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International Journal of Biological Macromolecules 74 (2015) 202–210

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

olyhydroxyalkanoates from Pseudomonas sp. using synthetic andlive mill wastewater under limiting conditions

. Kourmentzaa,b,∗, I. Ntaikoub, G. Lyberatosb,c, M. Kornarosa

Laboratory of Biochemical Engineering & Environmental Technology (LBEET), Department of Chemical Engineering, University of Patras, 1 Caratheodorytr., 26500 Patras, GreeceInstitute of Chemical Engineering Sciences, Foundation for Research & Technology Hellas (ICE-HT/FORTH), 26504 Patras, GreeceSchool of Chemical Engineering, National Technical University of Athens (NTUA), Zografou Campus, 15780 Athens, Greece

r t i c l e i n f o

rticle history:eceived 5 November 2014eceived in revised form 9 December 2014ccepted 10 December 2014vailable online 24 December 2014

eywords:

a b s t r a c t

The present study aimed at investigating the ability of bacteria isolated from an enriched mixed culture toproduce polyhydroxyalkanoates (PHAs) and examining the effect of nitrogen and dual nitrogen–oxygenlimitation on PHAs production, by using both synthetic and olive mill wastewater (OMW). PHAs produc-tion was performed through batch experiments using both the enriched culture and the isolated strains(belonging to the genus of Pseudomonas) aiming to compare PHAs accumulation capacity, yields andrates. The use of enriched culture and synthetic wastewater under nitrogen limitation resulted in the

olyhydroxyalkanoates (PHAs)seudomonaslive mill wastewateritrogen oxygen limitation

highest PHA accumulation, i.e. 64.4% g PHAs/g of cell dry mass (CDM). However, when OMW was used,PHAs accumulation significantly decreased, i.e. 8.8% g PHAs/g CDM. The same trend was followed by theisolated strains, nevertheless, their ability to synthesize PHAs was lower. Although, dual nitrogen–oxygenlimitation generally slowed down PHAs biosynthesis, in certain strains PHAs production was positivelyaffected.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Polyhydroxyalkanoates (PHAs) are biodegradable polyestershat have been reported to be synthesized by various microor-anisms. Those polyesters are accumulated as intracellular carbonnd energy source, in an amorphous state, in the form of gran-les bearing a diameter of 0.2–0.9 �m. Usually, PHAs production

s performed under stress conditions caused by the limitation ofn essential nutrient that restricts bacterial growth [1]. Apart fromhe fact that PHAs can be characterized as bio-derived materialshey also attract interest because they bear similar physicochem-cal properties to conventional polymers such as polyethylenePE/LDPE) and polypropylene (PP) [2].

Recognizing the fact that PP and PE have gained a major positionn the global plastics market, which accounts for around 50% of theverall plastics demand in Europe [3], PHAs are representing an

∗ Corresponding author. Present address: Department of Chemistry, Faculdade deiências e Tecnologia, Universidade Nova de Lisboa (FCT/UNL), 2829-516 Caparica,ortugal. Tel.: +351 212948300, ext: 10947; fax: +351 212948550.

E-mail addresses: ckourmentza@gmail.com, c.kourmentza@fct.unl.ptC. Kourmentza).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.12.032141-8130/© 2014 Elsevier B.V. All rights reserved.

attractive alternative to those petrochemically derived materials,while their use can minimize the detrimental impact of persistentplastics on the environment. However, their high production costis still the major drawback for their commercialization. Among theways being proposed for decreasing PHAs high production cost, theuse of mixed microbial consortia grown on synthetic feedstocks,such as sugars, as well as the use of pure cultures grown on wastestreams have been reported to result in the reduction of the overallproduction cost by 30–40% [4].

Olive mill wastewater is a by-product generated by the olive oilindustry. In Greece, the majority of olive oil extraction industriesuse three-phase centrifugation systems (decanters) which produce,as by-product, a stream of Olive Mill Wastewater (OMW). OMW ischaracterized by its high organic load containing large amountsof proteins, lipids, polysaccharides, a high C/N ratio and phyto-toxic components, such as phenolic compounds, that may inhibitmicrobial degradation [5]. Biopolymers production via microbialtransformation of OMW was first proposed by Dionisi et al. [6] fol-lowed by Beccari et al. [7] and Ntaikou et al. [8,9] who investigated

the feasibility of anaerobically fermented olive mill effluents forPHAs production. In those cases PHAs were accumulated due tovolatile fatty acids (VFAs) consumption, as OMW was previouslyacidified in order to convert its carbohydrates content to VFAs.

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C. Kourmentza et al. / International Journal

his additional process step is considered as a prerequisite sincetorage of carbohydrates as glycogen is more favored energeticallyompared to storage of PHAs in open mixed cultures [10].

In general, the key for successful PHAs production via mixedultures is their enrichment with bacteria that are characterizedy high PHAs storage capacity. The enrichment strategies takelace either under alternating anaerobic/aerobic or feast/famineonditions [11]. In addition, the selection of other operational andnvironmental parameters such as sludge retention time (SRT),H, organic loading rate (OLR), C/N ratio, temperature etc. playn important role in the enrichment process and the ability of theulture to produce PHAs [6,7,11].

