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Anaerobic acidogenic digestion of olive mill wastewatersin biofilm reactors packed with ceramic filters or granularactivated carbon

Lorenzo Bertin a,*, Silvia Lampis b, Daniela Todaro c, Alberto Scoma a, Giovanni Vallini b,Leonardo Marchetti a,c, Mauro Majone d, Fabio Fava a

aDepartment of Applied Chemistry and Material Science (DICASM), Faculty of Engineering, University of Bologna, via Terracini 28,

40131 Bologna, ItalybDepartment of Biotechnology, University of Verona, Strada Le Grazie 15 e Ca’ Vignal, I-37134 Verona, Italyc INCA e Interuniversitary Consortium “Chemistry for the Environment”, via delle Industrie 21/8, I-30175 Marghera (VE), ItalydDepartment of Chemistry, Sapienza University of Rome, Piazzale Aldo Moro 5, I-00185 Rome, Italy

a r t i c l e i n f o

Article history:

Received 15 April 2010

Received in revised form

4 June 2010

Accepted 11 June 2010

Available online 18 June 2010

Keywords:

Olive mill wastewaters

Acidogenesis

Packed bed biofilm reactors

Vukopor S10 ceramic cubes

Polyhydroxyalkanoates

Microbial speciation

* Corresponding author. Tel.: þ39 051 209031E-mail addresses: lorenzo.bertin@unibo.i

unibo.it (A. Scoma), giovanni.vallini@univr(M. Majone), fabio.fava@unibo.it (F. Fava).0043-1354/$ e see front matter ª 2010 Elsevdoi:10.1016/j.watres.2010.06.025

a b s t r a c t

Four identically configured anaerobic packed bed biofilm reactors were developed and

employed in the continuous acidogenic digestion of olive mill wastewaters to produce

volatile fatty acids (VFAs), which can be exploited in the biotechnological production of

polyhydroxyalkanoates. Ceramic porous cubes or granular activated carbon were used as

biofilm supports. Aside packing material, the role of temperature and organic loading rate

(OLR) on VFA production yield and mixture composition were also studied. The process

was monitored through a chemical, microbiological and molecular biology integrated

procedure. The highest wastewater acidification yield was achieved with the ceramic-

based technology at 25 �C, with an inlet COD and an OLR of about 17 g/L and 13 g/L/day,

respectively. Under these conditions, about the 66% of the influent COD (not including its

VFA content) was converted into VFAs, whose final amount represented more than 82% of

the influent COD. In particular, acetic, propionic and butyric acids were the main VFAs by

composing the 55.7, 21.5 and 14.4%, respectively, of the whole VFA mixture. Importantly,

the relative concentrations of acetate and propionate were affected by the OLR parameter.

The nature of the packing material remarkable influenced the process performances, by

greatly affecting the biofilm bacterial community structure. In particular, ceramic cubes

favoured the immobilization of Firmicutes of the genera Bacillus, Paenibacillus and Clos-

tridium, which were probably involved in the VFA producing process.

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction similar to those of some petroleum-derived plastics [e.g.

Polyhydroxyalkanoates (PHAs) are promising microbial

biopolymers,mainly because: a) they showphysical properties

7; fax: þ39 051 2090322.t (L. Bertin), silvia.lampis@.it (G. Vallini), leonardo.

ier Ltd. All rights reserved

the copolymer poly(3-hydroxybutyrate/3-hydroxyvalerate)

[P3-(HB/HV)] can replace polypropylene in a wide range of

applications (Lee, 1996)]; b) they can be produced by means of

univr.it (S. Lampis), dan.t@libero.it (D. Todaro), alberto.scoma2@marchetti@unibo.it (M. Marchetti), mauro.majone@uniroma1.it

.

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 94538

renewable resources (Serafim et al., 2008); c) they are

completely biodegradable (Braunegg et al., 1998) and biocom-

patible (Mochizuki, 2002). To date, industrial PHA productions

have taken advantage only of pure bacterial cultures andwell-

defined synthetic media (Reddy et al., 2003; Khanna and

Srivastava, 2005; Philip et al., 2007). PHA production

processes carried out under such well-defined conditions can

provide excellent results, but are not economically competi-

tivewith those of petroleum-derived plastics (Noda et al., 2005;

Philip et al., 2007). Thus, the exploitation of alternative low-

cost feeding stocks (e.g. organic wastes) and mixed bacterial

consortia (i.e. highly biodiverse microbial populations selec-

tively enriched for the ability of producing PHA) is of great

relevance to get an economically feasible PHA production

process. In this respect, many papers have dealt with PHAs

production by activated sludgemicroorganisms fromdifferent

waste (Reis et al., 2003; Chua et al., 2003; Rhu et al., 2003;

Dionisi et al., 2005a; Albuquerque et al., 2007; Salmiati et al.,

2007; Bengtsson et al., 2008a,b). In particular, Dionisi et al.

(2005a) proposed the exploitation of olive mill wastewaters

(OMWs) in a three-stage integrated anaerobic-aerobic PHA

producing process: in the first anaerobic stage, the organic

waste is fermented under acidogenic conditions to obtain an

effluent rich in volatile fatty acids (VFAs), which are suitable

substrates for the PHA biological synthesis occurring in the

following second and third aerobic steps. Although this

process has been already assessed for the performances of the

aerobic phases (Dionisi et al., 2004, 2005b, 2006), a little has

been done so far to optimize the acidogenic step. In this

respect, the employment of reactors capable of supporting

high flow rate feedings, such as Packed Bed Biofilm Reactors

(PBBRs) (Bertin et al., 2004), can allow to minimize the VFA-

consuming methanogenic activity, which is normally medi-

ated by bacteria with very low specific growth rates. Thus, we

recently developed a biofilm reactor packed with Ceramic

Cubes (CCs) with the aim of fermenting OMWs to generate

a VFA-rich effluent employed in the production of PHAs

(Beccari et al., 2009). However, no efforts to optimize the

process in terms of concentration and relative amounts of

produced VFAs were made in the previous study.

