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Chemical Engineering Journal 172 (2011) 28– 36

Contents lists available at ScienceDirect

Chemical Engineering Journal

jo u r n al hom epage: www.elsev ier .com/ locate /ce j

ffect of upflow velocity and hydraulic retention time in anaerobic fluidized-bedeactors used for hydrogen production

ristiane Marques dos Reis, Edson Luiz Silva ∗

epartment of Chemical Engineering, Federal University of São Carlos, São Carlos, São Paulo, Brazil

r t i c l e i n f o

rticle history:eceived 23 December 2010eceived in revised form 22 April 2011ccepted 4 May 2011

eywords:

a b s t r a c t

The present study evaluated the influence of upflow velocity (Vup) applied to an anaerobic fluidized-bedreactor used for hydrogen production. For comparison, two reactors were used with different velocities:1.24 cm s−1 (R124) and 1.88 cm s−1 (R188). Expanded clay was used as a support material for immobiliza-tion along with synthetic wastewater containing glucose as the main carbon source (5000 mg L−1). Thereactor R124, which operated at the minimum fluidization velocity (1.24 cm s−1), had the best values for

−1 −1

ydrogen productionthanolnaerobic fluidized-bed reactorpflow velocityydraulic retention time

hydrogen production. The maximum hydrogen production rate was 2.21 L h L for a hydraulic reten-tion time (HRT) of 1 h, while the best hydrogen yield was 2.55 mol H2 mol−1 glucose for an HRT of 2 h.The hydrogen content in the biogas was around 40.53–67.57%. A high amount of ethanol was produced,suggesting that a metabolic pathway via ethanol was preferable. In general, the reactor under the min-imum velocity fluidization (R124) presented a greater production of hydrogen than R188 showing thatshould have a limit point in Vup till the hydrogen production is maximized.

. Introduction

Hydrogen is a potential alternative fuel. It is the most abundantlement on earth and is present in all materials. Hydrogen can bebtained from renewable sources, and it does not emit pollutantsuring combustion or when it is used in fuel cells. The disadvan-age is that hydrogen is an expensive element that is not freelyound in nature in a usable form. In addition, portions of hydrogenroduction require substantial amounts of energy, which is oftenrovided in the form of fossil fuels. The need for more economicalnd environmentally friendly methods of obtaining hydrogen hased researchers to investigate the possibility of obtaining hydrogenas from biomass fermentation.

The fermentation of organic matter is an appropriate and sus-ainable method for producing hydrogen. Besides using materialshat are easy to obtain, fermentation minimizes the problemselated to the disposal of tailing wastes. During hydrogen pro-uction, different substrates can be used, such as industrial andomestic waste, which is rich in carbonaceous matter [1]. Throughermentation, these wastes are degraded with less pollution. Thebility to conduct the process under ambient pressure and temper-

ture is another important factor because it reduces the operatingxpenses for hydrogen production.

∗ Corresponding author. Tel.: +55 16 33518264; fax: +55 16 33518266.E-mail address: edsilva@power.ufscar.br (E.L. Silva).

385-8947/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.cej.2011.05.009

© 2011 Elsevier B.V. All rights reserved.

In glucose fermentation, the formation of hydrogen is accom-panied by the production of acetic acid, butyric acid and ethanol.Depending on which metabolic pathway is used, one product oranother is formed in greater quantities. Glucose fermentation canalso generate propionic acid; however, propionic acid is an unde-sirable product because hydrogen is necessary for its formation.The traditional pathway for ethanol production from glucose gen-erates only alcohol and CO2. This pathway could be a useful methodbecause hydrogen acts as an electron donor. Studies in which bothhydrogen and ethanol were generated were found in the literature[2–4]. Hwang et al. [5] showed alternative pathways that generatehydrogen, ethanol and acetic acid.

Hydrogen production via dark fermentation has been studiedwith various types of reactors, such as fixed-bed reactors [6,7], con-tinuous stirred-tank reactors [8,9], and upflow anaerobic sludgeblanket reactors (UASB) [10]. Another type of reactor configurationthat was recently used for treating wastewater is the anaerobicfluidized-bed reactor (AFBR) [2,3,11–20].

Fluidized-bed reactors have the advantage of being more com-pact than other reactors. Additionally, certain characteristics ofan AFBR make this reactor configuration beneficial to the anaer-obic process. These characteristics include the use of particles ina fluidized state, which increases the effective area for depositingmicroorganisms that degrade organic matter in the residues that

are being treated; the mixing conditions of the fluidization-relatedmass transfer efficiency; and easy operation. Still, compared withother reactors, fluidized-bed reactors have a higher biomass adhe-sion, a high rate of organic operation, a low hydraulic retention time

C.M. dos Reis, E.L. Silva / Chemical Engineering Journal 172 (2011) 28– 36 29

(Ap

frdtatatwc

utivhoitmldp

tthscvc

2

2

w

Table 1Hydrodynamic data applied to the reactors.

Reactor Vup (cm s−1) Vmf ratio

Fig. 1. Schematic diagram of the anaerobic fluidized bed reactor (AFBR).

HRT) and better mixing conditions [14,15]. Research conducted onFBRs has demonstrated the efficiency of the reactor for hydrogenroduction [2,12,14,15,18–20].

