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Journal of Biotechnology 151 (2011) 66–76

Contents lists available at ScienceDirect

Journal of Biotechnology

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ixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acidVFA)-rich streams: Effect of substrate composition and feeding regime on PHAroductivity, composition and properties

.G.E. Albuquerquea, V. Martinob, E. Polletb, L. Avérousb, M.A.M. Reisa,∗

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

r t i c l e i n f o

rticle history:eceived 1 July 2010eceived in revised form 2 September 2010ccepted 15 October 2010

a b s t r a c t

In this study, the possibility of manipulating biopolymer composition in mixed culture polyhydrox-yalkanoate (PHA) production from fermented molasses was assessed by studying the effects of substratevolatile fatty acid (VFA) composition and feeding regime (pulse wise versus continuous). It was foundthat the use of a continuous feeding strategy rather than a pulse feeding strategy can not only help mit-igate the process constraints of the pulse-feeding strategy (resulting in higher specific and volumetric

eywords:olyhydroxyalkanoatesixed cultures

ermented molasseseeding regimenolatile fatty acids

productivities) but also be used as means to broaden the range of polymer structures. Continuous feedingincreased the hydroxyvalerate content by 8% relatively to that obtained from the same feedstock usingpulse wise feeding. Therefore, the feeding strategy can be used to manipulate polymer composition. Fur-thermore, the range of PHA compositions, copolymers of P(HB-co-HV) with HV fraction ranging from 15to 39%, obtained subsequently resulted in different polymer properties. Increasing HV content resultedin a decrease of the average molecular weight, the glass transition and melting temperatures and also in

linity

a reduction in the crystal

. Introduction

Polyhydroxyalkanoates (PHAs) are biologically synthesizedolyesters that are fully biodegradable and can be produced fromenewable sources, thus allowing for a lower environmental impacthan conventional chemically synthesized polymers. Moreover,HAs present a very high replacement potential over conventionalolyolefins due to interesting thermoplastic properties. Dependingn the type and relative proportion of HA monomers, these biopoly-ers present a broad range of structural, thermal and mechanical

roperties. Although they are already industrially produced, theirommercialization remains limited to high-value applications dueo their high production costs. To date, industrial PHA productions carried out using pure microbial culture fermentation technol-gy with high costs associated with carbon substrate (refined sugarubstrates), fermentation operation and downstream processing.

In the last decade, research has focused on the development of

lternative production processes aiming to decrease these produc-ion costs. Such alternative processes include not only the use ofenetic/metabolic engineering strategies to optimize pure cultureermentations, but also that of mixed microbial cultures (MMC),

∗ Corresponding author.E-mail address: amr@dq.fct.unl.pt (M.A.M. Reis).

168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2010.10.070

degree from a semi-crystalline material to an amorphous matrix.© 2010 Elsevier B.V. All rights reserved.

requiring lower investment and operating costs due to the use ofopen systems which do not require sterile conditions, coupled tothat of wastes/surplus based feedstocks. It has recently been sug-gested, based on Life Cycle Analysis (LCA), that PHA productionusing mixed cultures may be more favorable than using pure cul-tures in both economic and environmental terms (Gurieff and Lant,2007).

Conditions of external substrate excess (feast) and limita-tion (famine) were shown to select for microbial populationswith enhanced capacity to store PHA (Majone et al., 1996; vanLoosdrecht et al., 1997). This type of process was designated as Feastand Famine (FF) or Aerobic Dynamic Feeding (ADF). Bacterial PHAsynthesis is known to occur under conditions in which growth isrestricted by either an external (lack of nutrient or electron accep-tor) or an internal factor (Daiger and Grady, 1982; Anderson andDawes, 1990; Gujer et al., 1999). It was proposed that mixed micro-bial cultures operated under Feast and Famine conditions weresubjected to an internal growth limitation arising from the alter-nate substrate availability, which compelled the organisms to aphysiological adaptation (Beccari et al., 1998). During this physio-

logical adaptation period, substrate uptake is mainly driven towardpolymer storage. Beun et al. (2002) have shown that following along starvation period, a mixed culture operated under ADF con-ditions channels around 70% (on a C-mole basis) of the carbonsubstrate uptake toward PHA storage.

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M.G.E. Albuquerque et al. / Journ

The key to the effectiveness (in terms of both storage capac-ty and productivity) of MMC PHA production processes relies onulture selection (enrichment in PHA accumulating organisms) byhe conditions imposed to the reactor. The use of MMC to pro-uce added value products (such as biochemicals and biomaterials)sing ecological selection principles to engineer the microbial con-ortium has been designated as eco-biotechnology (Kleerebezemnd van Loosdrecht, 2007). Research has focused first on the usef synthetic volatile fatty acids (as reviewed by Dias et al., 2006)nd, more recently, on the use of low cost agro-industrial surpluseedstocks (as reviewed by Serafim et al., 2008a; e.g. fermented

olasses – Albuquerque et al., 2007, 2010a,b; Bengtsson et al.,010a,b; fermented paper mill effluents – Bengtsson et al., 2008;

ndustrial wastewaters – Dionisi et al., 2006; fermented olive oilill effluents – Dionisi et al., 2005; Beccari et al., 2009).Although MMC FF systems have demonstrated a good poten-

ial for PHA production reaching high specific productivities, witholymer yields on substrate and maximum PHA contents similaro those attained by pure cultures (Serafim et al., 2004; Johnsont al., 2009) these systems still present lower performances thanure culture fermentations in terms of volumetric productivity.his is due to the considerably lower cell concentrations obtainedn ADF mixed culture processes than those reached in pure cul-ure systems. Most studies on MMC PHA-accumulating cultureelection reactors (operated under Feast and Famine conditions)eport cell concentrations lower than 10 g/L of volatile suspendedolids (Serafim et al., 2004; Dionisi et al., 2005, 2006; Bengtssont al., 2008; Johnson et al., 2009; Beccari et al., 2009; Albuquerquet al., 2007, 2010a,b), whereas, in pure culture fermentations, val-es above 100 g/L are often reported (Lee et al., 1999). This resultsrom the conditions of alternate substrate availability under whichulture selection is carried out, which limit the culture’s primaryetabolism (internal growth limitation induced by the Feast and

amine conditions). Developing strategies to improve volumetricroductivity of MMC PHA production processes is essential to makehis process competitive with that of pure cultures.

Besides, it is also important to determine whether the qual-ty of the biopolymers produced by MMC can meet the standardsequired for use in common plastic applications (which has alreadyeen demonstrated for PHA produced using pure culture fermenta-ion from refined substrates), particularly considering the polymersroduced from waste/surplus based feedstocks. Although the num-er of studies dedicated to mixed microbial culture PHA productionas increased considerably in recent years, only few and recenttudies investigated the polymer characteristics (Serafim et al.,008b; Bengtsson et al., 2010b; Patel et al., 2009).

The most commonly investigated polyhydroxyalkanoate is theomopolymer poly-3-hydroxybutyrate, P(3HB). This biopolymerhows a melting temperature close to 180 ◦C (Kunioka and Doi,990) and a glass transition temperature around 4 ◦C (Mitomot al., 1999). But P(3HB) is a highly crystalline polymer (55–80%)esulting in fairly stiff and brittle materials, somewhat limiting itspplications (e.g. elongation to break is about 2–10% comparedo up to 400% for some polyolefins). Another important limita-ion comes from narrow window of processability. PHA thermalnd mechanical properties depend directly on the polymer com-osition and structure. The incorporation of different monomerypes reduces polymer crystallinity by disturbing the crystal lat-ice. For instance, poly-3-hydroxybutyrate-co-3-hydroxyvalerate,(3HB-co-3HV), copolymers exhibit lower crystallinity. Comparedo P(3HB), copolymers of hydroxybutyrate and hydroxyvalerate

resent improved mechanical properties with decreased stiff-ess and brittleness, increased flexibility (higher elongation toreak), increased tensile strength and toughness, with preservediodegradability. Moreover, P(3HB-co-3HV) melting and glass tran-ition temperatures steadily decrease from 0 to 30 mol% fraction

iotechnology 151 (2011) 66–76 67

of 3HV (Bluhm et al., 1986; Feng et al., 2002). Because the tem-perature at which degradation and decomposition of PHA occur israther insensitive to the polymer composition, the lower meltingtemperatures of these copolymers allow for a wider temperatureprocessing window and thus increase its processability.

