i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 9 6 3 0 e9 6 3 5

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/he

The influence of the degree of back-mixing on hydrogen

production in an anaerobic fixed-bed reactor

Daniel Moureira Fontes Lima, Marcelo Zaiat*

Laboratório de Processos Biológicos (LPB), Centro de Pesquisa, Desenvolvimento e Inovação em Engenharia Ambiental, Escola de Engenharia
de São Carlos (EESC), Universidade de São Paulo (USP), Engenharia Ambiental - Bloco 4-F, Av. João Dagnone, 1100 - Santa Angelina,
13.563-120, São Carlos, SP, Brazil

article info abstract

Article history: This paper reports on results obtained from experiments carried out in an acidogenic
Received 23 November 2011 anaerobic reactor aiming at the optimization of hydrogen production by altering the degree
Received in revised form of back-mixing. It was hypothesized that there is an optimum operating point that maxi-
9 March 2012 mizes the hydrogen yield. Experiments were performed in a packed-bed bioreactor by
Accepted 19 March 2012 covering a broad range of recycle ratios (R) and the optimum point was obtained for an R
Available online 9 April 2012 value of 0.6. In this operating condition the reactor behaved as 8 continuous stirred-tank
reactors in series and the maximum yield was 4.22 mol H2 mol sucrose
1. Such optimum
Keywords: point was estimated by deriving a polynomial function fitted to experimental data and it
Acidogenic bioreactor was obtained as the conjugation of three factors related to the various degrees of back-
Hydrogen production mixing applied to the reactor: mass transfer from the bulk liquid to the biocatalyst,
Recycle ratio liquid-to-gas mass transfer and the kinetic behavior of irreversible reactions in series.
Anaerobic up-flow fixed-bed reactor Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

1. Introduction technically simpler because the hydrogen (along with volatile

acids and alcohols) is mainly derived from the carbohydrates
Fossil fuels are non-renewable sources of energy, and they are present in wastewater, it requires no light or pure cultures,
becoming increasingly scarce. In addition, their combustion and the operating costs are relatively low [4].
products contribute to global warming [1]. Due to these Several factors, such as temperature, pH, the type and
problems, researchers are looking for possible sources of operating conditions of the reactor, the form of inoculation,
energy to replace these fuels. Of the several existing alterna- the inoculum source, the treatment of the inoculums
tive energy sources, thermonuclear, solar, wind and (thermal, acid or alkaline), and the characteristics of the
geothermal energy can be highlighted. However, hydrogen wastewater, are known to affect hydrogen production in
remains the best alternative due to its unique characteristics anaerobic reactors [5]. The degree of back-mixing is one of the
compared to other fuels [2]. main factors that affect hydrogen production because it
The biological production of hydrogen can use the organic affects reaction rates and mass-transfer fluxes. The degree of
fraction present in wastewater and is less harmful to the mixing can be altered in a fixed-bed bioreactor by changing
environment because it does not consume fossil fuels [3]. The the external circulation of the liquid phase. The flow patterns
production of hydrogen from this type of process can occur in in fixed-bed reactors, which are intended to behave as plug-
two ways, photosynthesis and fermentation. The latter is flow reactors, can be changed into mixed flows by applying

* Corresponding author. Tel.: þ55 (16) 33738360; fax: þ55 (16) 33739550.
E-mail address: [email protected] (M. Zaiat).
0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2012.03.097
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 9 6 3 0 e9 6 3 5 9631

