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Effect of hydraulic retention time on biohydrogen and volatilefatty acids production during acidogenic digestion ofdephenolized olive mill wastewaters

Alberto Scoma, Lorenzo Bertin*, Fabio Fava

DICAM-Unit of Environmental Biotechnology and Biorefineries, Faculty of Engineering, University of Bologna, via Terracini 28, 40131

Bologna, Italy

a r t i c l e i n f o

Article history:

Received 21 February 2012

Received in revised form

8 August 2012

Accepted 23 October 2012

Available online 23 December 2012

Keywords:

Biological hydrogen production

VFA

Biofilm

Vukopor S10�

Acidogenic microbial consortium

Polyphenols

* Corresponding author. Tel.: þ39 051 209031E-mail addresses: alberto.scoma2@unibo

0961-9534/$ e see front matter ª 2012 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2012.10.

a b s t r a c t

The influence of Hydraulic Retention Time (HRT) on the performances of a recently

developed biotechnological anaerobic acidogenic process fed with dephenolized Olive Mill

Wastewater (OMW) was investigated. The study was carried out under mesophilic condi-

tions in Packed Bed Biofilm Reactors (PBBRs), filled with ceramic cubes and inoculated with

a characterized and acclimated acidogenic microbial consortium. The PBBRs were fed with

a HRT of 7, 5, 3 or 1 day, which corresponded to Organic Loading Rates (OLRs) of about 5.5,

7.8, 12.9 and 38.8 g L�1 d�1, respectively. A significant production of a H2-rich biogas was

observed when shorter HRTs were applied: in particular, H2 relative amount and produc-

tivity increased from 3% to 32% and from 0.20 to 6.10 dm3 m�3 h�1, respectively, by

decreasing the HRT from 7 to 1 day. On the contrary, shorter HRTs turned into a lower

accumulation of Volatile Fatty Acids (VFAs), whose highest amounts were found with HRTs

of 7 and 5 days (about 18.4 and 19.7 g L�1 COD equivalents, respectively). The highest

conversion yield of COD into VFAs (36%) was obtained with a HRT of 5 days, when VFAs

represented about 78% of the effluent COD. HRT also influenced the composition of the VFA

mixture: acetic, propionic and butyric acid were the most prominent VFAs, being their

relative amounts higher when PBBRs were operated with shorter HRTs (up to 19, 12 and

42% of the whole mixture, respectively, when HRT was 1 day).

ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The management of agroindustrial residues represents to

date a serious environmental and economic concern [1].

However, the possibility of employing such matrices as

renewable feedstock [2e4] has fostered the development of

the biorefinery concept as a sustainable approach to exten-

sively exploit some of these residues for production of

biomolecules [5e7], biofuels [8e10] and bio-based materials

[11].

7; fax: þ39 051 2090322..it (A. Scoma), lorenzo.berier Ltd. All rights reserved028

For instance, recent studies have shown that a number of

integrated processes can be applied to the case study of Olive

MillWastewaters (OMWs), allowing a wide exploitation of this

effluent for the sustainable recovery of natural antioxidants

such as polyphenols [12] and other natural compounds [13],

and for the production of biofuels (CH4 [14,15] or photo-

heterotrophically generated H2 [16]) and biopolymers such as

polyhydroxyalkanoates (PHAs) [17]. In this latter case, OMWs

need to be previously digested under acidogenic conditions,

since Volatile Fatty Acids (VFAs) are the major substrates for

tin@unibo.it (L. Bertin), fabio.fava@unibo.it (F. Fava)..

ofilm

Reactors

(PBBRs),a

ndofthepro

duce

deffl

uents

accord

ingto

tions(s.d.)are

alsoreported. P

roce

ssyields

sity

VFAOUT/C

OD

INVFAOUT/C

OD

OUT

COD

/VFA

COD

removed

s.d.

gcm

3gL�1COD

eq.

gL�1COD

eq.

