Accepted Manuscript

Title: Production of polyhydroxyalkanoates in open, mixed cultures from a waste sludge stream containing high levels of soluble organics, nitrogen and phosphorus Authors: Fernando Morgan-Sagastume, Anton Karlsson, Peter Johansson, Steven Pratt, Nico Boon, Paul Lant, Alan Werker PII: DOI: Reference: To appear in: S0043-1354(10)00430-6 10.1016/j.watres.2010.06.043 WR 8098 Water Research

Received Date: 5 January 2010 Revised Date: 11 May 2010 Accepted Date: 15 June 2010

Please cite this article as: Morgan-Sagastume, F., Karlsson, A., Johansson, P., Pratt, S., Boon, N., Lant, P., Werker, A. Production of polyhydroxyalkanoates in open, mixed cultures from a waste sludge stream containing high levels of soluble organics, nitrogen and phosphorus, Water Research (2010), doi: 10.1016/j.watres.2010.06.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 *Corresponding author. Tel.: +46 46182159; fax: +46 46133201. E-mail address: fernando.morgan@anoxkaldnes.com (F. Morgan-Sagastume). Fernando Morgan-Sagastumea*, Anton Karlssona, Peter Johanssona, Steven Prattb, Nico Boonc, Paul Lantb, and Alan Werkera
a

Production of polyhydroxyalkanoates in open, mixed cultures from a waste sludge stream containing high levels of soluble organics, nitrogen and phosphorus

AnoxKaldnes AB, Klosterngsvgen 11A, 226 47 Lund, Sweden Advanced Water Management Centre, The University of Queensland, Level 4, Gehrmann

Laboratories Building (60), Brisbane QLD 4072, Australia

c

Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure

Links 653, B-9000 Ghent, Belgium

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2 Keywords Polyhydroxyalkanoates (PHAs), fermentation, thermal hydrolysis, waste activated sludge, PHA production ABSTRACT In this study, the production of polyhydroxyalkanoates (PHAs) from waste activated sludge (WAS) was evaluated. PHAs were produced from fermented WAS pretreated via highpressure thermal hydrolysis, a stream characterised by high levels of nutrients (approximately 3.5 g N L-1 and 0.5 g P L-1) and soluble organics. PHA-storing organisms were successfully enriched at high organic loading rates (6 gCODsol L-1d-1) under aerobic dynamic feeding in sequencing batch reactors at a sludge retention time of 6 d with a short feast length less than 20% of the cycle, and a maximum substrate concentration during feast of 1 g CODVFA L-1. The biomass enrichment, characterised by a decrease in species evenness based on Lorenz curves, provided a biomass that accumulated 25% PHA on a dry-biomass basis with yields on VFA of 0.4 Cmol Cmol-1 in batch tests. The PHA consisted of ~70 mol% 3-hydroxybutyrate and ~30 mol% 3-hydroxyvalerate, and presented high thermal stability (Td = 283-287C) and a molecular mass ranging from 0.7 to 1.0x106 g mol-1. Overall PHA storage was comparable to that achieved with other complex substrates; however, lower PHA storage rates (0.04-0.05 CmolPHA-1 CmolX-1 h-1) and productivities (3-4 Cmmol PHA L-1h-1) were probably associated with a biomass-growth and high-respiration response induced by high levels of non-VFA organics (40-50% of CODsol in feed) and nutrients. PHA production is feasible from pretreated WAS, but the enrichment and accumulation process require further optimisation. A milder WAS pretreatment yielding lower levels of non-VFA organics and readily available nutrients may be more amenable for improved performance.

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1. Introduction Polyhydroxyalkanoates (PHAs) are a family of biodegradable polymers with a broad range of applications (Philip et al., 2007). Particularly, the copolymer of 3-hydroxybutyrate and 3hydroxyvalerate, P(3HB-3HV), has a high potential to substitute conventional plastics since it has thermoplastic properties comparable to those of petroleum-based polyolefins, i.e. polypropylene and polyethylene (Lee, 1996). PHAs are intracellular carbon and energy storage molecules that can be produced by many bacteria aerobically under external/internal growth-limiting conditions (Sudesh et al., 2000) or by phosphorus- or glycogen-accumulating bacteria under alternating anaerobic-aerobic conditions (Mino et al., 1998). Industrial PHA production is based on pure culture fermentation and has high operating costs associated with renewable but refined substrates and sterilisation (Lee, 1996). These high costs have impeded PHA commercial development (Reis et al., 2003), despite current commercial PHA availability (Jacquel et al., 2008). Open, mixed cultures represent an economic alternative strategy for producing PHAs from renewable, low-cost substrates (Dias et al., 2006, Serafim et al., 2008a). Mixed-culture PHA production relies on enriching PHA-storing bacteria by applying an environmental selective pressure via dynamic conditions (Dionisi et al., 2004a, Serafim et al., 2004). Under dynamic conditions with respect to carbon substrate, i.e., aerobic dynamic feeding (ADF) or feast-famine, bacteria are subjectedto alternating high (feast) and low/none (famine) substrate concentrations. Since in mixed cultures volatile fatty acids (VFAs) are directly converted into their respective acyl-CoA and further converted into PHAs (Luengo et al., 2003), acidogenic fermentation has been widely applied in mixed-culture PHA production (Dias et al., 2006). Consequently, a 3-stage process (Dionisi et al., 2004a) has preferentially been used: (i) acidogenic fermentation for VFA production, (ii) enrichment of PHA-storing organisms in

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sequencing batch reactors (SBRs), (iii) Batch PHA accumulation in the selected biomass. The PHA product is extracted and purified in a final fourth stage. Mixed culture PHA production offers the possibility of material recycling by using renewable organics from wastes and industrial effluents. Research studies have demonstrated the technical feasibility of producing PHAs in mixed cultures using single and simple VFA mixtures (Dionisi et al., 2004a, Dionisi et al., 2005a) and more complex substrates, including several fermented waste streams: food waste (Rhu et al., 2003), olive oil (Dionisi et al., 2005b, Beccari et al., 2009) and palm oil (Md Din et al., 2006) mill effluents, sugar-cane molasses (Albuquerque et al., 2007), paper mill effluent (Bengtsson et al., 2008), fruit (Gurieff, 2007) and tomato (Liu et al., 2008) cannery effluents, and municipal sludge (Coats et al., 2007, Mengmeng et al., 2008). Nevertheless, only a few studies have systematically evaluated both the enrichment of PHA-accumulating organisms and PHA accumulation with the same fermented effluent: sugar cane molasses (Albuquerque et al., 2007), cannery effluents (Gurieff, 2007), paper mill wastewater (Bengtsson et al., 2008), and olive mill effluent (Beccari et al., 2009). Assessing the technical feasibility of producing PHAs from complex waste streams entails evaluating the effects of non-VFA organic matter and high levels of organics and nutrients on the phenotypic environment inducive to PHA production, aspects which may be relevant to several organic wastes of interest. Primary and waste activated sludge (WAS) have only recently been considered as substrates for PHA production, despite being abundant sources of waste organics inducing high treatment and disposal costs. Fermented primary sludge has been widely considered in connection with two-phase anaerobic digestion and nutrient removal, but it has also recently shown potential as substrate in PHA production (Coats et al., 2007, Gurieff, 2007). PHA production from fermented waste organics may represent not only an alternative, but also a complementing process approach to anaerobic digestion from similar wastes. Fermented

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WAS has been successfully used to accumulate PHA in an unacclimated activated sludge biomass (Mengmeng et al., 2008); however, both enrichment and accumulation have not yet been tested with fermented WAS. WAS pretreated with high-pressure thermal hydrolysis (HPTH) has been little characterized and has never been studied for the production of PHAs. HPTH-prehydrolysed sludge displays high fermentability potential and organic strength, which increases the economic feasibility of PHA production (Gurieff & Lant, 2007); however, the associated high organic levels may compromise the ADF regime during biomass selection and high nutrient levels may promote a growth rather than an accumulation response during PHA accumulation. Therefore, the aim of this study was to evaluate the technical potential for the enrichment of PHA-storing organisms and the PHA-storage capacity of the enriched biomass using fermented HPTH sludge with inherently high organic and nutrient contents. In conjunction, the stability and consistency of the biomass feast-famine response, PHA-accumulating capacity, and PHA properties were studied.

2. Materials and methods Two sets of experiments were conducted, in which the enrichment of PHA-accumulating organisms was conducted in two parallel, sequencing batch reactors (SBRs) operated under ADF using as substrate fermented sludge pretreated via HPTH. In the first experiments, here in referred to as organic loading rate (OLR) experiments, the SBRs (SBRs 1 and 2) were operated for 90 days at two differently high OLRs. The second set of experiments, here in referred to as stability experiments, was conducted to demonstrate the stability of the enrichment reactors and further test improvements in the operating conditions, such as increased aeration during the feast. In this case, the SBRs (SBRs 2A and 2B) were operated for 86 days at a loading rate selected based on the results from the first set of experiments. In both sets of experiments, the PHA accumulation capacity of the enriched biomass was

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evaluated aerobically in fed-batch with consecutive pulses of substrate controlled by respirometry. Mixtures of synthetic VFAs without nutrients were used for reference accumulations without the potential effects of nutrients. In the stability experiments, a twoVFA mixture was used to further simplify the evaluation of accumulation performance with less nutrient addition. Details for each specific set of experiments are given below according to the outlined section. 2.1. Fermented HPTH sludge used as substrate The feed used in both enrichment experiments was sludge pretreated with HPTH via the Cambi process and fermented semi continuously to produce VFAs. The Cambi process desintegrates the sludge organic material and lyses cells by subjecting the sludge to highpressure steam and temperatures in a 3 step process described elsewhere (Kepp et al., 2000). The sludge feed for the OLR experiments was first collected from the Cambi plant at the Fredericia Central Wastewater Treatment Plant (Fredericia, Denmark), and then fermented and daily centrifuged (4000xg for 12 min, twice with in-between decanting), stored for 2 days at 4C and finally at -4C for 3 months. For the stability experiments, HPTH sludge was collected from the Cambi plant at Nstved's Central Treatment Plant (Nstved, Denmark) and pre-centrifuged (4000xg, 8min) before fermentation for most of the operating period. The fermented sludge was also centrifuged and stored at -4C before use. The supernatants from fermented HPTH sludge from Fredericia and Nstved are further referred to as fermented HPTH centrate, and their characteristics are presented in Table 1. The fermentation was conducted semi-continuously at 42C, OLRs of 20-30 g soluble COD (CODsol) L-1d-1, and 1-2 d retention time with yields of 0.4-0.6 gCODVFA.g CODsol-1; more fermentation details and results are provided elsewhere (Morgan-Sagastume et al., 2009). The sludge treated at the Fredericia plant corresponded to a mixture of primary and municipal/industrial WAS and to municipal WAS at the Nstved plant.

