Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: Effect of substrate composition and feeding regime on PHA productivity, composition and properties

Journal of Biotechnology 151 (2011) 66–76

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Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: Effect of substrate composition and feeding regime on PHA productivity, composition and properties M.G.E. Albuquerque a , V. Martino b , E. Pollet b , L. Avérous b , M.A.M. Reis a,∗ a b

CQFB-Requimte, FCT-UNL, Lisbon, Portugal ECPM-LIPHT (EAc CNRS 4379), Université de Strasbourg, 25 Rue Becquerel, 67087 Strasbourg Cedex 2, France

a r t i c l e

i n f o

Article history: Received 1 July 2010 Received in revised form 2 September 2010 Accepted 15 October 2010

Keywords: Polyhydroxyalkanoates Mixed cultures Fermented molasses Feeding regimen Volatile fatty acids

a b s t r a c t In this study, the possibility of manipulating biopolymer composition in mixed culture polyhydroxyalkanoate (PHA) production from fermented molasses was assessed by studying the effects of substrate volatile fatty acid (VFA) composition and feeding regime (pulse wise versus continuous). It was found that the use of a continuous feeding strategy rather than a pulse feeding strategy can not only help mitigate the process constraints of the pulse-feeding strategy (resulting in higher specific and volumetric productivities) but also be used as means to broaden the range of polymer structures. Continuous feeding increased the hydroxyvalerate content by 8% relatively to that obtained from the same feedstock using pulse wise feeding. Therefore, the feeding strategy can be used to manipulate polymer composition. Furthermore, the range of PHA compositions, copolymers of P(HB-co-HV) with HV fraction ranging from 15 to 39%, obtained subsequently resulted in different polymer properties. Increasing HV content resulted in a decrease of the average molecular weight, the glass transition and melting temperatures and also in a reduction in the crystallinity degree from a semi-crystalline material to an amorphous matrix. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Polyhydroxyalkanoates (PHAs) are biologically synthesized polyesters that are fully biodegradable and can be produced from renewable sources, thus allowing for a lower environmental impact than conventional chemically synthesized polymers. Moreover, PHAs present a very high replacement potential over conventional polyolefins due to interesting thermoplastic properties. Depending on the type and relative proportion of HA monomers, these biopolymers present a broad range of structural, thermal and mechanical properties. Although they are already industrially produced, their commercialization remains limited to high-value applications due to their high production costs. To date, industrial PHA production is carried out using pure microbial culture fermentation technology with high costs associated with carbon substrate (refined sugar substrates), fermentation operation and downstream processing. In the last decade, research has focused on the development of alternative production processes aiming to decrease these production costs. Such alternative processes include not only the use of genetic/metabolic engineering strategies to optimize pure culture fermentations, but also that of mixed microbial cultures (MMC),

∗ Corresponding author. E-mail address: [email protected] (M.A.M. Reis). 0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.10.070

requiring lower investment and operating costs due to the use of open systems which do not require sterile conditions, coupled to that of wastes/surplus based feedstocks. It has recently been suggested, based on Life Cycle Analysis (LCA), that PHA production using mixed cultures may be more favorable than using pure cultures in both economic and environmental terms (Gurieff and Lant, 2007). Conditions of external substrate excess (feast) and limitation (famine) were shown to select for microbial populations with enhanced capacity to store PHA (Majone et al., 1996; van Loosdrecht et al., 1997). This type of process was designated as Feast and Famine (FF) or Aerobic Dynamic Feeding (ADF). Bacterial PHA synthesis is known to occur under conditions in which growth is restricted by either an external (lack of nutrient or electron acceptor) or an internal factor (Daiger and Grady, 1982; Anderson and Dawes, 1990; Gujer et al., 1999). It was proposed that mixed microbial cultures operated under Feast and Famine conditions were subjected to an internal growth limitation arising from the alternate substrate availability, which compelled the organisms to a physiological adaptation (Beccari et al., 1998). During this physiological adaptation period, substrate uptake is mainly driven toward polymer storage. Beun et al. (2002) have shown that following a long starvation period, a mixed culture operated under ADF conditions channels around 70% (on a C-mole basis) of the carbon substrate uptake toward PHA storage.

M.G.E. Albuquerque et al. / Journal of Biotechnology 151 (2011) 66–76

The key to the effectiveness (in terms of both storage capacity and productivity) of MMC PHA production processes relies on culture selection (enrichment in PHA accumulating organisms) by the conditions imposed to the reactor. The use of MMC to produce added value products (such as biochemicals and biomaterials) using ecological selection principles to engineer the microbial consortium has been designated as eco-biotechnology (Kleerebezem and van Loosdrecht, 2007). Research has focused first on the use of synthetic volatile fatty acids (as reviewed by Dias et al., 2006) and, more recently, on the use of low cost agro-industrial surplus feedstocks (as reviewed by Serafim et al., 2008a; e.g. fermented molasses – Albuquerque et al., 2007, 2010a,b; Bengtsson et al., 2010a,b; fermented paper mill effluents – Bengtsson et al., 2008; industrial wastewaters – Dionisi et al., 2006; fermented olive oil mill effluents – Dionisi et al., 2005; Beccari et al., 2009). Although MMC FF systems have demonstrated a good potential for PHA production reaching high specific productivities, with polymer yields on substrate and maximum PHA contents similar to those attained by pure cultures (Serafim et al., 2004; Johnson et al., 2009) these systems still present lower performances than pure culture fermentations in terms of volumetric productivity. This is due to the considerably lower cell concentrations obtained in ADF mixed culture processes than those reached in pure culture systems. Most studies on MMC PHA-accumulating culture selection reactors (operated under Feast and Famine conditions) report cell concentrations lower than 10 g/L of volatile suspended solids (Serafim et al., 2004; Dionisi et al., 2005, 2006; Bengtsson et al., 2008; Johnson et al., 2009; Beccari et al., 2009; Albuquerque et al., 2007, 2010a,b), whereas, in pure culture fermentations, values above 100 g/L are often reported (Lee et al., 1999). This results from the conditions of alternate substrate availability under which culture selection is carried out, which limit the culture’s primary metabolism (internal growth limitation induced by the Feast and Famine conditions). Developing strategies to improve volumetric productivity of MMC PHA production processes is essential to make this process competitive with that of pure cultures. Besides, it is also important to determine whether the quality of the biopolymers produced by MMC can meet the standards required for use in common plastic applications (which has already been demonstrated for PHA produced using pure culture fermentation from refined substrates), particularly considering the polymers produced from waste/surplus based feedstocks. Although the number of studies dedicated to mixed microbial culture PHA production has increased considerably in recent years, only few and recent studies investigated the polymer characteristics (Serafim et al., 2008b; Bengtsson et al., 2010b; Patel et al., 2009). The most commonly investigated polyhydroxyalkanoate is the homopolymer poly-3-hydroxybutyrate, P(3HB). This biopolymer shows a melting temperature close to 180 ◦ C (Kunioka and Doi, 1990) and a glass transition temperature around 4 ◦ C (Mitomo et al., 1999). But P(3HB) is a highly crystalline polymer (55–80%) resulting in fairly stiff and brittle materials, somewhat limiting its applications (e.g. elongation to break is about 2–10% compared to up to 400% for some polyolefins). Another important limitation comes from narrow window of processability. PHA thermal and mechanical properties depend directly on the polymer composition and structure. The incorporation of different monomer types reduces polymer crystallinity by disturbing the crystal lattice. For instance, poly-3-hydroxybutyrate-co-3-hydroxyvalerate, P(3HB-co-3HV), copolymers exhibit lower crystallinity. Compared to P(3HB), copolymers of hydroxybutyrate and hydroxyvalerate present improved mechanical properties with decreased stiffness and brittleness, increased flexibility (higher elongation to break), increased tensile strength and toughness, with preserved biodegradability. Moreover, P(3HB-co-3HV) melting and glass transition temperatures steadily decrease from 0 to 30 mol% fraction