Given the key role played by the relative content of

carboxylic acids containing even or odd number of carbon

atoms on the properties of resulting PHAs (Bengtsson et al.,

2008b), this research was undertaken to evaluate the influ-

ence of some key process parameters, such as temperature

and Organic Loading Rate (OLR), on concentration and relative

amounts of produced VFAs by the recently developed OMW

acidogenic process (Beccari et al., 2009). Moreover, the role of

the packing material was also evaluated by developing

parallel reactors packed with Granular Activated Carbon

(GAC), a biomass carrier which was already employed in the

biomethanization of OMWs (Bertin et al., 2004), and by

determining the microbial speciation of the biofilms gener-

ated onto the surface of CC and GAC samples collected at the

end of the study.

To the very best of our knowledge, this is the first work in

which an immobilized cell-based acidogenic anaerobic diges-

tion of OMWswas studied in terms of the influence of itsmain

process parameters and assessed through an integrated

chemical,microbiologicalandmolecularbiologymethodology.

2. Materials and methods

2.1. Olive mill wastewaters

TwoOMWs, namedOMW1andOMW2,were kindly purchased

by the Sant’Agata d’Oneglia (Imperia, Italy) and Grassanese

(Matera, Italy) three phase olive mills, respectively, and

employed in the research. Their COD was about 25 and 35 g/L,

respectively, partially due to VFAs (about 10 and 12 gCOD/L,

respectively) and phenols (about 2 g/L in both OMWs). Total

and volatile suspended solids were 20 and 12 g/L, respectively,

in OMW1; 32 and 13 g/L, respectively, in OMW2. Their pH

values were 4.3 and 4.6, respectively.

2.2. Packed bed biofilm reactors

Four identically configured up-flow PBBRs were developed

following the approach reported elsewhere (Beccari et al.,

2009) and employed as described in Section 2.3. Each PBBR

consisted of a 2.5 L-hermetically closed glass column (diam-

eter: 80 mm; height: 450 mm) wrapped with a silicon tubing

serpentine continuously recycling thermostated water and

equipped with a recycle line. The recycling ratio, expressed as

the ratio between the recycled broth flow and the whole flow

entering the column, was about 0.97. The liquid and gas

effluents were collected in a tank, connected to a “Mariotte”

bottle through which the produced biogas volume was

determined. A pH probe (81-04 model, ATI Orion, Boston, MA)

was placed at the top of the bioreactor. Two of the reactors

were packed with Ceramic Cubes (CCs, Vukopor S10 product,

Lanik, Boskovice, CZ) whose dimensions, porosity and density

were 25 � 25 � 18 mm, 10 ppi and 2.38 g/mL, respectively (CC-

PBBRs), while the other two with Granular Activated Carbon

(GAC, CP4-60 product, Chemviron Carbon, Feluy, Belgium),

consisting of cylinders of about 3 mm diameter and 10 mm

length, whose density was 1.32 g/mL (GAC-PBBRs). As a result

of support addition, the reaction volumes of CC-PBBRs became

2.25 and 2.28 L (CC1 and CC2, respectively), while the ones of

GAC-PBBRs became 1.50 and 1.80 L (GAC1 and GAC2,

respectively).

2.3. Olive mill wastewater digestion experiments

Where not differently described, the reactor influent flows

consisted of OMWs diluted with an equal volume of tap water

and amended with urea (0.45 g/L) and with a 10 N NaOH

solution (added to correct their pH to 5.5).

One PBBR per group, i.e. CC1 and GAC1, where thermo-

stated at 55 �C, whereas the other two, i.e. CC2 and GAC2, at

35 �C. They were all filled anaerobically with amended OMW1,

which was previously inoculated at 10% v/v with the same

deoxygenated, high-density suspension of microbial biomass

employed to develop the process described in Beccari et al.

(2009), which did not harbor any detectable taxa belonging

to archaeal domain. The main detected bacteria occurring in

the inoculum were a strain belonging to the Flexibacter-Cyto-

phaga-Bacteroides group and a Syntrophus sp.. The reactor

medium was replaced with deoxygenated fresh amended

OMW1 for three successive two-weeks batch cycles. The

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 9 4539

reactors were then forced to operate under continuous mode.

Five experiments of about one-month long were carried out

with the aim of studying the effect of temperature and loading

conditions on process performances. Steady state conditions

were considered to be attained when VFAs, COD and phenols

concentrations along with produced biogas were appreciably

constant (with standard deviations generally not exceeding

15%) for at least a week (i.e. at least 4 times each process HRT).

Steady state was typically achieved within two weeks after

a new loading condition was set.

The main process working parameters are reported in

Table 1. The first experiment, through which information on

the influence of temperature on process performances was

attained, was carried out by feeding both thermophilic

(experiment No. 1a) and mesophilic (experiment No. 1b)

reactors with the amended OMW1. The four successive

experiments, through which the former information was

integrated and the influence of OLR was studied, were per-

formed with CC2 and GAC2 reactors, which were fed with the

amended OMW1 (experiments No. 2 and 3) or OMW2 (exper-

iments No. 4 and 5). Experiments No. 3, 4 and 5 were carried

out by thermostating the reactors at 25 �C. PBBRs were fed at

an OLR of about 9 (experiments No. 1 and 3) and 13 (experi-

ments No. 2 and 4) g/L/day. During the last experiment, OMW2

was not diluted with tap water, so that the influent COD was

doubled respect to the one of experiment No. 4 (Table 1).

Moreover, during the last experiment the pH of GAC2 inlet

flow was not modified by means of NaOH amendment.