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2.2. Reactors for the enrichment of PHA-accumulating organisms In both sets of experiments, the SBRs were operated in cycles consisting of feeding, aeration (feast and famine) and wasting, and regulated via a programmable logic controller. The reactors were inoculated with municipal activated sludge (~3 g TSS/L) from Kllby and Klagshamn municipal wastewater treatment plants (Lund and Malm, respectively, Sweden). The SBRs were glass made and operated at 35C, which was regulated via water jackets connected to water baths (Landa E100). Continuous mixing was provided by magnetic stirring plates (400 rpm) during the whole cycle. Aeration was manually controlled and supplied by air pumps (~10-20 L/min) connected to stone air diffusers. To avoid water evaporation from the reactors, air was prehumidified by bubbling it into two consecutive, heated water bottles at 36-38 and 35C. The SRT was similar to the HRT, and it was controlled by the amount of solids wasted via the mixed liquor volume directly collected from the reactors every cycle. Both pH and DO (pH/Oxi 340i, WTW Wellheim) were monitored during some cycles; pH was not controlled. SBR performance was monitored at least in triplicate during different cycle studies under the same operating conditions. Although similar SBR operating conditions were pursued during the OLR and stability experiments, slight differences in feed pH, innocula, biomass history and time of acclimation occurred. OLR experiments. SBRs 1 and 2 had an operating volume of 0.75 L and their feed contained 1 % v/v antifoaming agent (Dow Corning, Antifoam RD Emulsion). The two SBRs were first operated at an OLR of 12 g COD L-1 d-1 (12.01.2 and 12.31.3 g COD L-1 d-1, respectively). The SBR cycle length was 4 h, including 2.5 min feeding and wasting periods. After 53 days of operation, the OLR of SBR 2 was reduced to 6 g COD L-1 d-1 (6.8 0.6 g COD L-1 d-1). The reduction in OLR was achieved by increasing the SBR cycle length to 8 h, which resulted in an extension of the famine phase of each cycle. SBR 1 was kept at 12 g COD L-1 d-1 as a reference although an erroneous short operation of 4 days at 6 g COD L-1 d-1

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occurred (Days 53-57). The SRTs for the 2 different OLRs (12 and 6 g COD L-1d-1) averaged 3.10.3 and 6.40.8 days, respectively, and the same maximum COD concentration of 1 g CODVFA L-1 was maintained during the different OLRs. Stability experiments. Two SBRs were operated targeting the best performance achieved with the conditions associated with the OLR of 6 g COD L-1 d-1 (OLR experiments). During the early period of operation, the SBRs experienced irregular operation due to initial trouble-shooting without optimised feeding and wasting control. These problems were overcome allowing for relatively stable operation from day 42 onwards. The SBRs had an average working volume of 2.60.3 L (SBR2A) and 2.50.3 L (SBR2B), and were equipped with foam disrupters, which allowed for intensive aeration and a DO of 3 mg L-1 during feast. The foam disrupters consisted of three 8.5 cm nylon whiskers intercalated at the lower end of a stainless steel shaft connected to a rotor on top the reactors' lids. The cycle length of 8 h was divided into non-aerated feeding (~7 min), aerobic feast and famine (7.75 h) and nonaerated wasting (~8 min). The wasting time included 5 min of only stirring for allowing level stabilisation and actual 2.5 min of pump wasting; stirring was continuous during the cycle. The HRT=SRT averaged 6.21.1 h and 6.00.8 h for SBR 2A and 2B, respectively. The organic loading rate to the SBRs was approximately 3 g CODVFA L-1d-1 and 7 g COD L-1d-1, targeting a maximum CODVFA concentration in a cycle of 1 g L-1. 2.3. PHA accumulation in batch Biomass enriched in PHA-storing organisms was harvested at the end of 3 consecutive SBR cycles, mixed, and its capacity to accumulate PHA was tested in fed-batch by subjecting the biomass to periodic substrate dosing. The accumulation tests were conducted in 2 parallel reactors with an initial working volume of 0.5 L under aerobic conditions at 35C. Prehumidified air was supplied by an air pump through a glass diffuser, the temperature was controlled via water jackets connected to water baths, and mixing was magnetically provided

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(400 rpm). Air flow rates, DO levels, and pH were monitored. DO levels were maintained above 1-2 mg L-1 to avoid oxygen limitation during the batch tests. The mixed liquor was first aerated until constant pH and DO levels were achieved (<30 min). Substrate feed aliquots were dosed at fixed volumes via a diaphragm pump (Grundfos DME) targeting a specific maximum substrate concentration based on a multiple-spiking respirometric control approach. The pH of the fermented HPTH centrate was close to 6.3 (Table 1) and, therefore, the pH of the synthetic substrate solutions was adjusted to 6.3 with 0.1 N NaOH. Foaming was controlled with few drops of antifoaming agent (Dow Corning, Antifoam RD Emulsion); however, foaming limited the levels of substrate provided via each spike since the higher aeration rates required to avoid oxygen limitation at higher substrate levels would have led to excessive foaming and biomass loss. The accumulation tests were run for 6-7 hours. OLR experiments. The biomass harvested from SBRs 1 and 2 was diluted with tap water to a volatile suspended solids (VSS) concentration of 1.8-3.3 g VSS L-1 and an initial volume of 0.48 L. Substrate dosing targeted a maximum substrate concentration of 400 mg CODVFA L-1 in the reactors. Feed pulsing was controlled by the DO trend after each substrate injection based on the DO upswing associated with substrate depletion. The batch accumulations were conducted with 3 substrates: fermented HPTH centrate, a synthetic feed with a similar VFA composition as the fermented HPTH centrate (5.2 g acetic acid L-1, 2.2 g propionic acid L-1, 0.7 g iso-butyric acid L-1, 2.1 g butyric acid L-1, 1.6 g iso-valeric acid L-1, 0.4 g valeric acid L-1 and 0.1 g caproic acid L-1), and a synthetic feed containing NH4+ and PO43- to a similar extent as the fermented HPTH centrate (2.5 g NH4Cl-N L-1 and 0.55 g K2HPO4-P L-1). The total volumes of substrate added ranged from 0.150 to 0.250 L among tests. Stability experiments. Volumes of biomass from SBRs 2A and 2B were also tested (0.5, 0.37 or 0.225 L), all supplemented to the working volume with tap water. Substrate

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pulses consisted of an acetic-propionic acid mixture (15 g CODVFA L-1 pH~6.3; 70:30 on COD basis) targeting a maximum substrate concentration of 100 to 200 mg CODVFA L-1 in the reactors. N and P were in excess ranging within 0.22-0.57 g NH4+-N L-1 and 0.09-0.22 g PO43--P L-1. Feed pulses were delivered manually based on a DO increase, and the total volumes of substrate added ranged from 0.050 to 0.100 L. pH ranged from 6.9 to 8.7. 2.5. Analytical techniques Process performance during given cycles in the SBRs and PHA accumulation tests was evaluated by measuring substrate uptake, PHA production, nutrient consumption, and solids levels. One biomass sample was used for measuring TSS and VSS based on standard methods (APHA et al., 2005). A second sample was filtered through a Munktell MGA micro glass fibre paper (1.6 m), and the filtrate was analysed with Hach-Lange cuvette kits for soluble COD (CODsol, kit # LCK114), total organic carbon (TOC, LCK381, LCK386), total inorganic carbon (TIC, LCK381), NH4+-N (LCK303 and 304), and PO43--P (LCK350). A Hach-Lange LT100 incubation block and a Xion 500 Hach-Lange spectrophotometer were used. Proper dilutions were conducted with distilled water. Temperature (ethanal), DO, pH (pH/Oxi 340i, WTW; Endress+Hauser Liquisys M, pH=4 and 7, Reagecon standards) were monitored inside the reactors. VFAs (acetic, propionic, butyric, iso-butyric, valeric, iso-valeric, and caproic acids) were analysed by gas chromatography (GC). An aliquot of the filtrate prepared as above was further centrifuged at 12000xg for 12 min. One-ml aliquots were stored at -20C in 2 mL GC vials with a Teflon lined screw cap for later analysis as per (Bengtsson et al., 2008). A known mixture of VFAs with their respective GC response factors was used as standard. The detection limit for each VFA was ~100 mg COD L-1. The PHA and polysaccharide contents in the biomass were measured from biomass samples (5-12 mL) previously centrifuged (3000xg, 5 min), decanted and stored at -20C.