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of 3HV (Bluhm et al., 1986; Feng et al., 2002). Because the temperature at which degradation and decomposition of PHA occur is rather insensitive to the polymer composition, the lower melting temperatures of these copolymers allow for a wider temperature processing window and thus increase its processability. An advantage of MMC PHA production relates to the wider range of PHA compositions obtained. MMC fed with fermented feedstocks (containing mixtures of organic acids such as acetate, propionate, butyrate and valerate) produce PHA polymers with a considerably high diversity of different HA monomers, containing monomers other than 3HB, such as 3-hydroxyvalerate (3HV), 3-hydroxy-2-methyl-valerate (3H2MV) or 3-hydroxyhexanoate (3HHx) (Takabatake et al., 2000; Lemos et al., 2006; Bengtsson et al., 2010b). For instance, co-polymers of P(HB-co-HV) with HV ranging from 17 to 85% and a termopolymer of 3HB:3HV:3H2MV with molar ratio of 6:58:24 were reported by Lemos et al. (2006) from acetate/propionate mixtures, whereas Takabatake et al. (2000) produced co-polymers of 3HB and 3HV, in which the 3HV molar fraction increased according to the propionate fraction in the mixture fed (up to 84% of 3HV from pure propionate). The fraction of monomers other than 3HB is considerably higher in polymers produced by mixed cultures from mixtures of VFA than what has been reported for pure cultures, typically fed with refined sugars, and which require large amounts of co-substrates (such as alcohols or organic acids) to produce polymers with relatively small fractions of monomers other than PHB. For instance, Lee et al. (2008) have reported the production of P(HB-co-HV) with only 2–8% HV from mixtures with equal mass amounts of a surplus carbon feedstock (vegetable oils) and a co-substrate used to induce HV storage (propionic acid, commercial grade). In MMC systems, polymer composition depends on the composition of the fermented effluent produced in the early anaerobic digestion step. MMC PHA production processes can be managed and controlled to produce copolymers with improved properties. As shown in Albuquerque et al. (2007), manipulating operating conditions of the anaerobic fermentation step (e.g. pH) used to produce the fermented volatile fatty acid (VFA)-rich stream can be used to control the biopolymer composition in the PHA production step. However, the degree of manipulation of the polymer composition will still be limited to the range of VFA concentrations obtained by fermentation for any given feedstock. Therefore, in order to broaden the range of copolymers produced from any given fermented effluent (with stable VFA composition), strategies to manipulate polymer composition (Villano et al., 2010 – effect of pH) and microstructure (Ivanova et al., 2009) in the batch production stage have also been investigated. In this study, the possibility to control polymer composition (and resulting properties) in mixed culture PHA production from fermented molasses was assessed by studying the effects of substrate VFA composition and feeding regime on polymer composition and structure. The different biopolymers produced were characterized in terms of chemical, structural and thermal properties. Finally, the copolymers produced from fermented molasses were compared in terms of molecular weight and thermal properties to PHA produced from synthetic VFA.

2. Materials and methods 2.1. Experimental setup The experimental setup consisted of three bench-scale reactor systems and a hollow fiber membrane filtration module. The molasses acidogenic fermentation (stage 1) was carried out in a continuous stirred tank reactor (CSTR) operated under anaerobic conditions. The reactor effluent was clarified by microfiltration

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and the clarified fermented molasses were used as a feedstock for culture selection and PHA batch accumulation. Selection of a PHAaccumulating culture (stage 2) was carried out in a Sequencing Batch Reactor (SBR) subjected to Aerobic Dynamic Feeding (ADF) conditions. PHA accumulation (stage 3) was carried out in a batch reactor inoculated with sludge from the culture enrichment SBR and fed with clarified fermented molasses produced in stage 1. 2.1.1. Continuous acidogenic fermentation reactor The CSTR with a working volume of 1140 ml was operated under anaerobic conditions using the conditions reported by Albuquerque et al. (2007), i.e., pH 6, 30 ◦ C, HRT kept at 10 h and influent substrate concentration of 10 g/L sugars. The sugars in the molasses, accounting for 75% of the molasses dissolved organic carbon (DOC) prior to fermentation, were fully exhausted (>99% consumption). The main fermentation products were VFA (acetate, propionate, butyrate and valerate), making up about 75% of the DOC of the fermented molasses. The sugar–VFA conversion yield was about 0.70 Cmol VFA/Cmol sugars. The CSTR feed was supplemented with NH4 Cl and KH2 PO4 using C/N/P ratios of 100/3/1. This ratio was previously optimized (Albuquerque et al., 2007) in order to result in low residual nutrients in the fermented molasses stream (<0.5 Nmmol/L and <0.2 Pmmol/L). The low residual nutrient concentration was designed to allow the 2nd and 3rd stages (culture selection and PHA production) to be operated under different nutrient conditions: the selection reactor, operated under excess nutrient conditions, was supplemented with N, P, while the batch production stage was run under nutrient limitation. The effluent was withdrawn by overflow and microfiltrated through a hollow fiber membrane module. The clarified effluent was kept at 4 ◦ C prior to its use in as a feedstock for PHA-accumulating culture selection or in PHA batch accumulation assays. 2.1.2. Culture selection A Sequencing Batch Reactor (SBR) – with a working volume of 800 ml – was operated according to the conditions previously described by Albuquerque et al. (2010a). SBR cycles were 12 h long and consisted of four discrete periods: (i) fill (5 min), (ii) aerobiosis (Feast and Famine) (11 h), (iii) settling (45 min) and (iv) draw (10 min). The hydraulic retention time (HRT) was kept at 1 day and the sludge retention time (SRT) at 10 days. The SBR was fed with clarified fermented molasses at an influent substrate concentration of 45 Cmmol VFA/L. Feed solution was kept at 4 ◦ C in a refrigerated vessel. A mineral nutrient solution, containing both ammonia and phosphate, was simultaneously added to the reactor, keeping the C/N/P ratios at 100/8/1. Thiourea (10 mg/L) was also added to the mineral nutrient solution to inhibit nitrification. Air was supplied by an air pump through a ceramic diffuser. Magnetic stirring was kept at 500 rpm. Feed pH was adjusted to 8 ± 0.05 prior to reactor feeding and pH was left uncontrolled during the reaction phase. The reactor stood in a temperature-controlled room (23–25 ◦ C). The SBR was operated under these conditions for a period of 10 consecutive months, selecting for a microbial culture highly enriched in PHA-storing organisms (88%) and showing stable PHA storage performance in the enrichment SBR and in batch production assays (Albuquerque et al., 2010a). 2.1.3. PHA production PHA accumulation assays were carried out in a 2 L reactor – with an initial working volume of 1.1 L – operated in batch mode, inoculated with the SBR enriched culture, which was collected at the end of the famine phase, and fed either with clarified fermented molasses produced in stage 1 or with chemically defined media simulating the fermented molasses effluent. Feed pH was adjusted to 8 before reactor feeding. In order to maximize PHA storage, assays were carried out under ammonia limitation (no ammonia