Several parameters from these studies were used as referencesor the study of hydrogen production (pH, temperature, type ofeactor, substrate and nutrient) as well as to analyze the fluidynamic behavior of the reactor and its influence on the fermenta-ion process. Studies [2,7,12,21] on hydrodynamic parameters suchs superficial velocity, mixing conditions and porosity have shownhat there may be an important link between hydrogen productionnd the hydrodynamic conditions applied to the reactor. However,here were few studies on this subject, and currently, it is not knownhether changes in the hydrodynamic conditions of the reactor

ould influence the anaerobic production of hydrogen.Among the hydrodynamic parameters that can be studied is the

pflow velocity (Vup). It is know that high velocities can favor massransfer among phases in anaerobic digestion. Otherwise, few stud-es deal with this subject. Wu et al. [2] found that the higher theelocity applied to an AFBR, the greater the hydrogen production;owever, they found no influence on the hydrogen yield. In a studyf fluidized-bed reactors it is essential to know the upflow veloc-ty that fluidizes the bed so that can be understood how far fromhis value an operation should be taken. According to the support

aterial used, this information can be acquired. Other parametersike degrees of packaging can lead to differences in hydrogen pro-uction, like Lin et al. [17] have evaluated. The higher the degree ofackaging, the smaller is the Vup applied.

Although the use of an AFBR for hydrogen production was effec-ive, studies on the best range of processing conditions for operatinghe reactor are necessary before AFBRs can be applied to full-scaleydrogen production. Therefore, in order to achieve a better under-tanding of the behavior of AFBRs for producing hydrogen, we haveonducted a study to determine the influence of applying upflowelocity during hydrogen production using synthetic wastewaterontaining glucose as the carbon source (5000 mg L−1).

. Materials and methods

.1. Anaerobic reactors

Two fluidized-bed reactors were used in this study (Fig. 1). Theyere constructed from acrylic and had an internal diameter of

R124 1.24 1.0R188 1.88 1.5

5.3 cm and a height of 190 cm, making a total volume working of4192 cm3. Fluidization was maintained with a recirculating pump,and the feed was introduced with a peristaltic pump.

The reactors were operated in a continuous mode for 217days using expanded clay for immobilization support. Expandedclay (diameter = 2.8–3.35 mm; density = 1.50 g cm−3; minimumfluidization velocity: Vmf = 1.24 cm s−1) was chosen because itprovides support, it is inert and it has physical characteristicssuch as roughness and porosity that are beneficial for microor-ganism deposits. The support occupied 60% of the reactor (bedheight = 115 cm). After fluidization, R124 reached 129 cm and R188reached 144 cm. Inoculum adaptation for the reactors was com-pleted in batch mode for 48 h. After this period, the reactors wereplaced in continuous mode. Both reactors started operating undera hydraulic retention time (HRT) of 8 h, which decreased until anHRT of 1 h was reached. The change from one stage to another wasperformed such that the data obtained on hydrogen production andglucose conversion was stabilized.

2.2. Synthetic wastewater

The reactors were fed with a synthetic wastewater that con-tained glucose at a concentration of 5000 mg L−1. The nutrientconcentrations were as follows (in mg L−1): CO(NH2)2 (125);NiSO4·6H2O (1); FeSO4·7H2O (5); FeCl3·6H2O (0.5); CaCl2·6H2O(47.0); CoCl2·2H2O (0.08); SeO (0.07); KH2PO4 (85.0); K2HPO4(21.7); Na2HPO4·2H2O (33.4). Hydrochloric acid (30%) and sodiumbicarbonate (0.84 g L−1) were also added to buffer the solution andavoid sudden changes in pH and maintain the pH inside the reactorbetween 4 and 5. The volume of HCl (30%) that was used corre-sponded to 1 mL of acid solution per liter of wastewater prepared.

2.3. Inoculum

The sludge from a UASB reactor that was used for treatment ofswine wastewater served as the inoculum for both reactors. Beforeinoculation, the sludge underwent a heat treatment to activate aci-dogenic cells according to the methodology of Kim et al. [22]. Itconsisted of heating the sludge for 10 min at 90 ◦C, followed by anice bath until the temperature reached 25 ◦C. A ratio of 7.5% byvolume was used for inoculating the reactors.

2.4. Start-up procedure

In order to analyze the influence of the upflow velocity (Vup), twodifferent Vup were applied to each reactor: 1.24 and 1.88 cm s−1.The reactors were named based on the velocity at which they wereoperated. R124 is the reactor with Vup of 1.24 cm s−1, and R188 is thereactor with Vup of 1.88 cm s−1. Table 1 shows the hydrodynamicconditions applied to each reactor.

2.5. Chemical analysis

Concentrations of volatile fatty acids (VFA) and alcohols were

also measured by gas chromatography (GC-2010, Shimadzu, Japan)equipped with FID and COMBI-PAL headspace injection (AOC5000 model) as well as a HP-INNOWAX column (30 m × 0.25 mmi.d. × 0.25 �m film thickness) [23].

30 C.M. dos Reis, E.L. Silva / Chemical Engineering Journal 172 (2011) 28– 36

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9HRT (h)

Glu

cose

con

vers

ion

(%)

R124

R188

ma1G

Pso

C(m[iD

3

3

rvrrH1

UVtwivt

tcttistfH

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9HRT (h)

H2 c

onte

nt (%

)

R12 4

R18 8

changed to 6 h. For an HRT of 4, 2 and 1 h, the H2 content remainedrelatively stable. Only H2 and CO2 were produced. CH4 was notproduced during the experiment. At an HRT of 8 h, R124 and R188

0,0

0,5

1,0

1,5

2,0

0 1 2 3 4 5 6 7 8 9

HPR

(Lh-1

L-1)

0

50

100

150

200

250

300

OLR

(kg.

CO

D.m

-3.d

-1)

R124

R188

OLR

Fig. 2. Glucose conversion for different HRTs in R124 and R188.

The biogas hydrogen content was determined by gas chro-atography (GC-2010, Shimadzu, Japan) using TCD with argon

s the carrier gas and a column packed with Supelco Carboxen010 Plot (30 m × 0.53 mm i.d.) [23]. A gas meter (TG1; Ritter Inc.,ermany) was used to quantify the amount of hydrogen generated.