Up-to-date, there have been few reports concerning the micro-ial composition of mixed cultures being subjected to enrichmentrocesses in order to promote growth of PHAs-forming bacteria.

n their studies Dionisi et al. [6,12] enriched a mixed culture withHA producers under feast/famine conditions. Using the techniquef denaturing gradient gel electrophoresis (DGGE) they were ableo analyze in detail the microbial consortium. Many bacteria ofifferent genera, including even small populations, were found toonstitute this mixed culture, such as strains of the genus Thauera,lcaligenes, Comamonas, Achromobacter, Xanthobacter, Curtobac-erium, Kluyvera, Pseudomonas, Acinetobacter, Methylobacteriaceaep., Flavobacterium and Candidatus Meganema perideroedes. In addi-ion, Lemos et al. [13] performed the enrichment process using

ixed cultures under aerobic feast/famine conditions which wereharacterized using RT-PCR (Reverse transcription-polymerasehain reaction). According to their results, the consortium consistedf bacteria belonging to Azoarcus, Amaricoccus and Thauera species.urthermore, Bengtsson and co-workers [14] as well as Pisco ando-workers [15] studied the enrichment of mixed cultures underlternating anaerobic/aerobic conditions. Using the technique ofuorescent in situ hybridization (FISH) they identified the pre-ominant strains which were Defluviicoccus vanus and Candidatusompetibacter phosphatis. However, none of those reports investi-ated the PHAs production efficiency of the isolates that had beendentified as the predominant strains of the mixed consortium, so,here is no comparison between the results obtained using thenriched culture and the isolated strains.

In the present study, the PHAs production efficiency of a mixedulture rich in PHA-forming bacteria, and bacterial strains iso-ated from it, was examined under stress conditions. Nitrogen andual nitrogen–oxygen limitation was applied, using both syntheticnd OMW, as the sole carbon source. The scope of those exper-ments was to compare the ability of those cultures to produceHAs and to investigate whether a PHA overproducing strain wassolated. Under stress conditions such as nitrogen limitation bacte-ial growth is restricted and carbon flux is therefore driven to PHAormation. On the other hand, oxygen limitation usually occurs dur-ng the fermentation process thus dual nitrogen–oxygen limitation

as applied in order to investigate the additional effect of oxygen

imitation in our study. In addition, we studied the valorization ofMW, a common waste stream in the Mediterranean region, via itsicrobial transformation to PHAs using the enriched culture and

ts isolates.

able 1dentification of strains isolated from the enriched to PHAs forming bacteria culture by pa

Isolate 1st match 2nd match

A Pseudomonas sp. ONBA Pseudomonas putidaB Pseudomonas putida ATCC 17453 Pseudomonas putidaC Pseudomonas sp. OCR7 Pseudomonas sp. K2D Pseudomonas sp. OCR7 Pseudomonas sp. K2E Pseudomonas putida ATCC 17390 Pseudomonas putidaF Pseudomonas putida RW-26 Pseudomonas putida

ogical Macromolecules 74 (2015) 202–210 203

2. Materials and methods

2.1. Microorganisms

An enriched culture in PHA-producing bacteria was developedusing activated sludge from the aerobic tank of the WastewaterTreatment Plant of Patras (Greece) as inoculum. The WastewaterTreatment Plant (WWTP) was an Enhanced Biological Phospho-rus Removal (EBPR) scheme coupled with a Carrousel system(oxidation ditch designed to effectively reduce the nutrients dis-charged). The strategy that was followed for the selection andenrichment of activated sludge in PHA-accumulating bacteria wasthe alternating cycling between limitation of carbon and nitro-gen substrates [16]. Following this procedure, PHA-accumulatingbacteria could adapt under stress conditions and bacteria withhigh PHAs-accumulation capacity were able to survive longer thantheir heterotrophic competitors after several cycles. The enrich-ment process took place in a draw n’ fill reactor, operated incycles of 23 h of continuous aeration and 1 h settling includingthe withdrawal of the supernatant. The feeding of the mediumwas taking place instantaneously at the start of each operationcycle, replacing 83% of the total working volume (2.4 L). The com-position of the basal synthetic medium used was as following:0.2 g/L MgSO4·7H2O, 0.0251 g/L CaCl2·2H2O, 5 g/L K2HPO4 and3 g/L KH2PO4 and 1.0 mL/L of a trace elements solution. The traceelements solution was of the following composition: 0.786 g/LCuSO4·5H2O, 5 g/L FeSO4·7H2O, 12.609 g/L NaMoO4·2H2O, 4.05 g/LNiCl2·6H2O, 4.398 g/L ZnSO4·7H2O, 2.453 g/L CoCl2·6H2O, 0.75 g/LKI, 3 g/L H3BO3, 5 g/L MnCl2·4H2O and 5 g/L EDTA. In eachcycle, either carbon or nitrogen source was added: 0.1715 g/LCH3CH2COOH, 0.256 g/L CH3COOH as carbon sources and 0.278 g/L(NH4)2SO4 as nitrogen source. The reactor was operated at roomtemperature, 28 ± 2 ◦C, and the stirring rate was kept at 250 rpm.

After several cycles of operation (approximately 30 cycles)under the above-mentioned conditions, the behavior and per-formance of the culture stabilized and therefore bacteria thatrepresented the ‘enriched’ culture were isolated. Bacterial isolateswere obtained by subsequent dilutions and further streaking onbasal medium (BM) agar plates. Medium’s composition was thesame as basal synthetic medium (as described above) including15 g/L agar. The plates were incubated at 28 ◦C for 48 h and coloniesfrom the plates were picked and further purified.

2.2. Genetic identification and phylogenetic affiliation of theisolated bacterial strains

According to the results, bacterial strains belonging to Pseu-domonas genus were isolated from the enriched culture, as shownin Table 1. All strains isolated in this study were identified byanalysis of the partial sequence of the gene encoding 16S rRNA.Primers fD1 and rD1 [17] were synthesized by Sigma Genosis (UK)

and used to amplify almost the full length of the 16S rRNA genefrom all strains. Fresh cultured colonies of each strain were lysedby the addition of 20 �L of NaOH (0.05 M)-SDS (0.25%, w/v) whichwas then boiled for 15 min. The volume of lysates was adjusted to

rtial 16S rRNA gene.

