde solution in phosphate buffer 0.03 M. Hybridisation was performed with 35% formamide solution at 46 8C for 2 h. Quantification was carried out as in Pijuan et al. [33].

3. Results and discussion 3.1. Comparison of the microbial community in both SBRs Both SBRs were inoculated with the same sludge from a nonEBPR municipal WWTP with a very low percentage of PAO. FISH

Fig. 1. CLSM micrographs of FISH. (A) Inoculum sludge; (B) sludge present in the Ac-SBR after 90 days of operation; (C) sludge present in the Pr-SBR after 90 days of operation. (a) Sludge hybridised with probe specific for PAO (in pink, Cy3-labelled PAOMIX probes) and probe for bacteria (in blue, Cy5-labelled EUBMIX probes). (b) Sludge hybridised with probe specific for GAO (in pink, Cy3-labelled GAOMIX probes) and probe for bacteria (in blue, Cy5-labelled EUBMIX probes). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 2. (A) Sudan Black stain of the Ac-SBR sludge withdrawn at the end of the anaerobic phase (black granules in a clear or lightly coloured back-ground indicate the presence of PHA). (B) Methylene Blue stain of the Ac-SBR sludge withdrawn at the end of the aerobic phase (a violet colouration against a dark blue background is positive for the presence of polyP). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

was performed in this initial sludge to detect the presence of PAO and GAO (Fig. 1A.a and A.b). A very low concentration (< 1%) of PAO and GAO was detected because of the not very favourable configuration of the WWTP for these microorganisms, a modified Ludzack–Ettinger nitrification–denitrification scheme without anaerobic reactor [38]. After the inoculation, the microbial communities developed in a different way. The Ac-SBR did not achieve a good EBPR microbial population as it did the Pr-SBR, where the stability of the system was higher. The Ac-SBR showed low EBPR efficiency when it reached stable operation, being the net P-removal almost undetectable in the reactor. FISH analysis performed after 90 days of operation (Fig. 1B.a and B.b) showed low abundance of Accumulibacter

(around 5%) and Competibacter (around 5%). Interestingly, a high number of bacteria containing PHA were detected when Sudan Black stain was performed in the Ac-SBR sludge withdrawn at the end of the anaerobic phase (Fig. 2A). That could be explained by the presence of other groups of PAO or GAO not targeted by the FISH probes used in this study. An important amount of not identified filamentous bacteria was observed in this reactor, causing a progressive deterioration in its settleability. Polyphosphate granules were observed in some of these filaments when Methylene Blue stain was carried out in this sludge (Fig. 2B). However, PHA was not detected at the end of the anaerobic period in these filaments, indicating they were not able to store substrate anaerobically (Fig. 2A).

Fig. 3. Sludge from Pr-SBR. (A) Sudan Black stain at the beginning of the anaerobic period. (B) Sudan Black stain at the end of the anaerobic period (black granules in a clear or lightly coloured back-ground indicate the presence of PHA). (C) Methylene Blue stain at the beginning of the anaerobic period. (D) Methylene Blue stain at the end of the anaerobic period (a violet colouration against a dark blue background is positive for the presence of polyP). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Table 1 Substrate-uptake rate and ratios obtained in the cycle studies performed with acetate and propionate in each SBR. Ratios were calculated using the initial and final concentration of each compound in the anaerobic phase. Rates/ratios

Ac-SBR Acetate

Substrate-uptake rate (mmol C/((g VSS) min)) P-release/substrate-uptake (mmol P/mmol C) Glycogen-degraded/substrate-uptake (mmol C/mmol C) PHA-produced/substrate-uptakea (mmol C/mmol C) % PHB % PHV % PH2MV Carbon recovery ratiob (mmol C/mmol C) a b

Pr-SBR Propionate

Acetate

Propionate

0.058

0.017

0.046

0.051

0.107 0.91 1.39 69.9 30.1 0.0 0.73

0.086 0.42 0.74 2.7 97.3 0.0 0.52

0.319 0.63 1.28 89.6 10.4 0 0.81

0.268 0.45 0.64 6.2 53.4 40.4 0.44

PHA (mmol C) = PHB + PHV + PH2MV. PHA-produced/(substrate-uptake + glycogen-degraded).