%%

0.022

ee

ee

0.023

47.4

61.2

31.7

22.6

0.044

50.7

77.8

36.0

34.8

0.017

41.9

60.4

24.6

30.5

0.004

30.8

39.7

10.1

22.5

b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 5 1e5 852

PHA-accumulating bacteria [18]. In this respect, a dedicated

continuous Packed Bed Biofilm Reactor (PBBR) performing

OMW organic matter bioconversion into VFAs was recently

developed [19]. According to the biorefinery approach

mentioned above, the feasibility of a preliminary recovery of

the polyphenolic fraction of OMWs before their submission to

acidogenic digestion was recently demonstrated [20]. Inter-

estingly enough, in that case, production of some H2 gas was

observed together with accumulation of VFAs.

All this considered, the aim of the present work was to

study the influence of Hydraulic Retention Time (HRT) on the

developed acidogenic process fed with a dephenolized OMW,

in the perspective of inducing the production of a H2-rich

biogas in concomitance with the acidification of the effluent.

Furthermore, the robustness of the most performing process

was assessed by operating the reactor for a considerably

longer experimental period with respect to the applied HRT.

The effectiveness of the acidogenic inoculum, whichwas fully

microbiologically characterized in a previous study [20], was

also evaluated by comparing the performances of acidogenic

digestion processes carried out in the presence and absence of

both the inoculum and the active OMW indigenous

microflora.

Table

1eChem

icalfeatu

resofthedephenolize

dolivem

illw

astewater(O

MW

deph)fedto

thePack

edBedBi

theappliedHydra

ulicRetentionTim

e(H

RT,days),alongwithth

erelatedpro

cess

yields.

Standard

devia

Pro

cess

effl

uents

VFAs

COD

pH

Den

mean

s.d.

mea

ns.d.

mea

ns.d.

mea

n

gL�1COD

eq.

gL�1COD

eq.

gL�1COD

eq.

gL�1COD

eq.

pH

value

pH

value

gcm

3

Feed

OMW

deph

8.92

1.42

38.79

1.60

4.43

0.09

0.985

PBBRs

HRT7

18.38

2.40

30.02

1.50

5.99

0.29

0.983

HRT5

19.67

3.68

25.28

3.14

5.94

0.11

0.986

HRT3

16.27

0.53

26.94

1.13

5.79

0.10

0.978

HRT1

11.93

2.78

30.08

0.76

5.04

0.10

0.994

2. Materials and methods

2.1. Dephenolized olive mill wastewater

The OMW employed in the present study was provided by the

Sant’Agata d’Oneglia (Imperia, Italy) three phase olive mill

during the olive oil production campaign of 2010/11. Poly-

phenols occurring in the OMW were removed according to

a solid phase extraction procedure previously developed [20].

The employed actual site OMW had the following character-

istics: COD, 51.66 � 1.97 g L�1; total phenols, 1.21 � 0.05 g L�1.

After phenols removal, the main features of the dephenolized

OMW (OMWdeph) were those reported in Table 1; OMWdeph

total phenols concentration was 0.37 � 0.04 g L�1.

2.2. Microbial consortium

The acidogenic microbial consortium employed as the inoc-

ulum was obtained and microbiologically characterized

within a previous investigation dedicated to the development

of an acidogenic biotechnological process fed with OMWdeph

[20]. The inoculumwas stored at 4 �C before being used in this

study.

2.3. Effectiveness of the inoculum in the acidogenicdigestion process

The effectiveness of the inoculum in the bioconversion of

OMWdeph organic matter into Volatile Fatty Acids (VFAs)

under anaerobic acidogenic conditions was preliminary

investigated in batch tests. In particular, its capability of

producing H2 and VFAs was evaluated at 35 �C and pH 7.0,

provided that such conditions were found to be ideal for the

acidogenic digestion of OMWdeph by the same inoculum [20].