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The PHA in the biomass was subjected to acid alcoholysis (butanol and HCl) followed by hexane extraction following (Werker et al., 2008), except that the samples were centrifuged at 3000xg for 20 min. Glucose and P(3HB) (Aldrich 36,350-2) were used as standards for polysaccharides and 3HB, respectively. P(3HB) was also used for 3HV standard with a response factor of one. After extraction, the organic phase was analysed in a Varian 3800 GC equipped with 2 parallel channels with FID and a Saturn 2000 GC/MS/MS detector (VF-1 ms columns 30 m x 0.25 mm, 1m thickness) (Werker et al., 2008). Polymer extraction and molecular and thermal characterization are detailed elsewhere (Bengtsson et al., 2010). Briefly, a chloroform-based method was used to extract PHA from biomass samples containing nominally 100 mg PHA, with methanol as precipitation agent. Weight average molecular and number average molecular weights and polydispersity indices were determined by size exclusion chromatography (SEC, Viscotek VE 1122). Glass transition temperature (Tg), melting temperature (Tm), and melting enthalpy (Hm) were analysed by differential scanning calorimetry (DSC, TA Instruments Q1000). 2.6. Microbial community analysis The microbial diversity was analysed in 7 biomass samples (within days 49 and 86) from each SBR during the stability experiments. Total DNA was extracted (Boon et al., 2002) and the crude extract (100 L) purified using Wizard PCR preps (Promega, Madison, Wis.) and stored at -20C. The extracted DNA (1L) was amplified by PCR with the bacteria specific 16S rRNA forward primer 338f (ACT CCT ACG GGA GGC AGC AG) containing a 40-base GC clamp and the reverse primer 518r (ATT ACC GCG GCT GCT GG) (Muyzer et al., 1993). PCR products were subjected to DGGE for 17 h at 38 V; 8 % w/v polyacrylamide gel with 45 - 60 % denaturing gradient in which 100 % denaturant contains 7 M urea and 40 % formamide (Boon et al., 2002). After electrophoresis, the gels were stained with SYBR Green I (1:10000 dilution; FMC BioProducts, Rockland, Maine) and photographed. Cluster 11

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analysis (WARD algorithm) of the DGGE patterns was performed with the Bionumerics software 2.0 (Applied Maths, Kortijk, Belgium). The ecological interpretation of the molecular data was conducted as per (Marzorati et al., 2008). The range-weighted richness (Rr) value is the total number of bands multiplied by the percentage of denaturing gradient needed to describe the total diversity of the sample analysed, according to the formula Rr = (N2 x Dg), where N is the total number of bands in the pattern, and Dg the denaturing gradient comprised between the first and the last band of the pattern; the functional organization concept is derived from the graphical representation of the structure of a bacterial community (species distribution) according to Lorenz evenness curves, as previously described (Mertens et al., 2005;). The evenness was described by the Gini coefficient (value between 0-1) calculated as twice the area above the Lorenz curve (Wittebolle et al., 2009). 2.7. Calculations The PHA content (3HB and 3HV) in the biomass was calculated as the fraction of g PHA per g TSS. The mass of polymer was converted to Cmol basis by using the following monomeric molecular weights: 86.1 g 3HB mol-1 and 100.13 g 3HV mol-1. PHA yields from accumulation tests were calculated by dividing the amount of PHA formed by either the total VFAs consumed (YP/VFA, Cmol PHA Cmol VFA-1) or the total organic C substrate consumed and measured as TOC (YP/S, Cmol PHA Cmol substrate-1). Other yields from accumulation tests were determined based on the amount of glucose formed per total organic C substrate consumed for the yield of polysaccharides (YPS/S), and based on the amount of active biomass formed per total organic C substrate consumed for the yield of active biomass (YX/S). The active biomass (X, Cmol) was estimated as X=VSS-PHA-PS, where PS corresponds to polysaccharides measured as glucose. A standard composition of the biomass, C5H7NO2, was used. The specific PHA production rates (qp, CmolPHA CmolX-1h-1) and specific VFA uptake rates (-qVFA, CmolVFA CmolX-1h-1) were calculated from the linear regression of

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PHA produced or VFA consumed divided by the corresponding active biomass (X) versus time, respectively. The performance parameters, such as productivities, calculated from the batch accumulation tests considered the dilution effects by substrate addition and volume decrease by sampling.

3. RESULTS 3.1. OLR experiments 3.1.1. Biomass enrichment performance under an OLR of 12 g COD L-1 d-1 Since the start, the biomass displayed a feast-famine response during the cycles at an OLR of 121 (n=9) g COD L-1 d-1 in both SBRs. Feast was defined as the period with VFA substrate availability and high respiration rates associated with DO levels below 1 mg O2 L-1. Famine was defined as the phase with higher DO levels in accordance to lower respiration rates associated with VFA substrate limitation. Immediately after feeding, the DO in the reactor dropped to close to zero mg L-1. DO started increasing at the end of the feast when VFAs became limiting, reaching their lowest concentrations in the mixed liquor. DO further increased to 7 mg L-1 during the famine (Fig. 1A). The length of the feast among different cycles varied approximately from 26 to 37% [306%, (n=3) in both SBRs] of the total cycle time (62-88 min). The VFAs were consumed at an almost constant rate (0.03-0.1 CmolVFA CmolX-1 h1

) during the feast and were depleted by the start of the famine (Fig. 1A). The CODsol also

decreased during the feast reflecting mostly VFA consumption; however, the CODsol continued to decrease slightly during the famine achieving final values of 10.20.8 (n=11) and 111 (n=26) gCODsol L-1 in each SBR, respectively. Approximately 45% of the total non-VFA COD fed per cycle was consumed (0.5 g of non-VFA COD L-1 per cycle), mostly during the famine period.

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N and P were consumed during both feast and famine (Fig. 1a). Approximately 50% of the consumed NH4+-N per cycle (1.9 g N L-1) was consumed during the feast and high levels of N [0.540.10 (n=11) and 0.590.12 (n=26) gNH4+-N L-1] and P [0.320.03 (n=9) and 0.320.02 (n=22) g PO43--P L-1] remained at the end of the famine. The biomass PHA concentration increased during the feast (Fig. 1A), reaching a maximum (0.01-0.03 g PHA g TSS-1) just before the start of the famine in agreement with the decrease in VFAs in the same period. During the famine, the biomass PHA content decreased to 0.008-0.002 g PHA g TSS1

, suggesting that not all the stored PHAs were degraded; only 41-44 % of the total stored

PHAs after the feast were degraded. Overtime, the PHA storage response of the biomass was lost during the feast, as indicated by a decrease in the biomass PHA content at the end of the feast from 0.03 to below 0.01 g PHA gTSS-1 (Fig. 2).

3.1.2. Biomass enrichment performance after changing operating conditions to an OLR of 6 g COD L-1 d-1 To stimulate a greater PHA-accumulating response of the biomass during the feast, the cycle length and SRT were increased by maintaining the same feed volume and maximum substrate COD per cycle constant, but decreasing the OLR to 6 [6.80.6 (n=5)] g COD L-1 d-1. The increase in cycle length from 4 to 8 h led to a decrease in the ratio of the feast compared to the total cycle length from 27-31% to 14-17%; however, the feast length remained unchanged (~1-1.3 h) (Fig. 1B). The operating conditions associated with the lower OLR led to a more defined feastfamine response of the biomass in the SBR in terms of VFA substrate consumption and PHA cycling (Fig. 1); however, increased non-VFA C occurred during the extended famine. Similar to the performance under the previous conditions, the VFAs were consumed at an almost constant rate (0.04 Cmol VFA Cmol X-1 h-1) during the feast and were completely

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consumed by the start of the famine (Table 2). During the longer famine, however, the final CODsol concentration decreased from 10.20.8 (n=11) to 8.10.3 (n=5) g L-1 (Fig. 1B), suggesting an increased consumption of non-VFA COD during the famine since the fed C substrate per cycle remained constant during the entire experiment. At least 2.1 g L-1 more non-VFA COD was degraded per cycle during the famine phase at the lower OLR conditions. The solids levels in the SBRs decreased from 11.50.7 (n=8) g TSS L-1 (84% VSS) to 9.80.5 (n=4) gTSS L-1 (79% VSS). A slight increase in N consumption per cycle from 76 to 80% was measured at the lower OLR, which coincided with the higher COD degradation per cycle, compared to those at high OLR. Nevertheless, the fraction of total NH4+-N removed during the feast decreased from 50 to 20-40%. The PHA-storage response of the biomass recovered after changing operating conditions. An extended PHA consumption during the famine and increased PHA accumulation during the feast were clearly measured at the lower OLR (Figs. 1 and 2). At the OLR of 6 g COD L-1 d-1, 78-91 % of the stored PHAs during the feast were consumed during the famine in comparison to only 41-44 % at the higher OLR conditions. The PHA-storage yield based on VFAs during the feast also increased from 0.14-0.26 Cmol Cmol-1 at the high OLR to 0.22 and 0.43 Cmol Cmol-1 at the low OLR (Table 2). By measuring the biomass PHA content at the end of the feast over time (~1 h after feeding), an increase in biomass PHA content was measured from 0.01 to 0.04 g PHA g TSS-1 under the low OLR conditions (Fig. 2).