Glucose concentration was measured with an enzymatic GOD-AP [18]. The chemical oxygen demand (COD), pH and volatileuspended solids (VSS) were analyzed according to Standard Meth-ds [24].

The attached biomass was measured according to Chen andhen [25]. Quantification of extracellular polymeric substancesEPSs) in protein form was performed in accordance with the

ethod proposed by Lowry et al. [26] and modified by Peterson27], using bovine serum albumin as a standard. Analysis of EPSsn carbohydrate form was carried out according to the methods ofubois et al. [28] using lactose as a standard.

. Results and discussion

.1. Glucose conversion

Fig. 2 illustrates the glucose conversion achieved by the twoeactors that were operated with different HRTs. The glucose con-ersion was above 80% in both reactors, R124 and R188, and iteached a maximum of 94.3% at an HRT of 6 h in R188. The resultsemained relatively constant throughout the experiment until anRT of 2 h was reached. Because of the change in the HRT from 2 to

h, there was a decrease in glucose conversion in both reactors.The glucose conversion was stable between an HRT of 2 and 8 h.

ntil an HRT of 2 h was reached, R188, which was operated with aup of 1.88 cm s−1, had an average conversion that was 9% higherhan R124 (1.24 cm s−1). At an HRT of 2 h, R124 became the reactorith a higher conversion. With an HRT of 1 h, there was a decrease

n the conversion for both reactors. This decrease in glucose con-ersion at an HRT of 1 h showed that for low hydraulic retentionimes, higher values of glucose degradation were not possible.

The adoption of Vup of 1.88 cm s−1, which was 1.5-fold higherhan Vmf in this experiment (1.24 cm s−1), improved the mixingonditions inside the reactor and diminished the resistance to massransfer between the liquid and the inner surface of the biofilm. Fur-hermore, the incremental increase in Vup implied that an increasen the bed height occurred as a result of its expansion. The inter-

titial space between the particles of the bed, along with higherurbulent conditions, could improve the parameters of mass trans-er involved in the diffusion of the substrate within the biofilm.owever, to confirm this hypothesis, mass transfer experiments

Fig. 3. H2 content for different HRTs in R124 and R188.

should be completed in order to evaluate the change in parame-ters under different configurations. This reversal of the results fromboth reactors may be related to other issues, such as the presenceof a dominant microbial culture or better adaptation of the reactor.

Overall, the results showed that the two different operating con-ditions yielded efficient substrate conversion; the results for bothreactors were above 80%. This finding confirmed previous studieson AFBRs in which the conversion values were around 90%.

Lin et al. [13] achieved conversions between 92% and 98% fromsucrose. Zhang et al. [14], who worked with glucose and AFBR,reached conversions of 99.47% with an HRT of 4 h and 71.44% withan HRT of 0.5 h. Shida et al. [18] achieved conversions between92.06% and 98.08%. Amorim et al. [19], who also used an AFBR,obtained conversions above 90%. Lin et al. [17] achieved conver-sions of 97% with HRTs of 6 and 4 h, as well as 72% with an HRTof 2 h. Barros et al. [20] achieved conversions between 70.50% and96.30%. These results were consistent with previous research onthe anaerobic fluidized-bed reactor.

3.2. Hydrogen production

The data obtained on the production of hydrogen in this studyare presented in Figs. 3–5. Fig. 3 shows the amount of H2 formed inthe biogas for different HRTs. The amount of H2 formed reached amaximum value at an HRT of 8 h but decreased when the HRT was

HRT (h)

Fig. 4. Hydrogen production rate and organic loading rate for each HRT applied toR124 and R188.

C.M. dos Reis, E.L. Silva / Chemical Engin

0

1

2

3

4

0 1 2 3 4 5 6 7 8 9

HRT (h)

HY

(mol

H 2

mol

-1 g

luco

se)

0

50

100

150

200

250

300

OLR

( kg

.CO

D.m

-3.d

-1)

R124R188OLR

Fig. 5. Hydrogen yield and organic loading rate for each HRT in R124 and R188.

sHwowH

cofH

agctO

oas4wHwairua

sgiht

dHHortvsR

obtained from each study. The table also contains the values for the

howed a H2 content of 67.57% and 61.82%, respectively. With anRT of 6 h, the H2 content fell to 43.03% and 40.60%. When the HRTas changed to 2 and 4 h, there was a small change to a minimum

f 36.04% for R124 at an HRT of 2 h. At an HRT of 1 h, a slight increaseas observed in R124 and R188, which reached a 40.53% and 41.54%2 content, respectively.

As shown in Fig. 3, the upflow velocity had no influence on the H2ontent in the biogas. However, Spagni et al. [29] studied the effectf varying organic loading rates (OLRs) on hydrogen production andound that higher organic loads led to a decrease in the amount of

2 in the biogas, despite the increasing hydrogen production rate.The hydrogen production rate (HPR) and the OLR for each HRT

re illustrated in Fig. 4. As long as the HRT decreased, the hydro-en production rate increased for both reactors. Since the substrateoncentration was fixed throughout the experiment in both reac-ors, the reduction in the HRT led to an increase in OLR. With a highLR, it was expected that more hydrogen should be produced.

The reactors generated similar values at the beginning of theperation when both reactors had an HRT of 8 h. In response to

decrease to an HRT of 6 h, R188 remained stable, while R124howed an increase in HPR. With further reduction in the HRT to, 2 and 1 h, the HPR in the reactors increased. The maximum HPRas achieved at an HRT of 1 h. The reactor that had the highestPR, R124 (1.22 L h−1 L−1), was operated with Vmf. Reactor R188,ith Vup that was 1.5-fold higher, had a lower HPR (0.93 L h−1 L−1)

nd a peak value that was 1.3-fold smaller than the productiv-ty for R124. In a previous study on AFBRs, the HPR values had aange between 1.80 and 2.36 L h−1 L−1. Lin et al. [13] obtained val-es around 2.27 L h−1 L−1, while Zhang et al. [14] obtained valuesround 2.36 L h−1 L−1. Lin et al. [17] reached 1.80 L h−1 L−1.