Liquid samples (collected on daily bases) were filtered on

0.22 mm cellulose-nitrate filters and then analyzed for COD,

VFAand total phenols (parameters related to influent flows are

reported with the subscript suffix “IN”, e.g. CODIN, while the

ones related to the effluents are reported with the subscript

suffix“OUT”, e.g.CODOUT).VFAamountswereexpressedasgof

equivalentCOD (gCOD) bymeansof stoichiometric calculations.

The net COD conversion into VFAs (COD/VFA) percentage,

representing the ratio between the net VFA production and

the influent COD excluding its VFA fraction, was employed to

define the process efficiency and calculated as follows:

COD/VFA ¼ VFAOUT � VFAIN

CODIN � VFAIN� 100 (1)

The whole process yield was calculated as the percentage of

the ratio between the effluent VFA total amount and the

influent COD:

Table 1 e List of performed experiments and relatedmainworkreactor couples (CC1 and GAC1, experiment No. 1a; CC2 and G

Exp. OMW T (�C) CODIN (g/L) VFAI

1a OMW1 55 13.52 � 0.43 6.1

1b OMW1 35 13.52 � 0.43 6.1

2 OMW1 35 11.71 � 0.26 6.4

3 OMW1 25 11.55 � 0.32 5.8

4 OMW2 25 16.71 � 0.52 7.9

5 OMW2 25 36.64 � 0.74 12.

a CC2.

b GAC2.

Process yield ¼ VFAOUT

CODIN� 100 (2)

The concentration of the total immobilized biomass

occurring at the top, the middle and the bottom of CC2 and

GAC2 was quantified at the end of the study by collecting

samples of about 1 g of support from the bottom, the middle

and the top of the fixed-beds (50, 200 and 380 mm from the

column bottom, respectively) and by subjecting them to

protein analysis. The biofilms immobilized on the surface of

the same samples together with the last experiment influent

and effluent flows were microbiologically characterized by

means of DNA extraction as described below.

2.4. Total DNA extraction, PCR amplification anddenaturing gradient gel electrophoresis

Total DNA extraction fromCCandGAC sampleswas carried as

reported elsewhere (Beccari et al., 2009). The 16S rRNA-genes

wereamplifiedbyPCRusingTaqDNApolymerasewithprimers

targeting conserved domains. Bacterial 16S rRNA genes were

selectively amplified using F8/R11 primers (Beccari et al., 2009)

with the following thermocycling program: initial denatur-

ation at 94 �C for 2 min; 30 cycles of denaturation at 94 �C for

45 s, annealing at 50 �C for 30 s, and extension at 72 �C for

2.5 min; final extension at 72 �C for 5min. Afterwards a nested

PCR was performed as described in Beccari’s work (2009).

Conditions were as above, except for number of cycles, 35, the

annealing temperature, 57 �C, and extension time, 35 s.

For Archaea, primers A109-f (Grosskopf et al., 1998) and

1510-r (Lane, 1991) were used for nearly complete 16S rRNA

gene amplification. Afterwards a nested PCR was performed

on the hypervariable V2-V3 region using primers A109(T)-f

and 515-GC-r (Roest et al., 2005), with a GC-clamp. The first

archaeal PCR reaction was performed with the following

thermocycle program: initial denaturation at 94 �C for 5 min;

30 cycles of denaturation at 94 �C for 45 s, annealing at 52 �Cfor 30 s, extension at 72 �C for 1 min; and final extension at

72 �C for 5 min. The nested PCR was as above but with 35

cycles. All primers were purchased from Sigma-Genosys

(Milan, Italy). The PCR products were quantified using Low

DNA MassTM Ladder (Celbio, Italy) in a 2.0% agarose gel.

DGGE analyses were performed on amplicons obtained

both for bacterial V3 and archaeal V2-V3 regions following the

procedures reported in Beccari’s et al.’s work (2009). Repre-

sentative DGGE bands were excised and incubated for 4 h in

ing parameters. Experiment No.1 was carried out with bothAC2, experiment No. 1b).

N (gCOD/L) PhenolIN (g/L) OLR (g/L/day)

83 � 0.29 1.034 � 0.08 9.66

83 � 0.29 1.034 � 0.08 9.62

65 � 0.25 1.009 � 0.04 12.5

12 � 0.14 1.399 � 0.06 8.17

24 � 0.29 0.978 � 0.03 13.3

52 � 0.46 2.393 � 0.04 22.3a 44.4b

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 94540

50 mL of sterile water. A total of 12 and 21 bands from ampli-

cons obtained respectively for Archaea and Bacteria regions

were sequenced.

2.5. Cloning, sequencing, and phylogenetic analysis

DGGE bands containing DNA to be sequenced were re-ampli-

fied. PCR amplification was carried out as described before,

except for the use of non-GC-clamped primers. PCR products

were transformed in Escherichia coli Xl1blue using the pGEM-T

vector system according to the manufacturer’s instructions

(Promega, Italy), sequenced on both strands, and finally

searched for homology using the BLASTN database (Altschul

et al., 1997).

The sequences were initially aligned using the multiple

alignment program CLUSTAL_X 1.83 (Thompson et al., 1997).

A phylogenetic tree was constructed using the neighbour-

joining method with the MEGA version 4.0 software package

(Kumar et al., 2008). Bootstrap analysis was performed from

1000 bootstrap replications.

2.6. Analytical methods

VFA concentration was monitored through a HP GC-5890

(Agilent, Milano, Italy) equipped with a FID detector and

a Supelcowax-10 column (SigmaeAldrich, Milano, Italy) under

the following conditions: initial temperature 60 �C; isothermal

for 1 min; temperature rate 25 �C/min; final temperature

150 �C; isothermal for 6 min; temperature rate 4 �C/min; final

temperature 180 �C; temperature rate 25 �C/min; final

temperature 240 �C; injector and FID temperature 280 �C;carrier gas flow rate (nitrogen) 17.6 mL/min. Before the anal-

yses, the samples were diluted with an equal amount of

a 60 mM oxalic acid solution. VFAs concentrations are

expressed as g of COD equivalents/L (gCOD/L). COD and total

phenol concentrations were determined spectrophotometri-

cally according to the following methods: Hach Mn(III) (Miller

et al., 2001) and Folin-Ciocalteu (Folin and Ciocalteu, 1927),

respectively. Total phenols were determined by employing

4-hydroxybenzoate as the calibration standard. Biogas

amount and composition were daily determined as reported

elsewhere (Bertin et al., 2004). The concentration of the total

biomass occurring at the bottom, themiddle and the top of the

reactor packed beds was quantified according to the Lowry

method applied in previous studies (Bertin et al., 2004).