An advantage of MMC PHA production relates to the widerrange of PHA compositions obtained. MMC fed with fermentedfeedstocks (containing mixtures of organic acids such as acetate,propionate, butyrate and valerate) produce PHA polymers with aconsiderably high diversity of different HA monomers, contain-ing 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 rang-ing from 17 to 85% and a termopolymer of 3HB:3HV:3H2MV withmolar ratio of 6:58:24 were reported by Lemos et al. (2006) fromacetate/propionate mixtures, whereas Takabatake et al. (2000) pro-duced co-polymers of 3HB and 3HV, in which the 3HV molarfraction increased according to the propionate fraction in the mix-ture fed (up to 84% of 3HV from pure propionate). The fractionof monomers other than 3HB is considerably higher in polymersproduced by mixed cultures from mixtures of VFA than what hasbeen reported for pure cultures, typically fed with refined sugars,and which require large amounts of co-substrates (such as alcoholsor organic acids) to produce polymers with relatively small frac-tions of monomers other than PHB. For instance, Lee et al. (2008)have reported the production of P(HB-co-HV) with only 2–8% HVfrom mixtures with equal mass amounts of a surplus carbon feed-stock (vegetable oils) and a co-substrate used to induce HV storage(propionic acid, commercial grade).

In MMC systems, polymer composition depends on the compo-sition of the fermented effluent produced in the early anaerobicdigestion step. MMC PHA production processes can be managedand controlled to produce copolymers with improved properties.As shown in Albuquerque et al. (2007), manipulating operatingconditions of the anaerobic fermentation step (e.g. pH) used toproduce the fermented volatile fatty acid (VFA)-rich stream canbe used to control the biopolymer composition in the PHA pro-duction step. However, the degree of manipulation of the polymercomposition will still be limited to the range of VFA concentra-tions obtained by fermentation for any given feedstock. Therefore,in order to broaden the range of copolymers produced from anygiven fermented effluent (with stable VFA composition), strategiesto manipulate polymer composition (Villano et al., 2010 – effect ofpH) and microstructure (Ivanova et al., 2009) in the batch produc-tion stage have also been investigated.

In this study, the possibility to control polymer composi-tion (and resulting properties) in mixed culture PHA productionfrom fermented molasses was assessed by studying the effects ofsubstrate VFA composition and feeding regime on polymer com-position and structure. The different biopolymers produced werecharacterized in terms of chemical, structural and thermal prop-erties. Finally, the copolymers produced from fermented molasseswere compared in terms of molecular weight and thermal proper-ties to PHA produced from synthetic VFA.

2. Materials and methods

2.1. Experimental setup

The experimental setup consisted of three bench-scale reac-tor systems and a hollow fiber membrane filtration module. Themolasses acidogenic fermentation (stage 1) was carried out in acontinuous stirred tank reactor (CSTR) operated under anaerobicconditions. The reactor effluent was clarified by microfiltration

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nd the clarified fermented molasses were used as a feedstock forulture selection and PHA batch accumulation. Selection of a PHA-ccumulating culture (stage 2) was carried out in a Sequencingatch Reactor (SBR) subjected to Aerobic Dynamic Feeding (ADF)onditions. PHA accumulation (stage 3) was carried out in a batcheactor inoculated with sludge from the culture enrichment SBRnd fed with clarified fermented molasses produced in stage 1.

.1.1. Continuous acidogenic fermentation reactorThe CSTR with a working volume of 1140 ml was operated

nder anaerobic conditions using the conditions reported bylbuquerque et al. (2007), i.e., pH 6, 30 ◦C, HRT kept at 10 h and

nfluent substrate concentration of 10 g/L sugars. The sugarsn the molasses, accounting for 75% of the molasses dissolvedrganic carbon (DOC) prior to fermentation, were fully exhausted>99% consumption). The main fermentation products were VFAacetate, propionate, butyrate and valerate), making up about 75%f the DOC of the fermented molasses. The sugar–VFA conversionield was about 0.70 Cmol VFA/Cmol sugars. The CSTR feed wasupplemented with NH4Cl and KH2PO4 using C/N/P ratios of00/3/1. This ratio was previously optimized (Albuquerque et al.,007) in order to result in low residual nutrients in the fermentedolasses stream (<0.5 Nmmol/L and <0.2 Pmmol/L). The low resid-

al nutrient concentration was designed to allow the 2nd and 3rdtages (culture selection and PHA production) to be operated underifferent nutrient conditions: the selection reactor, operated underxcess nutrient conditions, was supplemented with N, P, whilehe batch production stage was run under nutrient limitation. Theffluent was withdrawn by overflow and microfiltrated through aollow fiber membrane module. The clarified effluent was kept at◦C prior to its use in as a feedstock for PHA-accumulating culture

election or in PHA batch accumulation assays.

.1.2. Culture selectionA Sequencing Batch Reactor (SBR) – with a working volume

f 800 ml – was operated according to the conditions previouslyescribed by Albuquerque et al. (2010a). SBR cycles were 12 h longnd consisted of four discrete periods: (i) fill (5 min), (ii) aerobio-is (Feast and Famine) (11 h), (iii) settling (45 min) and (iv) draw10 min). The hydraulic retention time (HRT) was kept at 1 day andhe sludge retention time (SRT) at 10 days. The SBR was fed withlarified fermented molasses at an influent substrate concentrationf 45 Cmmol VFA/L. Feed solution was kept at 4 ◦C in a refrigeratedessel. A mineral nutrient solution, containing both ammonia andhosphate, was simultaneously added to the reactor, keeping the/N/P ratios at 100/8/1. Thiourea (10 mg/L) was also added to theineral nutrient solution to inhibit nitrification. Air was supplied

y an air pump through a ceramic diffuser. Magnetic stirring wasept at 500 rpm. Feed pH was adjusted to 8 ± 0.05 prior to reactoreeding and pH was left uncontrolled during the reaction phase.he reactor stood in a temperature-controlled room (23–25 ◦C).

The SBR was operated under these conditions for a period of0 consecutive months, selecting for a microbial culture highlynriched in PHA-storing organisms (88%) and showing stable PHAtorage performance in the enrichment SBR and in batch produc-ion assays (Albuquerque et al., 2010a).

.1.3. PHA productionPHA accumulation assays were carried out in a 2 L reactor – with

n initial working volume of 1.1 L – operated in batch mode, inoc-lated with the SBR enriched culture, which was collected at the

nd of the famine phase, and fed either with clarified fermentedolasses produced in stage 1 or with chemically defined media

imulating the fermented molasses effluent. Feed pH was adjustedo 8 before reactor feeding. In order to maximize PHA storage,ssays were carried out under ammonia limitation (no ammonia

iotechnology 151 (2011) 66–76

was added and residual ammonia concentration in the sludge col-lected from the SBR was less than 0.1 Nmmol/L). Aeration was keptat 400 ml/min and stirring at 300 rpm. The reactor was kept in atemperature controlled room (23–25 ◦C). The reactor was equippedwith two gas mass flowmeters (Smart MassFlow 5850S, BrooksInstruments) to measure the inlet and outlet air flow rates. The con-centration 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 inassays A1–A3 carried out using a VFA mixture designated as VFAprofile A (acetate, propionate, butyrate and valerate in fractionsof 30/20/30/20 Cmol/100 Cmol VFA) either in pulse wise feedingmode (A1 and A2) – feeding several pulses of VFA profile A withconcentrations between 60 and 80 Cmmol VFA/L – or using a con-tinuous supply of carbon substrate (A3). The first test A1 was carriedout leaving the pH uncontrolled (as used in the enrichment SBR),while the second pulse wise feeding test (A2) and the continuousfeeding test (A3) were operated with pH control, respectively at8.2 and 8.4. For the later, the pH setpoint was used as a meansof controlling substrate addition to the reactor (pH-stat). In thiscase, the VFA feed solution was used as acid solution for the pHcontroller. In all cases, a concentrated feed solution (1000 CmmolVFA/L) was used in order to minimize the dilution resulting fromsubstrate addition.