When we discuss in terms of organic loading rate, it can beeen that when the OLR was above 132.6 kg COD m−3 d−1, therowth rate of hydrogen production decreased and stabilized. Thus,ncreasing the organic load to levels above 132.6 kg COD m−3 d−1

ad no effect on the production of hydrogen under the experimen-al conditions applied in this study.

Fig. 5 illustrates the hydrogen yield (HY) related to the substrateegradation and OLR for each reactor at different HRTs. When theRT was reduced, the average HY increased in both reactors. TheY varied between 0.9 and 2.6 mol H2 mol−1 glucose for an HRTf 2 h. At an HRT of 1 h, there was a decrease in the HY for botheactors. Similar to the change in HPR, the HY values increased ashe HRT decreased until an HRT of 2 h was reached. The best HY

alues were obtained at an HRT of 2 h for both reactors, which wasimilar to the trend for HPR values. However, at HRTs of 8 and 6 h,188 had a better HY than R124. At an HRT of 1 h, the effect of the

eering Journal 172 (2011) 28– 36 31

Vup was not verified because of the similarity between the results.There was also a drop in the HY for both reactors at an HRT of 1 h.

The Vup of 1.88 cm s−1 (R188) did not influence the hydrogenyield obtained in the reactor operated at Vup of 1.24 cm s−1 (R124).The maximum HY in R124 (2.55 mol H2 mol−1 glucose) was 18%higher than the yield obtained with R188 (2.16 mol H2 mol−1 glu-cose). Likewise, the maximum HPR in R124 (1.22 L h−1 L−1) was 30%higher than the HPR for R188 (0.93 L h−1 L−1). Additionally, at anHRT above 6 h, the hydrogen yield tended to be better when thereactor was operated at a lower Vup. During reactor operation witha low HRT, there was a better performance of the reactor operatedat high Vup. Wu et al. [2] studied the influence of Vup and did notobserve a clear effect of the superficial velocity on the hydrogenyield. An increase of 33% in the velocity (from 0.55 to 0.73 cm s−1)led to a decrease in yield by 58% (from 1.04 to 0.44 mol H2 mol−1

glucose). When there was a 25% increase in Vup (from 0.73 to0.91 cm s−1), unlike the previous result, an increase greater than100% in the HY was observed. Operating a reactor at a low HRTrequires smaller Vup because of the greater mixing conditions thatexist compared with operating a reactor at a high HRT. Reactoroperation at low HRTs requires higher upflow velocities in order tocircumvent the lowest agitation conditions for the medium in thereactor. In this experiment, that behavior was not observed.

In the same way, a reasonable explanation can be taken fromthe OLR applied to the system.

The HY increased when the OLR increased. However, when theOLR exceeded 60 kg COD m−3 d−1, the increase was small and a rel-atively constant HY occurred with increasing OLRs.

Ren et al. [30] also verified an increase in the specific hydrogenproduction and in the HY when the organic loading rate varied from6.32 to 68.21 kg COD m−3 d−1. At 68.21 kg COD m−3 d−1, hydrogenproduction declined because of the accumulation of volatile fattyacids in the system.

As noted, the data that were obtained were similar to resultsfrom previous studies in the literature. Although there is currentlyno consensus on the real reasons for the reduction in the HY as theresult of high OLRs, the yield may be inhibited by high organic loads.Several studies have observed these effects [31,32], yet other stud-ies have shown that increasing the OLR can lead to higher hydrogenproduction yields [13,22].

We found that there was a limit point for producing hydrogen.After that point was reached, the HPR and the HY remained con-stant because of the continued increase in the organic loading rateapplied. This trend was also observed by Van Ginkel and Logan [32]and Kramer et al. [33].

Van Ginkel and Logan [32] attributed this trend to an inhibi-tion caused by organic acids nondissociated in the medium. Theseacids would act by inhibiting the hydrogenase enzyme that wasresponsible for producing hydrogen. However, Kramer et al. [33]stated that the acids were not the only compounds responsiblefor this inhibition. Their argument was that the reduction in HYwas accompanied by a reduction in the quantities of acids thatwere produced. Another issue, raised by Van Ginkel and Logan [32],was the low concentration of hydrogen that occurred with lowOLRs. This hypothesis suggested that there would be no limitationon hydrogen production because there would be no inhibition ofhydrogenase. However, low concentrations would be necessary inorder for there to be significant changes in the thermodynamicsinvolved with hydrogenase [33].

Table 2 provides a comparative analysis of the HPR, the HY andthe biogas content from previous studies using AFBRs for hydrogenproduction. The results shown in Table 2 are the maximum values

Vup employed in those studies.The HPR in AFBRs varied between 0.93 and 2.36 L h−1 L−1, but

Zhang et al. [14] reached productivity values of 7.60 L h−1 L−1. The

32 C.M. dos Reis, E.L. Silva / Chemical Engineering Journal 172 (2011) 28– 36

Table 2Comparison of the main results with other studies on AFBRs from the literature.