Acidogenic digestion was studied in statically incubated

b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 5 1e5 8 53

anaerobic 100 mL-Pyrex bottles equipped with 2 stacked sili-

cone stoppers (thickness 2.5 mm each), tightly closed to the

bottleneck through a modified Pyrex-cap to allow gas

sampling. Three experimental conditionswere set up, in order

to evaluate the acidogenic potential of: a) the microbial

consortium used as inoculum; b) the active microflora origi-

nally occurring in OMWdeph; c) both consortia when combined

together in the process. Thus, under nitrogen gas sparging,

flasks were filled with, respectively: a) 5 mL of inoculum and

45 mL of sterile OMWdeph (sterilized in autoclave at 121 �C for

20 min); b) 50 mL of OMWdeph (without inoculum addition); c)

5 mL of inoculum and 45 mL of OMWdeph. Static incubation

was provided for over 12 days. Biogas production and

composition was determined every 2e3 days. After biogas

sampling, flasks were opened under nitrogen gas sparging,

1 mL of liquid phase sampled (for chemical analyses), and

pH controlled and corrected to 7.0 via addition of some drops

of a NaOH solution (whose concentration was 400 g L�1).

Results are the mean value of at least two independent

replicates.

2.4. Packed bed biofilm reactors (PBBRs)

The effects of Hydraulic Retention Time (HRT) on acidogenic

digestion of OMWdeph were evaluated under continuous

mode of operation in anaerobic PBBRs, consisting of a glass

column (40 cm in height, 5 cm outer diameter) with an empty

volume of 0.796 � 0.052 L. Ceramic cubes of Vukopor S10�

(Lanik, Boskovice, CZ), which were previously described [19],

were used as cells carriers to fill up reactors. As a result of

support addition (150.9 � 3.0 g), reactors working volume

became 0.693 � 0.020 L. PBBRs were fed according to an up-

flow scheme with HRTs equal to 7, 5, 3 or 1 day, corre-

sponding to Organic Loading Rates (OLRs) of 5.54, 7.76, 12.93

and 38.79 g L�1 d�1, respectively. PBBRs were anaerobically

loaded with a mixture of OMWdeph and microbial inoculum

(added at 10%, v:v). Generation of a biofilm by passive

immobilization was achieved by recycling such a liquid

phase under batch conditions for 1 week. Thereafter, reac-

tors were operated under continuous mode. After steady-

state conditions in terms of VFA production were achieved,

PBBRs loaded with HRTs of 7 and 5 d were operated for more

than 3 weeks, while PBBRs loaded with HRTs of 3 and 1 days

were operated for 2 and 1 week, respectively. Process

temperature was set at 35 �C, pH maintained to 7.03 � 0.01 by

periodically dropping a NaOH solution (whose concentration

was 400 g L�1). The process was monitored on a daily basis

for biogas production and composition, VFAs and COD

concentrations.

Finally, the process allowing the highest H2 production

(namely, the PBBR loaded with HRT of 1 d) was tested again

under the same conditions for a prolonged time of operation

(42 days).

2.5. Analytical procedures and process yield calculation

Concentrations of VFAs, COD and total phenols were

measured as reported by Bertin and co-workers [19]. VFAs

concentration was always reported as COD equivalents (g L�1

COD eq.) by means of stoichiometric calculations. Biogas

production was measured as reported elsewhere [14] by

employing a Mariotte system, while its composition was gas-

chromatographically determined [20].

Bioconversion of OMWdeph organic matter into VFAs

(COD / VFA) was calculated as the ratio between produced

VFAs and influent net COD not due to VFAs already occur-

ring in the process feeding, according to the following

equation:

COD/VFA ¼ ðVFAOUT � VFAINÞ=ðCODIN � VFAINÞ � 100 (a)

Other process parameters were also evaluated, namely: 1)

VFAOUT/CODIN, which expresses the amount of VFAs released

in the effluent with respect to the amount of COD fed to the

reactor; 2) VFAOUT/CODOUT, which defines the fraction of the

effluent COD due to VFAs.

3. Results

3.1. Effectiveness of the inoculum in the acidogenicdigestion of OMWdeph

Biogas cumulative production observed during preliminary

batch investigations are reported in Fig. 1.