3rd match % Homology

ATCC 17390 Pseudomonas sp. HR 13 99 ATCC 17514 – 99

Pseudomonas sp. WBC-2 99 Pseudomonas sp. WBC-2 98

ATCC 17514 Pseudomonas putida OW-27 99 5Asal – 98

2 of Biological Macromolecules 74 (2015) 202–210

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Table 2Typical characteristics of synthetic and acidified OMW medium used for PHAsproduction.

Parameters Synthetic OMW

TSS (g/L) – –VSS (g/L) – –pH 6.90 ± 0.10 6.80 ± 0.20Acetate (g/L) 0.80 ± 0.00 0.90 ± 0.05Propionate (g/L) 0.80 ± 0.00 0.80 ± 0.00Butyrate (g/L) 0.80 ± 0.00 0.80 ± 0.00Iso-butyrate (g/L) – 0.10 ± 0.02Valerate (g/L) – 0.10 ± 0.02Iso-valerate (g/L) – 0.10 ± 0.02Soluble carbohydrates (g/L) – 1.10 ± 0.10

+

04 C. Kourmentza et al. / International Journal

00 �L using sterile water and centrifuged at 2500 × g for 5 min in table top centrifuge. 4 �L of cleared lysates were used as templateor amplification. PCR was performed by adding, to the lysates, 1×CR Gold buffer (Applied Biosyetms, Germany), 1.5 mM L−1 MgCl2Applied Biosystems, Germany), 200 mM L−1 dNTPs (Roche Molec-lar Biochemicals, Germany), 20 pmol of each primer, and 1 U ofmpli-Taq Gold polymerase (Applied Biosystems, Germany). Thenal volume of the reaction tubes was adjusted to 50 �L. Reactionsere run in a Perkin Elmer GeneAmp PCR system 2400 (Perkin

lmer, Norwalk, USA). The temperature profile was the one pre-iously described by Vinuesa et al. [18], except for the extensionf the initial denaturation step to 7 min, as required by Ampli-Taqold polymerase. Amplification products were run on 1% agaroseels and the bands were purified using the Quiaex II kit (Quia-en, Germany). The nucleotide sequence of the purified bandsas determined by the dideoxy chain-termination method, using

he ABI-PRISM Big Dye Terminator Cycle Sequencing Ready Reac-ion kit (Perkin Elmer, USA) and automated sequencer ABI PRISM100 Avant Genetic Analyzer (Applied Biosystems, Germany). Theequenced fragment analyzed corresponded to the first 650 bp ofhe 16S-rRNA gene, comprising hypervariable regions V1, V2 and3 [19].

DNA sequences were analyzed using biocomputing tools pro-ided on-line by the European Bioinformatics Institute [20]. TheLAST [21] program was used for preliminary sequence similaritynalysis. The Clustal X v.1.8 software [22] was used for sequencelignment, and generation of the distance matrix. Phylogenetic andolecular evolutionary analyses were conducted with MEGA ver-

ion 2.1 [23], using the Neighbor-Joining method and the bootstrapest [22,24].

.3. Feedstocks

A mixed culture derived from anaerobically digested sludge wassed for the acidification of a three-phase OMW. The acidificationrocess was performed in a mesophilic (35 ◦C) CSTR reactor with00 mL active volume operating at a hydraulic time (HRT) of 27 h.he reactor was fed with raw OMW that was previously diluted1:4) with tap water [8,25,26].

The acidified effluent of the mesophilic reactor was collectednd pretreated prior to its use as substrate for PHAs production. Inore detail, the acidified OMW was centrifuged and then filtered

n order to remove solid residues. The pH of the filtrated liquid washen adjusted to ∼7.0 by adding a phosphate buffer solution con-isting of 5 g/L K2HPO4 and 3 g/L KH2PO4. Besides pH adjustment,he particular buffer addition served also as phosphorus source.olutions were sterilized separately by autoclaving at 1 bar and21 ◦C for 20 min, mixed and fed to the reactors as PHAs productionedium.The synthetic wastewater was nitrogen-free and its compo-

ition is described in Table 2. The medium consisted also of.2 g/L MgSO4·7H2O, 0.0251 g/L CaCl2·2H2O, 5 g/L K2HPO4 and 3 g/LH2PO4 and 1.0 mL/L of a trace elements solution, prepared as men-

ioned previously.In both types of wastewater used in this study (i.e. synthetic

edium and acidified OMW), the precursors for the productionf PHAs were acetate, propionate and butyrate. The concentra-ions used in the synthetic one were selected to match those inhe acidified OMW in order to draw comparable conclusions fromhe bacterial growth in these two different liquid media.

.4. Experimental setup

Batch experiments were conducted in order to estimate theccumulation percentages, rates and yields of PHAs in the enrichedulture and all isolates. Both synthetic wastewater and acidified

N–NH4 (mg/L) – 1.20 ± 0.30

Values represent the mean of two replicates ± standard deviation.

OMW were used as substrates for PHAs production. Batch exper-iments were conducted at room temperature with continuous airsupply provided through sterile filters of 0.2 �m pore size, underoxygen sufficient (DO > 2 ppm) and oxygen limited conditions(DO < 2 ppm), and constant stirring at 250 rpm. Total suspendedsolids (TSS), cell dry mass (CDM), determined as VSS, Volatile FattyAcids (VFAs) concentration and pH were monitored versus time,while the PHAs content in biomass was estimated at the beginningand end of each experiment.

The isolated strains were grown in the basal medium describedabove, containing both nitrogen and carbon sources, under sterileconditions. The medium was thermally sterilized for 20 min at 1 barand 121 ◦C and air supply was provided to the cultures via sterilefilters with 0.2 �m pore size. When nitrogen was fully consumedthe microbial biomass was left to settle and then used as inoculumin the batch experiments.