was added and residual ammonia concentration in the sludge collected from the SBR was less than 0.1 Nmmol/L). Aeration was kept at 400 ml/min and stirring at 300 rpm. The reactor was kept in a temperature controlled room (23–25 ◦ C). The reactor was equipped with two gas mass flowmeters (Smart MassFlow 5850S, Brooks Instruments) to measure the inlet and outlet air flow rates. The concentration of O2 in the off-gas was measured with a gas analyser (Tandem, Magellan Instruments). The effects of feeding regimen and pH control were assessed in assays A1–A3 carried out using a VFA mixture designated as VFA profile A (acetate, propionate, butyrate and valerate in fractions of 30/20/30/20 Cmol/100 Cmol VFA) either in pulse wise feeding mode (A1 and A2) – feeding several pulses of VFA profile A with concentrations between 60 and 80 Cmmol VFA/L – or using a continuous supply of carbon substrate (A3). The first test A1 was carried out leaving the pH uncontrolled (as used in the enrichment SBR), while the second pulse wise feeding test (A2) and the continuous feeding test (A3) were operated with pH control, respectively at 8.2 and 8.4. For the later, the pH setpoint was used as a means of controlling substrate addition to the reactor (pH-stat). In this case, the VFA feed solution was used as acid solution for the pH controller. In all cases, a concentrated feed solution (1000 Cmmol VFA/L) was used in order to minimize the dilution resulting from substrate addition. To test the effect of substrate composition on PHA composition, two additional VFA mixtures designated as VFA profile B (acetate, propionate, butyrate and valerate in fractions of 60/15/20/05 Cmol/100 Cmol VFA) and VFA profile C (acetate, propionate, butyrate and valerate in fractions of 60/10/25/05 Cmol/100 Cmol VFA), were used to carry out pulse wise feeding tests (B1 and C1). VFA mixtures A and B simulated the fermented molasses VFA composition obtained at two different acidogenic fermentation operating pH (5 and 6, as reported in Albuquerque et al., 2007), while VFA profile C simulated the fermented molasses currently fed to the enrichment SBR. Finally, to confirm that the results obtained with the VFA mixtures could serve to predict those that could be obtained when using real fermented molasses, the later assay (VFA profile C) was repeated using real fermented molasses (test C2). The exact VFA profiles A1, A2, B1, C1 and C2 are listed in Table 1. 2.2. Analytical procedures Biomass concentration was determined using the volatile suspended solid (VSS) procedure described in Standard Methods (APHA, 1995). Volatile fatty acids – namely acetate, propionate, butyrate and valerate – concentrations were determined by high performance liquid chromatography (HPLC) using a Merck-Hitachi chromatographer equipped with a UV detector and Aminex HPX87H pre-column and column from BioRad (USA). Sulphuric acid 0.01 M was used as the eluent at a flow rate of 0.6 ml/min and 50 ◦ C operating temperature. The detection wavelength was set at 210 nm. The organic acids (acetate, propionate, butyrate and valerate) concentrations were calculated through calibration curves using 25–1000 mg/L standards (Merck, analytical grade). Polyhydroxyalkanoate concentrations were determined by gas chromatography using the method adapted from Serafim et al. (2004). Lyophilized biomass was incubated for methanolysis in chloroform and a 20% sulphuric acid in methanol solution. After the digestion step, the organic phase (methylated monomers dissolved in chloroform) of each sample was extracted and injected into a gas chromatograph coupled to a Flame Ionization Detector (GC-FID Varian CP-3800). A ZBWax-Plus column was used at a flow rate of 1 ml/min. Split injection at 280 ◦ C with a split ratio of 10 was used. The oven temperature program was as follows: 40 ◦ C;

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Table 1 Summary of batch tests conducted by feeding the SBR enriched culture (selected using a fermented molasses feedstock) with either real or simulated fermented molasses (with different VFA compositions: A–C) using either pulse wise or continuous feeding. Batch

Feedstock

VFA profile Hac/Hprop/Hbut/Hval in Cmol/100 Cmol VFA

Simulated fermented molasses

A

A1 A2

32/19/28/21

A3a B1a

Pulse feeding

30/20/28/22

B

C1 C2a

Feeding regime

C Fermented molasses

pH control

YPHA/VFA (Cmol/Cmol)

Maximum PHA content (%)

PHA composition (%HB:%HV)

No 8.3–8.9

0.77b (0.07; 18); 0.53c (0.13; 26) 0.78b (0.05; 13); 0.51c (0.14; 22) 0.80c (0.02; 10) 0.74b (0.07; 16); 0.59c (0.12; 24) 0.75b (0.04; 15); 0.57c (0.12; 19) 0.80b (0.07; 22); 0.65c (0.15; 27)

65 (4)

69:31 (1.7; 12)

65 (4)

70:30 (1.2; 13)

77 (3) 68 (6)

61:39 (0.3; 10) 80:20 (1.8; 10)

66 (5)

83:17 (1.2; 11)

56 (4)

85:15 (0.8; 18)

Yes 8.2

31/18/29/22 60/16/20/04

Continuous Pulse feeding

Yes 8.4 No 8.2–9.0

59/09/26/06

Pulse feeding

No 8.3–9.0

60/09/25/06

Pulse feeding

No 8.4–9.1

(st dev; sample number); (error associated with the listed value). a Samples (A3, B1, and C2) were characterized in terms of structural and thermal properties. b Yields of polymer on substrate were calculated accounting for all the PHA formed (sum of PHA formed in each pulse). c Also considering the difference between final and initial PHA concentration. The later value is lower than the first due to PHA consumption during stops between pulses.

then 20 ◦ C/min until 100 ◦ C; then 3 ◦ C/min until 175 ◦ C; and finally 20 ◦ C/min until 220 ◦ C. The detector temperature was set at 250 ◦ C. Hydroxybutyrate and hydroxyvalerate concentrations were calculated using two calibration curves, one for hydroxybutyrate and one for hydroxyvalerate, using standards (0.1–2 mg/ml) of a commercial P(HB-HV) (88%/12%) (Sigma) and corrected using a heptadecane internal standard (concentration of approximately 1 mg/ml). 2.3. Biopolymer recovery and characterization 2.3.1. Biopolymer recovery At the end of each batch accumulation assay, mixed liquor was discharged and cells were separated from the exhaust supernatant by centrifugation (15 min at 8000 rpm). The concentrated sludge cake was washed, filtered and lyophilized (to completely remove all water content). Lyophilized samples were then suspended in chloroform and left to dissolve for a period of 3 days at 37 ◦ C. The chloroform solution was then filtered to remove all non dissolved material and used to fill glass petri dishes. Finally, chloroform was evaporated from the petri dishes to allow polymer recovery in the form of a thin film. 2.3.2. Biopolymer characterization Polymer samples from batch tests A3, B1 and C2 were characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and nuclear magnetic resonance (NMR), respectively. Thermogravimetric analysis (TGA) was performed on a TA Instruments TGA Q5000 (USA). Samples were heated from room temperature up to 500 ◦ C at 10 ◦ C/min under inert gas atmosphere. TGA was used to determine the thermal stability of the polymer samples according to the maximum degradation rate temperature (Tmax ). Differential scanning calorimetry (DSC) was conducted on a TA Instruments DSC 2910 (USA) under nitrogen atmosphere using both hermetic and non-hermetic aluminium pans. The materials were exposed to successive thermal cycles (heat–cool–heat) between −70 ◦ C and 180 ◦ C at 10 ◦ C/min. DSC was used to determine the polymers’ thermal properties. Glass transition temperature (Tg ) and melting temperature (Tm ) were estimated during the second eating scan, while crystallization temperature (Tc ) was determined during cooling. Size exclusion chromatography (SEC) measurements were performed using a Shimadzu liquid chromatograph apparatus (Japan) equipped with a RID-10A refractive index detector and a SPD-M10A diode array UV detector. The columns set used was composed of

a 50 mm PLgel Guard 5 ␮m column, two 300 mm PLgel MixedC 5 ␮m columns and a 300 mm PLgel 5 ␮m-100 A column. The calibration was realized with polystyrene standards from 580 to 1.6 × 106 g mol−1 . Chloroform was used as the mobile phase and the analyses were carried out at 25 ◦ C with a solvent flow rate of 0.8 ml/min. SEC analysis was used to determine the polymers’ average molar masses (Mn , Mw ) and polydispersity index (PDI). X-ray diffraction (XRD) and more precisely wide angle X-ray scattering (WAXS) were used to determine the crystallinity of the biopolymers. Diffraction patterns of PHBV films were recorded with a powder diffractometer Siemens D-5000 (Munich, Germany) using Cu K␣ radiation source ( = 0.1546 nm). The incidence angle was varied between 5 and 60◦ with step size of 0.06◦ and step time of 4 s. The degree of crystallinity was estimated by considering the area under the crystalline peaks related to that of the amorphous halo. Some samples were also analyzed using a polarized light optical microscope (POM) to observe the crystallization behavior. 2.4. Calculation of kinetic and stoichiometric parameters of PHA production The sludge PHA content was calculated as a percentage of VSS on a mass basis (%PHA = g PHA/g VSS × 100), where VSS includes active biomass (X) and PHA. Active biomass was calculated by subtracting PHA from VSS. The maximum specific substrate uptake (−qS in Cmol VFA/Cmol X h) and PHA storage rates (qP in Cmol PHA/Cmol X h) were determined by adjusting a linear function to the experimental data of VFA and PHA concentrations plotted over time, calculating the first derivative at time zero (taking the slope of the fitting) and dividing the value thus obtained by the active biomass concentration at that point. VFA concentration corresponds to the sum of all the organic  acid concentrations (VFA, in terms of Cmmol/L, is equal to HAc, HProp, HBut, HVal in Cmmol/L). PHA concentration (in Cmmol/L) corresponds to the sum of HB and HV monomer concentrations (in Cmmol/L). The yields of PHA (YPHA/VFA in Cmol PHA/Cmol VFA) on substrate consumed were calculated by dividing the amount of PHA formed by the total amount of organic acids consumed, respectively. 3. Results and discussion 3.1. Effect of substrate VFA profile on polymer composition, yield and maximum PHA content An important aspect related to the optimization of MMC PHA production systems, particularly from fermented feedstocks,