To test the effect of substrate composition on PHA com-position, two additional VFA mixtures designated as VFAprofile B (acetate, propionate, butyrate and valerate in frac-tions of 60/15/20/05 Cmol/100 Cmol VFA) and VFA profile C(acetate, propionate, butyrate and valerate in fractions of60/10/25/05 Cmol/100 Cmol VFA), were used to carry out pulsewise feeding tests (B1 and C1). VFA mixtures A and B simulatedthe fermented molasses VFA composition obtained at two differ-ent acidogenic fermentation operating pH (5 and 6, as reported inAlbuquerque et al., 2007), while VFA profile C simulated the fer-mented molasses currently fed to the enrichment SBR. Finally, toconfirm that the results obtained with the VFA mixtures could serveto predict those that could be obtained when using real fermentedmolasses, the later assay (VFA profile C) was repeated using realfermented molasses (test C2). The exact VFA profiles A1, A2, B1, C1and C2 are listed in Table 1.

2.2. Analytical procedures

Biomass concentration was determined using the volatile sus-pended solid (VSS) procedure described in Standard Methods(APHA, 1995).

Volatile fatty acids – namely acetate, propionate, butyrateand valerate – concentrations were determined by high per-formance liquid chromatography (HPLC) using a Merck-Hitachichromatographer equipped with a UV detector and Aminex HPX-87H pre-column and column from BioRad (USA). Sulphuric acid0.01 M was used as the eluent at a flow rate of 0.6 ml/min and50 ◦C operating temperature. The detection wavelength was set at210 nm. The organic acids (acetate, propionate, butyrate and valer-ate) concentrations were calculated through calibration curvesusing 25–1000 mg/L standards (Merck, analytical grade).

Polyhydroxyalkanoate concentrations were determined by gaschromatography using the method adapted from Serafim et al.(2004). Lyophilized biomass was incubated for methanolysis inchloroform and a 20% sulphuric acid in methanol solution. Afterthe digestion step, the organic phase (methylated monomers dis-

solved in chloroform) of each sample was extracted and injectedinto a gas chromatograph coupled to a Flame Ionization Detector(GC-FID Varian CP-3800). A ZBWax-Plus column was used at a flowrate of 1 ml/min. Split injection at 280 ◦C with a split ratio of 10was used. The oven temperature program was as follows: 40 ◦C;

M.G.E. Albuquerque et al. / Journal of Biotechnology 151 (2011) 66–76 69

Table 1Summary 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 profileHac/Hprop/Hbut/Hvalin Cmol/100 Cmol VFA

Feeding regime pH control YPHA/VFA

(Cmol/Cmol)Maximum PHAcontent (%)

PHA composition(%HB:%HV)

A1Simulatedfermentedmolasses

A32/19/28/21

Pulse feedingNo 8.3–8.9 0.77b (0.07; 18);

0.53c (0.13; 26)65 (4) 69:31 (1.7; 12)

A2 30/20/28/22 Yes 8.2 0.78b (0.05; 13);0.51c (0.14; 22)

65 (4) 70:30 (1.2; 13)

A3a 31/18/29/22 Continuous Yes 8.4 0.80c (0.02; 10) 77 (3) 61:39 (0.3; 10)B1a B 60/16/20/04 Pulse feeding No 8.2–9.0 0.74b (0.07; 16);

0.59c (0.12; 24)68 (6) 80:20 (1.8; 10)

C1C

59/09/26/06 Pulse feeding No 8.3–9.0 0.75b (0.04; 15);0.57c (0.12; 19)

66 (5) 83:17 (1.2; 11)

C2a Fermentedmolasses

60/09/25/06 Pulse feeding No 8.4–9.1 0.80b (0.07; 22);0.65c (0.15; 27)

56 (4) 85:15 (0.8; 18)

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st dev; sample number); (error associated with the listed value).a Samples (A3, B1, and C2) were characterized in terms of structural and thermalb Yields of polymer on substrate were calculated accounting for all the PHA formc Also considering the difference between final and initial PHA concentration. The

hen 20 ◦C/min until 100 ◦C; then 3 ◦C/min until 175 ◦C; and finally0 ◦C/min until 220 ◦C. The detector temperature was set at 250 ◦C.ydroxybutyrate and hydroxyvalerate concentrations were calcu-

ated using two calibration curves, one for hydroxybutyrate and oneor hydroxyvalerate, using standards (0.1–2 mg/ml) of a commer-ial P(HB-HV) (88%/12%) (Sigma) and corrected using a heptade-ane internal standard (concentration of approximately 1 mg/ml).

.3. Biopolymer recovery and characterization

.3.1. Biopolymer recoveryAt the end of each batch accumulation assay, mixed liquor was

ischarged and cells were separated from the exhaust supernatanty centrifugation (15 min at 8000 rpm). The concentrated sludgeake was washed, filtered and lyophilized (to completely removell water content). Lyophilized samples were then suspended inhloroform and left to dissolve for a period of 3 days at 37 ◦C. Thehloroform solution was then filtered to remove all non dissolvedaterial and used to fill glass petri dishes. Finally, chloroform was

vaporated from the petri dishes to allow polymer recovery in theorm of a thin film.

.3.2. Biopolymer characterizationPolymer samples from batch tests A3, B1 and C2 were charac-

erized by thermogravimetric analysis (TGA), differential scanningalorimetry (DSC), size exclusion chromatography (SEC), anduclear magnetic resonance (NMR), respectively.

Thermogravimetric analysis (TGA) was performed on a TAnstruments TGA Q5000 (USA). Samples were heated from roomemperature up to 500 ◦C at 10 ◦C/min under inert gas atmosphere.GA was used to determine the thermal stability of the polymeramples according to the maximum degradation rate temperatureTmax).

Differential scanning calorimetry (DSC) was conducted on a TAnstruments DSC 2910 (USA) under nitrogen atmosphere usingoth hermetic and non-hermetic aluminium pans. The materi-ls were exposed to successive thermal cycles (heat–cool–heat)etween −70 ◦C and 180 ◦C at 10 ◦C/min. DSC was used toetermine the polymers’ thermal properties. Glass transition tem-erature (Tg) and melting temperature (Tm) were estimated duringhe second eating scan, while crystallization temperature (Tc) was

etermined during cooling.

Size exclusion chromatography (SEC) measurements were per-ormed using a Shimadzu liquid chromatograph apparatus (Japan)quipped with a RID-10A refractive index detector and a SPD-M10Aiode array UV detector. The columns set used was composed of

rties.m of PHA formed in each pulse).value is lower than the first due to PHA consumption during stops between pulses.

a 50 mm PLgel Guard 5 �m column, two 300 mm PLgel Mixed-C 5 �m columns and a 300 mm PLgel 5 �m-100 A column. Thecalibration was realized with polystyrene standards from 580 to1.6 × 106 g mol−1. Chloroform was used as the mobile phase andthe analyses were carried out at 25 ◦C with a solvent flow rateof 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-rayscattering (WAXS) were used to determine the crystallinity of thebiopolymers. Diffraction patterns of PHBV films were recorded witha powder diffractometer Siemens D-5000 (Munich, Germany) usingCu K� radiation source (� = 0.1546 nm). The incidence angle wasvaried 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 areaunder the crystalline peaks related to that of the amorphous halo.Some samples were also analyzed using a polarized light opticalmicroscope (POM) to observe the crystallization behavior.

2.4. Calculation of kinetic and stoichiometric parameters of PHAproduction

The sludge PHA content was calculated as a percentage of VSS ona mass basis (%PHA = g PHA/g VSS × 100), where VSS includes activebiomass (X) and PHA. Active biomass was calculated by subtractingPHA from VSS.