On the contrary, the Pr-SBR showed a good net P-removal after 15 days of the start-up and increased progressively until stable operation was achieved around 30 days after the start up. Methylene Blue stain of this sludge (Fig. 3C and D) showed an important PolyP storage. FISH analyses performed after 90 days of reactor operation (Fig. 1C.a and C.b) show around 50% of PAOMIXbinding cells (Accumulibacter) and just a few cells (< 1%) of Competibacter. This supports the hypothesis that CompetibacterGAO are less competitive than PAO when propionate is used as substrate [22,24,29]. 3.2. PHA synthesis with single substrates The response of each SBR to different individual substrates was analysed after 90 days of operation. A typical cycle study of each reactor with the default carbon source is shown in Fig. 4. In both cases, the substrate was consumed during the anaerobic period. Phosphorus, PHA and glycogen were also monitored along the cycle. Net P-removal was observed in both systems although the EBPR efficiency in the Pr-SBR was much higher than in the Ac-SBR. PHA synthesis was also corroborated by Sudan Black stain in the Ac-SBR sludge (Fig. 2A) and the Pr-SBR sludge (Fig. 3A and B). It has been hypothesised [30] that PAO may need a period of acclimation to achieve significant VFA uptake and PHA synthesis with other VFA species differing from the one used in cultivation. However, Pijuan et al. [22] found that no acclimation period was necessary between acetate and propionate. Accordingly, the behaviour with a different substrate was firstly tested shifting the carbon source in one cycle study, so the Ac-SBR was supplied with propionate and the Pr-SBR was fed with acetate. Table 1

summarises the rates and ratios obtained in the different cycle studies performed in both SBRs with these substrates. The P-release to substrate-uptake ratio is a good indicative of the activity of an EBPR system. Pr-SBR presented the highest ratio with both VFAs tested compared to the Ac-SBR. This observation agreed with the higher presence of Accumulibacter detected in this reactor (Fig. 1C.a). The Ac-SBR contained the same abundance of Accumulibacter and Competibacter but the ratios obtained when acetate was used as substrate indicate a predominance of the GAO metabolism. This fact suggests that other groups of GAO different from Competibacter were present in this SBR and were contributing to the storage of acetate into PHA. Glycogen requirements for storing propionate as PHA were lower than acetate in both reactors, in accordance with the literature [22–24]. This lower glycogen degradation had a direct effect on the PHA production, which was lower than the experiments performed with acetate. Table 1 shows the total PHA-produced for each substrate and its molar percentages. PHA synthesis was evaluated in terms of PHA (sum of PHB, PHV and PH2MV) produced during the anaerobic phase per substrate consumed (mmol C/mmol C). The results show that PHA composition was strongly influenced by the carbon source and was different for both SBRs. Acetate uptake resulted in the highest PHA synthesis. Similar results were obtained by Satoh et al. [15] and Lemos et al. [9]. It was mainly stored as PHB in the PrSBR, while it was stored as PHB and PHV in the Ac-SBR. Propionate was mainly stored as PHV and PH2MV in the Pr-SBR, while it was mainly stored as PHV in the Ac-SBR. To evaluate the effect of other VFA on PHA synthesis, butyrate was supplied as the only carbon source. The results in Fig. 5 and

Fig. 4. Typical cycle studies performed in the Ac-SBR and in the Pr-SBR with the initial concentration shown in each plot (*, substrate; & , phosphorus; *, glycogen; ! , PHB; ~, PHV).

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Fig. 5. Experimental profiles of different compounds during the anaerobic/aerobic cycle study with butyrate as carbon source with the initial concentration shown in each plot (*, substrate; & , phosphorus; *, glycogen; !, PHB; ~, PHV).

Table 2 show that butyrate was consumed in both reactors but with a lower rate than the other VFA tested. In the Ac-SBR, 0.92 mmol CPHA to mmol C-butyrate was produced being 0.46 in the Pr-SBR. Butyrate was stored mainly as PHB in the Pr-SBR, while it was stored as PHB and PHV in the Ac-SBR and PH2MV was produced in both but with much lower quantities. PHA synthesis was lower than the observed for the same reactor with acetate or propionate. These observations would agree with the literature where some authors suggest that butyrate is less efficient than acetate or propionate due to the lower substrate consumption rate and less PHA synthesis [9]. However, for this carbon source, the gas chromatogram of the analysis of PHA showed another peak. This non-identified peak suggests the synthesis of a different PHA monomer. It appeared in both reactors when butyrate was used and it seemed the main monomer synthesised in the Pr-SBR because of the high peak area detected in the analysis. This peak was not present with the other substrates. Therefore, a proper identification and quantification of this PHA monomer would lead to a higher measured PHA production than previously reported for butyrate. Glucose was also tested in both SBRs (Table 2 and Fig. 6) and although it was consumed during the anaerobic period in both cases, there was low PHA production with this carbon source. The PHA-produced was a mixture of PHB and PHV with a higher content in this last form. The main difference with the VFAs tested was glycogen synthesis in the anaerobic period in addition to the low PHA storage. Moreover, glucose was consumed slower in the Pr-SBR, but higher glycogen synthesis during the anaerobic phase