Reference Substrate Materialsupport

Vup (cm s−1) HPRmax

(L h−1 L−1)/HRT(h)

HYmax

(mol H2 mol−1 glucose)/HRT (h)

H2max (%)

Wu et al. [12] Sucrose Alginate-gel 0.85 0.93/2 2.67 25Lin et al. [13] Sucrose Silicone-gel – 2.27/2.2 4.98/8.9 >40

Wu et al. [2] Glucose Immobilized-cells

0.55 0.90a 1.04 –

0.73 0.49a 0.44 –0.91 1.30a 0.91 –

Wu et al. [2] Sucrose Immobilized-cells

0.55 1.09a 0.64 –

0.73 1.39a 0.61 –0.91 1.44a 0.51 –

Wu et al. [2] Fructose Immobilized-cells

0.55 0.20a 0.23 –

0.73 0.10a 0.09 –0.91 0.81a 0.56 –

Zhang et al. [14] Glucose Activatedcarbon

2.08 2.36/0.5 1.19/1 61

Zhang et al. [15] Glucose Activatedcarbon

– 7.60/0.25 1.70/0.5 48

Koskinen et al. [3] Glucose Celite R-633 – 0.15a/3.1 0.80/3.1 46Lin et al. [17] Sucrose Ethylene–vinyl

acetatecopolymer

– 1.80/2 4.26/6 44

Shida et al. [18] Glucose Expanded clay 1.61 1.28/1 2.29/2 37Amorim et al. [19] Glucose Expanded clay 1.61 0.97/1 2.49/2 35Barros et al. [20] Glucose Expanded clay 1.61 1.21/1 2.49/2 51

.24

.88

Htbrbrsf

a[sbpwaaa

aswe1amuVledtrV

R124 (this study) Glucose Expanded clay 1R188 (this study) Glucose Expanded clay 1

a Based on data from article.

Y was between 0.44 and 2.55 mol H2 mol−1 glucose (11–64% of theheoretical maximum from glucose through the acetic pathway),etween 0.51 and 4.98 mol H2 mol−1 sucrose (6–62% of the theo-etical maximum from sucrose through the acetic pathway) andetween 0.09 and 0.51 mol H2 mol−1 fructose (2–12% of the theo-etical maximum from fructose through the acetic pathway) in atudy by Wu et al. [2]. With the exception of fructose, the resultsor glucose and sucrose were in the same range of hydrogen yield.

The optimum HRTs, which varied among the different studies, islso displayed in Table 2. Studies by Shida et al. [18], Amorim et al.19] and Barros et al. [20] had a large HPR for an HRT of 1 h that wasimilar to our experiments with R124 and R188. In contrast, studiesy Zhang et al. [14] and Zhang et al. [15] demonstrated optimumroduction at HRTs of 0.5 and 0.25 h, respectively. Optimal HPRsere achieved at a higher HRT by Wu et al. [2], Koskinen et al. [3]

nd Lin et al. [17], who achieved their best rates an HRT of 2, 3.1nd 2 h, respectively. However, with the exception of Wu et al. [12],ll of the studies found the best productivity for the shortest HRTs.

A conjoint analysis of the studies is presented in Table 2. Thenalysis showed that the velocity applied to the reactors in thistudy had a positive effect on HPR until a velocity of 1.61 cm s−1

as reached, which was the same upflow velocity applied by Shidat al. [18], Amorim et al. [19] and Barros et al. [20]. At Vup of.88 cm s−1, there was a decrease in hydrogen production. Thisnalysis took into consideration the fact that the same supportaterials (expanded clay) and the same substrates (glucose) were

sed in those studies. However, Zhang et al. [14], who worked withup of 2.08 cm s−1, obtained a HPR of 2.36 L h−1 L−1, which was the

argest among the studies shown in Table 2. Thus, despite the influ-nce of Vup on the parameters of hydrogen production, the use of

ifferent support materials should be taken into account becausehe support materials modify the fluidization level in the fluidizedeactor. Table 2 shows that hydrogen production improved whenup increased. Regarding the results for HY and the biogas content,

1.22/1 2.55/2 570.93/1 2.26/2 62

it was not possible to verify the influent characteristic of applyingdifferent Vup from the studies available in the literature.

There may also be an upflow velocity limit for acceptable hydro-gen production from microorganisms in the reactors operated byShida et al. [18], Amorim et al. [19] and Barros et al. [20]. At avelocity of 1.61 cm s−1, which was the Vup applied in those stud-ies (and the same operational conditions used in the current study)and which corresponded to a velocity that was 1.3-fold larger thanVmf, the reactors had a similar HY to the yield obtained in thisstudy for R124, the reactor that was operated with Vmf. Perhapsthis gap between the Vmf and 1.3 Vmf is the best operation range forAFBRs such that controlled hydrogen production can occur withoutproduction inhibition.

The application of high superficial velocities maintained goodmixing conditions inside the reactor [2]. Furthermore, the highsuperficial velocities allowed the biogas that was produced to beeasily removed. Kraemer and Bagley [34] observed the effect ofH2 saturation when they investigated using nitrogen aspersionto dilute the concentration of H2 in the reaction media for batchreactors. The HY increased from 1.3 to 1.8 mol H2 mol−1 glucose.However, the authors stated that the yield improved as a result ofdiluting the concentration of the substrate; the aspersion was notable to affect the levels of dissolved H2 present in the media.

It is believed that a high concentration of hydrogen in the reac-tion media may inhibit the production of new biogas. In contrast,when the reactor is operated at a high superficial velocity, biomassdetachment can occur, which could lead to a reduction in themicrobial community inside the reactor. Consequently, hydrogenproduction decreases. However, in regard to H2 saturation, newexperimental tests would have to be carried out so that the fac-

tors involved in mass transfer and the influence of dissolved biogascould be evaluated.

In order to study the limits of H2 production based on saturationof the media, Kraemer and Bagley [33,34] conducted an experiment

C.M. dos Reis, E.L. Silva / Chemical Engineering Journal 172 (2011) 28– 36 33

Table 3Molar distribution of the major soluble metabolite products (SMP) for different HRTs in reactors R124 and R188.