The maximum specific substrate uptake (−qS in CmolVFA/Cmol X h) and PHA storage rates (qP in Cmol PHA/Cmol X h)were determined by adjusting a linear function to the experimentaldata of VFA and PHA concentrations plotted over time, calculatingthe first derivative at time zero (taking the slope of the fitting) anddividing the value thus obtained by the active biomass concentra-tion at that point. VFA concentration corresponds to the sum ofall the organic acid concentrations (VFA, in terms of Cmmol/L, isequal to

∑HAc, HProp, HBut, HVal in Cmmol/L). PHA concentra-

tion (in Cmmol/L) corresponds to the sum of HB and HV monomerconcentrations (in Cmmol/L).

The yields of PHA (YPHA/VFA in Cmol PHA/Cmol VFA) on substrateconsumed were calculated by dividing the amount of PHA formedby the total amount of organic acids consumed, respectively.

3. Results and discussion

3.1. Effect of substrate VFA profile on polymer composition, yieldand maximum PHA content

An important aspect related to the optimization of MMCPHA production systems, particularly from fermented feedstocks,

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elates to the ability to manipulate polymer composition in theccumulation stage. It had been previously shown by Albuquerquet al. (2007) that the fermented molasses VFA profile could beanipulated through the acidogenic fermentation pH. The authors

eported the use of both fermented molasses (with differentFA profiles) and that of synthetic VFA mixtures simulating the

ermented molasses feedstock for PHA production using an acetate-elected culture. No significant variations were reported in termsf PHA yield on substrate, maximum PHA content and PHA com-osition between the synthetic and fermented feedstocks. Thus, inhe present study, different synthetic VFA mixtures (A, B and C –ee Table 1) were used as substrate for PHA batch production by aermented molasses-enriched culture through pulse wise feedingn order to assess the effect of substrate composition on polymeromposition and subsequent properties. Three assays were carriedut with simulated fermented molasses (A1, B1 and C1), with VFArofiles simulating those obtained at different acidogenic reactorH (Albuquerque et al., 2007). One assay was carried out with realermented molasses aiming at confirming that the molasses matrixill not introduce significant variations on the final result.

In terms of PHA storage efficiency, maximum PHA contentsetween 56 and 77% were attained, with the fermented molasseseed and synthetic feedstock, respectively. However, it is importanto point out that the later assay was run until PHA synthesis satura-ion was attained, while the batch assay using fermented molassesas not. Due to differences in kinetic rates of substrate uptake and

HA storage, assays run with the real fermented stream take longerhan those carried out with synthetic VFA mixtures, accountingor the observed differences in maximum PHA content reached.espite the lower value attained with fermented molasses, this

s still a comparatively high value for fermented feedstocks (55%ere reported by Dionisi et al., 2005 using olive oil mill efflu-

nts, 54% by Bengtsson et al., 2008, using paper mill effluents and2–37% by Bengtsson et al., 2010a for fermented molasses). Theigher maximum PHA content obtained with the synthetic feed-tock (77%) represents a comparatively high value for mixed cultureHA production. The maximum value for MMC PHA production waseported by Johnson et al. (2009), with a maximum PHA content of9% obtained from pure acetate. High PHA yields on substrate werelso obtained, ranging from 0.74 to 0.80 Cmol PHA/Cmol VFA, theigher values observed for the fermented molasses feedstock.

In all cases, copolymers of hydroxybutyrate and hydroxyvaler-te were obtained (confirmed by unimodal curve in SEC). The HVontent ranged from 15 to 31% as a function of the amount ofFA with odd number of carbon atoms (propionate and valer-te) in the feed (which ranged from 15 to 40% Cmol/Cmol VFA)Table 1). This is in accordance with what had been previouslyeported by Albuquerque et al. (2007) for an acetate selectedulture fed with fermented molasses produced at different aci-ogenic fermentation pH (with different VFA profiles). It is also

n agreement with results by Bengtsson et al. (2010a) whichbtain co-polymers containing five types of monomers, namely-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2-ethylbutyrate (3H2MB), 3-hydroxy-2-methylvalerate (3H2MV)

nd the medium chain length monomer 3-hydroxyhexanoate3HHx) from fermented molasses with different VFA profiles. Theuthors found that composition of the PHA was dependent on theFA composition of the fermented molasses and was 56–70 mol%HB, 13–43 mol% 3HV, 1–23 mol% 3HHx and 0–2 mol% 3H2MB andH2MV. Despite the fewer types of monomers obtained in thistudy, similarly to the report by Bengtsson et al. (2010a), (3H)B was

he monomer with the higher fraction and the presence of other

onomers was directly dependent on the propionate and valerateraction of the feed.

The different co-polymers produced were then characterizedsee Section 3.3) in terms of molecular weight and thermal prop-

iotechnology 151 (2011) 66–76

erties in order to assess whether the manipulation of the polymercomposition through substrate VFA profile could be an appropriatemeans to manipulate polymer properties.

An interesting result is that the polymer composition is not sig-nificantly affected by using real fermented molasses instead of thesimulated fermented molasses (Table 1). This result is in agree-ment with previous observations using fermented molasses andsynthetic VFA mixtures fed to an acetate-selected PHA storing cul-ture (Albuquerque et al., 2007) and also concordant with findingsreported by Albuquerque (2009) and Pardelha et al. (submitted forpublication) which have compared kinetic aspects of PHA produc-tion from either synthetic VFA mixtures or fermented molasses.This indicates that the molasses matrix does not introduce signifi-cant variation on the polymer composition. The yield of PHA on VFAwas slightly higher with fermented molasses due to the presence ofresidual non VFA carbon existing in the fermented molasses whichwas used for PHA production. The likely use of a carbon source otherthan the measured VFA for PHA production was confirmed by anal-ysis of the soluble TOC consumed over several production cycles.It was shown that the sTOC consumed was in each case higherthan the total VFA quantified (Albuquerque et al., 2010a). How-ever, and despite attempts to characterize the fermented effluent,the nature of this additional carbon source was not identified. Theslightly lower HV content obtained with fermented molasses (15%)relatively to that obtained with the simulated VFA feed (17%) couldindicate that this residual organic fraction contributes to the HBcontent of the co-polymer. The lower maximum PHA content (56%)obtained with the fermented molasses compared to that producedfrom the simulated feed (66%) relates to the slightly lower rates ofsubstrate uptake and PHA storage observed with the real effluent,which result in a lower kinetic performance and thus, for a similarfermentation period, in lower polymer content. These results showthat although the molasses matrix has a slight impact on kineticperformance it does not significantly influence polymer composi-tion and thus, results obtained in the remaining tests can be usedto predict possible outcome when using real fermented molasses.

3.2. Effect of feeding regimen on PHA productivity andcomposition

In previous batch PHA accumulation studies using fermentedmolasses (Albuquerque et al., 2007, 2010a,b), a multiple pulseaddition of the fermented substrate was used in order to over-come potential substrate inhibition (shown in Serafim et al., 2004;Albuquerque et al., 2007). However, this feeding strategy poseda constraint on process productivity. The high number of pulsessupplied (due to the use of relatively low carbon substrate concen-trations per pulse) and the need to supply a considerable volumeof feed for each new pulse (resulting from the relatively dilutedfermented feedstock) resulted in a considerable loss of productiv-ity. For each new pulse, the reaction had to be stopped, biomassdecanted, exhaust supernatant withdrawn and new medium fed,which resulted in stops between pulses and on some partial con-sumption of the PHA synthesized between consecutive pulses.Therefore, the first goal of this study was to assess the use of acontinuous feeding strategy to operate the batch PHA productionstage in order to overcome the constraints associated with pulsewise feeding.

The continuous feeding was carried out using the VFA feed solu-tion (VFA profile A) as acid solution to control reactor pH. In thisassay, A3, the continuous addition of substrate was carried out by

supplying a first pulse of feed solution (60 Cmmol VFA/L), and henceon keeping the pH controlled (at 8.4) by continuous addition of theVFA feed (pH-stat).