was observed. However, there was a high aerobic increase of glycogen in the Ac-SBR not observed in the Pr-SBR. Table 3 shows a comparison of the substrate-uptake rate of each specie tested respect the rate obtained with the substrate normally used in each reactor. The uptake rate of propionate and butyrate by the microbial community in Ac-SBR was 70% lower than the rate observed with acetate. Interestingly, glucose was the fastest substrate consumed in this reactor. On the other hand, the microbial community in Pr-SBR was able to uptake acetate almost at the same rate than propionate and it was able to uptake butyrate and glucose but at a lower rate. It needs to be taken into account that the consumption rates of each substrate tested could be different when using sludge previously adapted to glucose or butyrate. However, depending on the length of the adaptation period, a change on the microbial population could occur and therefore, the results obtained with each one of the four substrates could be very different. The low carbon recovery ratio observed for some substrates suggests that, apart from the CO2 production, there are some polymers or subproducts that are not considered in the usual measurements in EBPR systems. This would lead to erroneous calculation of ratios involving for example PHA productivity. 3.3. PHA synthesis with mixed substrates A combination of the substrates was used as a feeding media in a cycle of each SBR. Fig. 7 shows the experimental profiles obtained

Table 2 Rates and ratios obtained in the cycle studies performed with butyrate and glucose in each SBR. Ratios were calculated using the initial and final concentration of each compound in the anaerobic phase. Rates/ratios

Substrate-uptake rate (mmol C/((g VSS) min)) P-release/substrate-uptake (mmol P/mmol C) Glycogen-degraded/substrate-uptake (mmol C/mmol C) PHA-produced/substrate-uptake (mmol C/mmol C) % PHB % PHV % PH2MV Carbon recovery ratio (mmol C/mmol C) a b c d

Ac-SBR

Pr-SBR

Butyrate

Glucose

Butyrate

Glucose

0.017 0.170 0.43 0.92b 46.7 48.9 4.4 0.64c

0.078 0.121 0.12a 0.44 29.9 70.1 0.0 0.77d

0.016 0.217 0.49 0.46b 78.5 16.4 5.1 0.31c

0.022 0.053 0.28a 0.36 44.1 55.9 0 0.64d

When glucose was used as substrate, glycogen was synthesised not degraded. When butyrate was used as substrate, an unknown PHA monomer was synthesised but was not quantified. PHA-produced/(substrate-uptake + glycogen-degraded). (PHA-produced + glycogen-produced)/substrate-uptake.

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Fig. 6. Experimental profiles of different compounds during the anaerobic/aerobic cycle study with glucose as carbon source with the initial concentration shown in each plot (*, substrate; & , phosphorus; *, glycogen; !, PHB; ~, PHV).

Fig. 7. Experimental profiles of different compounds during the cycle study performed with a mixture of four substrates (*, acetate; *, propionate; !, butyrate; ~, glucose; & , phosphorus).

for the substrates and phosphate analysed, while Table 4 shows the different rates and ratios obtained. Pr-SBR showed a better anaerobic substrate-uptake, obtaining a total depletion of all substrates in 60 min. On the other hand, AcSBR did not obtain total depletion of acetate and butyrate during the 120 min of anaerobic phase. The main differences observed in substrate-uptake rate with respect to the cycle studies with single substrate are the lower acetate uptake rate in both SBR and the lower glucose uptake rate in the Ac-SBR. Dai et al. [39] found that acetate utilization rate decreased dramatically in the presence of propionate in an enriched D. vanus related GAO community. This could explain the decrease observed in the acetate uptake rate in the mixed substrate experiment in both SBRs. The other substrateuptake rates were similar to the ones previously obtained. Table 3 Substrate-uptake rate of each substrate tested in mmol C/((g VSS) min), expressed as a percentage of the substrate-uptake rate used to enrich the microbial community in each reactor.

Acetate Propionate Butyrate Glucose

Ac-SBR, % of acetate uptake rate

Pr-SBR, % of propionate uptake rate

100 30 29 135

90 100 31 43

The values obtained in the P-release vs. substrate-uptake ratio (0.120 and 0.199 mmol P/mmol C, respectively, for Ac-SBR and PrSBR) were similar to the ones calculated considering the P-release vs. substrate-uptake ratios obtained in the individual substrate experiments and multiplying thereby the amount of each substrate taken up (0.119 and 0.224 mmol P/mmol C, calculated values). Therefore, it seems that for both SBR no synergic effect for mixed substrates exists in this ratio, i.e. the energy requirements for PHA storage depend uniquely on the amount of each substrate taken up. With respect to the aerobic P-uptake, net P-removal was slightly observed in the Pr-SBR but not in the Ac-SBR. The cycle studies carried out with mixed substrate presented the lowest ratios of glycogen consumed to substrate-uptake for any VFA tested. This could be because when glucose was consumed, glycogen was formed and although the other substrates implied glycogen degradation, the overall glycogen consumption was lower in this case. The different types of PHA synthesised could also contribute to the reduction of the reducing power requirements, as not all the PHA monomers require the same amount of reducing power to be synthesised. Table 4 also summarises the percentages of the different PHA monomers synthesised in the mixed substrate cycles. The productivity obtained was not very high and only PHB and PHV were quantified, despite that another peak was detected as when butyrate was used as sole carbon source. PH2MV was not detected