liquid recycle rates (R). According to Levenspiel [6], the recycle

reactor is a convenient method for approaching a mixed flow
with what is essentially a plug-flow device.
Besides to provide the achievement of different degree of
back-mixing in a fixed-bed reactor, recycling affects the mass
transfer rates from the bulk liquid to the biocatalyst and the
liquid-to-gas mass transfer, mainly for poorly soluble gases,
such as hydrogen [7]. While the liquid-to-solid mass transfer
resistances can reduce the organic matter uptake rates and
worsen the performance of the reactor [8], the liquid-to-gas
mass transfer resistance may result in the over-
concentration of poorly soluble gases, such as hydrogen, in
the liquid medium.
The positive effect achieved by increasing the degree of
mixing in packed-bed reactors can be contrasted with the
disadvantage of having a mixture when the desired product is
an intermediate in the reaction, such as hydrogen. The
quantitative treatment of irreversible reactions in series in
ideal reactors demonstrates that plug-flow reactors always
require less time than mixed reactors for achieving the
Fig. 1 e The up-flow anaerobic fixed-bed reactor.
maximum concentration of the intermediate. Moreover, the
maximum obtainable concentration of the intermediate in
a plug-flow reactor is always greater than that which is 2.3. Synthetic wastewater
obtainable in a mixed reactor [6].
Thus, by considering the conflicting effects of altering the The reactors were fed with a synthetically prepared affluent,
degree of back-mixing in the reactor, this study hypothesized with a chemical oxygen demand (COD) of 2000 mg L
1, sucrose
that a fixed-bed reactor has an optimum operating point for (1781.24 mg L
1)as the main carbon source, urea (11.50 mg L
1)
the flow pattern, which will maximize the production of as the nitrogen source and an optimal carbon-to-nitrogen (C/
hydrogen. This optimum point can be achieved by varying the N) ratio of 140, as determined by Rojas [10]. Micronutrients
recycle ratio that is applied to the bioreactor. were amended according to Del Nery [9], as: nickel sulfate
In this context, the main objective of this study was to (0.50 mg L
1), ferrous sulfate (2.50 mg L
1), ferric sulfate
evaluate the influence of the recycle ratio on the biological (0.25 mg L
1), calcium chloride (2.06 mg L
1), cobalt chloride
hydrogen production in an up-flow anaerobic fixed-bed (0.040 mg L
1), selenium oxide (0.036 mg L
1), potassium
reactor fed with sucrose-based synthetic wastewater. This phosphate monobasic (5.36 mg L
1), potassium phosphate
study aimed to achieve the optimum operation point as dibasic (1.30 mg L
1) and sodium phosphate dibasic
a function of the degree of back-mixing in the reactor. (2.70 mg L
1). In addition, sodium bicarbonate (500 mg L
1) and
hydrochloric acid (0.45 ml L
1/10 mol L
1) were added to adjust
the influent pH to 6.5.
2. Material and methods
2.4. The inoculum
2.1. The up-flow anaerobic fixed-bed reactor
The inoculum was obtained by the natural fermentation of the
This study was performed in a bench-scale anaerobic fixed- feed, which was prepared with tap water, and stored in an open
bed reactor (Fig. 1). The reactor was built using acrylic tubes container for three days. This process favored the fermentation of
with an inner diameter of 80 mm, an outer diameter of 88 mm the feed by the microorganisms present in the atmosphere [11].
and a length of 750 mm, a bed volume of 2.5 L and a total
volume of 3.5 L. Each reactor was divided into three 2.5. The operation of the reactor
compartments (the entrance of the influent, the effluent exit
and the fixed-bed), which were separated by a stainless steel Before starting the operation of the bioreactor, hydrodynamic
mesh (5-mm aperture) that was fixed using 5-mm rods made stimuluseresponse studies were performed for a wide range
from the same material. The reactor was fed using a positive of recycle ratios (R), which are the relationships between the
displacement pump (Prominent Concept Plus), and the liquid flow rates of the liquid returned to the reactor entrance and
was recycled from the top to the bottom of the reactor using the flow rates leaving the reactor, in order to choose a suitable
the same type of pump. range of R based on the changes in the hydrodynamic pattern.
A theoretical hydraulic retention time (HRT) of 2 h was
2.2. The support material for biomass immobilization applied, and recirculation rates of 0, 0.25, 0.5, 1.0, 2.0 and 3.0
were evaluated in abiotic experiments. The reactors were fed
The material used to attach the biomass was recycled low- with tap water, and the flow patterns were assessed by
density polyethylene, a non-porous material with a surface analyzing the response curves obtained from a pulse input of
area of approximately 7.94 m2 g
1. blue dextran, using the technique presented by Levenspiel [6].
9632 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 9 6 3 0 e9 6 3 5