Because the pulse wise feeding assay (A1) was run withoutpH control (pH varying for each pulse from about 8.3 to 8.9), a

al of Biotechnology 151 (2011) 66–76 71

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M.G.E. Albuquerque et al. / Journ

ew experiment with pulse wise feeding and pH control (A2) wasarried out in order to access the pH impact on the polymer com-osition and productivity. Stoichiometric and kinetic parametersbserved in the three batch assays are compiled in Table 2. Resultshow that pH control has no significant effect on polymer yield onubstrate, composition and PHA content. Results of the continuouseeding test (A3) are compared with pulse wise feeding tests A1nd A2.

Villano et al. (2010) assessed the effect of operating pH on PHAtorage efficiency and composition using a mixture of syntheticcetate (85% on a COD basis) and propionate (15%), simulating theffluent of the first fermentation stage in a WWTP, to carry out bothhe culture enrichment and PHA accumulation stages in a 2-stageHA production process. The authors carried out PHA batch accu-ulation studies at different controlled pH values (7.5–9.5) and

ave observed somewhat lower PHA storage rates (and increasedV content) in co-polymers of P(HB-co-HV) synthesized at pH 9.5

elatively to the those observed at pH 8.5 (same pH as used fornrichment). The authors suggested that this may be due to aigher maintenance requirement at higher pH. However, unlikehat occurred in the reported study (in which the culture was

nriched using controlled pH, at 8.5), in this case, the culture wasnriched without pH control. Moreover, the pH variation duringach pulse was only in the range of 0.5–0.7 pH units.

However, a slight distinction was observed between assays A1nd A2. In the first assay (A1), the instant volumetric PHA storageate seemed to gradually decrease throughout each pulse of sub-trate, whereas in test A2, carried out at a controlled pH of 8.2,or each pulse of substrate fed, the PHA storage rate seemed toemain fairly constant throughout most of each pulse, decreasingater and more steeply. Thus, for assay A1 there seemed to be aradual decrease of the PHA storage rate, which was thought toe associated with the decrease of external substrate concentra-ion (as was previously suggested in Albuquerque et al., 2010a,b),hereas for assay A2 – with controlled pH – the decrease of the

HA storage rate appeared to be less gradual and thus seemed toe rather associated with near full depletion of the carbon sub-trate. Therefore, the results from the two assays may suggest thathe gradual decrease observed in the assay run without pH control

ay be attributable, at least in part, to the increasing pH.Although a clear correlation between increasing pH and

ecreased PHA storage rate cannot be inferred from these twossays (A1 and A2), the slight distinction between the two is enougho suggest that further clarification on the effect of pH on PHA stor-ge efficiency is required. Notwithstanding this distinction in therofiles of the instant PHA storage rates, in both cases, the volu-etric productivity is greatly affected by the use of a pulse wise

eeding strategy.Fig. 1a and b shows PHA production using pulse wise feeding

un without and with pH control (A1 and A2), respectively. In assay1, stops between pulses represented 16% of the total batch dura-

ion and some partial consumption of the PHA synthesized occurredetween consecutive pulses (33% of the PHA formed was consumedetween pulses). The consumption of formed PHA reduced thelobal PHA yield on VFA substrate from 0.77 Cmol PHA/Cmol VFA (ifll the PHA was accounted) to 0.53 Cmol PHA/Cmol VFA. The timeoss and polymer consumption – resulted in a 43% decrease of volu-

etric productivity relatively to what would have been obtained ifhe stops between pulses (and respective PHA consumption) coulde avoided.

It was also observed in both pulse wise feeding assays (Fig. 2a

nd b), that for each pulse of substrate fed, the rates of sub-trate uptake and PHA storage were maximum at the beginning ofhe pulse, but as substrates were depleted these rates decreased30 and 35% decrease for the substrate uptake and PHA stor-ge rates, respectively, in assay A1). The dependence of rates Ta

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

F pulsea

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ig. 1. Batch accumulation assays with VFA mixture: (a) pulse-wise feeding A1, (b)ssays A1–A3.

f substrate uptake and PHA storage on substrate concentrationas been reported for the enrichment reactor operating cyclesAlbuquerque et al., 2010a,b), for which an affinity constant (kS) ofbout 18.5 Cmmol VFA/L was reported (Albuquerque et al., 2010b).

Fig. 1c shows the results from the continuous feeding assay3. This system demonstrated a considerable improvement of theHA storage performance relatively to both pulse-wise feedingests using the same medium (A1 and A2). In test A3, the maxi-

um specific substrate uptake (0.64 Cmol VFA/Cmol X h) and PHAtorage (0.40 Cmol PHA/Cmol X h) rates were higher than thosebtained in both pulse wise feeding tests (0.46 and 0.43 CmolFA/Cmol X h and 0.31 and 0.35 Cmol PHA/cmol X h, in A1 and A2,espectively). This can be explained by the different initial substrate

oncentrations used (60 Cmmol VFA/L used for A3 rather than the0 Cmmol VFA/L used in A1 and A2). A considerable decrease inubstrate uptake and polymer storage rates was shown to occur forhe same fermented molasses-selected culture for concentrations

ig. 2. PHA productivity (in g PHA/L h), instant PHA storage rate (in g PHA/L h) and PHA cithout and with pH control – and continuous feeding assay A3 (c); (d) PHA productivity

wise feeding A2 and (c) continuous feeding A3; (d) PHA fraction in the three batch

higher than 90 Cmmol VFA/L (Albuquerque et al., 2007). A gradualdecrease of these rates has also been shown to occur in the rangeof 60–90 Cmmol VFA/L (Albuquerque, 2009).

More importantly, a much steadier PHA storing activity (at max-imum level) was observed for the continuous feeding assay A3, asopposed to the two pulse feeding assays, A1 and A2, where a slow-down of specific rates was observed for each pulse. This is visiblein Fig. 1d in which the fraction of PHA per active biomass is plot-ted for the three assays. The continuous feeding strategy allowedthe specific rates of substrate uptake and PHA storage to stay veryclose to maximum levels until about 6 h (0.54 Cmol VFA/Cmol X hand 0.39 Cmol PHA/Cmol X h, respectively), at which point the cul-ture had already reached a PHA content of 72%. After this point

onward, the specific rates of substrate uptake and PHA storagedecrease, due to proximity to the saturation level, which causesthe average specific rates determined for the full length of testA3 (substrate uptake rate of 0.47 Cmol VFA/Cmol X h and PHA stor-

ontent (%) over time for pulse wise feeding assays A1 (a) and A2 (b) – respectively(in g PHA/L h) as a function of PHA content (%) for assays A1–A3.

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M.G.E. Albuquerque et al. / Journ

ge rate of 0.26 Cmol PHA/Cmol X h, Table 2) to decrease relativelyo the rates observed 6 h into the test. Nonetheless, these aver-ge rates were still considerably higher than the average rates ofoth pulse-wise feeding tests (0.32 and 0.27 Cmol VFA/Cmol X h and.20–0.17 Cmol PHA/Cmol X h, in A1 and A2, respectively), none ofhich having attained the same proximity to the saturation level

since the maximum PHA contents observed were about 65% inoth A1 and A2).

The continuous feeding strategy seemed to be an effective wayf improving process productivity (Fig. 2). A volumetric productiv-ty of 1.2 g PHA/L h was observed 6 h into the continuous feedingest (at 72% PHA content) (Fig. 2c), which represents a 2.5–4 foldncrease relatively to the productivities obtained in assays A1 and2 at 65% PHA content (0.3–0.5 g PHA/L h) (Fig. 2a and b). In fact,

he 1.4 fold decrease of the instant volumetric PHA storage ratebserved in the continuous feeding test during this first 6 h periodFig. 2c) is almost consistent with the dilution factor (1.4) resultingrom the new influent being fed to the reactor. The steep decreasebserved in both the instant volumetric PHA storage rate and theolumetric productivity about 6 h into the test directly reflects thelowdown of specific PHA storage rates associated with proximityo the saturation level.