As for the ‘enriched’ culture, microbial biomass was collectedand used as inoculum at the end of the carbon limitation phase, i.e.growth of biomass by consuming intracellular PHAs and extracel-lular nitrogen source in the form of (NH4)2SO4.

2.5. Analytical methods

TSS, VSS, N–NH4+ and dissolved COD (d-COD) were deter-

mined according to Standard Methods [27]. For the quantificationof volatile fatty acids (acetate, propionate, butyrate) acidified sam-ples with H2SO4 (20% v/v) were analyzed on a Varian CP-3800gas chromatograph equipped with a flame ionization detector(FID) and a HP-FFAP (Agilent technologies INC. 30 m × 0.53 mmI.D. × 1.00 �m film) capillary column. For soluble carbohydratesdetermination, a colored sugar derivative was produced throughthe addition of l-tryptophan, sulphuric and boric acid which wassubsequently measured colorimetrically at 520 nm (MILTON ROY-Spectronic 601) [28].

For the extraction and purification of PHAs, cells were harvestedand centrifuged at 4000 × g for 20 min (DUPONT-Econospin),washed twice with distilled water and then lyophilized using aCryodos Telstar freeze-dryer unit. PHAs were extracted from thelyophilized cells using a Soxhlet apparatus (VELP SCIENTIFICA-SER148/6) in which chloroform was applied as solvent (50 mL solventfor each g of CDM) for 4 h at 100 ◦C. In order to remove cellu-lar debris, the chloroform solution after extraction was filteredvia Whatman® glass microfiber filters, Grade GF/F. The dissolvedin chloroform PHAs were afterwards purified by precipitationwith a tenfold volume quantity of ice-cold methanol. The precipi-tated polymer was separated by centrifugation at 4000 × g and the

formed chloroform–methanol mixture was decanted. The collectedPHAs were subsequently dried in vacuum to completely remove theresidual solvents until constant weight. The biomass PHA content

C. Kourmentza et al. / International Journal of Biological Macromolecules 74 (2015) 202–210 205

Table 3PHAs production characteristics using synthetic medium as carbon source.

Culture Incubation period YX/S qX YPHAs/S qPHAs % PHAs Reference

Nitrogen limited conditionsMixed 34 0.68 ± 0.02 53.8 ± 3.2 0.68 ± 0.02 53.8 ± 3.5 64.4 ± 2.0 This studyA 54 0.30 ± 0.02 14.9 ± 1.4 0.09 ± 0.01 4.4 ± 0.3 23.8 ± 1.1 [16]B 33 0.23 ± 0.01 17.5 ± 2.2 0.04 ± 0.00 3.2 ± 0.2 13.6 ± 0.7 [16]C 60 0.11 ± 0.01 6.1 ± 0.5 0.01 ± 0.00 0.6 ± 0.0 5.0 ± 0.3 This studyD 41 0.39 ± 0.02 30.4 ± 1.8 0.03 ± 0.00 2.1 ± 0.1 4.7 ± 0.3 This studyE 40 0.15 ± 0.01 12.2 ± 1.0 0.04 ± 0.00 2.9 ± 0.2 11.5 ± 0.6 This studyF 27 0.18 ± 0.01 26.5 ± 1.6 0.02 ± 0.00 2.0 ± 0.1 5.1 ± 0.4 This study

Nitrogen and oxygen limited conditionsMixed 43 0.46 ± 0.02 11.0 ± 0.9 0.18 ± 0.02 4.3 ± 0.2 18.2 ± 0.8 This studyA 305 0.08 ± 0.00 0.7 ± 0.0 0.08 ± 0.00 0.7 ± 0.0 50.6 ± 1.9 [16]B 100 0.21 ± 0.02 4.5 ± 0.3 0.03 ± 0.00 0.7 ± 0.0 11.2 ± 0.6 [16]C 76 0.10 ± 0.01 3.8 ± 0.3 0.01 ± 0.00 0.3 ± 0.0 3.5 ± 0.3 This studyD 65 0.14 ± 0.01 7.0 ± 0.6 0.01 ± 0.00 0.5 ± 0.0 3.3 ± 0.2 This studyE 56 0.11 ± 0.01 6.5 ± 0.4 0.04 ± 0.00 2.3 ± 0.1 13.8 ± 0.5 This studyF 38 0.09 ± 0.00 8.7 ± 0.6 0.01 ± 0.00 0.8 ± 0.0 4.6 ± 0.2 This study

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ncubation period [=] hours; YX/S [=] g VSS/g VFAs; qX [=] mg VSS L−1 h−1; YPHAs/S [=]

ata represent the average of two fermentations ± standard deviation.

as thus determined as the percentage of PHA in the cell dry massCDM) [29].

The monomeric composition of the produced PHAs was iden-ified by measuring its methyl-ester derivatives using a VarianP-3800 gas chromatograph equipped with a flame ioniza-ion detector (FID) and a DB-5MS (Agilent technologies INC.0 m × 0.25 mm I.D. × 0.25 �m film) capillary column. Samples andtandards, for constructing the calibration curves, were preparedccording to the method described by Oehmen et al. [30]. Thereeze-dried biomass was weighted and placed into pyrex screw-apped tubes, with PTFE liner. 2 mL of chloroform and 2 mL ofcidified methanol (3% v/v sulphuric acid, 2.5 g/L methyl benzoate)ere added. Methyl benzoate was used as an internal standard.

he samples were kept at 100 ◦C for 4 h and then left to cool atoom temperature. 1 mL of distilled water was added in the sam-les which were then vigorously shaken for 1 min. After achievinghase separation, 1 �L of the organic phase was injected into theas chromatograph, under split injection mode with a split ratio0:1. Helium was used as the carrier gas while N2 was used as theake-up gas. The gas flow and the inlet temperature were set at

.5 mL/min and 250 ◦C respectively, while the detector tempera-ure was at 300 ◦C. The column temperature was set at 60 ◦C for

min and then gradually increased up to 160 ◦C by 15 ◦C/min.