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relates to the ability to manipulate polymer composition in the accumulation stage. It had been previously shown by Albuquerque et al. (2007) that the fermented molasses VFA profile could be manipulated through the acidogenic fermentation pH. The authors reported the use of both fermented molasses (with different VFA profiles) and that of synthetic VFA mixtures simulating the fermented molasses feedstock for PHA production using an acetateselected culture. No significant variations were reported in terms of PHA yield on substrate, maximum PHA content and PHA composition between the synthetic and fermented feedstocks. Thus, in the present study, different synthetic VFA mixtures (A, B and C – see Table 1) were used as substrate for PHA batch production by a fermented molasses-enriched culture through pulse wise feeding in order to assess the effect of substrate composition on polymer composition and subsequent properties. Three assays were carried out with simulated fermented molasses (A1, B1 and C1), with VFA profiles simulating those obtained at different acidogenic reactor pH (Albuquerque et al., 2007). One assay was carried out with real fermented molasses aiming at confirming that the molasses matrix will not introduce significant variations on the final result. In terms of PHA storage efficiency, maximum PHA contents between 56 and 77% were attained, with the fermented molasses feed and synthetic feedstock, respectively. However, it is important to point out that the later assay was run until PHA synthesis saturation was attained, while the batch assay using fermented molasses was not. Due to differences in kinetic rates of substrate uptake and PHA storage, assays run with the real fermented stream take longer than those carried out with synthetic VFA mixtures, accounting for the observed differences in maximum PHA content reached. Despite the lower value attained with fermented molasses, this is still a comparatively high value for fermented feedstocks (55% were reported by Dionisi et al., 2005 using olive oil mill effluents, 54% by Bengtsson et al., 2008, using paper mill effluents and 32–37% by Bengtsson et al., 2010a for fermented molasses). The higher maximum PHA content obtained with the synthetic feedstock (77%) represents a comparatively high value for mixed culture PHA production. The maximum value for MMC PHA production was reported by Johnson et al. (2009), with a maximum PHA content of 89% obtained from pure acetate. High PHA yields on substrate were also obtained, ranging from 0.74 to 0.80 Cmol PHA/Cmol VFA, the higher values observed for the fermented molasses feedstock. In all cases, copolymers of hydroxybutyrate and hydroxyvalerate were obtained (confirmed by unimodal curve in SEC). The HV content ranged from 15 to 31% as a function of the amount of VFA with odd number of carbon atoms (propionate and valerate) in the feed (which ranged from 15 to 40% Cmol/Cmol VFA) (Table 1). This is in accordance with what had been previously reported by Albuquerque et al. (2007) for an acetate selected culture fed with fermented molasses produced at different acidogenic fermentation pH (with different VFA profiles). It is also in agreement with results by Bengtsson et al. (2010a) which obtain co-polymers containing five types of monomers, namely 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2methylbutyrate (3H2MB), 3-hydroxy-2-methylvalerate (3H2MV) and the medium chain length monomer 3-hydroxyhexanoate (3HHx) from fermented molasses with different VFA profiles. The authors found that composition of the PHA was dependent on the VFA composition of the fermented molasses and was 56–70 mol% 3HB, 13–43 mol% 3HV, 1–23 mol% 3HHx and 0–2 mol% 3H2MB and 3H2MV. Despite the fewer types of monomers obtained in this study, similarly to the report by Bengtsson et al. (2010a), (3H)B was the monomer with the higher fraction and the presence of other monomers was directly dependent on the propionate and valerate fraction of the feed. The different co-polymers produced were then characterized (see Section 3.3) in terms of molecular weight and thermal prop-

erties in order to assess whether the manipulation of the polymer composition through substrate VFA profile could be an appropriate means to manipulate polymer properties. An interesting result is that the polymer composition is not significantly affected by using real fermented molasses instead of the simulated fermented molasses (Table 1). This result is in agreement with previous observations using fermented molasses and synthetic VFA mixtures fed to an acetate-selected PHA storing culture (Albuquerque et al., 2007) and also concordant with findings reported by Albuquerque (2009) and Pardelha et al. (submitted for publication) which have compared kinetic aspects of PHA production from either synthetic VFA mixtures or fermented molasses. This indicates that the molasses matrix does not introduce significant variation on the polymer composition. The yield of PHA on VFA was slightly higher with fermented molasses due to the presence of residual non VFA carbon existing in the fermented molasses which was used for PHA production. The likely use of a carbon source other than the measured VFA for PHA production was confirmed by analysis of the soluble TOC consumed over several production cycles. It was shown that the sTOC consumed was in each case higher than the total VFA quantified (Albuquerque et al., 2010a). However, and despite attempts to characterize the fermented effluent, the nature of this additional carbon source was not identified. The slightly lower HV content obtained with fermented molasses (15%) relatively to that obtained with the simulated VFA feed (17%) could indicate that this residual organic fraction contributes to the HB content of the co-polymer. The lower maximum PHA content (56%) obtained with the fermented molasses compared to that produced from the simulated feed (66%) relates to the slightly lower rates of substrate uptake and PHA storage observed with the real effluent, which result in a lower kinetic performance and thus, for a similar fermentation period, in lower polymer content. These results show that although the molasses matrix has a slight impact on kinetic performance it does not significantly influence polymer composition and thus, results obtained in the remaining tests can be used to predict possible outcome when using real fermented molasses. 3.2. Effect of feeding regimen on PHA productivity and composition In previous batch PHA accumulation studies using fermented molasses (Albuquerque et al., 2007, 2010a,b), a multiple pulse addition of the fermented substrate was used in order to overcome potential substrate inhibition (shown in Serafim et al., 2004; Albuquerque et al., 2007). However, this feeding strategy posed a constraint on process productivity. The high number of pulses supplied (due to the use of relatively low carbon substrate concentrations per pulse) and the need to supply a considerable volume of feed for each new pulse (resulting from the relatively diluted fermented feedstock) resulted in a considerable loss of productivity. For each new pulse, the reaction had to be stopped, biomass decanted, exhaust supernatant withdrawn and new medium fed, which resulted in stops between pulses and on some partial consumption of the PHA synthesized between consecutive pulses. Therefore, the first goal of this study was to assess the use of a continuous feeding strategy to operate the batch PHA production stage in order to overcome the constraints associated with pulse wise feeding. The continuous feeding was carried out using the VFA feed solution (VFA profile A) as acid solution to control reactor pH. In this assay, A3, the continuous addition of substrate was carried out by supplying a first pulse of feed solution (60 Cmmol VFA/L), and hence on keeping the pH controlled (at 8.4) by continuous addition of the VFA feed (pH-stat). Because the pulse wise feeding assay (A1) was run without pH control (pH varying for each pulse from about 8.3 to 8.9), a

(st dev; sample number); (error associated with the listed value). a VFA profile HAc/HProp/HBut/HVal in fractions of Cmol/100 Cmol VFA. b Yields of polymer on substrate were calculated accounting for all the PHA formed (sum of PHA formed in each pulse). c Also considering the difference between final and initial PHA concentration. The later value is lower than the first due to PHA consumption during stops between pulses. d −qS and qP are the average specific rates of substrate uptake and PHA storage, in Cmol VFA/Cmol X h and Cmol PHA/Cmol X h, respectively, calculated for the entire batch.

0.32 (0.006) 0.35 (0.005) 0.47 (0.005) 0.77b (0.02); 0.53c (0.01) 0.78b (0.02); 0.51c (0.01) 0.80c (0.02) A1 A2 A3

Pulses Pulses Continuous

No (8.3–8.9) Yes 8.2 Yes 8.4

469 254 497

32/19/28/21 30/20/28/22 31/18/29/22

1.0 1.0 1.4

6.3 (0.03) 4.0 (0.02) 6.8 (0.03)

65 (4) 65 (4) 77 (3)

69:31 (1.7; 12) 70:30 (1.2; 13) 61:39 (0.3; 10)

−qS d

0.20 (0.010) 0.17 (0.007) 0.26 (0.008)

71

YPHA/VFA (Cmol PHA/Cmol VFA) PHA comp. (%HB:%HV) Max. PHA (%) VSS (g/L) Dil. factor VFA profilea Substrate fed (Cmmol VFA/L) pH control Feeding reg.