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Table 4 Rates and ratios obtained in the cycle studies performed with a mixture of substrates in each SBR. Ratios were calculated using the initial and final concentration of each compound in the anaerobic phase. Rates/ratios

Ac-SBR

Pr-SBR

Substrate-uptake rate (mmol C/((g VSS) min))

[0.019 (Prop) 0.009 (Buty)], [0.010 (Acet) 0.019 (Gluc)]

[0.057 (Prop) 0.020 (Buty)], [0.015 (Acet) 0.018 (Gluc)]

P-release/substrate-uptake (mmol P/mmol C) Glycogen-degraded/substrate-uptake (mmol C/mmol C) PHA-produced/substrate-uptake (mmol C/mmol C) % PHB % PHV % PH2MV Carbon recovery ratiob (mmol C/mmol C)

0.120 0.26 0.67a 21 79 0 0.53

0.199 0.29 0.45a 64 36 0 0.35

a b

An unknown PHA monomer was synthesised, probably because of the presence of butyrate, but was not quantified. PHA-produced/(substrate-uptake + glycogen-degraded).

in the mixed substrate experiments. Ac-SBR provided 21%PHB– 79%PHV and Pr-SBR 64%PHB–36%PHV, showing the different utilization of substrates by each microbial community.

de Educacio´n y Ciencia’’, project CTM2004-02569/TECNO. The authors are members of the GENOCOV group (‘‘Grup de Recerca Consolidat de la Generalitat de Catalunya’’).

4. Conclusions Appendix A. Nomenclature The main conclusions obtained from this study are as follows:  The SBR working with propionate as carbon source presented the best net P-removal capacity in the system. FISH analyses demonstrated that a higher amount of PAO was present in the Pr-SBR sludge and almost no Competibacter-GAO was detected.  PHA composition was different for the sludge of both SBRs and was strongly influenced by the carbon source.  The highest PHA production per substrate consumed for both AcSBR and Pr-SBR was obtained using acetate as carbon source. Different composition of PHA was measured: 70%PHB–30%PHV for Ac-SBR and 90%PHB–10%PHV for Pr-SBR.  Propionate used as carbon source produced PHA with 97% of PHV in the Ac-SBR. Pr-SBR with propionate produced a copolymer composition of 6%PHB–53%PHV–40%PH2MV. This was the only PHA obtained in this study with a significant amount of PH2MV.  Butyrate used as carbon source produced more PHA in the Ac-SBR than in the Pr-SBR. The composition obtained in the AcSBR was 47%PHB–49%PHV–4%PH2MV. However, another nonidentified monomer unit was detected in the chromatographic analysis.  Glucose used as carbon source produced PHB–PHV copolymer and glycogen. It gave the lowest ratio PHA-produced/substrateuptake of all the carbon sources tested.  The productivity obtained using mixed carbon sources was not very high and PHB and PHV were mainly obtained. Ac-SBR provided 21%PHB–79%PHV and Pr-SBR 64%PHB–36%PHV. The non-identified monomer unit attributed to PHA storage from butyrate was also detected in the chromatographic analysis.  The results of PHA composition, where some monomers not always analysed have an important contribution, show that not considering these PHA polymers for some substrates can lead to erroneous ratios of PHA-produced/substrate-uptake, P-release/ PHA-produced or P-uptake/PHA-oxidized.  The low carbon recovery ratio for some substrates suggests that, apart from the CO2 production, there are some polymers or subproducts that are not considered in the usual measurements in EBPR systems. Acknowledgments M. Pijuan acknowledges ‘‘Generalitat de Catalunya’’ for her PhD grant FI0036. This work was supported by the Spanish ‘‘Ministerio

Acet Ac-SBR ATU Buty DO EBPR FISH GAO Gluc HRT ORP PAO PE PHA PHB PHV PH2MV PLC polyP PP Prop Pr-SBR SBR SRT TSS VFA VSS WWTP

acetic acid SBR developed with acetate as sole carbon source allylthiourea butyric acid dissolved oxygen enhanced biological phosphorus removal fluorescence in situ hybridisation glycogen accumulating organisms glucose hydraulic residence time oxidation–reduction potential polyphosphate accumulating organisms polyethylene polyhydroxyalkanoate polyhydroxybutyrate polyhydroxyvalerate polyhydroxy-2-methylvalerate programmable logic controller polyphosphate polypropylene propionic acid SBR developed with propionate as sole carbon source sequencing batch reactor sludge residence time total suspended solids volatile fatty acid volatile suspended solids wastewater treatment plant

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