Anaerobicreactor

HRT (h) EtOH/SMP (%)

HAc/SMP (%)

HPr/SMP (%)

HBu/SMP (%)

MetOH/SMP (%)

TVFA(mmol L−1)

SMP(mmol L−1)

HAc/HBua

HAc/EtOHa

R124

8 60.15 19.39 3.28 10.34 6.84 11.18 33.88 1.87 0.326 50.15 20.60 12.57 11.37 5.30 17.74 39.82 1.81 0.414 46.88 24.77 15.58 4.72 8.06 23.52 52.20 5.25 0.532 68.67 13.50 11.51 2.22 4.11 11.86 43.57 6.09 0.201 68.66 12.39 9.39 2.58 6.98 9.05 37.15 4.81 0.18

R188

8 58.80 11.81 0.47 6.31 22.61 5.94 31.97 1.87 0.206 50.92 15.64 7.85 6.32 19.28 11.56 38.80 2.47 0.314 56.47 17.65 7.86 6.63 11.39 15.29 47.56 2.66 0.31

33

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study, however, we verified that the higher the ratio, the greaterthe hydrogen production, but there was also a high production ofethanol that had no effect on hydrogen production in fluidized reac-

Table 4Ethanol yield in previous studies on AFBRs.

Reference Substrate Dependentvariable

EtOH yield(mol EtOH mol−1

substrate)

Wu et al. [2] Sucrose Vup (cm s−1)0.55 0.240.73 0.380.91 0.49

Wu et al. [2] Fructose 0.55 0.160.73 0.500.91 0.65

Wu et al. [2] Glucose 0.55 0.290.73 0.290.91 0.33

Zhang et al. [14] Glucose HRT (h)4 0.45a

2 0.44a

1 0.42a

0.5 0.39a

Koskinen et al. [16] Glucose pH5.3 0.286 0.656.8 0.69

2 49.40 27.09 7.99 4.1 49.31 18.49 8.09 4.

a Molar ratio.

o examine of the influence of sprayed nitrogen on the mass trans-er parameters. The authors studied the optimum range of the N2praying rate so that there would be an increase in the hydrogenield. However, the authors argued that the application of nitrogenspersion to full-scale hydrogen production would be an impor-ant cost factor because it requires more energy to perform thespersion.

.3. Production of organic acids and alcohols

In order to evaluate the effect of the HRT on the production ofetabolites for each reactor, the ratio of each metabolite over the

um of all soluble metabolite products (SMP) formed was calcu-ated. Table 3 shows the molar distribution of the major metabolitesroduced in the reactors for each HRT that was studied.

The main metabolites were ethanol (EtOH) and acetic acid (HAc),hich represented 80% of the metabolites formed at certain stages

f the experiment. The presence of butyric acid (HBu), propioniccid (HPr) and methanol (MetOH) was also reported. Regarding theata obtained from R124 with Vmf, there was a predominance ofthanol over other metabolites.

For all the HRTs, the EtOH molar percent was over 50% of theMP. The maximum EtOH percentage occurred at HRTs of 2 and

h when the percentage reached 68.7% of the SMP. The minimumercentage occurred at an HRT of 4, which was 46.9% of the SMP.he HAc production was relatively constant for HRTs of 8, 6 and 4 h19.4%, 20.6% and 24.7% of the SMP) and fell to 12% at HRTs of 2 and

h. HBu decreased as the HRT decreased; it is possible that it wasashed from the reactor during the decrease in the HRT. MetOH

emained between 4.1% and 8.1% of the SMP, while propionic acidas between 3.28% and 15.6% of the SMP.

For R188, also shown in Table 3, a large EtOH presence, rang-ng between 49.3% and 58.8% of the SMP, was observed for eachRT. There was no variation in EtOH production when the HRT washanged. The HAc production also remained relatively constant andanged between 11.8% and 27.1% of the SMP. Unlike other studiesn the literature, the MetOH presence was relevant in this experi-

ent. At an HRT of 8 h, MetOH production accounted for 22.6% ofhe SMP then fell to 19.3%, 11.4%, 11.1% and 19.2% of the SMP as theRT decreased. The production of both HBu and HPr acid did notxceed 9% of the SMP.

Higher amounts of ethanol occurred at HRTs for which theydrogen production was maximal (an HRT of 2 h). This fact demon-trated the possibility that there is another metabolic pathwayhere there is no competition between the formation of ethanol

nd hydrogen. R124 reached its best hydrogen yield at an HRT of

h.

The HAc/HBu ratio increased in both R124 and R188 after thehange of the HRT from 8 to 2 h, as shown in Table 3. Although theAc/HBu ratio shows that hydrogen production is favored, other

11.19 18.94 48.05 6.25 0.5519.90 14.20 46.12 4.39 0.38

factors may be involved in the high ethanol production observedin this study. In fact, the largest HAc/HBu ratio was obtained atthe HRTs for which the HPR and the HY were higher. However, theamounts of HAc and HBu were lower than those amounts obtainedby Shida et al. [18], Amorim et al. [19] and Barros et al. [20], whichare shown in Table 4. These studies were conducted with samesubstrate (glucose) and used expanded clay particles that were thesame size as the ones used in the current study. The HAc/EtOH ratio,which reflects the preference for the formation of solvents, rangedbetween 0.18 and 0.53 for R124 and between 0.20 and 0.55 forR188. So, the lower the ratio, the greater the production of solvents.Koskinen et al. [16] also reported high ethanol production in a studyof a pure culture of thermophilic bacteria that were isolated forhydrogen production in an AFBR. They also found HAc/EtOH valuesbetween 0.27 and 0.38.

Wu et al. [31] pointed out that HAc/HBu that may not be theonly parameter for indicating the efficiency of hydrogen produc-tion. According to authors of other previous studies, the higher theHAc/Hbu ratio, the lower the production of hydrogen. In the current

This study Glucose Vup (cm s−1)1.24 2.021.88 1.55

a Based on data from the article.