Table 2 e COD, VFA and phenol concentrations in PBBR effluenperformed experiments.

Exp. CC-PBBRs

CODOUT

(g/L)VFAOUT

(gCOD/L)CH4/CODDEP

a

(L/g)PhenolOUT

(g/L)

1a 10.93 � 0.37 6.138 � 0.11 0.197 � 0.02 0.774 � 0.10

1b 9.350 � 0.26 6.207 � 0.52 0.334 � 0.05 0.797 � 0.07

2 10.33 � 0.30 6.232 � 0.30 0.380 � 0.06 1.007 � 0.03

3 11.20 � 0.16 6.121 � 0.32 0.010 � 0.00 1.120 � 0.06

4 15.47 � 0.43 13.73 � 0.49 0.015 � 0.00 0.830 � 0.05

5 28.52 � 0.89 13.67 � 0.42 0.024 � 0.01 2.256 � 0.06

a Depleted COD.

3. Results

Significant amounts of VFAswere found to be generated in the

bioreactors only when the anaerobic treatment was per-

formed in CC2. The main results of the five successive

experiments performed with the CC- and GAC-PBBRs under

continuousmode of operation are summarized in Table 2. The

highest VFAs production was observed when CC2 was ther-

mostated at 25 �C and fed with an OLR of about 13 g/L/day

(experiment No. 4): under such conditions, the total VFAs

concentration in the effluent was 13.73 gCOD/L and it was

mainly due to acetic, propionic and butyric acids (representing

the 55.7, 21.5 and 14.4% of the whole detected VFAs, respec-

tively, Fig. 1a). The net COD conversion into VFAs was about

the 66%. VFAs total amount represented about the 89% of the

overall effluent COD (Table 3), while the process yield was

about 82% (Table 3). The pH of such a VFA-rich effluent was

5.13 � 0.04.

3.1. Effect of packing material on PBBRs performances

The effect of packing material on process performances was

studied throughout the first four experiments performed with

the parallel CC- and GAC-PBBR under the same temperature

and loading conditions (CC1 and GAC1, employed in the sole

experiment No. 1; CC2 and GAC2, Table 1). Higher VFA

concentrations were always attained in the CC-PBBRs than in

the GAC-PBBRs, where a large part of the influent VFA load

was conversely depleted (Table 2). Compatible pH values for

biological acidogenic processes were alwaysmeasured for CC-

PBBRs (5.23 averagely), while in GAC-PBBRs such a parameter

was in the range 6.43e6.97. High methane productions

occurred in the latter reactors, where in particular CH4

production yields close to the maximum theoretical ones (i.e.

0.35 L of CH4 produced per g of COD removed)were obtained in

the first two experiments (Table 2). At the same time, GAC-

PBBRs provided very high COD removals which ranged

between 58 and 86% of the initial COD. Conversely, COD

removals were very low (between 3 and 31%) in CC-PBBRs,

where a negligible methanogenic activity occurred during the

experiments carried out at 25 �C.Different mixtures of VFAs accumulated in the two packed

bed reactor types: acetic, propionic and butyric acids were the

main VFAs generally occurring in CC-PBBR effluents, whereas

ts along with CH4 production yields related to all the

GAC-PBBRs

CODOUT

(g/L)VFAOUT

(gCOD/L)CH4/CODDEP

a

(L/g)PhenolOUT

(g/L)

2.827 � 0.16 0.837 � 0.18 0.391 � 0.04 0.071 � 0.04

2.133 � 0.06 0.501 � 0.04 0.339 � 0.06 0.076 � 0.02

1.677 � 0.02 0.189 � 0.03 0.324 � 0.02 0.287 � 0.03

1.787 � 0.14 0.553 � 0.06 0.261 � 0.01 0.389 � 0.03

5.396 � 0.35 1.497 � 0.10 0.116 � 0.01 0.207 � 0.06

15.27 � 1.05 4.808 � 0.63 0.135 � 0.02 0.954 � 0.16

Acetic

Propionic

i-Butyric

Butyric

i-Valeric

ValericOthers

1a1b2

34

5

0

20

40

60

80

100

A

Acetic

Propionic

i-Butyric

Butyric

i-Valeric

ValericOthers

1a1b2

34

5

0

20

40

60

80

100

B

Fig. 1 e Single VFA relative amounts with respect to the

total VFA amounts detected in CC-PBBR (A) and GAC-PBBR

(B) effluents in all the performed experiments (expressed

by their identificative number).

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 9 4541

only the former two were prominent in GAC-PBBRs (Fig. 1),

where propionic acid concentration was averagely higher

than in CC-PBBRs (28 and 23.5%, respectively).

The two reactor systems also exhibited different ability to

remove the OMW phenols: such compounds tent to persist in

all CC-effluents while theywere depleted up tomore than 90%

within the GAC-PBBRs (Table 2).