In the continuous feeding assay A3, the volumetric produc-ivity and instant volumetric PHA storage rate remained higherhroughout the batch test and subjected to less fluctuations thanhe respective curves obtained for assays A1 and A2. In both pulseise feeding studies (A1 and A2), the instant volumetric PHA stor-

ge rates decreased (either gradually – A1 – or more steeply – A2)or each pulse of substrate fed and, as a result, the volumetric pro-uctivities decreased considerably throughout the two batch testsFig. 2a and b). The higher average specific rates of substrate uptakend PHA storage observed in the continuous feeding assay (A3) rel-tively to those reported for the two pulse wise feeding tests (A1nd A2) seem to be related, on one hand with:

(i) the fact that some of the constraints associated with pulse-feeding had effectively been removed (namely, the stopsbetween pulses which represent time and polymer losses) and

ii) more importantly, the higher residual substrate concentra-tion observed throughout the entire batch test (never below20 Cmmol VFA/L, see Fig. 1b), which allowed the specific ratesof substrate uptake and PHA storage to stay close to maximumlevels (almost until the saturation level was reached).

It can be argued that during the pulse wise feeding tests, theiomass was adversely affected metabolically during the lag peri-ds between pulses. Whether this eventual adverse metabolicffect was caused solely by the stops between pulses or may bessociated with the slow-down of metabolic rates observed nearhe end of each pulse (as carbon substrate concentration becomesimiting) is not completely clear from the results obtained. How-ver, results seem to indicate that the use of a continuous feedingtrategy has the advantage of keeping the culture at constant max-mum rates (high metabolic activity throughout the batch test),

hile the pulse wise feeding strategy (either due to the volumeeplacement regimen used or associated with the pulse wise feed-ng itself) seems to result in a slow-down of metabolic activity at thend of every pulse, causing the average performance to decrease.

The main constraint on volumetric productivity imposed by theontinuous feeding strategy was the dilution resultant from the

ontinuous supply of new medium (a dilution factor of 1.4 wasbserved in this test). However, this may be overcome through these of a more concentrated feed (making sure the residual substrateoncentration is kept bellow inhibiting values) or a microfiltrationembrane coupled to the batch production reactor.

iotechnology 151 (2011) 66–76 73

As can be seen in Fig. 2d which shows the volumetric produc-tivity as a function of PHA content, for the same PHA content theproductivity was higher in the continuous feeding assay than usingpulse wise feeding. A much more gradual decrease of the volumetricproductivity was observed in the continuous feeding assay rela-tively to the pulse wise feeding tests, where a very steep decreaseis observed almost from the beginning of the each batch test. Onlyclose to the saturation level, does the productivity greatly decreaseas a function of PHA content, but only for values of PHA contenthigher that 72%.

The effect on feeding regimen on polymer composition was alsoevaluated. The use of continuous feeding increased the HV contentrelatively to pulse wise feeding from 31 to 39% in the case of feedsolution with VFA profile B. This is most likely explained by the factthat in pulse feeding assays, propionate and valerate, whose frac-tions were significantly lower than acetate and butyrate fractions,were first exhausted and the amount of HV formed was limitedby the availability of these organic acids (Fig. 1a and b). In fact, inpulse feeding assays A1 and A2, for each pulse of substrate fed, HBsynthesis is observed throughout the full extent of the pulse, whileHV synthesis stops once propionate and valerate are exhausted.Because these two organic acids are present in lower quantity thanacetate and butyrate, they are exhausted before the later two, thuscausing HV synthesis to stop before the end of the pulse. On thecontrary, in continuous feeding assay A3, the continuous supply offresh medium, kept the residual concentrations of acetate and pro-pionate constant (see Fig. 1c), thus ensuring HB and HV synthesisthroughout the full test. In the continuous feeding assay (A3), theHB:HV ratio was thus determined by the ratio between HB andHV synthesis rates rather than by the availability of the appro-priate precursors. Therefore, although hydroxybutyrate was stillthe major component of the copolymer produced, the HV contentconsiderably increased as a result of feeding regimen alone.

These results indicate that the continuous feeding strategy,apart from being an interesting alternative to effectively increaseprocess productivity, may also be used to manipulate the polymercomposition, increasing the range of polymer compositions whichcan be synthesized from a given fermented effluent.

3.3. Effect of polymer composition on polymer structural andthermal properties

The copolymers of hydroxybutyrate (HB) and hydroxyvalerate(HV) obtained in the batch studies previously described (A3, B1 andC2) were characterized in terms of structural and thermal proper-ties. These particular samples were selected in order to cover thewide range of HV contents obtained (15–39%) and also to includeat least one co-polymer produced from real fermented molasses(sample C2).

3.3.1. Structural propertiesSize exclusion chromatography (SEC) was performed and the

results are reported (Table 3). Reasonably low PDI (2.3–2.7) wasobserved for all samples (A3, B1 and C2). The SEC traces obtainedwith either the RID or UV/Vis detectors did not show additionalpeaks at low elution times, suggesting the absence of high molec-ular weight impurities. Weight average molecular weights of thedifferent copolymers P(HB-co-HV) produced decreased, from 6.5 to2.2 × 105, with increasing HV content (15–39%). This may be due tothe incorporation of a higher amount of HV units causing a disrup-tion on polymer chains (Organ, 1993). Notwithstanding the lower

molecular weights, the higher HV content did not result in lowerPDI. In fact, samples with higher and lower HV contents showedsimilar 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

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

Table 3Summary 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 C2 B1 A3Substrate Fermented molasses Simulated Fermented molassesFeeding regimen Pulse feeding Pulse feeding Continuous feeding

Polymer composition HV (%) 15 21 39

Structural properties

Mn 2.75 × 105 1.44 × 105 0.95 × 105

Mw 6.46 × 105 3.86 × 105 2.15 × 105

PDI 2.3 2.7 2.3Crystallinity Marked crystallization in the first cooling Crystallization was

only observed in thesecond thermal cycle

Almost amorphous

ctas2bso(iomsrpuaotaiPm

silaoTvcms

ss

3

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Thermal propertiesTmax (◦C) 247Tg (◦C) −1Tm (◦C) 134/147

lose to those reported for PHA produced by other mixed cul-ures, namely to that of a P(HB-co-HV) copolymer produced byn acetate selected culture using fermented molasses as sub-trate for PHA accumulation (Mw of 8.5 × 105, Albuquerque et al.,007) and also to those of PHA co- and ter-polymers producedy glycogen-accumulating organisms (GAO) enriched cultureselected using either fermented molasses (3.5–9.0 × 105 and PDIf 1.8–3.9, Bengtsson et al., 2010b) or acetate/propionate mixtures3.9–5.6 × 105, Dai et al., 2008). The molecular weights obtainedn this study were, however, slightly lower (between half and onerder of magnitude lower) than those reported for a PHB homopoly-er (1.0–3.0) × 106 produced by a mixed culture using acetate as

ole carbon source (Serafim et al., 2008a,b) and also lower than thateported by Patel et al. (2009) for a copolymer of P(3HB-co-3HV)roduced by a mixed microbial culture of nitrogen-fixing bacteriasing acetate as sole carbon source for enrichment and fed withcetate–propionate mixtures during the accumulation stage (Mn

f 2.5 × 106). The PDI was either higher or in the higher range ofhose reported in the two previous studies (1.3, Patel et al., 2009)nd (1.3–2.2, Serafim et al., 2008b). Because this difference is slight,t can also safely be argued that the difference between the Mw andDI reported here relatively to values commonly reported for bothixed and pure cultures.X-ray diffraction (XRD) was used to characterize a copolymer

ample with 30% HV content. The sample crystallinity was approx-mately 40 ± 5%. The sample was also analyzed using a polarizedight optical microscope (POM). It was observed that immediatelyfter melting and cooling no crystals were formed. However, afterne day, a great amount of very small crystals could be observed.he crystallinity degree determined for this sample falls within thealues reported by Patel et al. (2009) for PHB and P(HB-co-HV) withrystallinity indices of 44% and 37%, respectively, produced by aixed culture of nitrogen fixing bacteria from two different carbon

ubstrates.The crystallinity of the remaining samples recovered in this

tudy (A3, B1 and C1) could not be quantified due to insufficientample amount available.