. Results and discussion

.1. Synthetic medium as carbon source

.1.1. Nitrogen limitationThe results of the PHAs production process using both the

nriched culture as well as the bacterial strains isolated from it,hen synthetic medium was used, under conditions of nitrogen

nd dual nitrogen–oxygen limitation are summarized in Table 3.Under nitrogen-limited conditions the enriched culture exhib-

ted the best performance compared to all isolates, as far asHAs yield, rate and accumulation percentage are concerned.he intracellular accumulation in PHAs reached 64.4% and washaracterized by a high YPHAs/S yield, i.e. 0.68 g PHAs/g VFAsnd the higher production rate qPHAs = 53.8 mg L−1 h−1. Then, byar, strain A follows. The accumulation percentage in this caseas nearly three times less, 23.8% g PHAs/g CDM, while both

he yield, YPHAs/S = 0.09 g PHAs/g VFAs and the production rate,PHAs = 4.4 mg L−1 h−1, significantly lower. In overall, it appearshat the isolated microorganisms showed lower PHA accumula-ion capacities, ranging from 4.7–23.8% g PHAs/g CDM. This fact

s/g VFAs; qPHAs [=] mg PHAs L−1 h−1; % PHAs [=] (g PHAs/g VSS) × 100.

may indicate synergism among bacteria that comprise the enrichedculture. According to Rittmann and McCarty, mixed culturesmicroorganisms are characterized by synergistic interactions inwhich all microorganisms benefit. Microorganisms that live intightly coupled synergistic relationships may never be isolated [31].

Although there is limited literature on the production of PHAsby strains of the genus Pseudomonas and Pseudomonas putida usinga mixture of short-chain volatile fatty acids it has been reportedthat those microorganisms are able to produce high amounts ofPHAs when longer chain fatty acids are utilized. For example, whenthe strain P. putida BM01 utilized octanoic acid it produced up to79.8% g PHAs/g CDM, in the absence of nitrogen source [32]. StrainP. putida CA-3 has been reported to produce PHAs using butyricacid under non-limiting conditions and PHAs accumulation capac-ity in this case reached up to 15 ± 1% g PHAs/g CDM, while the yieldcoefficient YPHAs/S was equal to 0.10 g PHAs/g butyric acid [33]. Inthe same study, alkanoic acids ranging from 5 to 10 carbon atomswere investigated as carbon substrates and the most efficient onesappeared to be heptanoic and octanoic acid. The intracellular PHAscapacities reached 59 ± 4% g PHAs/g CDM in both cases, while theiryield coefficients YPHAs/S were 0.64 g PHAs/g heptanoic acid and0.57 g PHAs/g octanoic acid.

According to Madison and Huisman [34] the biosynthetic mech-anism of microorganisms belonging to the genus of Pseudomonas ismore active toward monomers that consist of 8–10 carbon atoms.For this reason, the most common carbon sources mentioned inthe literature, in addition to glucose and other carbohydrates, arealkanoic acids with 8–12 carbon atoms length as well as oleic acid.

It is expected that under conditions of absence of a nitrogensource any gravimetric biomass increase would correspond to thestorage of intracellular products and not to bacterial growth. Asshown in Fig. 1, in the case of the enriched culture, the biomassincrease (�X) is entirely attributed to PHAs formation. Also, inTable 3, the yield coefficient YX/S equals the yield coefficient YPHAs/S,as far as the enriched culture is concerned. On the other hand,in all cases of isolated strains differences were observed betweenthe concentrations of �X and PHAs as well as the values of thosetwo yield coefficients (YX/S versus YPHAs/S). This is an indicationthat, apart from PHAs, accumulation of other intracellular macro-molecules may be taking place under the conditions followed inthis study.

According to the literature, bacteria are able to produce otherintracellular products as well. Glycogen, PHAs and lipids in theform of Single Cell Oils (SCOs) and Poly-Unsaturated Fatty Acids(PUFAs), can be regarded as potential reserve materials due to their

206 C. Kourmentza et al. / International Journal of Biological Macromolecules 74 (2015) 202–210

F under( on.

fgouae[

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ig. 1. Biomass increase (�X) and PHAs concentrations using (a) synthetic mediumc) OMW under nitrogen limitation, (d) OMW under dual nitrogen–oxygen limitati

ollowing properties: (a) their accumulation is observed when cellrowth is restricted due to nitrogen limitation, while there is excessf carbon source, (b) the bacteria that accumulate those productsse them for maintenance when medium nutrients are exhausted,nd (c) those carbon storages can be used for the synthesis of nec-ssary cellular components under conditions favorable for growth35].