As a matter of fact, results obtained when the OMWdeph

indigenous microflora and the acidogenic consortium used as

inoculumwere operated together did not represent the sum of

what obtained when tested separately. On the one hand, total

biogas volumes were substantially comparable. H2 accumu-

lation was initially slow in the presence of only one consor-

tium, although a much higher H2 production was finally

achieved in the presence of the sole acidogenic inoculumwith

respect to what obtained when the indigenous OMWdeph

microflora was employed. Nevertheless, when consortia were

operated together, H2 accumulation in the first days (T ¼ 2

days) was significantly high (14.6 vs. 2.3 and 0.0mL 100mL�1 of

working volume with both consortia, inoculum and OMWdeph

microflora, respectively), and it reached a plateau already

after 5 days. Methane production was negligible at all the

tested conditions, particularly when tested with the OMWdeph

microflora, and roughly one order of magnitude lower than H2

or CO2. Finally, most of the produced biogas was CO2, which

was always released at a higher pace when using both

consortia.

As regards VFAs concentration, the most prominent ones

(namely acetic, propionic and butyric acid) were noted at all

the tested conditions (Fig. 2).

However, a higher number of VFAs, including valeric and

caproic acid, accumulated in the presence of both consortia,

whereas both these acids were not detected when consortia

were tested alone. Furthermore, aside the accumulation of

a wider range of VFAs, the highest amount of VFAs was

produced when operating both consortia together. Notwith-

standing this, after 9 days of incubation the amount of VFAs

produced by the inoculum alone was equivalent to 2/3 of the

total amount of VFAs producedwhen operating both consortia

together, and roughly one order of magnitude higher than

what found when the acidogenic digestion of OMWdeph was

carried out by its own microflora.

Fig. 1 e Overall biogas (A), H2 (B), CH4 (C), and CO2 (D) cumulative productivities in batch tests where the active microflora

was represented by the native microflora occurring in the dephenolized Olive Mill Wastewater (OMWdeph) (white keys), the

acclimated acidogenic inoculum (grey keys), or both consortia together (black keys).

b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 5 1e5 854

3.2. Effect of hydraulic retention time on acidogenicdigestion of OMWs

Reduction of HRT from 7 to 1 day was reflected in a relevant

increase in biogas production, namely from 150 to

460 dm3m�3 d�1, respectively, with a peak of productionwhen

HRT was regulated to 3 days (475 dm3 m�3 d�1) (Fig. 3). Most

interestingly, biogas was significantly enriched in H2 (from 3%

to 32%, when HRT was shortened from 7 to 1 day, respec-

tively), whereas CO2 concomitantly declined from 93 to 64% of

the produced biogas, respectively (Fig. 3).

As a net result, H2 release increased from 0.20 to

6.10 dm3 m�3 h�1 (i.e., more than 30 times). On the other hand,

methane relative presence was never higher than 4% in all the

tested HRTs. These results are consistent with what observed

under batch conditions, where methane release was always

very low (Fig. 1c) and H2 and CO2 were produced at a similar

rate (Fig. 1b and d, respectively), especially in the first part of

the experiment.

In Table 1, process yields and effluent features are re-

ported. Reduced HRTs turned into lower accumulations of

VFAs and, consequently, lower conversion efficiencies (as

defined in Section 2.5). The highest yields and final produc-

tivities were achieved with a HRT equal to 5 days, although in

these conditions the highest removal of organic matter

(evaluated as chemical oxygen demand, COD) was also

observed.

Relative composition of the VFAs mixture occurring in the

effluents of the PBBRs operating with different HRTs was also

evaluated (Fig. 4). In general, the most produced VFAs were

acetic, propionic, butyric, valeric and caproic acid, in accor-

dance with what observed in the preliminary screening

(Fig. 2c). However, their relative presence was affected by

changes in HRT. For instance, among the short chain fatty

acids monitored, the relative amount of some of the longer

ones (i.e., isovaleric, isocaproic and eptanoic acid)was reduced

by prolonged HRTs, whereas the relative amount of some of

the other VFAs (namely, valeric and caproic acid) concomi-

tantly increased. On the contrary, acetic, propionic and

butyric acid were the most prominent VFAs when operating

PBBRs with a HRT of 1 day (19, 12 and 42%, respectively), their

relative amount being slightly reduced when longer HRTs

were applied.