3.2. Effect of temperature on PBBRs performances

The effect of temperature on VFAs and biogas production was

studied throughout experiments No. 1 (a and b) and 3, by

feeding each of the reactors with very similar OLRs and by

differently thermostatingeach reactor couple (CC1andGAC1at

Table 3 e Net COD conversions into VFAs, amount of VFAs withwith process yields related to all the performed experiments. A

Exp. CC-PBBRs

COD/VFA (VFA/COD)IN (VFA/COD)OUT Process yield

1a �0.61 45.7 56.2 45.4

1b 0.33 45.7 66.4 45.9

2 �4.44 55.2 60.3 53.2

3 5.38 50.3 54.6 53.0

4 66.1 47.4 88.7 82.2

5 4.77 34.2 47.9 37.3

55 �C; CC2 and GAC2 at 35 or 25 �C, Table 1). Concerning CC-

PBBRs, an accumulation of VFAs was observed in CC2 at 25 �C(experiment No. 3), while no appreciable differences among

VFA concentrations occurring in influent and effluent flows

were observed at both 55 �C (experiment No. 1a) and 35 �C(experiment No. 1b) (Tables 1 and 2). Accordingly, methano-

genesis was almost absent at 25 �C (Table 2). In addition, no

significant COD depletions were measured for CC2 under the

latter experimental conditions. At the opposite, high methane

productions along with high COD depletions were always

attained within GAC-PBBRs (Table 2). No effects related to

temperatureonVFAdistributionwereobserved inbothCC-and

GAC-systems.

3.3. Effect of organic loading rate on PBBRsperformances

The effect of OLR on process performances was studied

throughout two couples of experiments in which CC2 and

GAC2 were fed with increasing OLRs (about 9 and 13 g/L/day).

In particular, experiments No. 1b and 2 were carried out at

35 �C, while experimentsNo. 3 and 4 at 25 �C.When exposed to

the higher OLR, both packingmaterials gave rise to higher VFA

productions at 25 �C but lower at 35 �C, where high meth-

anogenic activities were observed (Table 2). Concerning the

effect on VFA mixture composition, the increase of OLR

generally caused the decrease of the relative abundance of

acetic acid and, at 25 �C, the increase of propionic acid in both

CC- and GAC-PBBRs (Fig. 1).

On the basis of the evidences observed within the first four

experiments, a fifth final experiment was carried out by

differently loading CC2 and GAC2. In particular, CC2 was fed

with a higher OLR (from 13.3 to 22.3 gCOD/L/day) by doubling

the incoming COD and lowering the flow rate: the aim of this

approach was to maintain the high net conversion yields

previously obtained by processing higher concentrated

wastewaters with a lower flow, this resulting in anaerobic

effluents with higher VFA concentrations. As concerns GAC2,

the last runwas aimed at limitingmethanogenesis and this by

strongly increasing the OLR (more than three times higher,

from 13.3 to 44.4 gCOD/L/day), by both increasing the flow rate

and the incoming COD concentration. Furthermore, to verify if

it was possible to operate with a lower pH with respect to the

one adopted in all the former experiments, GAC2was fed with

a pH which was not increased by NaOH amendment. A

marked decrease of VFAs production was observed in CC2,

respect to the COD in the influent and effluent flows alongll data are expressed as percentages.

GAC-PBBRs

COD/VFA VFAIN/CODIN VFAOUT/CODOUT Process yield

�72.9 45.7 29.6 6.19

�77.4 45.7 23.5 3.70

�120 55.2 11.3 1.61

�91.6 50.3 30.9 4.79

�73.1 47.4 27.7 8.96

�32.0 34.2 31.5 13.1

Fig. 2 e Top: DGGE fingerprints of the eubacterial communities. Samples collected from top, middle and bottom sections of

the reactors are marked with T, M and B, respectively. OMWIN refers to the inlet flow, CC2OUT and GAC2OUT to the effluents

discharged under steady state conditions. Roman numerals (I and II) correspond to different replicates. Letters indicate

bands that have been excised, cloned and sequenced. Bottom: Similarity dendrograms indicate relationships among the

different DGGE profiles.

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 94542

which probably suffered from the high-applied OLR. However,

in agreement with the above described trend, acetic and

propionic acid relative amounts further decreased and

increased, respectively. Concerning GAC2, methane produc-

tion yield was still comparable to the one observed in exper-

imentNo. 4 although the influent flowpHwas not increased to

5.5. However, the highest tested loading condition led to the

lowest COD consumption ever detected in such a reactor and

the VFAs concentration was correspondently the highest

obtained within GAC-PBBRs. Nevertheless, VFAs were yet

significantly consumed as a result of the methanogenesis

(Table 2).

3.4. Microbiological features of the biofilms

The total amount of immobilized biomass in CC2 and GAC2,

along with its microbial composition, was analyzed at the end

of the study, when the two reactors were sacrificed and

samples of both packing materials were collected at three

different heights of the reactor packed beds. The total

immobilized biomass (quantified as mg of dried biomass/g of

dried support) found in the reactor packed beds collected from

the bottom, the middle and the top of the reactors was 12.6,

15.2 and 18.1, respectively, for CC2, and 1.8, 3.5 and 2.6,

respectively, for the GAC2. Considering the dry weight of the

support initially introduced in the GAC- and CC-PBBR (739.8

and 1228.1 g, respectively), it was estimated that the total

immobilized biomass available in the CC- and GAC-PBBR was

11.31 and 3.215 g, respectively.

The composition of both Bacteria and Archae cenoses

enriched at the end of the study in CC2 and GAC2 reactors and

in their inlet and outlet flows was monitored through PCR-

DGGE analysis. All the evidences gathered for Bacteria in

relation to either packing matrices or influent and effluent

wastewaters indicate the presence of heterogeneous

communities, quite rich in bacterial biodiversity (Fig. 2,

Table 4 e Taxonomic characterization of themajor bands in the DGGE profiles obtained with primers for Eubacteria relatedto the biofilm samples collected from CC2 and GAC2 and to the last experiment influent flow (OMWIN) and effluentsdischarged under steady state conditions from CC2 (CC2OUT) and GAG2 (GAC2OUT). Different sampling positions along theprofile of the reactor are marked with T (top), M (middle), and B (bottom).