As has been recently discovered, the strain Pseudomonaspp. RRL-28 has the ability to accumulate intracellular lipidsSCOs/PUFAs) up to 42.7% per cell dry weight using glucose underitrogen-limited conditions (ratio C/N = 50), which were proveno promote lipid formation [36]. The particular microorganism isne of the few Gram-negative bacteria characterized as oleagi-ous as Acinetobacter sp. 211, Alkanivorax borkumensis SK2 andseudomonas aeruginosa 44T1 [37]. Acetyl-CoA is the key enzymend precursor for both PHAs and SCOs/PUFAs biosynthesis [38]. Tohere biosynthesis will be driven, i.e. PHAs or SCOs, depends on the

train used and the culture conditions. Zevenhuizen and Ebbink35] are the only ones to date having studied the interrelationsetween glycogen, PHAs (PHB) and lipids accumulation in Pseu-omonas V-19 using sodium acetate and sodium hydroxybutyrates carbon sources. They observed that the profile of intracellular

roducts accumulated in the case of sodium acetate was 1.3% glyco-en, 1.3% PHB and lipids 20.4% g/g dry cell weight, while in thease of sodium hydroxybutyrate 9.45% glycogen, 33.4% PHB and4% lipids were produced.

nitrogen limitation, (b) synthetic medium under dual nitrogen–oxygen limitation,

3.1.2. Dual nitrogen–oxygen limitationLow concentrations of dissolved oxygen (DO) have been

reported to suppress the formation of proteins that are necessaryfor microbial growth [39]. Under those conditions microorganismsare able to consume organic substrates by supplying energy viaoxidative degradation of these organic substrates. If oxygen sup-ply is sufficient, the microorganisms are able to obtain sufficientenergy for the production of proteins, glycogen and other cellularcomponents with simultaneous consumption of organic substrates[40].

In the present study, the effect of oxygen limitation(DO < 2 ppm), along with nitrogen absence in the medium, wasexamined in the production process of PHAs. It has been reportedthat in some strains such as Azotobacter beijerinckii, limitation ofoxygen appears to significantly increase the production of PHB [41].

According to our findings, it seems that when dualnitrogen–oxygen limitation was applied, lower organic sub-strate consumption rates were obtained leading thus to lowerrates of PHA biosynthesis as batch duration was extended in time(Figs. 2b and 3b). This phenomenon has also been observed byLefebvre et al. [42] and the strain Alcaligenes eutrophus.

Most studies on the effect of dissolved oxygen concentration on

PHAs production have been conducted using the microorganismA. beijerinckii [41,43–45]. In biopolymers production acetyl-CoAplays an important role. It can be either oxidized via the TCA cycleor used for PHAs biosynthesis. The followed pathway of acetyl-CoA

C. Kourmentza et al. / International Journal of Biological Macromolecules 74 (2015) 202–210 207

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e(gpcit6

ig. 2. VFA consumption profiles obtained from the enriched culture. Concentratioedium under (a) nitrogen and (b) dual nitrogen–oxygen conditions and OMW un

onsumption depends on the environmental conditions and espe-ially on oxygen limitation because in that case the NADH/NADatio increases. The activities of citrate synthase and isocitrateehydrogenase are inhibited by NADH and in return acetyl-CoAannot participate to the same extent in the TCA cycle. Acetyl-CoAs then converted into acetoacetyl-CoA by 3-ketothiolase, which ishe first essential enzyme for the biosynthesis of PHAs.

Due to the importance of the effect of dissolved oxygen con-entration and the presence of the cell respiratory protectionechanism, the model that describes the effect of oxygen limi-

ation in the strains that belong to Azotobacter genus may not bepplicable for the biosynthesis of PHAs by other microorganisms.or example, the reduced oxygen concentration did not result inHAs accumulation in the strain Pseudomonas sp. K [46]. Culturesf the microorganism A. eutrophus H16 seemed however capable toccumulate PHB under limiting oxygen conditions, but were char-cterized by a lower rate of biosynthesis compared to other limitingonditions [47].

It therefore appears that the effect of oxygen is not completelylucidated and it does not have a commonly accepted explanationespecially on a biochemical basis) and varies depending on theenus a strain belongs to. According to the results obtained in theresent study, oxygen limitation severely affected the enriched

ulture, leading to lower PHAs production efficiencies and yields,.e. 18.2% g PHAs/g CDM and 0.18 g PHAs/g VFAs compared tohe respective ones obtained under nitrogen limiting conditions,4.4% g PHAs/g CDM and 0.68 g PHAs/g VFAs. Furthermore, the

acetic (�), propionic ( ), butyric ( ) acid and total VFAs ( ) using: synthetic nitrogen and (d) dual nitrogen–oxygen conditions.

additional limitation of oxygen led to the production of non-PHAintracellular products in the enriched culture, which was notobserved under conditions of sufficient aeration (DO > 2 ppm).

PHAs accumulation percentages (% g PHAs/g CDM) in strains B,C, D, E, F as well as the yield coefficients (YPHAs/S) were quite lowand similar to the respective ones in the case that oxygen sup-ply was sufficient. On the contrary, in strain A, oxygen limitationseemed to have a positive impact on the accumulation of PHAs asit increased significantly from 23.8 to 50.6% g PHAs/g CDM withoutthough having the same effect on PHAs yield, 0.09 and 0.08 g PHAs/gVFAs under nitrogen and dual nitrogen–oxygen limiting conditions,respectively.

3.2. Acidified olive mill wastewater as carbon source

3.2.1. Nitrogen limitationThe results of the PHAs production process using acidified OMW

under conditions of nitrogen and dual nitrogen–oxygen limita-tion are shown in Table 4. The use of OMW instead of a mixtureof synthetic VFAs as carbon source generally led to smaller PHAsaccumulation yields, rates and capacities [25].

Based on the results shown in Table 4, the enriched cul-ture exhibited the higher PHAs accumulation efficiency,

reaching 8.8% g PHAs/g CDM, the higher yield coefficientYPHAs/S = 0.07 g PHAs/g VFAs and the higher production rateqPHAs = 1.8 mg L−1 h−1 compared to the other cultures. Thoseresults are in agreement with the ones that had been obtained

208 C. Kourmentza et al. / International Journal of Biological Macromolecules 74 (2015) 202–210

Fig. 3. VFA consumption profiles obtained from strain A. Concentrations of acetic (�), propionic ( ), butyric ( ) acid and total VFAs ( ) using: synthetic medium under( and (d

ieip9u

TP

ID

a) nitrogen and (b) dual nitrogen–oxygen conditions and OMW under (c) nitrogen

n our previous study [8]. PHAs production efficiency of thenriched culture in batch mode is the same with the one obtained

n continuous mode using an SBR reactor. This proves that thearticular enriched culture can store, in maximum, approximately% g PHAs/g CDM using olive mill wastewater as carbon sourcender conditions of nitrogen limitation.

able 4HAs production characteristics using acidified OMW as carbon source.