3.1.3. PHA accumulation in the selected biomass In general, the biomass selected under the conditions associated with the OLR of 6 g COD L-1 d-1 displayed a higher PHA storage capacity (higher YP/VFA) than the biomass selected under an OLR of 12 g COD L-1 d-1 (Table 3). Only in PHA accumulations with the VFA mixture

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without nutrients, the YP/VFA from the high-OLR-selected biomass was high and comparable to those obtained with the biomass selected under a low OLR. The YP/VFA and biomass PHA content from the low-OLR-selected biomass were similar independent from the feed used (Table 3) and N/P content. In contrast, the YP/VFA and biomass PHA content from the highOLR-selected biomass were hindered by the presence of nutrients and/or non-VFA C substrates (fermented HPTH centrate and VFAmix with N/P). Not only PHAs but also polysaccharides were produced from the C substrates fed during the PHA accumulation tests (YPS/S, Table 3). Overall, high polysaccharide yields (0.24-0.32 Cmol Cmol-1) were associated with low PHA storage yields (YP/VFA=0.18-0.25 and YP/S=0.09-0.23) in specific tests; however, no trends between polysaccharide content and biomass type or feed were observed. The polymer produced by both types of selected biomass was composed of 3HV and 3HB (Table 3); however, the 3HV content of the polymer produced by the biomass enriched under conditions associated with the low OLR was higher (30-48% 3HV) than that of the other biomass (16-17% 3HV). Lower biomass growth was associated with accumulations conducted with nutrient limitation (VFA mix, Table 3) and the highest growth with accumulations using fermented HPTH sludge, which contained high N/P levels and non-VFA COD available. Minimum DO profiles during the accumulation tests support these observations, since decreasing DO profiles were associated with high nutrient feeds due to higher OURs compared to increasing DO profiles when using VFAs alone due to lower OUR from a PHA storage response.

3.2. Stability experiments 3.2.1. Biomass performance during enrichment Overall, both enrichment SBRs performed similarly removing 100% of the VFAs and around 70% of CODsol and TOC per cycle (Table 4). N and P were removed with 75 and 60%

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efficiencies, respectively. Biomass from both SBRs displayed similar DO profiles during a cycle reflecting similar extents of feast (low DO) and famine (high DO). The feast based on VFA consumption lasted 13-20% of the cycle length (1-1.5 h; Fig. 3); the branched VFAs, i.e., iso-butyric and iso-valeric acids, were consumed more slowly and disappeared last from the medium. In addition, other C substrates were further consumed during the rest of the 8-h cycle, as indicated by the continued decrease in TOC during a cycle (Fig.3d). TOC consumption during the feast was due exclusively to VFA C consumption, but up to approximately 20% of the total TOC consumed corresponded to non-VFA C consumed during the feast. The lowest DO point and the onset of DO increase during a cycle corresponded to a pH peak (up to 6.8 or 7.3) and to the lowest VFA concentrations and VFA depletion, indicating the end of the feast and the start of the famine. Famine as defined on VFA depletion was characterised by an exponential increase in DO levels and an exponential decrease in pH (Data not shown). The PHA content of the biomass from both SBRs increased during the feast, and decreased during the famine (Fig. 3), suggesting an enrichment of PHA-storing organisms with the fermented HPTH centrate as substrate. The peaks in PHA concentrations coincided with VFA depletion, indicating that VFAs were the main substrate for PHA formation. Slight performance differences were observed over time in both SBRs, which suggested that biomass continued adapting after 17 days of stable operation (after day 42). For example, the VFA consumption rates during the feast increased over time from 0.13-0.15 (Day 58) to 0.21-0.30 CmolVFA CmolX-1h-1(Day 82), and the VFAs were consumed within 1 h at the end of the experiments (Day 82). Nevertheless, similar amounts of substrate were consumed in both SBRs per cycle (3914 and 307 Cmmol L-1; 343 and 315 CmmolVFA L-1) and similar levels of PHA accumulated after the feast (0.190.02 and 0.210.04 gPHA gTSS-1) and remained at the end of the cycle (0.170.03 and 0.180.06 gPHA gTSS-1) in both

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SBRs (Table 2). Biomass in SBR2B displayed slightly more consistent performance and a higher YP/VFA of 0.160.08 Cmol Cmol-1 compared to SBR2A (YP/VFA=0.120.02 Cmol Cmol-1). N and P were in high levels in the feed (Table 3) and although they were removed during the cycles, 0.2-0.7 g L-1 remained as soluble NH4+-N and PO43--P in the effluents (Table 4). Most NH4+-N was simultaneously removed with VFAs and other C substrates (Fig. 3). Active biomass yields corresponded to approximately 0.3-0.4 CmolX Cmol substrate-1 per cycle in both SBRs.

3.2.2. PHA accumulation The overall PHA accumulation capacity improved over time in parallel with the improved feast-famine response of the biomass during enrichment (after day 58), indicating continued phenotypic acclimation. At the end of the experiments, the biomass was able to accumulate as PHA 33% of the Cmol VFA fed despite lower yields 14 days before (YP/VFA=0.08 Cmol PHA Cmol VFA-1). The final biomass PHA content achieved in the batch accumulations was 233% and 251% g PHA g TSS-1 in SBR2A and 2B, respectively, from an initial PHA content of 14 gPHA gTSS-1 at the start of the tests (Table 3). PHA was accumulated at an almost constant rate during the accumulation tests (6-7 h) (qp = 0.04 and 0.05 CmolPHA CmolX-1 h-1). The deviation of carbon into other storage compounds, such as glycogen or extracellular polymeric substances, was limited as indicated by the low polysaccharides yields (YPS/S, Table 3). Carbon was not exclusively used for PHA storage. Biomass growth occurred together with PHA storage as indicated by the yields of active biomass on substrate (YX/S=0.08 and 0.17; Table 3) and 373% and 484% removal of N and 384% and 3410% removal of P in SBRs 1 and 2, respectively. All the added VFAs and part of other originally present substrates were consumed during PHA accumulation, as attested by the lower final COD,

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TOC, and VFA concentrations at the end of the tests in comparison to the initial concentrations (Table 4).

3.3. PHA composition and properties from both experiments The produced polymer presented stable properties despite slight variations in biomass performance during enrichment and accumulation. Overall, the PHAs produced under similar OLR conditions displayed similar high thermal stability, as indicated by similar decomposition temperatures (Td = 283-287C), which for the high OLR case were slightly lower (Table 5). The composition and chain-length distribution of the PHA produced in both experiments were similar; however, some differences in molecular weights and microstructure were detected (Table 5). The PHA accumulated in both the OLR and stability experiments consisted of a mixture of 3HV and 3HB in a 30:70 molar ratio (Table 3), and displayed similar chain length distributions (PDI=1.5-2.5). One similar Tg was detected in all PHA samples, except for one sample (OLR experiments, low OLR) that exhibited two Tgs. Some variability was detected in molecular mass but within the range of 700 000 to 1 000 000 g mol-1 among the different PHAs; specifically, the molecular mass of the polymer during the stability experiments was higher (870 000 g mol-1) in earlier than in later accumulation tests (350 000-430 000 g mol-1) (Table 5) without any apparent reason. A polymer with lower Hm and crystallinity was produced from the low-OLR than from the high-OLR biomass during the OLR experiments. Furthermore, the polymers produced during the stability experiments partly with acetate and propionate as feed displayed only one or no Tm, indicating the completely amorphous character of the polymer. Overall, the low Tg (-2.4 1.9C) and low or no crystallinity suggest a tendency for producing an amorphous and rubbery polymer.

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3.4. Microbial community of the enriched biomass The enriched biomass from both experiments was dominated by free-swimming bacteria, small flocs (~<100 m) with an open structure and microcolonies. Protozoa were observed as both free-swimming and stalked ciliates, and as amoebae in tandem with some rotifers. Less free-swimming organisms were observed when the conditions changed by decreasing the OLR from 12 to 6 g COD L-1d-1. The biomass had little settling capacity and was present at concentrations ranging from 4-7 g TSS L-1 (90% VSS) during the stability experiments, and 111.9 and 9.80.5 g TSS L-1 (84% VSS) during the high and low OLR experiments, respectively. The analysis of microbial diversity in the SBR biomass during the stability experiments was based on PCR-DGGE band richness (as the number of species or genotypes) and genotypes evenness via the Gini coefficients (as the relative abundance of species or genotypes). This analysis (Fig. 4) indicated that a limited amount of species became more abundant over time, i.e., a decrease in evenness (increase in Gini coefficients) occurred over time in both SBRs, which agrees with the proceeding microbial enrichment. Higher evenness in both reactors (lower Gini coefficients) was quantified during acclimation (Days 49, 58, and 65), in which consistent operating conditions were kept after an initially unstable operating period. Lower evenness was measured in the last 4 (SBR1) or 3 (SBR2) biomass samples, in agreement with the more stable operating conditions that tend to select for PHA-storing organisms. This lower evenness agrees with the improved PHA-storing capacity of the biomass assessed at the end of the operating period (Day 85).