Table 2 Stoichiometric and kinetic parameters of PHA storage as a function of feeding regime and pH control.

new experiment with pulse wise feeding and pH control (A2) was carried out in order to access the pH impact on the polymer composition and productivity. Stoichiometric and kinetic parameters observed in the three batch assays are compiled in Table 2. Results show that pH control has no significant effect on polymer yield on substrate, composition and PHA content. Results of the continuous feeding test (A3) are compared with pulse wise feeding tests A1 and A2. Villano et al. (2010) assessed the effect of operating pH on PHA storage efficiency and composition using a mixture of synthetic acetate (85% on a COD basis) and propionate (15%), simulating the effluent of the first fermentation stage in a WWTP, to carry out both the culture enrichment and PHA accumulation stages in a 2-stage PHA production process. The authors carried out PHA batch accumulation studies at different controlled pH values (7.5–9.5) and have observed somewhat lower PHA storage rates (and increased HV content) in co-polymers of P(HB-co-HV) synthesized at pH 9.5 relatively to the those observed at pH 8.5 (same pH as used for enrichment). The authors suggested that this may be due to a higher maintenance requirement at higher pH. However, unlike what occurred in the reported study (in which the culture was enriched using controlled pH, at 8.5), in this case, the culture was enriched without pH control. Moreover, the pH variation during each pulse was only in the range of 0.5–0.7 pH units. However, a slight distinction was observed between assays A1 and A2. In the first assay (A1), the instant volumetric PHA storage rate seemed to gradually decrease throughout each pulse of substrate, whereas in test A2, carried out at a controlled pH of 8.2, for each pulse of substrate fed, the PHA storage rate seemed to remain fairly constant throughout most of each pulse, decreasing later and more steeply. Thus, for assay A1 there seemed to be a gradual decrease of the PHA storage rate, which was thought to be associated with the decrease of external substrate concentration (as was previously suggested in Albuquerque et al., 2010a,b), whereas for assay A2 – with controlled pH – the decrease of the PHA storage rate appeared to be less gradual and thus seemed to be rather associated with near full depletion of the carbon substrate. Therefore, the results from the two assays may suggest that the gradual decrease observed in the assay run without pH control may be attributable, at least in part, to the increasing pH. Although a clear correlation between increasing pH and decreased PHA storage rate cannot be inferred from these two assays (A1 and A2), the slight distinction between the two is enough to suggest that further clarification on the effect of pH on PHA storage efficiency is required. Notwithstanding this distinction in the profiles of the instant PHA storage rates, in both cases, the volumetric productivity is greatly affected by the use of a pulse wise feeding strategy. Fig. 1a and b shows PHA production using pulse wise feeding run without and with pH control (A1 and A2), respectively. In assay A1, stops between pulses represented 16% of the total batch duration and some partial consumption of the PHA synthesized occurred between consecutive pulses (33% of the PHA formed was consumed between pulses). The consumption of formed PHA reduced the global PHA yield on VFA substrate from 0.77 Cmol PHA/Cmol VFA (if all the PHA was accounted) to 0.53 Cmol PHA/Cmol VFA. The time loss and polymer consumption – resulted in a 43% decrease of volumetric productivity relatively to what would have been obtained if the stops between pulses (and respective PHA consumption) could be avoided. It was also observed in both pulse wise feeding assays (Fig. 2a and b), that for each pulse of substrate fed, the rates of substrate uptake and PHA storage were maximum at the beginning of the pulse, but as substrates were depleted these rates decreased (30 and 35% decrease for the substrate uptake and PHA storage rates, respectively, in assay A1). The dependence of rates

qP d

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Fig. 1. Batch accumulation assays with VFA mixture: (a) pulse-wise feeding A1, (b) pulse wise feeding A2 and (c) continuous feeding A3; (d) PHA fraction in the three batch assays A1–A3.

of substrate uptake and PHA storage on substrate concentration has been reported for the enrichment reactor operating cycles (Albuquerque et al., 2010a,b), for which an affinity constant (kS ) of about 18.5 Cmmol VFA/L was reported (Albuquerque et al., 2010b). Fig. 1c shows the results from the continuous feeding assay A3. This system demonstrated a considerable improvement of the PHA storage performance relatively to both pulse-wise feeding tests using the same medium (A1 and A2). In test A3, the maximum specific substrate uptake (0.64 Cmol VFA/Cmol X h) and PHA storage (0.40 Cmol PHA/Cmol X h) rates were higher than those obtained in both pulse wise feeding tests (0.46 and 0.43 Cmol VFA/Cmol X h and 0.31 and 0.35 Cmol PHA/cmol X h, in A1 and A2, respectively). This can be explained by the different initial substrate concentrations used (60 Cmmol VFA/L used for A3 rather than the 80 Cmmol VFA/L used in A1 and A2). A considerable decrease in substrate uptake and polymer storage rates was shown to occur for the same fermented molasses-selected culture for concentrations

higher than 90 Cmmol VFA/L (Albuquerque et al., 2007). A gradual decrease of these rates has also been shown to occur in the range of 60–90 Cmmol VFA/L (Albuquerque, 2009). More importantly, a much steadier PHA storing activity (at maximum level) was observed for the continuous feeding assay A3, as opposed to the two pulse feeding assays, A1 and A2, where a slowdown of specific rates was observed for each pulse. This is visible in Fig. 1d in which the fraction of PHA per active biomass is plotted for the three assays. The continuous feeding strategy allowed the specific rates of substrate uptake and PHA storage to stay very close to maximum levels until about 6 h (0.54 Cmol VFA/Cmol X h and 0.39 Cmol PHA/Cmol X h, respectively), at which point the culture had already reached a PHA content of 72%. After this point onward, the specific rates of substrate uptake and PHA storage decrease, due to proximity to the saturation level, which causes the average specific rates determined for the full length of test A3 (substrate uptake rate of 0.47 Cmol VFA/Cmol X h and PHA stor-

Fig. 2. PHA productivity (in g PHA/L h), instant PHA storage rate (in g PHA/L h) and PHA content (%) over time for pulse wise feeding assays A1 (a) and A2 (b) – respectively without and with pH control – and continuous feeding assay A3 (c); (d) PHA productivity (in g PHA/L h) as a function of PHA content (%) for assays A1–A3.

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age rate of 0.26 Cmol PHA/Cmol X h, Table 2) to decrease relatively to the rates observed 6 h into the test. Nonetheless, these average rates were still considerably higher than the average rates of both pulse-wise feeding tests (0.32 and 0.27 Cmol VFA/Cmol X h and 0.20–0.17 Cmol PHA/Cmol X h, in A1 and A2, respectively), none of which having attained the same proximity to the saturation level (since the maximum PHA contents observed were about 65% in both A1 and A2). The continuous feeding strategy seemed to be an effective way of improving process productivity (Fig. 2). A volumetric productivity of 1.2 g PHA/L h was observed 6 h into the continuous feeding test (at 72% PHA content) (Fig. 2c), which represents a 2.5–4 fold increase relatively to the productivities obtained in assays A1 and A2 at 65% PHA content (0.3–0.5 g PHA/L h) (Fig. 2a and b). In fact, the 1.4 fold decrease of the instant volumetric PHA storage rate observed in the continuous feeding test during this first 6 h period (Fig. 2c) is almost consistent with the dilution factor (1.4) resulting from the new influent being fed to the reactor. The steep decrease observed in both the instant volumetric PHA storage rate and the volumetric productivity about 6 h into the test directly reflects the slowdown of specific PHA storage rates associated with proximity to the saturation level. In the continuous feeding assay A3, the volumetric productivity and instant volumetric PHA storage rate remained higher throughout the batch test and subjected to less fluctuations than the respective curves obtained for assays A1 and A2. In both pulse wise feeding studies (A1 and A2), the instant volumetric PHA storage rates decreased (either gradually – A1 – or more steeply – A2) for each pulse of substrate fed and, as a result, the volumetric productivities decreased considerably throughout the two batch tests (Fig. 2a and b). The higher average specific rates of substrate uptake and PHA storage observed in the continuous feeding assay (A3) relatively to those reported for the two pulse wise feeding tests (A1 and A2) seem to be related, on one hand with: (i) the fact that some of the constraints associated with pulsefeeding had effectively been removed (namely, the stops between pulses which represent time and polymer losses) and (ii) more importantly, the higher residual substrate concentration observed throughout the entire batch test (never below 20 Cmmol VFA/L, see Fig. 1b), which allowed the specific rates of substrate uptake and PHA storage to stay close to maximum levels (almost until the saturation level was reached). It can be argued that during the pulse wise feeding tests, the biomass was adversely affected metabolically during the lag periods between pulses. Whether this eventual adverse metabolic effect was caused solely by the stops between pulses or may be associated with the slow-down of metabolic rates observed near the end of each pulse (as carbon substrate concentration becomes limiting) is not completely clear from the results obtained. However, results seem to indicate that the use of a continuous feeding strategy has the advantage of keeping the culture at constant maximum rates (high metabolic activity throughout the batch test), while the pulse wise feeding strategy (either due to the volume replacement regimen used or associated with the pulse wise feeding itself) seems to result in a slow-down of metabolic activity at the end of every pulse, causing the average performance to decrease. The main constraint on volumetric productivity imposed by the continuous feeding strategy was the dilution resultant from the continuous supply of new medium (a dilution factor of 1.4 was observed in this test). However, this may be overcome through the use of a more concentrated feed (making sure the residual substrate concentration is kept bellow inhibiting values) or a microfiltration membrane coupled to the batch production reactor.