Culture Incubation period YX/S qX

Nitrogen limited conditionsMixed 60 0.29 ± 0.01 3.8 ± 0.2

A 62 0.31 ± 0.02 12.9 ± 0.4

B 60 0.20 ± 0.01 12.3 ± 0.3

C 97 0.24 ± 0.01 6.3 ± 0.2

D 50 0.39 ± 0.02 19.6 ± 0.5

E 58 0.19 ± 0.01 6.0 ± 0.2

F 52 0.40 ± 0.03 21.0 ± 0.5

Nitrogen and oxygen limited conditionsMixed 70 0.35 ± 0.03 7.5 ± 0.2

A 328 0.19 ± 0.02 1.4 ± 0.0

B 110 0.17 ± 0.01 3.5 ± 0.1

C 127 0.31 ± 0.02 6.2 ± 0.1

D 77 0.23 ± 0.02 7.4 ± 0.2

E 74 0.09 ± 0.00 2.3 ± 0.1

F 69 0.21 ± 0.01 7.8 ± 0.2

ncubation period [=] hours; YX/S [=] g VSS/g VFAs; qX [=] mg VSS L−1 h−1; YPHAs/S [=] g PHAsata represent the average of two fermentations ± standard deviation.

) dual nitrogen–oxygen conditions.

However, these values were considerably smaller when OMWwas used for PHAs production compared to the synthetic medium.

PHAs production ranged from 0.9 to 6.2% g PHAs/g CDM in the iso-lates using OMW under nitrogen limited conditions. When OMWwas used the enriched culture seemed to accumulate non-PHAintracellular products, as can be concluded from the difference

YPHAs/S qPHAs % PHAs Reference

0.07 ± 0.00 1.8 ± 0.1 8.8 ± 0.3 This study0.04 ± 0.00 1.1 ± 0.1 6.2 ± 0.3 This study0.03 ± 0.00 1.2 ± 0.1 3.8 ± 0.2 This study0.01 ± 0.00 0.1 ± 0.0 1.2 ± 0.1 This study0.03 ± 0.00 1.4 ± 0.1 3.5 ± 0.1 This study0.02 ± 0.00 0.7 ± 0.0 3.4 ± 0.1 This study0.01 ± 0.00 0.3 ± 0.0 0.9 ± 0.0 This study

0.03 ± 0.00 0.7 ± 0.0 3.2 ± 0.2 This study0.13 ± 0.00 1.0 ± 0.1 11.5 ± 0.5 This study0.02 ± 0.00 0.8 ± 0.0 3.4 ± 0.2 This study0.01 ± 0.00 0.1 ± 0.0 1.3 ± 0.1 This study0.02 ± 0.00 0.6 ± 0.0 3.2 ± 0.2 This study0.02 ± 0.00 0.4 ± 0.0 3.1 ± 0.1 This study0.01 ± 0.00 0.1 ± 0.0 0.6 ± 0.0 This study

/g VFAs; qPHAs [=] mg PHAs l−1 h−1; % PHAs [=] (g PHAs/g VSS) × 100.

C. Kourmentza et al. / International Journal of Biological Macromolecules 74 (2015) 202–210 209

Table 5PHA monomeric composition of the examined cultures using synthetic and OMW medium under nitrogen and dual nitrogen–oxygen limitation.

Culture Medium Limitation PHA monomeric composition (mol%)

HB HV HHx

Enriched culture Synthetic N 81 ± 1 19 ± 1 –N, O2 77 ± 1 23 ± 1 –

OMW N 78 ± 1 22 ± 1 –N, O2 74 ± 1 26 ± 1 –

A Synthetic N 66 ± 1 23 ± 2 11 ± 1N, O2 71 ± 1 9 ± 1 20 ± 2

OMW N 70 ± 1 22 ± 2 8 ± 1N, O2 74 ± 1 7 ± 1 19 ± 2

B Synthetic N 71 ± 1 20 ± 1 9 ± 1N, O2 73 ± 1 10 ± 1 17 ± 1

OMW N 72 ± 1 18 ± 1 10 ± 1N, O2 75 ± 2 10 ± 1 15 ± 2

C Synthetic N 69 ± 1 19 ± 2 12 ± 1N, O2 75 ± 2 7 ± 1 18 ± 1

OMW N 64 ± 1 22 ± 1 14 ± 1N, O2 72 ± 1 6 ± 1 22 ± 1

D Synthetic N 69 ± 1 18 ± 2 13 ± 1N, O2 71 ± 1 9 ± 1 20 ± 2

OMW N 69 ± 2 21 ± 1 10 ± 1N, O2 74 ± 2 8 ± 1 18 ± 1

E Synthetic N 73 ± 2 15 ± 1 12 ± 1N, O2 73 ± 2 4 ± 1 23 ± 2

OMW N 74 ± 2 16 ± 1 10 ± 1N, O2 76 ± 2 5 ± 1 19 ± 2

F Synthetic N 72 ± 1 18 ± 2 10 ± 1N, O2 75 ± 2 5 ± 1 20 ± 1

OMW N 70 ± 1 17 ± 2 13 ± 1

D

bn

Kabdgwr

Ksduewioa

cpwaeMV

3

i

N, O2

ata represent the average of two fermentations ± standard deviation.

etween the YX/S and YPHAs/S yield coefficients. Because of the oilyature of the waste, accumulation of lipids seems possible.