4. DISCUSSION 4.1. Operating conditions ensuring enrichment with a high-strength substrate under ADF A long-enough famine appears critical for ensuring the selection of PHA storing organisms with high-strength substrates under ADF. The famine phase has to be long enough to ensure

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that PHA-storing organisms utilise the stored carbon for growth and maintenance, whereas the other organisms starve and maintain a lower growth rate. Since high-strength substrates entail high OLRs if applied undiluted, as would be the preference in real appications, the length of the feast has to remain much shorter than the famine to ensure a selective pressure. In this case, changing the operating conditions associated with the high OLR of 12 g COD L-1 d-1 to those of a low OLR of 6 g COD L-1 d-1 lowered the feast-to-cycle-length ratio from 0.27-0.31 to 0.14-0.17, resulting in higher PHA-storage capacity during the enrichment. A feast phase under 20 % of the total cycle length has been shown as critical for selecting for PHA-storing organisms (Dionisi et al., 2007). A combination of interrelated operating parameters ensures the enrichment of PHAstoring organisms in an SBR. Besides the feast-to-cycle ratio, the maximum substrate concentration during the feast and sludge age appear to be critical. The substrate concentration at the start of the feast is directly related to the cycle length and OLR, and it should be high enough to trigger PHA storage but low enough to avoid biomass inhibition. Here, a maximum VFA concentration during the feast of 1 g CODVFA L-1 was used in agreement with most studies with mixed substrates in which a successful enrichment was achieved with a substrate concentration below 1.1 g CODVFA L-1 (Dionisi et al., 2005a, Dionisi et al., 2005b, Albuquerque et al., 2007, Gurieff, 2007, Liu et al., 2008). Loss of PHA-storing capacity was observed in a biomass exposed to a long feast with short cycle lengths under a relatively high OLR with VFA concentrations during the feast of 3.9 CODVFA L-1 (Albuquerque et al., 2007). The SRT also influences the biomass PHA storage capacity and selection since short SRTs tend to lead to higher growth rates and less substrate storage (Dias et al., 2006). By decreasing the OLR in this study, the SRT (similar to the HRT) was increased from approximately 3 to 6 d with the observed improved enrichment. When treating high-strength substrates, the inherent high OLR tend to select for free-swimming

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biomass with poor settleability; therefore, the SRT cannot be uncoupled from the HRT based on gravity separation. The results from this study suggest that the SRT should be high enough to ensure enrichment without disregarding other operating parameters discussed above. Successful enrichment with mixed substrates with an HRT=SRT= 1 d and high OLRs of 8.5 and 12.5 g CODVFA L-1 d-1 has only been reported with a mixture of acetic, propionic and lactic acids (Dionisi et al., 2004a, Dionisi et al., 2005a, Dionisi et al., 2005b), but not with complex nutrient-rich substrates like the fermented HPTH sludge.

4.2 Feast-famine response and PHA storage under the presence of non-VFA carbon and high nutrient levels Despite the feast-famine response and PHA accumulation during the feast (to a maximum of 19-20%), the selective pressure appeared compromised in the SBRs. A higher biomass PHA content at the end of the famine during the stability experiments (17-18% g PHA g VSS-1) than previously observed with the same feed during the OLR experiments (<1%)(Table 2) and in other studies [2-10%; (Dionisi et al., 2004a, Dionisi et al., 2005a, Dionisi et al., 2005b, Albuquerque et al., 2007, Liu et al., 2008)] suggested that the biomass was unable to utilise completely the stored C for growth during famine. Furthermore, although the stored PHA was almost completely consumed during a cycle (78-91%) in the OLR experiments at the lower OLR, the maximum biomass PHA levels achieved were relatively low (0.04 g PHA g TSS-1) compared to those reported in other SBR studies with complex feeds (0.12 and 0.08 g PHA g VSS-1; (Coats et al., 2007, Liu et al., 2008). Also, the YP/VFA during the SBR cycles was variable in both experiments (0.12-0.43 Cmol Cmol-1) with some values lower than those previously reported [0.37-0.45; (Dionisi et al., 2004a, Dionisi et al., 2005a, Dionisi et al., 2005b, Albuquerque et al., 2007)], pointing to a slightly weaker storage response by the biomass under enrichment and possibly a stress physiology.

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The presence of degradable non-VFA organic substrates may have dampened the ADF selective pressure for PHA-storing organisms. The enrichment profiles (Figs. 1 and 3) indicate that although all VFAs were consumed at the beginning of a cycle (feast), other organic C substrates were further consumed during the rest of the cycle, although at lower rates. These non-VFA substrates remained unconverted to VFAs from the previous fermentation step and were consumed aerobically in the SBRs. These non-VFA substrates could hinder the enrichment since they can be utilised (i) for growth by non-PHA-storing organisms during the whole cycle, (ii) as extra substrate by PHA-storing organisms during the VFA-based famine instead of stored PHA, and/or (iii) as storage substrate but in the form of polysaccharides during the feast. Non-VFA substrates in fermented molasses were reported to shorten the famine phase and compromise enrichment in highly loaded SBRs with fermented molasses (Albuquerque et al., 2007). Exponential DO increments associated to a decrease in SOUR during the VFA-base famine as observed in this study were previously related to the utilization of slowly degradable C and of stored PHA (Majone et al., 1999). In addition, some of these compounds may be more energetically favourable for glycogen than PHA storage, as for the case of glucose in open mix cultures (Dircks et al., 2001). The contribution of non-VFA substrates to PHA formation as observed in the case of a fermented olive mill effluent (Dionisi et al., 2005b) was less likely in this study since the maximum PHA level in a cycle occurred at the end of the feast when VFAs were depleted and the YP/S were similar or slightly lower than the YP/VFA (Table 3). Simultaneous VFA and NH4+-N consumption during the feast (Figs. 1 and 3) suggests that biomass growth occurred in tandem with PHA storage. The steady decrease in DO during the feast (stability experiments) supports the idea of an increased microbial respiration associated with PHA storage in combination with growth of possibly both PHA-storing and non PHA-storing organisms during the feast. The continued NH4+-N consumption during the

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rest of the cycle, although at somehow lower rates and in tandem with the decrease in other C substrates and PHA, also suggests active biomass growth. Growth of PHA-storing biomass during famine based on PHA is expected, and lower N consumption rates in the famine than in the feast have been related to biomass growth on PHAs during famine (Albuquerque et al., 2007). The slow decrease in oxygen requirement over famine is in accordance with a shift into a lower respiration on storage compounds than on soluble substrates (Dionisi et al., 2004). The continued consumption of excess N during famine, however, may still indicate growth of non-PHA-storing biomass. Nevertheless, biomass growth appeared restricted during the stability experiments when higher PHA content in the enriched biomass along with the limited use of stored C as PHAs were measured, compared to those from the OLR experiments under a similar OLR of 6 g COD L-1 d-1 (Table 2). Lower biomass levels in the stability experiments could be partially explained by the lower PHA cycling; however, influences of lower pH and higher DOs on biomass metabolism during the stability experiments remained undetermined.

4.3. PHA storage capacity under high nutrient levels The biomass enriched in both experiments under an OLR of 6 g COD L-1 d-1 displayed similar PHA accumulation capacities; although during the stability experiments the biomass displayed lower accumulation rates. A similar biomass PHA content was achieved in both experiments and the PHA yields (YP/VFA and YP/S) were comparable between experiments (Table 3) despite a relatively high PHA content of the biomass (0.14 g PHA g TSS-1) from the stability experiments at the start of the accumulations. Although the overall storage capacity of the selected biomass was not compromised by the high PHA content at the end of the famine, the specific rates of PHA storage (qp), VFA uptake (-qVFA) and productivities may have been affected. The polysaccharide yields (YPS/S), qp and -qVFA, and therefore PHA

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productivity, were lower in the stability experiments, reflecting the relatively high levels of PHA already present in the biomass. The differences in performance between the OLR and stability experiments may have arisen from the changes in operating conditions aimed at improving performance, such as lower substrate concentrations per spike and the simpler VFA mixture in the accumulation tests, and/or increased aeration during enrichment, i.e., suspected oxygen limitation during the OLR experiments (Fig. 1) versus oxygen excess (stability experiments). The overall PHA accumulation performance of the enriched biomass using fermented HPHT sludge centrate (Table 3) is comparable to that of some complex substrates despite some parameters being in the low range of performance with respect to some studies. The YP/VFA are in the range of those reported for fermented paper mill effluent with nutrient excess [0.33 Cmol Cmol-1 at a C:N = 100:12 (Bentgsson et al., 2008)] and for a mixture of acetic, propionic and lactic acids [0.4 Cmol Cmol-1 (Dionisi et al., 2004b)], but slightly lower than those obtained with fermented molasses [0.44 Cmol Cmol-1 at a C:N=100:1(Albuquerque et al., 2007)]. The PHA productivities are lower than those reported for some fermented wastes [13 and 15 Cmmol PHA L-1h-1 (Albuquerque et al., 2007, Mengmeng et al., 2008)], but similar to those obtained with fermented olive oil mill effluent [5 Cmmol PHA L-1h-1; (Dionisi et al., 2005b)]. The qp are lower than those reported in systems with higher PHA productivity; however, they are comparable to those of biomass enriched with other complex substrates, such as fermented pulp mill wastewater [0.04-0.06 CmolPHA CmolX-1h-1; (Bentgsson et al., 2008)] and fermented fruit cannery wastewater [0.02-0.1 CmolPHA CmolVSS-1h-1; (Gurieff, 2007)]. Finally, the enriched biomass accumulated less PHAs at the end of the tests compared to other studies with fermented sludge [0.57 gPHA gTSS-1 (Mengmeng et al., 2008)], cannery wastewater [0.20-0.39 gPHA gTSS-1 (Gurieff, 2007)], fermented paper mill wastewater [0.32-0.48 gPHA gTSS-1 (Bengtsson et al., 2008)] and