73

As can be seen in Fig. 2d which shows the volumetric productivity as a function of PHA content, for the same PHA content the productivity was higher in the continuous feeding assay than using pulse wise feeding. A much more gradual decrease of the volumetric productivity was observed in the continuous feeding assay relatively to the pulse wise feeding tests, where a very steep decrease is observed almost from the beginning of the each batch test. Only close to the saturation level, does the productivity greatly decrease as a function of PHA content, but only for values of PHA content higher that 72%. The effect on feeding regimen on polymer composition was also evaluated. The use of continuous feeding increased the HV content relatively to pulse wise feeding from 31 to 39% in the case of feed solution with VFA profile B. This is most likely explained by the fact that in pulse feeding assays, propionate and valerate, whose fractions were significantly lower than acetate and butyrate fractions, were first exhausted and the amount of HV formed was limited by the availability of these organic acids (Fig. 1a and b). In fact, in pulse feeding assays A1 and A2, for each pulse of substrate fed, HB synthesis is observed throughout the full extent of the pulse, while HV synthesis stops once propionate and valerate are exhausted. Because these two organic acids are present in lower quantity than acetate and butyrate, they are exhausted before the later two, thus causing HV synthesis to stop before the end of the pulse. On the contrary, in continuous feeding assay A3, the continuous supply of fresh medium, kept the residual concentrations of acetate and propionate constant (see Fig. 1c), thus ensuring HB and HV synthesis throughout the full test. In the continuous feeding assay (A3), the HB:HV ratio was thus determined by the ratio between HB and HV synthesis rates rather than by the availability of the appropriate precursors. Therefore, although hydroxybutyrate was still the major component of the copolymer produced, the HV content considerably increased as a result of feeding regimen alone. These results indicate that the continuous feeding strategy, apart from being an interesting alternative to effectively increase process productivity, may also be used to manipulate the polymer composition, increasing the range of polymer compositions which can be synthesized from a given fermented effluent. 3.3. Effect of polymer composition on polymer structural and thermal properties The copolymers of hydroxybutyrate (HB) and hydroxyvalerate (HV) obtained in the batch studies previously described (A3, B1 and C2) were characterized in terms of structural and thermal properties. These particular samples were selected in order to cover the wide range of HV contents obtained (15–39%) and also to include at least one co-polymer produced from real fermented molasses (sample C2). 3.3.1. Structural properties Size exclusion chromatography (SEC) was performed and the results are reported (Table 3). Reasonably low PDI (2.3–2.7) was observed for all samples (A3, B1 and C2). The SEC traces obtained with either the RID or UV/Vis detectors did not show additional peaks at low elution times, suggesting the absence of high molecular weight impurities. Weight average molecular weights of the different copolymers P(HB-co-HV) produced decreased, from 6.5 to 2.2 × 105 , with increasing HV content (15–39%). This may be due to the incorporation of a higher amount of HV units causing a disruption on polymer chains (Organ, 1993). Notwithstanding the lower molecular weights, the higher HV content did not result in lower PDI. In fact, samples with higher and lower HV contents showed similar PDI (samples A3 and C2 both with PDI of 2.3). The weight average molecular weights (2.2–6.5) × 105 and PDI (2.3–2.7) of the P(HB-co-HV) characterized in this study were

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Table 3 Summary of structural and thermal properties of PHA produced by a fermented molasses selected culture using either fermented molasses or synthetic VFA feedstock. Production

Batch test Substrate Feeding regimen

C2 Fermented molasses Pulse feeding

B1 Simulated Fermented molasses Pulse feeding

A3

Polymer composition

HV (%)

15

21

39

Structural properties

Mn Mw PDI Crystallinity

2.75 × 105 6.46 × 105 2.3 Marked crystallization in the first cooling

0.95 × 105 2.15 × 105 2.3 Almost amorphous

Thermal properties

Tmax (◦ C) Tg (◦ C) Tm (◦ C)

247 −1 134/147

1.44 × 105 3.86 × 105 2.7 Crystallization was only observed in the second thermal cycle 242 −10 121/137

close to those reported for PHA produced by other mixed cultures, namely to that of a P(HB-co-HV) copolymer produced by an acetate selected culture using fermented molasses as substrate for PHA accumulation (Mw of 8.5 × 105 , Albuquerque et al., 2007) and also to those of PHA co- and ter-polymers produced by glycogen-accumulating organisms (GAO) enriched cultures selected using either fermented molasses (3.5–9.0 × 105 and PDI of 1.8–3.9, Bengtsson et al., 2010b) or acetate/propionate mixtures (3.9–5.6 × 105 , Dai et al., 2008). The molecular weights obtained in this study were, however, slightly lower (between half and one order of magnitude lower) than those reported for a PHB homopolymer (1.0–3.0) × 106 produced by a mixed culture using acetate as sole carbon source (Serafim et al., 2008a,b) and also lower than that reported by Patel et al. (2009) for a copolymer of P(3HB-co-3HV) produced by a mixed microbial culture of nitrogen-fixing bacteria using acetate as sole carbon source for enrichment and fed with acetate–propionate mixtures during the accumulation stage (Mn of 2.5 × 106 ). The PDI was either higher or in the higher range of those reported in the two previous studies (1.3, Patel et al., 2009) and (1.3–2.2, Serafim et al., 2008b). Because this difference is slight, it can also safely be argued that the difference between the Mw and PDI reported here relatively to values commonly reported for both mixed and pure cultures. X-ray diffraction (XRD) was used to characterize a copolymer sample with 30% HV content. The sample crystallinity was approximately 40 ± 5%. The sample was also analyzed using a polarized light optical microscope (POM). It was observed that immediately after melting and cooling no crystals were formed. However, after one day, a great amount of very small crystals could be observed. The crystallinity degree determined for this sample falls within the values reported by Patel et al. (2009) for PHB and P(HB-co-HV) with crystallinity indices of 44% and 37%, respectively, produced by a mixed culture of nitrogen fixing bacteria from two different carbon substrates. The crystallinity of the remaining samples recovered in this study (A3, B1 and C1) could not be quantified due to insufficient sample amount available. 3.3.2. Thermal properties Thermogravimetric analysis (TGA) was carried out to determine the degradation temperature of the copolymers produced. The thermograms of samples from batch tests A3, B1 and C2 showed a volatilization temperature below 75 ◦ C, indicating that these samples still contained some residual solvent (mass losses between 1% and 14%), and a small degradation step centered at 120 ◦ C, which extent seemed to increase with increasing HV content. This behavior could be due to the presence of some residual raw material used in the fermentation process (low molecular weight impurities). The following degradation step, between 190 ◦ C and 280 ◦ C, with Tmax centered between 242 ◦ C and 247 ◦ C, corresponds to P(HB-co-HV)