Results obtained when operating with a HRT of 1 day were

confirmed throughout a final experimental set, during which

a PBBR was continuously fed with OMWdeph for more than 40

days. As a whole, H2 production was effectively maintained at

7.07� 1.12, while VFAs concentration in the effluent remained

quite low, being acetic, propionic and butyric acids the

predominant ones (data not shown).

Density of effluents was also evaluated each day. It was

noted that this value was generally lower than 1 g cm3, which

may be probably due to the fact that residual lipid compo-

nents remained in the wastewater.

Fig. 2 e Volatile Fatty Acids (VFAs) net productions in batch tests where the active microflora was represented by (A) the

native microflora occurring in the dephenolized Olive Mill Wastewater (OMWdeph) (white bars), (B) the acclimated acidogenic

inoculum (grey bars), or (C) both consortia together (black bars), along with total amounts of produced VFAs (D).

b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 5 1e5 8 55

4. Discussion

The use of multidisciplinary approaches for the extensive

exploitation of agroindustrial residues is to date a widely

investigated field. Sequential integrated strategies can be

addressed to the obtainment of a single specific final product

(e.g., biohydrogen; for a review, see [21]) or aimed at a multi-

purpose valorization of the residue, in accordance with the

biorefinery concept of the waste management [20].

In this respect, the present work intended to enhance the

potentialitiesof aphysicochemical-biotechnological integrated

process dedicated to the exploitation of OliveMillWastewaters

(OMWs). In the latter, a fully biocompatible approach for the

recovery of OMWnatural antioxidants, either as a mixture [22]

or a single selected compound [23], was coupled to the

productionof aneffluent enriched inVolatile FattyAcids (VFAs)

[20], which can be employed for the biotechnological produc-

tion of polyhydroxyalkanoates (PHAs) [17]. In particular, the

possibility of coupling wastewater acidogenesis to the

production of a H2-rich biogas was evaluated by testing the

influence of Hydraulic Retention Time (HRT).

Dark fermentative H2 release is an anaerobic ubiquitous

phenomenon. When bacteria grow on organic substrates,

hydrolysis of proteins, lipids and carbohydrates provides

building blocks and metabolic energy for growth. Oxidation

of such compounds generates electrons which need to be

disposed via the production of fermentation products,

including VFAs and H2 [for a review, see [24]. As known,

these processes are the first to take place during anaerobic

digestion. Consistently, reduced HRTs shifted the biotech-

nological treatment of OMWdeph from VFAs to biogas

production, with a remarkable increase in the H2 content, as

already observed with other residues [25]. In particular,

when shortening the HRT from 7 to 1 day, H2 production rate

increased 30 times, reaching values higher than

7 dm3 m�3 h�1 when a HRT equal to 1 d was assessed for than

40 days. On the other hand, methane production was found

to be always very low at all the tested HRTs, meaning that

methanogenic bacteria were adversely affected by the

conditions employed. Most likely, such a high H2 produc-

tivity was also due to the consistent number of Clostridium

species as found in the inoculum employed [20], provided

that these Firmicutes are known as some of the most H2-

producing living forms [26e32]. Increased H2 productions

achieved by shorter HRTs confirmed observation by Scoma

and co-workers [20] that longer HRTs may favour hydro-

genotrophic activities.

Fig. 3 e Changes in biogas production and composition

during the acidogenic digestion of dephenolized Olive Mill

Wastewater (OMWdeph) in Packed Bed Biofilm Reactors

(PBBRs) as a consequence of the application of different

Hydraulic Retention Times (HRTs): (A) total biogas

production rate and (B) biogas composition in H2 (white

bars), CH4 (grey bars) and CO2 (black bars).

b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 5 1e5 856

Biological H2 production using OMWs has been already

carried out with photosynthetic purple bacteria [16,33]. The

latter entailed a strong dilution rate, due to both high organic

content and dark colour of this residue (preventing light

penetration), whichmakes this approach unpractical. In some

cases, a first dark fermentation aimed at increasing the

Fig. 4 e Single VFA relative amounts (%) detected in the

effluents of the PBBRs fed with different Hydraulic

Retention Times (HRTs).

concentration of the substrate (i.e., VFAs) for a following

photo-fermentative step was successfully conducted [34,35].