.3.2. Thermal propertiesThermogravimetric analysis (TGA) was carried out to deter-

ine the degradation temperature of the copolymers produced.he thermograms of samples from batch tests A3, B1 and C2 showedvolatilization temperature below 75 ◦C, indicating that these sam-les still contained some residual solvent (mass losses between 1%nd 14%), and a small degradation step centered at 120 ◦C, which

xtent seemed to increase with increasing HV content. This behav-or could be due to the presence of some residual raw material usedn the fermentation process (low molecular weight impurities). Theollowing degradation step, between 190 ◦C and 280 ◦C, with Tmax

entered between 242 ◦C and 247 ◦C, corresponds to P(HB-co-HV)

242 243−10 −16121/137 113/138

main degradation. Sample C1 (produced from fermented molassesrather than simulated feed) showed the highest degradation tem-perature (Tmax of 247 ◦C), but also a further small degradation stepcentered at 389 ◦C. Regarding the amount of residues left at 500 ◦C,very low quantities were detected in all cases. Despite the pres-ence of some residual solvent and possibly fermentation products,which reflect the fact that the extraction and recovery procedurewas not the main focus of this study and still requires some furtheroptimization, the degradation temperatures of the polymers werequite consistent with values described in the literature for P(HB-co-HV) either from pure or mixed cultures, enabling a wide processingtemperature window (Bengtsson et al., 2010a,b).

Other thermal properties such as melting and glass transitiontemperature were inferred from DSC thermograms obtained forsamples A3, B1 and C2 (Fig. 3a, b and c, respectively). For sampleC2 (sample with the lowest HV content), a glass transition temper-ature (Tg) of −1 ◦C was observed (during the second eating scan),a crystallization peak was observed at 77 ◦C (during cooling) and adouble melting peak was observed at 134 ◦C and 147 ◦C. For sampleB1 (sample with the intermediate HV content), Tg, was observed at−10 ◦C, a crystallization peak was observed at 72 ◦C (crystallizationtemperature was determined during the second eating, since nopeak was observed during cooling) and a double melting peak at121 ◦C and 137 ◦C. For sample A3 (sample with the higher HV con-tent), Tg value was observed at −16 ◦C, the crystallization peak wasnot well defined (in either the cooling or the second eating scan)but a slight exothermic event appeared at 67 ◦C. In addition a broadand low intensity double melting peak could be detected at 113 ◦Cand 138 ◦C.

Thermal characterizations (Table 3) showed values of glasstransition temperature between −1 and −16 ◦C and melting tem-peratures between 137 ◦C and 147 ◦C (main peak). Such values areslightly lower than those reported for P(HB-co-HV) produced bypure cultures and in the same range as those reported for P(HB-co-HV) produced by other fully aerobic mixed cultures (Serafimet al., 2008a,b) (Fig. 4). Moreover, since the onset of thermal degra-dation was between 190 ◦C and 200 ◦C, these polymers show arelatively wide processing thermal window, since the differencebetween melting temperature and degradation temperature isapproximately 50 ◦C.

Although the crystallinity of samples A3, B1 and C2 could notbe quantified, a reduced kinetic of crystallization was observedwith increasing HV contents from the DSC analyses. For 15% HVcontent (sample C2), a marked crystallization was observed dur-ing cooling. For 20% HV content (sample B1), a crystallization peak

could also be observed but only during the second heating. A fur-ther increase in the HV content to 39% (sample A3) conducted toalmost an amorphous matrix.

The observed variation of the thermal properties (melting andglass transition temperature) with the polymer composition (HV

M.G.E. Albuquerque et al. / Journal of Biotechnology 151 (2011) 66–76 75

67.44°C

-15.68°C

113.24°C

a Sample A3 (39% HV)

137.95°C

Heat Flow (W/g)

-50 0 50 100 150Temperature (°C)Exo Up

71.77°C

-9.83°C

120.62°C

137.40°C

b Sample B1 (21% HV)

Heat Flow (W/g)

-50 0 50 100 150Temperature (°C)Exo Up

77.46°C

-1.18°C

133.56°C

146.52°C

Heat Flow (W/g)

-50 0 50 100 150

c Sample C2 (15% HV)

cfee20

Temperature (°C)Exo Up

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

ontent) is in accordance with reports from the literature (Fig. 4)

or other mixed and pure cultures (Chua and Yu, 1999; Serafimt al., 2008b; Reis et al., 2003; Punrattanasim, 2001; Savenkovat al., 2000; Sudesh et al., 2000; Yoshie et al., 2001; Yamada et al.,001). Namely, to results obtained by Dai et al. (2008) (Tg: −8 to◦C and Tm: 70–161 ◦C) and Bengtsson et al. (2010a,b) (Tg: −14 to

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 prop-erties of PHA polymers produced by GAO enriched cultures werecontrolled in broad ranges by the monomer composition. How-ever, in the present study the range of compositions was narrowerthan that observed in the two previously mentioned studies, whichcan be explained by the higher monomer variability observedin the two later cases (four and five different monomers werereported by Dai et al. and Bengtsson et al., respectively). Bengtssonet al. (2010a,b) also observed that the decomposition temperatures(277.2–294.9 ◦C) were independent of monomer composition andmolecular weight.

4. Conclusions and perspectives

Strategies to improve the PHA accumulation stage – both interms of productivity and possibility to control polymer composi-tion – in a three-stage PHA production process from sugar molasseswere investigated. It was found that the use of a continuous feedingstrategy rather than a pulse feeding strategy can, not only help mit-igate the process constraints of the pulse-feeding strategy (stopsbetween pulses often with polymer consumption), but also allowhigher rates of substrate uptake and polymer storage to be main-tained due to constant residual substrate concentration. Thus, aconsiderable increase of volumetric productivity was obtained as aresult of the use of a continuous feeding regimen in the PHA pro-duction stage of a 3 stage PHA process from an industrial residualfeedstock.

Moreover, the continuous feeding strategy can also be used asa means to increase the HV content by 8–9% relatively to thatobtained from the same feedstock using pulse wise feeding. Thiscan be an interesting alternative for the manipulation of the poly-mer composition, increasing the range of polymer structures whichcan be synthesized from a given fermented effluent.

The range of PHA compositions obtained (PHB-co-HV with HVfraction ranging from 15 to 39%) subsequently resulted in differentpolymer properties (molecular weight and thermal properties). Theproduced polymers presented slightly lower molecular weightsthan those generally obtained with other fully aerobic mixed cul-tures but still high enough for thermoplastic processing. Moreimportantly, it was demonstrated by DSC that these polymerspresent similar thermal properties relatively to PHA copolymersproduced by both mixed and pure cultures, showing a relatively

wide window of processability. The Mw and PDI obtained are withinthe range of values reported in the literature for PHA producedboth by pure and mixed cultures. Although mechanical propertieswere not determined in this study, the molecular weights and PDIobtained, as well as the thermal properties observed, are compa-

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6 M.G.E. Albuquerque et al. / Journ

able to those reported in the literature for PHA, which have beenhown to be adequate for thermoplastic processing. Thus, it cane expected that these PHA would also be suited for thermoplasticrocessing.