Sample Phylogenetic group TAXON Identity (%)

CC2

CC2T-a Firmicutes Bacillus sp. 100

CC2T-b Firmicutes Paenibacillus sp. 100

CC2M-c Firmicutes Uncultured Clostridium sp. 100

CC2M-d Firmicutes Clostridiaceae bacterium 99

CC2B-e Firmicutes Clostridium sp. 100

CC2M-f Firmicutes Pasteuriaceae bacterium 100

GAC2

GAC2T-h Uncultured bacterium clone 100

GAC2T-i Firmicutes Uncultured Clostridiales bacterium 98

GAC2T-l Uncultured Chloroflexi 98

GAC2T-m Actinobacteria Eggerthella sinensis 99

GAC2T-n b-Proteobacteria Comamonas sp. 2009I4 98

GAC2T-o b-Proteobacteria Uncultured Massilia sp. 98

GAC2M-p g-Proteobacteria Acinetobacter sp. 100

GAC2M-q g-Proteobacteria Acinetobacter baumannii 98

GAC2M-r a-Proteobacteria Uncultured Alphaproteobacteria 99

Flows

OMWIN-s Firmicutes Lactobacillus suebicus 100

OMWIN-t Firmicutes Lactobacillus camelliae 100

OMWIN-u a-Proteobacteria Acetobacter pasteurianus 99

OMWIN-v a-Proteobacteria Acetobacter sp. 100

OMWIN-z Firmicutes Uncultured Lactobacillus sp. 98

CC2OUT-j Uncultured bacterium isolate 97

CC2OUT-y Firmicutes Uncultured Clostridium sp. 100

GAC2OUT-x b-Proteobacteria Aquabacterium sp. 98

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 9 4543

Panel B). The dendrogram obtained through UPGMA method

shows low similarity values (<0.2) among the profiles corre-

sponding to samples collected within the differently packed

reactors. In fact, it is particularly worth noting a taxonomical

grouping in two distinct clusters according to samples drawn

from the two reactors. As far as the profile of the influent

OMW is concerned, a bacterial community composition closer

to that revealed in CC2 compared to the GAC-digester speci-

ated one was found, even if in the presence again of a low

similarity (<0.2). The effluents collected from both CC2 and

GAC2 presented a bacterial composition similar to those

observedwithin the reactors they came from. Nevertheless, in

the case of CC loaded reactor, the microbial cenosis structure

of the effluent shows a higher similarity (>0.8) to that accli-

mated in the corresponding digester with respect to what

observed between GAC loaded reactor and its effluent (simi-

larity value equal to 0.2).

Major bands in DGGE gels were excised, cloned and

sequenced. Results from sequencing (Table 4, Fig. 3) demon-

strated the presence of Lactobacillus sp. and Acetobacter sp. in

the OMW fed to the reactors. Meanwhile, the bacterial

community in CC2 has resulted mainly composed by Firmi-

cutes belonging to the genera Bacillus, Paenibacillus, and Clos-

tridium. On the other hand, GAC2 has revealed a prevailing

presence of Proteobacteria, distributed among alpha, beta and

gamma sub-classes. In particular, the prominent genera

found were Acinetobacter, Comamonas and Massilia. A selective

speciation towards bacterial strains belonging to the phyla of

Firmicutes and Proteobacteria was finally recorded in CC and

GAC effluents, respectively.

As far as the community structure of Archaea is concerned,

DGGE profiles neatly demonstrated distinct speciations inside

the two differently packed reactors as well as in the corre-

sponding effluents, with dominant, restricted microbial con-

sortia represented bymajor bandsmigrating in the gels (Fig. 4,

Panel A). Even in the case of Archaea, amarked diversity in the

composition of the communities within reactors packed with

CC or GAC filling materials was observed. The formation of

two different clusters corresponding to samples from the two

reactors was actually observed. Interestingly, the DGGE

profiles obtained for the inlet OMW have shown an archaeal

composition strongly close (similarity value equal to 0.7) to

that recorded for the CC filled reactor, opposite the situation

found in GAC-packed digester. No significant differences have

been detected between the archaeal populations in effluents

from both CC and GAC packed digesters and those acclimated

inside the respective reactor. Once again, the similarity was

much higher in the case of CC2.

Sequencing of major bands from DGGE gel (Table 5, Fig. 5)

has evidenced a dominance of the Methanobacterium genus

(Methanobacteria family) in the OMW fed to the reactors. The

presence of strains belonging to the genera Methanobacterium

and Methanobrevibacter was revealed in both CC2 and the

corresponding outlet wastewater. On the other hand, GAC

filled reactor resulted to be populated mostly by Meth-

anomicrobia such as Methanosarcina sp. and Methanocella sp..

Fig. 3 e Neighbour-joining tree based on the sequence of the hypervariable V3 region of the 16S rRNA gene, showing the

phylogenetic relationship of different microbial components (DGGE bands marked with bold letters) and related species

within the domain Eubacteria. Bootstrap values are shown for nodes that had >50% support in a bootstrap analysis of 1000

replicates. The scale bar indicates 0.02 substitutions per nucleotide position.

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 94544

4. Discussion

The utilization of pure cultures and synthetic media in PHA

production strongly contributes to the final high costs of the

biopolymer (Noda et al., 2005; Philip et al., 2007). The use of

cheap organic wastes as feedstock, along with the

employment of enriched PHA producing mixed consortia in

tailored biotechnological processes, can markedly lower such

costs. In this respect, the acidogenic anaerobic digestion of

a COD-richwaste (such as Olive Mill Wastewater, OMW) could

be employed to generate VFAs, which can be used to feed PHA

producing bacteria (Dionisi et al., 2004).

Fig. 4 e Top: DGGE fingerprints of the archaebacterial communities. Samples collected from top, middle and bottom sections

of the reactors are markedwith T, M and B, respectively. OMWIN refers to the inlet flow, CC2OUT and GAC2OUT to the effluents

discharged under steady state conditions. Roman numerals (I and II) correspond to different replicates. Letters indicate

bands that have been excised, cloned and sequenced. Bottom: Similarity dendrograms indicate relationships among the

different DGGE profiles.