According to Ribera et al. [48], who examined the strain P. putidaT2442, it was observed that the particular microorganism wasble to accumulate 1.52% g PHAs/g CDM while the rate of PHAsiosynthesis was qPHAs = 0.56 mg L−1 h−1. This study is the only oneealing with the production of PHAs by bacteria of the Pseudomonasenus from olive mill wastewater (alpechin). On the other hand,hen the same strain was grown in oleic acid PHAs accumulation

eached 23% g PHAs/g CDM [49].It has been reported that in the case of recombinant P. putida

T2442 PHAs content reached up to 65% g PHAs/g CDM whenodium octanoate was provided as carbon source but PHAs contentropped to ∼3.5% g PHAs/g CDM when olive mill wastewater wassed [50]. The results of the present study are similar to those of Rib-ra et al. [48] although different strains of the Pseudomonas genusere examined. Low PHAs production efficiency in the case of acid-

fied OMW could be attributed to the complexity of the wastewaterr to inhibitory effect of ‘toxic’ compounds present in OMW, suchs polyphenols.

The complex nature of OMW which was used as carbon sourceaused differences on the production process parameters com-ared to the synthetic medium. However, this is not in agreementith the findings of Dionisi et al. [6]. When acidified OMW was fed

gainst synthetic medium to an enriched mixed culture, no differ-nce in the PHAs yield coefficient (0.54 g PHAs/g VSS) was observed.oreover, the authors concluded that the culture, apart from the

FAs, consumed other carbon substrates, such as alcohols.

.2.2. Dual nitrogen–oxygen limitationThe behavior of all cultures was identical with the one observed

n the case of synthetic medium when they were facing dual

73 ± 2 5 ± 1 22 ± 2

nitrogen–oxygen limitation. When the enriched culture was usedPHAs production decreased from 8.8% to 3.2% g PHAs/g CDMwhereas, strains B, C, D, E and F showed no significant differenceon the PHAs accumulation percentages, yields and rates. StrainA seemed to respond positively to the low oxygen concentrationaccumulating 11.5% versus 6.2% g PHAs/g CDM under conditionsof sufficient oxygen supply. In strain A the yield coefficient YPHAs/Ssignificantly increased probably due to the preference of themicroorganism to accumulate PHAs against other intracellularmacromolecules under those conditions. This behavior is inaccordance with the one observed under dual nitrogen–oxygenlimitation using synthetic wastewater as substrate.

Despite the fact that strain A showed the higher PHAs contentin this case, production of PHAs using the enriched culture seemedbeneficial due to shorter batch duration. The enriched culture accu-mulated 3.2% g PHAs/g CDM, while stain A accumulated almost athree-fold amount, 11.5% g PHAs/g CDM, but in 328 h.

To our knowledge, there is no report to date that deals with theproduction of PHAs from OMW under simultaneous nitrogen andoxygen limitation.

3.3. Monomeric composition of polyhydroxyalkanoates

The monomeric composition of the PHAs produced, by theenriched culture as well as the isolated strains, using synthetic andolive mill wastewater under nitrogen and dual nitrogen–oxygenlimitation is shown in Table 5.

In general, mixed cultures derived from activated sludge are able

to produce PHAs that, apart from HB and HV units, may also con-tain 3-hydroxy-2-methylvalerate (3H2MV), which can be detectedby GC-FID as HHx, which is its isomer. PHAs that contain 3H2MVunits have never been found in any bacterial pure cultures [51].

2 of Biol

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10 C. Kourmentza et al. / International Journal

ccording to the results the enriched culture produced a copolymeronsisting of HB and HV fractions while in all isolates hydroxy-exanoate (HHx), was also present. According to the consumptionreference profile of the enriched culture, as depicted in Fig. 2,utyrate was consumed first, resulting in the production of HB, fol-

owed by propionate and acetate, which could form both HB and HVnits. In the case of the isolated strains acetate was consumed first,ollowed by propionate and butyrate. In Fig. 3 the VFAs consump-ion profiles of strain A are shown as an example, for each case,hich is representative to the behavior of all isolated strains. It

eems that the combined consumption of propionate and butyrateavors the production of HHx units.

Under conditions of dual nitrogen–oxygen limitation the HB andV fractions were slightly differentiated. In the case of the isolates

t seems that HB fraction remains almost the same but HV fractionsecrease while HHx fractions increase. This could be attributed tohe simultaneous consumption of propionate and butyrate underhose conditions as it can be seen in Fig. 3b and d.

. Conclusions

Compared to isolates, the enriched culture exhibited the highestHAs accumulation efficiency indicating the presence of a PHA-verproducer which, however, was not isolated. The use of mixedultures for PHAs production is considered promising, since wasteaterial could be fermented under non-aseptic conditions lead-

ng to significant cost reduction. Oxygen limitation lengthenedatch duration and affected the PHAs biosynthesis rate, yields andccumulation capacities in all cultures studied. PHAs productionfficiency in batch mode is in agreement with the one obtained inontinuous mode [8], indicating that the maximum PHAs capacityf the specific enriched culture can reach up to ∼9% g PHAs/g CDM,hen OMW is used as carbon source.

cknowledgments

The authors wish to thank the European Union, ProjectPOLYVER COOP-CT-2006-032967′ and the Research Committee ofhe University of Patras, Program ‘Constantin Carathéodory No.144′, for funding the above study, as well as the Environmentalicrobiology Research Group of the Institute of Water Research,niversity of Granada, for contributing to the identification of theicrobial consortium.

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