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fermented molasses [0.30 gPHA gTSS-1 (Albuquerque et al., 2007)]. The lowest qp from the stability tests may reflect some level of PHA saturation in the biomass achieved around 0.25 g PHA g TSS-1 since the initial PHA content was higher in these experiments and/or kinetic substrate limitation due to the relatively lower substrate concentrations achieved with each spike (100-200 mg CODVFA L-1) due to aeration constraints by foaming ensuring no DO limitation. The slow PHA storage response could also be related to the presence of longerchain C VFAs, non-VFA C substrates and/or excessive nutrient levels originating from mixed liquor background from the enriched biomass, in addition to the still suboptimal enrichment. Longer-chain C VFAs (e.g., propionic, valeric and caproic acids) can be decarboxylated during PHA synthesis resulting in a lower C-based PHA yield (Serafim et al., 2008a). Excessive nutrient levels and availability of non-VFA C distinctively associated with the fermented HPTH sludge could have hindered PHA accumulation by promoting a growth response, most notably with the biomass from the high OLRs of 12 g COD L-1 d-1, which presented a poor feast-famine response and was likely poorly enriched. This biomass displayed a higher PHA storage yields with the synthetic VFA mixture than with the other feeds containing excess nutrients (Table 3). High nutrient levels were the major difference between these tests and those in the literature. Biomass growth during PHA accumulation, as supported by YX/S=0.1-0.3 Cmol X Cmol TOC-1 (Table 3) and ~50% removal of N and P, would have deviated C substrate away from PHA storage. NH4+ consumption under high OLRs has been related to biomass growth, and increased NH4+ consumption rates and SOURs have been indicative of an increase in biomass growth over PHA storage (Dionisi et al., 2004). Also PHA accumulations with excess N have consistently led to relatively lower YP/VFA in other studies (Albuquerque et al., 2007, Bengtsson et al., 2008). Excessive nutrient levels could have also hindered PHA accumulation due to increased C-based respiration. High respiration levels on C substrate are plausible in this

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study since less than 50% of the Cmol of substrate was either stored as PHAs or polysaccharides or used for cell growth. High oxygen and ATP consumption rates have been associated with energy-spilling reactions, such as, futile cycles of ammonium, incurred by bacterial cells under high concentrations of ammonium, which was the case in this study (Russell & Cook, 1995).

4.4. Product properties and process reproducibility PHA production from fermented HTPH sludge centrate was reproducible, as shown by similar performances during biomass enrichment and PHA accumulation in 2 independent experiments in parallel reactors. However, slight differences in biomass PHA content at the end of the SBR cycles and polymer molecular weight occurred, and PHA accumulations were variable among experiments. The relative decrease in evenness and richness in the SBR biomass (Fig. 4) is in agreement with the overall increase in PHA-capacity of the biomass over time, i.e., a community with lower evenness and expectedly enriched in PHA-storing organisms with a higher PHA-storage capacity. The final polymer was consistently composed of a mixture of 3HB and 3HV despite some differences in the feed during the accumulation tests, and the performance of the PHA production process was comparable to that observed with other complex fermented wastes. In addition, the 3HV content of the produced polymer was in the range of commercial PHA from pure cultures [Biopol 0.-24% 3HV; (Salehizadeh & Van Loosdrecht, 2004)]. 3HV content was expected since propionic, valeric/caproic acids were present in the feed to the accumulation tests, and the SBRs, from which part of the stored PHAs were transferred to the batch accumulations (stability tests). Biomass enriched under ADF and subjected to PHA accumulation with complex fermented wastes has repeatedly produced a mixture of 3HV and 3HB (Serafim et al., 2008a) probably due to the broad VFA presence these wastes. The monomeric composition of PHAs is

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determined both by the VFA substrate composition and by the type of enriched biomass (Lemos et al., 2006). In fact, the difference in 3HV composition of the polymer produced by the biomass enriched differently during the OLR experiments but fed-batch similarly supports the role that biomass enrichment had on influencing the polymer composition during accumulation in contrast to findings by (Gurieff, 2007). The Mw (between 350,000 and 1,040,000 g mol-1; Table 5) and polydispersity indices (PDI = 1.5-2.6) of the produced polymer were similar to those reported in the literature when using synthetic substrates on ADF-selected pure and mixed cultures (Serafim et al., 2008b). Tgs, Tms, and Hm of the polymers produced during the OLR experiments were in the range of those reported for polymers produced by pure and mixed cultures with similar 3HB/3HV composition (Serafim et al., 2008b). An exception were the polymers from the stability experiments which had very low melting enthalpies or were completely amorphous, suggesting the presence of a third monomer in the co-polymer. Nevertheless, no identificaiton peak for such a monomer was detected in the GCMS spectra. Although the relatively higher levels of PHA accumulated by this biomass in the enrichment SBR may have influenced the polymer properties, further work is required to determine the causes for these observations. Also, the polymer produced with the VFA mixture and nutrients under the low OLR (Table 5) presented two Tgs, suggesting a blend of two compositionally different 3HB3HV co-polymers. In spite of this performance, the overall process appears challenged by the high strength and composition, and foaming tendency of the fermented HPTH sludge. Due to the incomplete removal efficiencies of TOC, CODsol and nutrients (Table 4), the treated effluent is still of high strength and requires post treatment before disposal. The increased sludge solubilisation by HPTH may lead to a higher fraction of recalcitrant solubilised organics requiring further treatment in order to be eliminated from the liquid stream. Increased levels

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of recalcitrant COD has been previously identified as a disadvantage of such harsh sludge pretreatment for wastewater treatment plants (Phothilangka et al., 2008). Although the foam disrupter effectively controlled the rising foam and allowed for intensive aeration, the higher DO levels ensuring no DO limitation during the feast did not improve the enrichment or PHA storage capacity. Foaming also made controlling the SRT via the PLC challenging, and therefore, the reactors' instability at the start of the stability experiments. Persistent foaming has been linked to aeration and to surface-active polypeptides (e.g., hydrophobic polypeptides) originating from autolysis and enzymatic production (Ghildyal et al., 1988, Drouin et al., 2008) or Maillard reaction products and denatured proteins (Ghildyal et al., 1988) from HPTH.

5. CONCLUSIONS PHA production is feasible in a reproducible fashion from a fermented waste stream rich in complex solubilised organics and nutrients via ADF-based selection of PHA-storing organisms and batch PHA accumulations. An improvement in feast-famine response during biomass enrichment at a high OLR of 6 gCOD L-1d-1 was achieved by lengthening the SBR cycles to 8 h ensuring a short feast-to-cycle-length < 0.2, under a fixed maximum substrate concentration during feast of 1 g CODVFA L-1 and an SRT= 6 d. PHA production was reproducible and consistent in terms of biomass feast-famine response, PHA storage capacity, and PHA properties. Nevertheless, slight differences in biomass PHA content and storage rates together with a decrease in microbial evenness and richness in the selected biomass indicated performance variability and optimisation of the enrichment is further required to maximise the selective pressure. Biomass selection and PHA accumulation appeared compromised by the high levels of solubilised non-VFA organics and nutrients associated with the fermented HPTH sludge due to a biomass-growth response and increased C-based

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respiration. Therefore, a less harsh pretreatment while ensuring high VFA production rates in the fermenter may lessen the unproductive load into the PHA production system.

Acknowledgements The authors would like to thank the technical advice from Simon Bengtsson, Per Magnusson and Stefan Erkselius at AnoxKaldnes AB. Betina Bjerregaard from the Fredericia Central Wastewater Treatment Plant and Rebecca V. Jensen from Nstved's Central Treatment Plant are thankfully acknowledged for their support in providing sludge samples and operating data. This study was part of the EU Neptune project (Contract No 036845, SUSTDEV-20053.II.3.2), which was financially supported by grants obtained from the EU Commission within the Energy, Global Change and Ecosystems Program of the Sixth Framework (FP6-2005Global-4).

References Albuquerque MGE, Eiroa M, Torres C, Nunes BR & Reis MAM (2007) Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses. Journal of Biotechnology 130: 411-421. APHA, AWWA & WEF (2005) Standard methods for the examination of water and wastewater. American Public Health Association, American Water Works Association, Water Environment Federation, Washington, D. C. Beccari M, Bertin L, Dionisi D et al. (2009) Exploiting olive oil mill effluents as a renewable resource for the production of biodegradable polymers through a combined anaerobicaerobic process. J. Chem. Technol. Biotechnol. 84: 901-908. Bengtsson S, Werker A, Christensson M & Welander T (2008) Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater. Bioresource Technology 99: 509-516. Bengtsson S, Pisco AR, Johansson P, Lemos PC & Reis MAM (2010) Molecular weight and thermal properties of polyhydroxyalkanoates produced from fermented sugar molasses by open mixed cultures. J Biotechnology In press. Boon N, De Windt W, Verstraete W & Top EM (2002) Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiology Ecology 39: 101-112. Coats ER, Loge FJ, Wolcott MP, Englund K & McDonald AG (2007) Synthesis of polyhydroxyalkanoates in municipal wastewater treatment. Wat. Environ. Res. 79: 2396-2403. 30