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 9 4545

Freely suspended cell bioreactors were already employed

for producing VFAs from several organic waste and effluents,

including OMWs (Ntaikou et al., 2009). The use of Packed Bed

Biofilm Reactor (PBBR) filled with Ceramic Cubes (CCs) was

also recently proposed by us for the same purpose (Beccari

et al., 2009); in the present study, the same process was

flunked by a GAC-packed technology with identical configu-

ration and both were assessed by evaluating the influence of

temperature and Organic Loading Rate (OLR) on concentration

and relative amounts of produced VFAs.

Packing material played a crucial role in the biofilm bio-

logical activities, as also evidenced by other authors (Ince

et al., 1995; Tay and Show, 1999; Picanco et al., 2001; Yang

et al., 2004). In particular, the biofilm generated on CCs

always gave rise to higher VFA concentrations than those

obtained in the parallel GAC reactors where, on the contrary,

a remarkable methanogenic activity was observed. Moreover,

GAC-PBBRs gave rise to much higher COD depletions, with

potential additional adverse effects on PAH production due to

the influent COD subtraction. Thus, the most favourable

conditions for a subsequent PHA production were achieved

with the CC-based process, where methanogenesis was

negligible or absent at all at 25 �C and no significant COD

removals occurred (Table 2). In particular, the latter evidence

allowed process yields, which link VFA amounts to the

influent COD (equation (2)), closed to the ratios between VFA

and COD concentrations in the effluents (Table 3). Packing

material played also a paramount role in the removal of

polyphenols, which were significantly depleted only in GAC-

PBBRs (Table 2). Since they are known to inhibit mostly

methanogenic populations (Dionisi et al., 2005a), their

persistence in CC-systems may have contributed to their

Table 5 e Taxonomic characterization of themajor bands in the DGGE profiles obtained with primers for Archaea related tothe biofilm samples collected from CC2 and GAC2 and to the last experiment influent flow (OMWIN) and effluentsdischarged under steady state conditions from CC2 (CC2OUT) and GAG2 (GAC2OUT). Different sampling positions along theprofile of the reactor are marked with T (top), M (middle), and B (bottom).

Phylogenetic group Taxon Identity (%)

CC2

CC2B-a Methanobacteria Methanobrevibacter acididurans 99

CC2B-b Methanobacteria Methanobrevibacter sp. 99

GAC2

GAC2T-c Methanobacteria Methanobacterium bryantii 99

GAC2T-d Methanomicrobia Uncultured Methanosarcinales archaeon 99

GAC2T-e Methanomicrobia Methanocella paludicola 97

Flows

OMWIN-f Actinobacteria Atopobium rimae 96

OMWIN-g Methanobacteria Methanobacterium sp. 99

OMWIN-h Methanobacteria Methanobacterium bryantii 99

GAC2OUT-i Methanobacteria Methanobacterium beijingenses 99

GAC2OUT-l Actinobacteria Uncultured Coriobacteriaceae bacterium 97

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 94546

higher efficiency in OMW acidification. On the contrary,

temperature and OLR did not affect the removal of such

compounds.

In accordance with the lower methanogenic activities,

acidogenic fermentation in CC-PBBRs raised proportionally by

decreasing temperature (experiments No. 1a, 1b and 3) by

achieving the best performance at 25 �C (5.38% of net COD

conversion into VFA, Table 3). Aside temperature, another

parameter typically affecting methanogenesis is OLR (Ince

Fig. 5 e Neighbour-joining tree based on the sequence of the hyp

phylogenetic relationship of different microbial components (D

within the domain Archaebacteria. Bootstrap values are shown f

1000 replicates. The scale bar indicates 0.05 substitutions per nu

here used for Archaea efficaciously functioned even in amplifica

et al., 1995; Grover et al., 2001; Kennedy et al., 2006). Meth-

anogenesis generally decreased when higher OLRs were

applied (Table 2). However, temperature played a key role in

determining to which extent this occurred, being at 25 �Cobtained the best results in term of methanogenesis reduc-

tion: once again, enhancements achieved with CCs support

were much greater than with GAC: the net COD conversion

into VFAs was one order of magnitude higher than what

previously obtained (up to about 66%), leading to a whole

ervariable V2-V3 region of the 16S rRNA gene, showing the

GGE bands marked with bold letters) and related species

or nodes that had >50% support in a bootstrap analysis of

cleotide position. It is worth noting that the specific primers

tion of 16S rRNA gene of Actinobacteria.

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 9 4547

process yield of about 82% (experiments No. 4, Table 3). Such

performances, which are the best among those obtained in

this study, were significantly better than those achieved

during the previous study performed with the same CC-based

technology and at the same temperature (25 �C) (Beccari et al.,2009), where the net COD conversion into VFAs and process

yield were about 13 and 29%, respectively. On the other hand,

when the OLR was further increased (experiment No. 5),

acidogenic fermentation was inhibited and the net COD

conversion into VFAs dropped back (Table 3). The high

concentration of toxic polyphenols present in the wastewater

could have contributed to this evidence (Kennedy et al., 2006)

together with the inability of CCs, differently from GAC, to

adsorb/desorb aromatic compounds, thus “buffering” such

toxic effects (Bertin et al., 2004). However, the results related

to the fifth experiment were closed to the ones achieved in the

previous study, which was carried out under similar loading

conditions (Beccari et al., 2009). Thus, even the previously

developed process was probably negatively influenced by

a high OLR.