These results indicate that the use of agricultural and indus-rial residual or surplus streams as feedstocks for PHA productiony mixed microbial cultures enriched in PHA-storing organismshrough culture selection under Aerobic Dynamic Feeding can beonsidered an interesting and possibly cost-effective alternative toure culture fermentations. This strategy allows for the productionf PHA polymers of similar quality and for which the monomeromposition may be manipulated through the acidogenic fermen-ation conditions and also through the PHA production feeding reg-men. This allows for a wide variety of polymer with different ther-

al properties to be produced from these complex surplus streams.

cknowledgments

Fundacão para a Ciência e Tecnologia (Portugal) is greatlycknowledged for funding this research through the individualrant SFRH/BD/17141/2004. The EU is also acknowledged for fund-ng through the project “Sustainable Microbial and Biocatalytic Pro-uction of Advanced Functional Materials”, EU Integrated Project,ontract no. 026515-2; 2006-2008. Refinaria de Acúcares ReúnidaRAR), Portugal, is acknowledged for the supply of the molasses.

eferences

lbuquerque, M.G.E., Eiroa, M., Torres, C., Nunes, B.R., Reis, M.A.M., 2007. Strategiesfor the development of a side stream process for polyhydroxyalkanoate (PHA)production from sugar cane molasses. J. Biotechnol. 130, 411–421.

lbuquerque, M.G.E., Torres, C., Reis, M.A.M., 2010a. Polyhydroxyalkanoate (PHA)production by a mixed microbial culture using sugar molasses: effect of theinfluent substrate concentration on culture selection. Water Res. 44, 3419–3433.

lbuquerque, M.G.E., Concas, S., Bengtsson, S., Reis, M.A.M., 2010b. Mixed culturepolyhydroxyalkanoates production from sugar molasses: the use of a 2-stageCSTR system for culture selection. Bioresour. Technol. 101, 7112–7122.

lbuquerque, M.G.E., 2009. Production of polyhydroxyalkanoates (PHA) from sugarcane molasses by mixed microbial cultures. PhD Thesis Universidade Nova deLisboa, Lisbon, Portugal.

PHA, Standard Methods for the Examination of Water and Wastewater, AmericanPublic Health Association, Washington DC, 1995.

nderson, A.J., Dawes, E.A., 1990. Biosynthesis and composition of bacterialpoly(hydroxyalkanoates). Int. J. Biol. Macromol. 12, 102–105.

eccari, M., Majone, M., Massanisso, P., Ramadori, R., 1998. A bulking sludge withhigh storage response selected under intermittent feeding. Water Res. 32,3403–3413.

eccari, 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 forproduction of biodegradable polymers through a combined anaerobic–aerobicprocess. J. Chem. Technol. Biotechnol. 84, 901–908.

engtsson, S., Werker, A., Christensson, M., Welander, T., 2008. Production ofpolyhydroxyalkanoates by activated sludge treating a paper mill wastewater.Bioresour. Technol. 99, 509–516.

engtsson, S., Pisco, A.R., Reisand, M.A.M., Lemos, P.C., 2010a. Production of poly-hydroxyalkanoates from fermented sugar cane molasses by a mixed cultureenriched in glycogen accumulating organisms. J. Biotechnol. 145, 253–263.

engtsson, S., Pisco, A.R., Johansson, P., Lemos, P.C., Reis, M.A.M., 2010b. Molec-ular weight and thermal properties of polyhydroxyalkanoates produced fromfermented sugar molasses by open mixed cultures. J. Biotechnol. 147, 172–179.

eun, J.J., Dircks, K., van Loosdrecht, M.C.M., Heijnen, J.J., 2002. Poly-b-hydroxybutyrate metabolism in dynamically fed mixed cultures. Water Res. 36,1167–1180.

luhm, T.L., Hamer, G.K., Marchessault, R.H., Fyfe, C.A., Veregin, R.P., 1986. Isodi-morphism in bacterial poly (beta-hydroxybutyrate-co-beta-hydroxyvalerate).Macromolecules 19, 2871–2876.

hua, H., Yu, P.H.F., 1999. Production of biodegradable plastics from chemicalwastewater – a novel method to reduce excess activated sludge generated from

industrial wastewater treatment. Water Sci. Technol. 39, 273–280.

aiger, G.T., Grady, P.L., 1982. The dynamics of microbial growth on soluble sub-strates: a unifying theory. Water Res. 16, 365–382.

ai, Y., Lambert, L., Yuan, Z.G., Keller, J., 2008. Microstructure of co-polymers of poly-hydroxyalkanoates produced by glycogen accumulating organisms with acetateas the sole carbon source. Process Biochem. 43, 968–977.

iotechnology 151 (2011) 66–76

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 polyhy-droxyalkanoate production by mixed aerobic cultures: from the substrate tothe 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 biodegradablepolymers. Water Res. 39, 2076–2084.

Dionisi, D., Majone, M., Levantesi, C., Bellani, A., Fuoco, A., 2006. Effect of feed lengthon settleability, substrate uptake and storage in a sequencing batch reactortreating 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(3-hydroxybutyrate-co-3-hydroxyhexanoate) and thermal characteristics of theirfractions. Biomacromolecules 3, 1071–1077.

Gujer, W., Henze, M., Mino, T., Van Loosdrecht, M.C.M., 1999. Activated sludge modelno. 3. Water Sci. Technol. 39, 183–193.

Gurieff, N., Lant, P., 2007. Comparative life cycle assessment and financial anal-ysis 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 com-position and microstructure of copolyesters P(3HB-co-3HV) analyzed by NMRand statistical analysis. Magn. Reson. Chem. 47, 497–504.

Johnson, K., Kleerebezem, R., Muyzer, G., Gand, M.C.M., van Loosdrecht, 2009. Enrich-ment of a mixed bacterial culture with a high polyhydroxyalkanoate storagecapacity. Biomacromolecules 10, 670–676.

Kleerebezem, R., van Loosdrecht, M.C.M., 2007. Mixed culture biotechnology forbioenergy 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-hydroxybutyrate-co-4-hydroxy butyrate). Macromolecules 23, 1933–1936.

Lee, S.Y., Choi, J., Wong, H.H., 1999. Recent advances in polyhydroxyalkanoate pro-duction 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 polyhydroxyalka-noate 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 polyhydroxyalkanoatesfrom different short-chain fatty acids by mixed cultures submitted to aerobicdynamic feeding. J. Biotechnol. 122, 226–238.

Majone, M., Masanisso, P., Carucci, A., Lindrea, K., Tandoi, V., 1996. Influence ofstorage 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 ofpoly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Burkholderia cepa-cia D1. Int. J. Biol. Macromol. 24, 311–318.

Organ, S.J., 1993. Variation in melting point with molecular weight for hydroxybu-tyrate/hydroxyvalerate copolymers. Polymer 34, 2175–2179.

Pardelha, F., Albuquerque, M.G.E., Reis, M.A.M., Oliveira, R., Dias, J.M.L., Metabolicmodeling of polyhydroxyalkanoates production from complex mixtures ofvolatile fatty acids by mixed microbial cultures. Submited for publication.

Patel, M., Gapes, D.J., Newman, R.H., Dare, P.H., 2009. Physico-chemical propertiesof polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria.Appl. Microbiol. Biotechnol. 82, 545–555.

Punrattanasim, W., 2001. The utilization of activated sludge polyhydroxyalkanoatesfor the production of biodegradable plastics. PhD Thesis. Virginia PolytechnicInstitute 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-hydroxyvaleratecontent on some physical and mechanical properties of polyhydroxyalkanoatesproduced by Azotobacter chroococcum. Process Biochem. 36, 445–450.

Serafim, L.S., Lemos, P.C., Oliveira, R., Reis, M.A.M., 2004. Optimization of polyhydrox-ybutyrate production by mixed cultures submitted to aerobic dynamic feedingconditions. Biotechnol. Bioeng. 87, 145–160.

Serafim, L.S., Lemos, P.C., Albuquerque, M.G.E., Reis, M.A.M., 2008a. Strategies forPHA production by mixed cultures and renewable waste materials. Appl. Micro-biol. Biotechnol. 81.

Serafim, L.S., Lemos, P.C., Torres, C., Reis, M.A.M., Ramos, A.M., 2008b. The influence ofprocess parameters on the characteristics of polyhydroxyalkanoates producedby mixed cultures. Macromol. Biosci. 8, 355–366.

Sudesh, K., Abe, H., Doi, Y., 2000. Synthesis, structure and properties of polyhydrox-yalkanoates: biological polyesters. Prog. Polym. Sci. 25, 1503–1555.

Takabatake, H, Satoh, H., Mino, T., Matsuo, T., 2000. Recovery of biodegradable plas-tics 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 storagepolymers 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 bymixed 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.