Continuous feeding

243 −16 113/138

main degradation. Sample C1 (produced from fermented molasses rather than simulated feed) showed the highest degradation temperature (Tmax of 247 ◦ C), but also a further small degradation step centered at 389 ◦ C. Regarding the amount of residues left at 500 ◦ C, very low quantities were detected in all cases. Despite the presence of some residual solvent and possibly fermentation products, which reflect the fact that the extraction and recovery procedure was not the main focus of this study and still requires some further optimization, the degradation temperatures of the polymers were quite consistent with values described in the literature for P(HB-coHV) either from pure or mixed cultures, enabling a wide processing temperature window (Bengtsson et al., 2010a,b). Other thermal properties such as melting and glass transition temperature were inferred from DSC thermograms obtained for samples A3, B1 and C2 (Fig. 3a, b and c, respectively). For sample C2 (sample with the lowest HV content), a glass transition temperature (Tg ) of −1 ◦ C was observed (during the second eating scan), a crystallization peak was observed at 77 ◦ C (during cooling) and a double melting peak was observed at 134 ◦ C and 147 ◦ C. For sample B1 (sample with the intermediate HV content), Tg , was observed at −10 ◦ C, a crystallization peak was observed at 72 ◦ C (crystallization temperature was determined during the second eating, since no peak was observed during cooling) and a double melting peak at 121 ◦ C and 137 ◦ C. For sample A3 (sample with the higher HV content), Tg value was observed at −16 ◦ C, the crystallization peak was not well defined (in either the cooling or the second eating scan) but a slight exothermic event appeared at 67 ◦ C. In addition a broad and low intensity double melting peak could be detected at 113 ◦ C and 138 ◦ C. Thermal characterizations (Table 3) showed values of glass transition temperature between −1 and −16 ◦ C and melting temperatures between 137 ◦ C and 147 ◦ C (main peak). Such values are slightly lower than those reported for P(HB-co-HV) produced by pure cultures and in the same range as those reported for P(HBco-HV) produced by other fully aerobic mixed cultures (Serafim et al., 2008a,b) (Fig. 4). Moreover, since the onset of thermal degradation was between 190 ◦ C and 200 ◦ C, these polymers show a relatively wide processing thermal window, since the difference between melting temperature and degradation temperature is approximately 50 ◦ C. Although the crystallinity of samples A3, B1 and C2 could not be quantified, a reduced kinetic of crystallization was observed with increasing HV contents from the DSC analyses. For 15% HV content (sample C2), a marked crystallization was observed during cooling. For 20% HV content (sample B1), a crystallization peak could also be observed but only during the second heating. A further increase in the HV content to 39% (sample A3) conducted to almost an amorphous matrix. The observed variation of the thermal properties (melting and glass transition temperature) with the polymer composition (HV

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75

a Sample A3 (39% HV)

Heat Flow (W/g)

-15.68°C 67.44°C 113.24°C 137.95°C

-50 Exo Up

0

50 100 Temperature (°C)

150

Fig. 4. Adapted from Dias et al., 2006 – comparison with literature (Chua and Yu, 1999; Serafim et al., 2008a,b; Reis et al., 2003; Punrattanasim, 2001; Savenkova et al., 2000; Sudesh et al., 2000; Yoshie et al., 2001; Yamada et al., 2001).

4.8 ◦ C and Tm : 89–174 ◦ C), which showed that the thermal properties of PHA polymers produced by GAO enriched cultures were controlled in broad ranges by the monomer composition. However, in the present study the range of compositions was narrower than that observed in the two previously mentioned studies, which can be explained by the higher monomer variability observed in the two later cases (four and five different monomers were reported by Dai et al. and Bengtsson et al., respectively). Bengtsson et al. (2010a,b) also observed that the decomposition temperatures (277.2–294.9 ◦ C) were independent of monomer composition and molecular weight.

b Sample B1 (21% HV)

Heat Flow (W/g)

71.77°C

-9.83°C

4. Conclusions and perspectives

120.62°C 137.40°C -50 Exo Up

0

50 100 Temperature (°C)

c Sample C2 (15% HV)

150

77.46°C

Heat Flow (W/g)

-1.18°C

133.56°C

-50 Exo Up

0

50 100 Temperature (°C)

146.52°C 150

Fig. 3. DSC thermograms from samples A3 (a), B1 (b) and C2 (c).

content) is in accordance with reports from the literature (Fig. 4) for other mixed and pure cultures (Chua and Yu, 1999; Serafim et al., 2008b; Reis et al., 2003; Punrattanasim, 2001; Savenkova et al., 2000; Sudesh et al., 2000; Yoshie et al., 2001; Yamada et al., 2001). Namely, to results obtained by Dai et al. (2008) (Tg : −8 to 0 ◦ C and Tm : 70–161 ◦ C) and Bengtsson et al. (2010a,b) (Tg : −14 to

Strategies to improve the PHA accumulation stage – both in terms of productivity and possibility to control polymer composition – in a three-stage PHA production process from sugar molasses were investigated. It was found that the use of a continuous feeding strategy rather than a pulse feeding strategy can, not only help mitigate the process constraints of the pulse-feeding strategy (stops between pulses often with polymer consumption), but also allow higher rates of substrate uptake and polymer storage to be maintained due to constant residual substrate concentration. Thus, a considerable increase of volumetric productivity was obtained as a result of the use of a continuous feeding regimen in the PHA production stage of a 3 stage PHA process from an industrial residual feedstock. Moreover, the continuous feeding strategy can also be used as a means to increase the HV content by 8–9% relatively to that obtained from the same feedstock using pulse wise feeding. This can be an interesting alternative for the manipulation of the polymer composition, increasing the range of polymer structures which can be synthesized from a given fermented effluent. The range of PHA compositions obtained (PHB-co-HV with HV fraction ranging from 15 to 39%) subsequently resulted in different polymer properties (molecular weight and thermal properties). The produced polymers presented slightly lower molecular weights than those generally obtained with other fully aerobic mixed cultures but still high enough for thermoplastic processing. More importantly, it was demonstrated by DSC that these polymers present similar thermal properties relatively to PHA copolymers produced by both mixed and pure cultures, showing a relatively wide window of processability. The Mw and PDI obtained are within the range of values reported in the literature for PHA produced both by pure and mixed cultures. Although mechanical properties were not determined in this study, the molecular weights and PDI obtained, as well as the thermal properties observed, are compa-

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rable to those reported in the literature for PHA, which have been shown to be adequate for thermoplastic processing. Thus, it can be expected that these PHA would also be suited for thermoplastic processing. These results indicate that the use of agricultural and industrial residual or surplus streams as feedstocks for PHA production by mixed microbial cultures enriched in PHA-storing organisms through culture selection under Aerobic Dynamic Feeding can be considered an interesting and possibly cost-effective alternative to pure culture fermentations. This strategy allows for the production of PHA polymers of similar quality and for which the monomer composition may be manipulated through the acidogenic fermentation conditions and also through the PHA production feeding regimen. This allows for a wide variety of polymer with different thermal properties to be produced from these complex surplus streams. Acknowledgments Fundac¸ão para a Ciência e Tecnologia (Portugal) is greatly acknowledged for funding this research through the individual grant SFRH/BD/17141/2004. The EU is also acknowledged for funding through the project “Sustainable Microbial and Biocatalytic Production of Advanced Functional Materials”, EU Integrated Project, contract no. 026515-2; 2006-2008. Refinaria de Ac¸úcares Reúnida (RAR), Portugal, is acknowledged for the supply of the molasses. References Albuquerque, M.G.E., Eiroa, M., Torres, C., Nunes, B.R., Reis, M.A.M., 2007. Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses. J. Biotechnol. 130, 411–421. Albuquerque, M.G.E., Torres, C., Reis, M.A.M., 2010a. Polyhydroxyalkanoate (PHA) production by a mixed microbial culture using sugar molasses: effect of the influent substrate concentration on culture selection. Water Res. 44, 3419–3433. Albuquerque, M.G.E., Concas, S., Bengtsson, S., Reis, M.A.M., 2010b. Mixed culture polyhydroxyalkanoates production from sugar molasses: the use of a 2-stage CSTR system for culture selection. Bioresour. Technol. 101, 7112–7122. Albuquerque, M.G.E., 2009. Production of polyhydroxyalkanoates (PHA) from sugar cane molasses by mixed microbial cultures. PhD Thesis Universidade Nova de Lisboa, Lisbon, Portugal. APHA, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Washington DC, 1995. Anderson, A.J., Dawes, E.A., 1990. Biosynthesis and composition of bacterial poly(hydroxyalkanoates). Int. J. Biol. Macromol. 12, 102–105. Beccari, M., Majone, M., Massanisso, P., Ramadori, R., 1998. A bulking sludge with high storage response selected under intermittent feeding. Water Res. 32, 3403–3413. Beccari, M., Bertin, L., Dionisi, D., Fava, F., Lampis, S., Majone, M., Valentino, F., Vallini, G., Villano, M., 2009. Exploiting olive oil mill effluents as a renewable resource for production of biodegradable polymers through a combined anaerobic–aerobic 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. Bioresour. Technol. 99, 509–516. Bengtsson, S., Pisco, A.R., Reisand, M.A.M., Lemos, P.C., 2010a. Production of polyhydroxyalkanoates from fermented sugar cane molasses by a mixed culture enriched in glycogen accumulating organisms. J. Biotechnol. 145, 253–263. Bengtsson, S., Pisco, A.R., Johansson, P., Lemos, P.C., Reis, M.A.M., 2010b. Molecular weight and thermal properties of polyhydroxyalkanoates produced from fermented sugar molasses by open mixed cultures. J. Biotechnol. 147, 172–179. Beun, J.J., Dircks, K., van Loosdrecht, M.C.M., Heijnen, J.J., 2002. Poly-bhydroxybutyrate metabolism in dynamically fed mixed cultures. Water Res. 36, 1167–1180. Bluhm, T.L., Hamer, G.K., Marchessault, R.H., Fyfe, C.A., Veregin, R.P., 1986. Isodimorphism in bacterial poly (beta-hydroxybutyrate-co-beta-hydroxyvalerate). Macromolecules 19, 2871–2876. Chua, H., Yu, P.H.F., 1999. Production of biodegradable plastics from chemical wastewater – a novel method to reduce excess activated sludge generated from industrial wastewater treatment. Water Sci. Technol. 39, 273–280. Daiger, G.T., Grady, P.L., 1982. The dynamics of microbial growth on soluble substrates: a unifying theory. Water Res. 16, 365–382. Dai, Y., Lambert, L., Yuan, Z.G., Keller, J., 2008. Microstructure of co-polymers of polyhydroxyalkanoates produced by glycogen accumulating organisms with acetate as the sole carbon source. Process Biochem. 43, 968–977.