3 Engineering Journal 172 (2011) 28– 36

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4 C.M. dos Reis, E.L. Silva / Chemical

ors. In Table 2, it is clear that the HPRs and HYs were similar tohose obtained by other authors who were using the same reac-or and the same substrate. Wu et al. [31] suggested in their studyhat these results occurred because of the different fermentationathways used by the microorganisms.

Skonieczny and Yargeau [35] affirmed that the ethanol produc-ion agreed with experiments on anaerobic fermentation, but theigh ethanol concentrations that were obtained showed a newirection in the biological production of hydrogen. Despite theompetition that ethanol production generated in the system, weannot ignore the possibility of different metabolic pathways inhich there is no inhibition of hydrogen production.

Zhu et al. [36] justified the study in which ethanol was producedlong with high levels of H2 by suggesting a pathway in which theroduction of hydrogen could benefit from ethanol production. Onef their suggestions was the following:

6H12O6 + H2O → C2H5OH + CH3COOH + 2H2 + 2CO2 (1)

n Eq. (1), ethanol production occurs concomitantly with the pro-uction of acetic acid and ethanol. For each mole of glucose that isegraded, 1 mol of EtOH, 1 mol of HAc and 2 mol of H2 were formed.imilar to the results of the current study, the main metabolites thatere obtained were HAc and EtOH. Thus, the equation suggested

y Zhu et al. [36] may be one of the pathways that were involvedn the present experiments.

Another study performed by Fang and Liu [37] on the effect ofH on hydrogen production assumed that a change in the metabolicathway would occur because of the influence of pH on the micro-ial community. Likewise, Zhu et al. [36] showed that for a pH below.5, the pathway that produces acetic acid is favored, along with anthanol pathway. At higher pH values, the authors found that all ofhese pathways acted concurrently in conjunction with the propi-nic pathway. In the present study, pH control was performed bydding NaHCO3 and HCl, which allowed the pH to be maintainedetween 4.5 and 5. The pH chosen for R124 and R188 was in the pHange for the ethanol and acetic pathways that was suggested byiu and Fang [37] and Zhu et al. [36], and the range worked well.

Another study in which ethanol fermentation was predominantas carried out by Ren et al. [30]. The authors noted that thereas a large production of metabolites that formed in accordanceith the OLR. Thus, a higher OLR applied to the reactor led to aigher production of metabolites, especially EtOH and HAc. Thisatio increased until a point was reached at which there was aecrease in the production of acids and alcohols. However, accord-

ng to the available literature, large amounts of metabolites cannhibit the metabolic activities performed by the cell. So, accord-ng to the authors, when a reactor was subjected to high organicoads, acid and CO2 saturation occurred and possibly inhibited theroduction.

Overall, R124 and R188 had a predominant ethanol-type pro-uction pathway. Alternative pathways for ethanol productionere likely engaged because the traditional pathway (Eq. (2)) doesot generate H2. Furthermore, it was verified that it is possible toroduce ethanol and hydrogen concurrently when specific condi-ions, such as low pH, are met.

6H12O6 → CH3CH2OH + 2CO2 (2)

In order to analyze the production of ethanol in a quantitativeay, the EtOH yield was calculated using calculations similar to

he ones for HY. Thus, the yield was calculated as a mol of ethanolormed per mol of glucose consumed, as illustrated in Fig. 6.

In Fig. 6, there was a slight increase in ethanol yield when theRT was reduced in both reactors. At HRTs of 8 and 4 h, the ethanolields were similar. At an HRT of 2 h, there was a higher tendencyor ethanol to form in R124.

HRT (h)

Fig. 6. Ethanol yield for each HRT in R124 and R188.

Table 4 shows the variation of the ethanol yield in multiplestudies in terms of certain variables such as upflow velocity, HRT,organic loading and pH. Wu et al. [2] varied the Vup from 0.55 to0.73 cm s−1 using three different substrates: glucose, sucrose andfructose. There was an increase in EtOH yield for all three substratesin response to an increase in Vup. In contrast, Zhang et al. [14] foundthat varying the HRT produced a reduction in the Vup. In the presentstudy, R124 and R188 had an increased EtOH yield when the HRTdecreased.

Koskinen et al. [16] also studied the influence of pH on the EtOHyield and found that at a pH of 6.9, a maximum ethanol yield of0.28 mol EtOH mol−1 glucose was achieved.

The effect of the upflow velocity in R124 and R188 on themetabolites that were formed cannot be described conclusivelybecause the hydrogen production did not show a trend for this vari-able. However, from previous studies by our group, some questionsabout the subject can be answered. Table 5 illustrates the distri-bution of metabolites formed in AFBRs as described in publishedpapers. All of the studies in the table used the same inoculum sourceand underwent the same heat treatment.

The metabolite distributions obtained in this study were dif-ferent from the ones obtained by Shida et al. [18], Amorim et al.[19] and Barros et al. [20]. There was no trend in the formationof specific metabolites when the upflow velocity in the reactorswas changed. Although the compatibility of data for hydrogen pro-duction was verified, there was significant variation between themetabolites generated by R124 and R188 as well as those pre-sented by Shida et al. [18], Amorim et al. [19] and Barros et al. [20].These results contribute to the hypothesis that a different path-way exists for ethanol production because there was no reductionin hydrogen production. Still, when all of the experiments wereanalyzed from Shida et al. [18], in which the glucose concentrationwas 2000 mg L−1 without an alkaline control; Amorim et al. [19],who worked with a glucose concentration of 4000 mg L−1 with theaddition of an alkaline agent; Barros et al. [20], who also workedwith a glucose concentration of 4000 mg L−1, there was a large pro-portion of acetic acid and butyric acid in the experiments from theliterature and relatively low amounts of ethanol.

3.4. Extracellular polymers and attached biomass

Extracellular polymeric substances (EPSs) produced by cellswere quantified in terms of protein and carbohydrates. The poly-

mers produced by cells are polysaccharides. These polymersrepresent most of the visible biomass in bioreactors. Nutrient lim-itation or changes in the microbial community could lead to theproduction of exopolysaccharides [38].