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Dias JML, Lemos PC, Serafim LS et al. (2006) Recent advances in polyhydroxyalkanoate production by mixed aerobic cultures: from substrate to the final product. Macromol. Biosci. 6: 885-906. Dionisi D, Majone M, Papa V & Beccari M (2004a) Biodegradable polymers from organic acids by using activated sludge enriched by aerobic periodic feeding. Biotechnol. Bioeng. 85: 569-579. Dionisi D, Renzi V, Majone M, Beccari M & Ramadori R (2004b) Storage of substrate mixtures by activated sludges under dynamic conditions in anoxic or aerobic environments. Water Res. 38: 2196-2206. Dionisi D, Majone M, Vallini G, Di Gregorio S & Beccari M (2007) Effect of the length of the cycle on biodegradable polymer production and microbial community selection in a sequencing batch reactor. Biotechnol. Prog. 23: 1064-1073. Dionisi D, Beccari M, Di Gregorio S, Majone M, Papini PM & Vallini G (2005a) Storage of biodegradable polymers by an enriched microbial community in a sequencing batch reactor operated at high organic load rate. J Chem Technol Biotechnol 80: 1306-1318. Dionisi D, Carucci G, Papini PM, Riccardi C, Majone M & Carrasco F (2005b) Olive oil mill effluents as a feedstock for production of biodegradable polymers. Wat. Res. 39: 20762084. Dircks K, Beun JJ, van Loosdrecht M, Heijnen JJ & Henze M (2001) Glycogen metabolism in aerobic mixed cultures. Biotechnology and Bioengineering 73: 85-94. Gurieff NB (2007) Production of biodegradable polyhydroxyalkanoate polymers using advanced biological wastewater treatment process technology. Ph. D. Thesis, The University of Queensland, Brisbane. Gurieff NB & Lant P (2007) Comparative life cycle assessment and financial analysis of mixed culture polyhydroxyalkanoate production. Bioresource Technol. 98: 3393-3403. Jacquel N, Lo C-W, Wei Y-H, Wu H-S & Wang SS (2008) Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochemical Engineering 39: 15-27. Kepp U, Machenbach N, Weisz N & Solheim OE (2000) Enhanced stabilisation of sewage sludge through thermal hydrolysis - three years of experience with full scale plant. Wat. Sci. Technol. 42: 89-96. Lee SY (1996) Bacterial polyhydroxyalkanoates. Biochemical Engineering 49: 1-14. Lemos PC, Serafim LS & Reis MAM (2006) Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding. J. Biotechnol. 122: 226-238. Liu H-Y, Hall PV, Darby JL, Coats ER, Green PG, Thompson DE & Loge FJ (2008) Production of polyhydroxyalkanoate during treatment of tomato cannery wastewater. Water Environment Research 80: 367-372. Luengo JM, Garcia B, Sandoval A, Naharro G & Olivera ER (2003) Bioplastics from microorganisms. Current Opinion in Microbiology 6: 251-260. Majone M, Dircks K & Beun JJ (1999) Aerobic storage under dynamic conditions in activated sludge processes. The state of the art. Wat. Sci. Technol. 39: 61-73. Marzorati M, Wittebolle L, Boon N, Daffonchio D & Verstraete W (2008) How to get more out of molecular fingerprints: practical tools for microbial ecology. Environmental Microbiology 10: 1571-1581. Md Din MF, Ujang Z, Van Loosdrecht MCM, Ahmad A & Sairan MF (2006) Optimization of nitrogen and phosphorus limitation for better biodegradable plastic production and organic removal using single fed-batch mixed cultures and renewable resources. Wat. Sci. Technol. 53: 15-20. Mengmeng C, Hong C, Qingliang Z, Shirley SN & Jie R (2008) Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

generated from alkaline excess sludge fermentation. Bioresource Technol. 100: 13991405. Mino T, Van Loosdrecht MCM & Heijnen JJ (1998) Microbiology and biochemistry of the enhanced biological phosphate removal process. Wat. Res. 32: 3193-3207. Morgan-Sagastume F, Pratt S, Karlsson A, Cirne D, Lant P & Werker A (2010) Fermentation of waste activated sludge pretreated with high-pressure thermal hydrolysis. Submitted to Bioresource Technol. Muyzer G, de Waal EC & Uitterlinden A (1993) Profiling of complex microbial populations using denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes encoding for 16S rRNA. Appl. Environ. Microbiol. 59: 695-700. Philip S, Keshavarz T & Roy I (2007) Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J. Chem. Technol. Biotechnol. 82: 233-247. Reis MAM, Serafim LS, Lemos PC, Ramos AM, Aguiar FR & Van Loosdrecht MCM (2003) Production of polyhydroxyalkanoates by mixed microbial cultures. Bioprocess. Biosyst. Eng. 25: 377-385. Rhu DH, Lee WH, Kim JY & Choi E (2003) Polyhydroxyalkanoate (PHA) production from waste. Wat. Sci. Technol. 48: 221-228. Salehizadeh H & Van Loosdrecht MCM (2004) Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance. Biotechnological Advances 22: 261-279. Serafim LS, Lemos PC, Oliveira C & Reis MAM (2004) Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions. Biotechnol. Bioeng. 87: 145-160. Serafim LS, Lemos PC, Albuquerque MGE & Reis MAM (2008a) Strategies for PHA production by mixed cultures and renewable waste materials. Appl. Microbiol. Biotechnol. 81: 615-628. Serafim LS, Lemos PC, Torres C, Reis MAM & Ramos AM (2008b) The influence of process parameters on the characteristics of polyhydroxyalkanoates produced by mixed cultures. Macromolecular Bioscience 8: 355-366. Sudesh K, Abe H & Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci. 25: 1503-1555. Werker A, Lind P, Bengtsson S & Nordstrm F (2008) Chlorinated-solvent-free gas chromatographic analysis of biomass containing polyhydroxyalkanoates. Wat. Res. 42: 2517-2526. Wittebolle L, Marzorati M, Clement L et al. (2009) Initial community evenness favours functionality under selective stress. Nature 458: 623-625.

32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Table 1. Characterization of the fermented centrates from HPTH pretreated sludge used as feeds to the SBRs during the 2 sets of experiments. Values are reported as averagestandard deviation (n = number of samples) or as single values from single measurements.

Table 2. Biomass enrichment performance in the different SBRs from the OLR and stability experiments based on cycle assessmente studies. Values are reported as averagestandard deviation (n = number of samples) or as single values. Initial and final values refere to the beginning and end of a cycle in the SBRs. The specific PHA production rates (qp), specific VFA uptake rates (-qVFA) and PHA yields based on VFAs (YP/VFA) correspond to the feast period.

Table 3. Biomass PHA accumulation performance in terms of PHA content, PHA composition and conversion yields during the batch PHA accumulation tests. Values are reported as averagestandard deviation (n = number of samples) or as single values from maximum performance. The specific PHA production rates (qp) and specific VFA uptake rates (-qVFA) correspond to the initial, maximum values during the OLR experiments since saturation was observed generally after 3 h. No PHA saturation was observed during the stability experiments.

Table 4. Overall performance of enrichment SBRs during the OLR and stability experiments. Data correspond to averagestandard deviation from cycle studies, except effluent values which were calculated based on averages for each cycle.

Table 5. Molecular and thermal properties of the produced PHAs in both experiments after fed-batch accumulations.

33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Fig. 1. Typical evolution of DO, VFAs, CODsol., NH4+-N, and PHA (P3HB and P3HV) in a cycle of (A) SBR1 under an OLR of 12 g COD L-1 d-1 after 21 days of operation, and (B) SBR2 under an OLR of 6 g COD L-1 d-1 after 25 days of lowering the OLR. Time 0 corresponds to the end of feeding. The dotted lines indicate the approximate end of the feast and start of the famine periods based on VFA consumption. VFA depletion may have occured between 1 and 1.5 h in the cycles shown. The delay in the VFA peak concentration (A) (15 min after feeding) was likely related to incomplete mixing. DOs are logged raw data points and the variability in the measurements reflect intense foaming. DO sudden decreases were due to aeration interruption during sampling.

Fig. 2. PHA content of the biomass in the SBRs at the end of the feast during the OLR experiments. The OLR was decreased from 12 to 6 g COD L-1 d-1 on day 53 in both reactors. Four days later (Day 57), the OLR was increased back to 12 g COD L-1 d-1 in the high OLR SBR, after a short recovery in PHA accumulation at the low OLR.

Fig. 3. Typical evolution of DO, VFAs, TOC, NH4+-N, PHAs (P3HB and P3HV) and biomass PHA content (PHA/TSS) in a cycle of (A, B) SBR2A (Day 75) and (C, D) SBR2B (Day 82). The dotted lines indicate the approximate end of the feast and start of the famine periods based on VFA consumption.

Fig. 4. .PCR-DGGE band patterns (A) and corresponding evenness as Gini coefficients (B) and band weighted richness (C) during the stability experiments in both SBRs. Full bars correspond to SBR2A and black bars to SBR2B. A Gini value = 0 represents total evenness.