The employment of the CC-PBBR technology allowed also

to obtain a higher OMW acidification with respect to that of

a conventional CSTR fed with different OLRs (Ntaikou et al.,

2009). In particular, the latter system higher process yield

was about 30%, due to an influent COD of 19.5 g/L fed at about

14 g/L/day and to a total effluent VFA amount of about

6 gCOD/L. Furthermore, the CC-based system performances

were higher than those of the study of Dionisi et al. (2005a),

where OMW was pre-treated by means of bentonite amend-

ment (even followed by centrifugation) and fermented under

batch conditions: in that case, comparable total VFA amounts

were achieved but with a high initial COD, this resulting

in lower process yields (higher process yield: 43.6%, corre-

sponding to an initial COD of 28.5 g/L and to a total final VFA

amount of 12.4 g/L).

The relative composition of the VFA mixture was mainly

affected by the parameter OLR: in particular, CC-biofilms

induced a decrease in the amount of acetate both at 35

(experiments No. 1b and 2) and 25 �C (experiments No. 3, 4 and

5) (Fig. 1a); within the experiments performed with CC2 at

25 �C, it was also reported a relative increase in the propionate

content while butyrate amount was maintained constant.

Thus, the OMW acidogenic digestion carried out in a CC-PBBR

operating at 25 �C (i.e. the best performing conditions) seems

to allow to control the acetateepropionate relative amounts

by regulating the applied OLR; however, the process optimi-

zation should take into consideration that the higher total VFA

production would not correspond to the higher propionate

production. On the contrary, lower hydraulic retention times

often led to a reduction of propionic acid, whose highest

production corresponded to the highest OMW acidification

(Ntaikou et al., 2009; Bengtsson et al., 2008b); however, the

opposite effect was also reported (Dinopoulou et al., 1988).

Themicrobiological investigations, regarding themicrobial

structure of both the biofilms within the two differently

packed bioreactors and the wastewaters, clearly indicated

a different speciation of either Bacteria and Archaea

depending on the two different support materials. This seems

to be in accordance with results concerning the performances

of the two bioreactors. In fact, the bacterial community that

has been selected within CC2 is exclusively composed by Fir-

micutes such as Clostridium, Bacillus, and strains belonging to

the Pasteuriaceae family. Actually, several bacterial species

belonging to these genera are known to show acidogenic

activity (Akao et al., 2007). In particular, some of the major

bands in the DGGE profiles (Fig. 2, bands c, d, e) revealed the

presence of strains closely related to C. tyrobutiricum as well as

Clostridium aminovalericum (Fig. 3). C. tyrobutiricum has been

shown to produce butyric and acetic acid as its major

fermentation products from glucose and xylose (Liu et al.,

2006). On the other hand, C. aminovalericum is capable to

anaerobically degrade 5-aminovalerate to valerate, acetate,

propionate, and ammonia (Barker et al., 1987).

Conversely, the bacterial community selected within the

GAC2 was mainly composed by Proteobacteria. Among the

different major bands in the DGGE profile, Acinetobacter is well

represented (Fig. 2, bands p, q). Nevertheless bacteria strains

belonging to this genus are common members of microbial

consortia involved in the biodegradation of biogenic and

xenobiotic compounds, and its high potential for the treat-

ment of phenol-containing wastewaters has been recently

elucidated (Liu et al., 2009).

As far as the Archaea speciation is concerned, the occur-

rence of species belonging mainly to the genus Methano-

brevibacter in the bioreactor packed with ceramic support is

consistent with the environmental conditions settled in such

digester. Striking VFA concentrations were in fact observed in

CC-PBBRs where acidic pHs (w5.0) might have favoured

methanogens to become well established, among those more

acidophilic (Savant et al., 2002; Rea et al., 2007). Moreover, as

Methanobrevibacter grows on a H2/CO2 gas mixture or, in

addition, can utilize formate, strict acidogenic conditions

such as those in CC-PBBRs are coherent with the activity of

possible consortia of incoming hydrogen-producing ace-

togens and this hydrogenotrophic methanogen (Fang, 2000).

Furthermore, the archaeal composition of the CC2 biofilm

was strongly closed to the one of the influent wastewater,

this suggesting that the immobilization of a large spectrum of

methanogenic consortia was not favoured in such a reactor.

The opposite situation found in GAC2, whereas a high

methanogenic activity was observed, seems to support the

latter hypothesis: the presence of mostly Methanomicrobia is

in agreement either with the pH conditions fluctuating nearly

around neutrality (w6.8), better conducive to the acclimation

of this kind of methanogens (Kendall and Boone, 2006), or

with the particular tendency of GAC to be colonized espe-

cially by Methanosarcinales (Schmidt and Ahring, 1999).

Interestingly, the sole member of Methanobacteria found in

GAC2, namely Methanobacterium bryantii, is a species which

shows an optimum pH range for growth between 6.9 and 7.2

(Boone, 1987). This picture fits well with recent findings on

bacterial speciation in GAC-PBBRs treating OMWs (Rizzi et al.,

2006).

5. Conclusions

An effective OMW acidification process was developed

through the employment of a PBBR technology which

employed porous ceramic cubes for supporting the biofilm

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 5 3 7e4 5 4 94548

generation. The highest COD conversion into VFAs was

observed when the reactor was thermostated at 25 �C and

loaded with an OLR of about 13 g/L/day, conditions under

which a total VFA concentration of about 14 g/L was ach-

ieved, this corresponding to about the 88 and the 82% of the

influent and effluent COD, respectively. The relative

amounts of the main VFAs can be controlled by regulating

the applied OLR, and this is of particular interest in the

perspective of feeding the acidogenic effluent to a biotech-

nological PHA producing process. The packing material

appeared as a crucial parameter able to influence the

process performances much more than temperature and

OLR, and this by greatly affecting the biofilm bacterial

community structure.

Acknowledgements

The authors thank the Olive mills Sant’Agata d’Oneglia

(Imperia, Italy) and Grassanese (Matera, Italy) for having

provided the OMWs. This research was financially supported

by the Italian Ministry of University and Research (PRIN 2005)

and partially by the Fondazione Del Monte di Bologna e Rav-

enna (Bologna, Italy).

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