C.M. dos Reis, E.L. Silva / Chemical Engineering Journal 172 (2011) 28– 36 35

Table 5Hydrogen production rate (HPR) and the distribution of soluble metabolite products (SMP) in studies on AFBR metabolites generated from glucose.

Reference Vup (cm s−1) HPR (L h−1 L−1) HY(mol H2 mol−1 glucose)

EtOH/SMP (%)

HAc/SMP (%)

HBu/SMP (%)

HPr/SMP (%)

MetOH/SMP (%)

R124 (this study) 1.24 1.22 2.55 68.67 13.50 11.51 2.22 4.11Shida et al. [18] 1.61 1.28 2.29 7.54 52.32 40.14 – –Amorim et al. [19] 1.61 0.97 2.49 7.0 53.3 39.7 – –Barros et al. [20] 1.61 1.21 2.49 15.27 45.06 37.30 2.37 –R188 (this study) 1.88 0.93 2.26 49.40 27.09 7.99 4.33 11.19

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ig. 7. Mass of the extracellular polymers excreted in the form of protein per gramf particles to the reactors R124 and R188.

Fig. 7 shows the production of polymers for each of the reactors.he control of protein production is important because the accu-ulation of exopolysaccharides in the biofilm structure could limit

he effects of mass transfer from the liquid to the bulk. Microorgan-sms start to synthesize exopolysaccharides in situations in whichhere are limited nutrients to maintain cellular activity.

As shown in Fig. 7, R188 achieved a higher EPSs production com-ared with R124 for all HRTs, except for the 4 h HRT. Still, a lowerRT resulted in lower EPSs production in R188. Increasing the mix-

ng conditions could have led to changes in cellular metabolism thated to hydrogen production instead of EPSs production.

Fig. 8 shows the amount of EPSs in the form of carbohydrateshat adhered to the immobilization support. The presence of car-

ohydrates showed that there was a direct relationship betweenolymers and the HRT; however, this relationship could not be ver-

fied because of the operational conditions that were used in this

0

0.02

0.04

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ig. 8. Mass of extracellular polymers excreted in the form of carbohydrates perram of particles in R124 and R188.

Fig. 9. Biomass attached to particles for each HRT for R124 and R188.

study. The HY tended to increase the mass of the carbohydratesexcreted from the cells in both of the reactors. During the experi-ment, more EPSs were released in the form of carbohydrates whenthe hydrogen production in the reactor was low.

As illustrated in Fig. 8, the reactor that had the highest excretionof carbohydrates was R188. Recalling the values from the hydrogenproduction rate, the same profile was observed for the hydrogenproduction, but in reverse order, with the R124 showing betterproduction of hydrogen. We assumed that a portion of the carbohy-drates that were measured were present because of the syntheticwastewater that was used in the experiment.

High concentrations of carbohydrates on the surface of thebiofilm may have led the substrate to penetrate the biofilm. Tak-ing this possibility into account, the gradient of the concentrationbetween the reaction media and the particle surface was low. Alarger amount of carbohydrates in the reticular network of thebiofilm would lead to lower feed rates, and only a small amountof substrate would be required for hydrogen production.

Another factor that may have contributed to the productionof polymers is a high concentration of substrate [39]. The EPSsproduction is required to maintain cell activity. Thus, exopolysac-charides are formed in order to maintain cell activity. Therefore,it was assumed that for reactors in which the Vup was small, themixing conditions were not favorable for the penetration of nutri-ents through the biofilm layer. As the degree of turbulence rosebecause of the increasing Vup in the reactor, there may have beenan improvement in the parameters of mass transfer as well asbetter transit of the biofilm/substrate. So, organisms might directtheir metabolism towards the production of hydrogen instead oftowards cellular maintenance. However, this is not a conclusivefinding because no mass transfer tests were performed to evaluatethese specific parameters.

Fig. 9 compares the biomass growth on the particles according

to the HRT. R188 had the highest values of attached biomass. Ingeneral, there was biomass growth that paralleled the increase inthe HRT, which demonstrates the efficiency of the particle chosen asa support material for microbial attachment. However, this biomass

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6 C.M. dos Reis, E.L. Silva / Chemical

rowth occurred until an HRT of 2 h was reached. Afterwards, thereas a decrease in biomass attached to the particles for both R124

nd R188. Higher turbulent conditions inside the reactor at a lowRT possibly caused the biomass detachment.

Rabah et al. [40] warned of this condition when they conductedn experiment on biomass detachment. The authors noted thatncreasing the superficial velocity led to an increase in shear stress.his increase, in turn, caused a high rate of detachment for theiofilm.

. Conclusions

The present study showed the applicability of AFBRs for hydro-en production. The results showed that an increase in the Vup hasot been effect to increase the hydrogen yield neither the hydro-en production rate. And it is suggested that it should have a pointill the velocity maximize the hydrogen production in AFBRs usingxpanded clay as support material.

The reactor under the Vmf (R124) had a maximum HPR of.22 L h−1 L−1 and a HY of 2.55 mol H2 mol−1 glucose at an HRT of

h. The biogas content was between 40.53% and 67.57%, and noethane was formed.EtOH represented almost 80% of the metabolites that were

ormed. HAc was also produced. We suggested that an alternativeetabolic pathway prevailed during the experiment because both

eactors had a high production of EtOH.The EPSs production was related to the velocity in the reactor

uch that a higher Vup would lead to low EPSs production. Changesn cellular maintenance could have led to this relationship. Also,

hen the velocity was higher, more biomass adhered to the supportaterial.

cknowledgment

The authors gratefully acknowledge the financial support ofNPq, FAPESP, and CAPES.

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