Dias, J.M.L., Lemos, P.C., Serafim, L.S., Oliveira, C., Eiroa, M., Albuquerque, M.G.E., Ramos, A.M., Oliveiraand, R., Reis, M.A.M., 2006. Recent advances in polyhydroxyalkanoate production by mixed aerobic cultures: from the substrate to the final product. Macromol. Biosci. 6, 885–906. Dionisi, D., Carucci, G., Petrangeli, M., Papini, P., Riccardi, C., Majone, M., Carrasco, F., 2005. Olive oil mill effluents as a feedstock for production of biodegradable polymers. Water Res. 39, 2076–2084. Dionisi, D., Majone, M., Levantesi, C., Bellani, A., Fuoco, A., 2006. Effect of feed length on settleability, substrate uptake and storage in a sequencing batch reactor treating an industrial wastewater. Environ. Technol. 27, 901–908. Feng, L.D, Watanabe, T., Wang, Y., Kichise, T., Fukuchi, T., Chen, G.Q., Doi, Y., Inoue, Y., 2002. Studies on comonomer compositional distribution of bacterial poly(3hydroxybutyrate-co-3-hydroxyhexanoate) and thermal characteristics of their fractions. Biomacromolecules 3, 1071–1077. Gujer, W., Henze, M., Mino, T., Van Loosdrecht, M.C.M., 1999. Activated sludge model no. 3. Water Sci. Technol. 39, 183–193. Gurieff, N., Lant, P., 2007. Comparative life cycle assessment and financial analysis of mixed culture polyhydroxyalkanoate production. Biores. Technol. 98, 3393–3403. Ivanova, G., Serafim, L.S., Lemos, P.C., Ramos, A.M., Reis, M.A.M., Cabrita, E.J., 2009. Influence of feeding strategies of mixed microbial cultures on the chemical composition and microstructure of copolyesters P(3HB-co-3HV) analyzed by NMR and statistical analysis. Magn. Reson. Chem. 47, 497–504. Johnson, K., Kleerebezem, R., Muyzer, G., Gand, M.C.M., van Loosdrecht, 2009. Enrichment of a mixed bacterial culture with a high polyhydroxyalkanoate storage capacity. Biomacromolecules 10, 670–676. Kleerebezem, R., van Loosdrecht, M.C.M., 2007. Mixed culture biotechnology for bioenergy production. Curr. Opin. Biotechnol. 18, 207–212. Kunioka, M., Doi, Y., 1990. Thermal degradation of microbial copolyesters – poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrateco-4-hydroxy butyrate). Macromolecules 23, 1933–1936. Lee, S.Y., Choi, J., Wong, H.H., 1999. Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review. Int. J. Biol. Macromol. 25, 31–36. Lee, W.H., Loo, C.Y., Nomura, C., Sudesh, K., 2008. Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors. Biores. Technol. 99, 6844–6851. Lemos, P.C., Serafim, L.S., Reis, M.A.M., 2006. Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding. J. Biotechnol. 122, 226–238. Majone, M., Masanisso, P., Carucci, A., Lindrea, K., Tandoi, V., 1996. Influence of storage on kinetic selection to control aerobic filamentous bulking. Water Sci. Technol. 34, 223–232. Mitomo, H., Takahashi, T., Ito, H., Saito, T., 1999. Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Burkholderia cepacia D1. Int. J. Biol. Macromol. 24, 311–318. Organ, S.J., 1993. Variation in melting point with molecular weight for hydroxybutyrate/hydroxyvalerate copolymers. Polymer 34, 2175–2179. Pardelha, F., Albuquerque, M.G.E., Reis, M.A.M., Oliveira, R., Dias, J.M.L., Metabolic modeling of polyhydroxyalkanoates production from complex mixtures of volatile fatty acids by mixed microbial cultures. Submited for publication. Patel, M., Gapes, D.J., Newman, R.H., Dare, P.H., 2009. Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria. Appl. Microbiol. Biotechnol. 82, 545–555. Punrattanasim, W., 2001. The utilization of activated sludge polyhydroxyalkanoates for the production of biodegradable plastics. PhD Thesis. Virginia Polytechnic Institute and State University, Blacksburg, USA. Reis, M.A.M., Serafim, L.S., Lemos, P.C., Ramos, A.M., Aguiar, F.R., van Loosdrecht, M.C.M., 2003. Bioprocess Biosyst. Eng. 23, 377. Savenkova, L., Gercberga, Z., Bibers, I., Kalnin, M., 2000. Effect of 3-hydroxyvalerate content on some physical and mechanical properties of polyhydroxyalkanoates produced by Azotobacter chroococcum. Process Biochem. 36, 445–450. Serafim, L.S., Lemos, P.C., Oliveira, R., Reis, M.A.M., 2004. Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions. Biotechnol. Bioeng. 87, 145–160. Serafim, L.S., Lemos, P.C., Albuquerque, M.G.E., Reis, M.A.M., 2008a. Strategies for PHA production by mixed cultures and renewable waste materials. Appl. Microbiol. Biotechnol. 81. Serafim, L.S., Lemos, P.C., Torres, C., Reis, M.A.M., Ramos, A.M., 2008b. The influence of process parameters on the characteristics of polyhydroxyalkanoates produced by mixed cultures. Macromol. Biosci. 8, 355–366. Sudesh, K., Abe, H., Doi, Y., 2000. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci. 25, 1503–1555. Takabatake, H, Satoh, H., Mino, T., Matsuo, T., 2000. Recovery of biodegradable plastics from activated sludge process. Water Sci. Technol. 42, 351–356. van Loosdrecht, M.C.M., Pot, M.A., Heijnen, J.J., 1997. Importance of bacterial storage polymers in bioprocesses. Water Sci. Technol. 35, 41–47. Villano, M., Beccari, M., Dionisi, D., Lampis, S., Miccheli, A., Vallini, G., Majone, M., 2010. Effect of pH on the production of bacterial polyhydroxyalkanoates by mixed cultures enriched under periodic feeding. Process Biochem. 45, 714–723. Yamada, S., Wang, Y., Asakawa, N., Yoshie, N., Inoue, Y., 2001. Macromolecules 34, 4659. Yoshie, N., Saito, M., Inoue, Y., 2001. Macromolecules 34, 8953.