34

pH Alkalinity (mmol/L) CODsol (g/L) TOCsol (g/L) VFAs (g COD/L) Total acids Acetic Propionic Isobutyric Butyric Isovaleric Valeric Caproic Solids (g/L) TS VS VS/TS (%) TSS VSS

Fredericia 6.3-6.4 110 10 (n =20) 40 4 (n =22) 14 1 (n =16) (n=29) 19.9 1.9 5.4 0.5 3.4 0.4 0.7 0.7 4.6 0.8 3.4 0.4 1.0 0.3 1.4 1.0 (n=2) 31 1 24 1 1.9 0.3 1.6 0.3 84 7

Nstved 6.4-6.8 120 31 3 (n =8) 11 1 (n =7) (n=4) 18.5 2.2 6.0 0.7 3.2 0.2 1.6 0.1 3.3 0.3 2.6 0.7 1.5 0.2 0.3 0.0 14 11 79 1.5 0.6 (n =4) 1.0 0.2 (n =4) 67 9 3.4 0.2 (n =4) 2.4 0.3 (n =8) 70 0.4 0.6 0.4 (n =3) 100 2.8 200:23:3 100:26:3

VSS/TSS (%) VSS/VS (%) Nitrogen (g/L) 3.7 0.3 (n =21) Ntot 2.5 0.2 (n =21) NNH4 NNH4/Ntot (%) 68 Phosphorus (g/L) 0.61 0.03 (n =20) Ptot 0.54 0.04 (n =16) PPO4 PPO4/Ptot (%) 89 Ratios (g basis) CODsol/TOCsol 2.9 COD: Ntot: Ptot 200:19:03 Molar TOC:Ntot:Ptot 100:23:2

Operating conditions Reactor

Conditions

VSS (g L ) (n=9 ) pH Initial NH4 +-N (mmol L-1 ) (n=3 ) C VFA:N-NH4 + (molar ratio) Min. DO (mg L-1 ) Initial/final PHA content (%, gPHA gTSS ) VFA-based feast length (Cycle fraction) Max. PHA content (Cmmol L-1) Max. PHA content (%, gPHA gTSS-1) qp (CmolPHA Cmol X-1 h-1) -qV FA (CmolVFA Cmol X h ) Y X/S (Cmol Cmol ) Y P/VFA (Cmol Cmol )
-1 -1 -1 -1 -1

-1

OLR Experiments OLR=12 gCODVFA L-1 d-1 SBR1 9.41.4 8.1-8.5 416 100:160 0 <2 0.27-0.31 13 1-3 0.0140.01 (n=3 ) 0.04 n.d. 0.200.06 (n=3 )

SBR2 7.50.9 8.0-8.3 386 100:160

Stability Experiments OLR=6 gCODVFA L-1 d-1 SBR2A SBR2B 5.00.8 6.00.6 6.0-7.2 6.0-7.3 499 4717 100:140 100:140 2-4.5 3.5-4 173.1 (n=3 ) 186 (n=3 ) 0.13-0.20 31-36 23-30 191.6 (n=2 ) 213.5 (n=2 ) 0.030.01 (n=3 ) 0.42 0.120.02 (n=3 ) 0.050.02 (n=3 ) 0.330.02 0.16-0.24 (n=3 ) 0.25-0.30

Performance

0 <1 0.14-0.17 20 4 0.020.01 (n=3 ) n.d. 0.22 and 0.43

OLR Experiments Operating conditions Susbstrate Biomass' reactor Final PHA content (gPHA gTSS-1) 3HV:3HB (molar basis) YP/VFA (Cmol Cmol-1) YP/S (Cmol Cmol-1) YPS/S (Cmol Cmol-1) YX/S (Cmol Cmol-1) qp (CmolPHA Cmol X h ) -qVFA (CmolVFA Cmol X-1 h-1) PHA productivity (CmmolPHA L h ) Initial NH4+-N (mmol L-1)
-1 -1 -1 -1

Performance parameter

OLR=12 gCODVFA L d Fermented HPTH (n=3) VFA mix (n=3) SBR1 0.080.04 0.290.12 18:827 13:872 0.070.03 0.270.08 0.060.03 0.140.13 0.550.06 0.100.01 0.570.18 61 80 0.290.08 0.170.13 0.33 0.150.09 0.730.21 73 80

-1

-1

VFAmix+N/P 0.13 15:85 0.18 0.15 0.14 0.13 0.42 8 8

OLR=6 gCODVFA L d Fermented HPTH (n=2) VFA mix (n=2) SBR2 0.190.02 0.230.02 26:742 29:716 0.360.15 0.360.05 0.280.07 0.150.13 0.880.09 0.100.00 0.310.16 115 134 0.320.13 0.100.01 0.240.13 0.120.01 0.320.09 103 134

-1

-1

Stability Experiments -1 -1 OLR=6 gCODVFA L d VFAmix+N/P VFAmix (acetic+propionic) SBR2A SBR2B 0.23 0.230.03 (n=3) 0.250.01 (n=3) 44:56 28:72 27:73 0.44 0.32 0.33 0.37 0.05 0.32 0.10 0.19 9 12 0.29 0.03 0.08 0.04 0.14 3 348 (n=3) 0.3 0.02 0.17 0.05 0.16 4 3113 (n=3)

Parameter CODsol CODVFA TOCsol NH4+-N PO43--P VSS

Low-OLR SBR2 Reduction Effluent (g/L) (%) 811 (n=5) 8.10.2 (n=5) 1000 0 801 (n=3) 2.90.1 (n=3) 814 (n=5) 0.480.09 (n=5) 413 (n=3) 0.350.00 (n=3) 395* 7.80.9 (n=4)

SBR2A Reduction Effluent (%) (n=3) (g/L) (n=3) 712 8.90.5 1000 0 732 3.00.2 755 0.660.11 593 0.250.02 280** 3.80.2

SBR2B Reduction Effluent (%) (n=3) (g/L) (n=3) 703 9.20.8 1000 0 712 3.10.2 788 0.530.20 6014 0.240.08 270** 3.70.9

* VSS increase based on average influent concentrations of 1.60.3 g L-1 (n=2). ** VSS increase based on average influent concentrations of 1.00.2 g L-1 (n=2).

OLR Experiments Operating conditions OLR=12 gCODVFA L d (SBR1) OLR=6 gCODVFA L-1 d-1 (SBR2) Stability Experiments Reactor (OLR=6 gCODVFA L d ) SBR2A
-1 -1 -1 -1

C substrate Fermented HPHT VFA mix VFAmix+N/P Fermented HPHT VFA mix VFAmix+N/P Day 71 76 85 71 76 85

Mw*

PDI

Tg (C) 0.71.8 0.11.1 1.9 -0.92.5 -0.90.4 -13.2/2.2 Tg (C) -1.2 -2.1 -1.9 -1.4 -2.4 -1.8

Tm1 (C) 1315 1313 136 1381 1403 139 Tm1 (C) 146 n.d. n.d. 142 n.d. n.d.

Tm2 (C) 1435 1443 148 1472 155 151 Tm2 (C) n.d. n.d. n.d. n.d. n.d. n.d.

Hm (J/g) 423 464 36 259 168 32 Hm (J/g) 17 n.d. n.d. 14 n.d. n.d.

Td-trans (C) 26830 2808 279 2872 2812 283 Td-trans (C) 287 284 284 287 286 284

(Mw/Mn) (g/mol) 610 000110 000 2.60.2 840 000110 000 2.50.5 920 000 2.1 820 00050 000 1.80.2 1 040 00030 000 1.50 880 000 2.5 Mw* (g/mol) 730 000 820 000 350 000 870 000 640 000 430 000 PDI (Mw/Mn) 2.2 1.5 1.7 2.4 2.1 1.6

SBR2B

Mw = weight average molar mass; PDI=polydispersity index; Mn=number average molar mass, all determined by SEC Td-trans = decomposition temperature determined by TGA from transition zone; Tg = glass-transition temperature, Tm = melting temperatures, and Hm = melting enthalpy determined by DSC *Measurements have a 50 000 g/mol accuracy n.d. = not detected

A
CODsol (g L-1), (gx10-1 L-1), DO (mg L-1)

CODsol

NH4-N
Feast

DO
Famine

VFA

PHA

12
10 8

40
35

25

6
4 2 0

20
15 10 5 0

NH4

+-N

0.5

1.5

2
Time (h)

2.5

3.5

B
9
CODsol (g L-1), (gx10-1 L-1), DO (mg L-1)

CODsol
Feast

NH4-N

DO
Famine

VFA

PHA

20 18

8 7 6 5

16
14 12 10

4
3 2 1 0 0 1 2 3 4 Time (h) 5 6 7 8

8 6 4 2 0

NH4

+-N

VFA, PHA (Cmol L -1)

VFA, PHA (Cmol L -1)

30

SBR1 brief ly at 6 gCOD L -1 d-1

4.5

SBR1 at 12 gCOD L -1 d-1 SBR2 at 6 gCOD L -1 d-1 Both SBRs at 12 gCOD L-1d -1

PHA content (%, g gTSS -1)

4.0
3.5

3.0
2.5 2.0 1.5 1.0
SBR 1 SBR 2

0.5
0.0 0 10 20 30 40 50 60 70 80 90 100 Time (d)

A
40
Feast

PHB

PHV

VFA Famine

PHA/TSS

B
TOC PHA
Famine

NH4-N

21 30 20 19 20 18 10 17 16 0 0 1 2 3 4 5 6 7 8 15

PHA content (%, g gTSS-1)

300

60

280

50

260

40

240

30

220 0 1 2 3 4 5 6 7 8

20

Time (h)

Time (h)

C
PHB PHV Famine VFA PHA/TSS

D
TOC PHA Famine NH4-N

40

20

VFA, PHA (Cmmol L-1)

gTSS-1)

300

35 30

30

TOC (Cmmol L-1)

18

280

16 20 14 10

PHA content (%, g

25 20

260 15

12

240

10 5

0 0 1 2 3 4 5 6 7 8

10

220

Time (h)

Time (h)

PHA (Cmmol L-1); NH4+ (Nmmol L-1)

Feast

Feast

PHA (Cmmol L-1); NH4+ (Nmmol L-1)

22

Feast

VFA, PHA (Cmmol L-1)

TOC (Cmmol L-1)

A
49
58

SBR 2A
65 71

SBR 2B
76 82 86 49 58 65 71 76 82 86

B
0.7 0.6

0.5

Gini value

0.4

0.3

0.2

0.1

0 2A-49 2A-58 2A-65 2A-71 2A-76 2A-82 2A-86 2B-49 2B-58 2B-65 2B-71 2B-76 2B-82 2B-86

SBR-Time (d)

C
35 30

Weighted richness

25

20

15

10

0 2A-49 2A-58 2A-65 2A-71 2A-76 2A-82 2A-86 2B-49 2B-58 2B-65 2B-71 2B-76 2B-82 2B-86

SBR-Time (d)