Such a two-stage process usually improves H2 productivities,

may decrease OMW dilution rate and leads to a better

exploitation of the residue. Notwithstanding this, direct dark

fermentative H2 production of by-products of the olive oil

agro-industry has been reported in literature only twice

[36,37]. It must be stressed that, contrary to the present paper,

in both these cases a dilution with tap water (representing up

to 75% of the experimental matrices employed) was carried

out. As a result, VFAs concentration in the effluents obtained

in the present investigation was more than 2 times higher

than what previously reported [36,37], while only slightly

lower H2 productivities were obtained (with respect to the

influent COD). In this respect, further reduction of HRTs may

lead to significant enhancements in H2 productivity [36,37]

and will be matter of future investigations.

Acidogenesis yields were also affected by HRTs. The

highest amount of VFAs (almost 20 g L�1 COD eq.) was

observed when the PBBR was operated with a HRT of 5 days,

which corresponded to the highest efficiency of bioconversion

of COD into VFAs (COD/VFA, 36%). The latter was slightly

higher than what obtained with a HRT of 7 days which,

notably, was equal to that observed in a previous investigation

(31.7 vs. 31.0%, respectively) operating under the same

conditions in a 3-time higher PBBR volume [20]. Hence, by

reducing the HRT, better performances were achieved in

a lesser time.

Importantly, HRT was found to influence also the relative

composition of the produced VFA mixture. Provided that the

chemical composition and, consequently, the properties of

PHAs depend on the different carbon sources supplied during

their biosynthesis, when carried out in aerobic PHA-producing

processes [38,39], HRT could be adjusted according to the

preferred relative amounts of VFAs, in order to finally obtain

PHAs with the desired features. In this respect, the successful

employment of OMWs for PHAs production has been already

reported [17].

Finally, the advantages associated with the employment of

the selected inoculum in combination with the microflora

occurring in the employed actual site OMW was demon-

strated. Together with a high content of organic matter,

OMWs usually carry their own microflora, represented by

a variable high number of bacteria, yeasts and fungi [27]. Such

an indigenous microflora is usually able to grow on that

wastewater [27], although survival of some species may not

guarantee the possibility to attain biotechnologically satis-

fying yields, when a selected process is performed. In previous

studies, a well-characterized and acclimated acidogenic

consortium was employed for the extensive bioconversion of

OMW organic matter into VFAs [19,20]. In the present inves-

tigation, batch tests showed that the acclimated inoculum

had the highest potential for H2 and VFAs production respect

to the microflora. However, when operated together these

bacterial communities positively interacted, leading to higher

H2 productivities (for short times of residence) and higher

VFAs net production (for longer ones) in agreement with what

observed for HRTs in continuous processes. In particular, the

latter result (i.e., VFAs production) was further endorsed by

the fact that a higher diversity of short chain fatty acids was

b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 5 1e5 8 57

found when operating these consortia in combination rather

than alone.

5. Conclusions

Themultipurpose valorization of agroindustrial residues such

as OMWs is to date a feasible option. The present contribution

showed that production of biohydrogen and VFAs, to be used

as feed for PHAs production, can be concomitantly obtained

by adjusting process parameters. These findings represent

a valuable new pathway for the integrated valorization of

OMWs, allowing the production of another final product (i.e.,

H2) without affecting the consolidated biorefinery scheme

previously proposed [20].

Acknowledgements

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

(Imperia,Italy) for having kindly provided OMWs. The

research leading to these results has received funding from

the European Community’s Seventh Framework Programme

(FP7/2007e2013) under the grant agreement no FP7-265669-

EcoBioCAP project”.

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