The system was operated continuously at a controlled of N ) to perfect mixing (low values of N ). The chosen values of
temperature of 25  C. The HRT was set for 2 h by applying R were 0.25, 0.5, 1.0 and 2.0, for which the value of N ranged
a flow rate of 21 mL min
1, and each experiment lasted for 60 from 16 to 4. The reactor was not evaluated without liquid
days. circulation, because this condition could not be compared to
the others. When the liquid phase is recycled, the feed is
2.6. Analytical methods subjected to intermediates (organic acids and solvents), other
than the primary substrate (sucrose); without the recycle
The flow rate of biogas was recorded using a MilliGascounter ratio, only sucrose fed the reactor.
(Ritter) gas meter. The composition of the biogas (hydrogen, Fig. 3 depicts the temporal variation of the sucrose
carbon dioxide and methane) were determined by gas chro- conversion (3A), the biogas flow rate (3B), the hydrogen molar
matography (GC 2010 Shimadzu) using a thermal conductivity flow (3C) and the hydrogen yield (3D). The sucrose conversion
detector and argon as the carrier gas. The temperatures of the showed average values of 67.2%, 79.2%, 70.8% and 70.3% for R
injector, detector and column were maintained at 30  C, 200  C equal to 0.25, 0.5, 1.0 and 2.0, respectively. As the recycle ratio
and 230  C, respectively. increased, the liquid superficial velocity in the bed also
The concentrations of the organic acids and the solvents increased, thereby improving the liquid- and solid-phase
were measured by gas chromatography (Shimadzu GC 2010, mass-transfer fluxes. Thus, the overall reaction rate tends to
model AOC 5000) and an HP-INNOWax column with a length increase as the mass transfer resistances are reduced. In
of 30 m, an internal diameter of 0.25 mm and a film thickness contrast, as the recycle ratio rises, the hydrodynamic pattern
of 25 mm. The chromatograph was equipped with a FID (flame shifts from a plug flow to a mixed flow, with a marked
ionization detector), which employed the static headspace tendency to decrease substrate conversion. Such conflicting
method with crotonic acid (a standard for the acids) and iso- phenomena resulted in superior conversion obtained when
butanol (a standard for the alcohol and acetone) in an acidic the recycle rate was increased from 0.25 to 0.5 and decreased
medium containing sulfuric acid. with higher values of R.
The sucrose concentrations were determined using Substrate conversion was used to define the ending of the
a colorimetric method [12]. The volatile suspended solids transient phase and the beginning of the steady-state phase of
(VSS), chemical oxygen demand (COD) and pH values were the process, which occurred at day 23 for each of the experi-
recorded in accordance with the Standard Methods for the ments. However, the behavior of the gas phase was quite
Examination of Water and Wastewater [13]. unstable, with the values of H2 and CO2 generation rising to
their maximum values and decreasing to zero by the end of
the operation. This instability conflicts with the stability
observed for sucrose conversion, and this behavior can be
3. Results and discussion
related to the development of hydrogen-consuming microor-
ganisms in the reactor. Given that methane was not detected
The recycle ratios that were applied in the experiments were
in the gas phase and that the sulfate concentration was low in
chosen via hydrodynamic studies. The results for the real HRT
the synthetic wastewater, it is improbable that the hydrogen
were 86 min, 75 min, 55 min, 59 min, 56 min and 54 min for R of
and carbon dioxide were consumed by the hydrogenotrophic
0, 0.25, 0.5, 1.0, 2.0 and 3.0, respectively. Such values are much
methanogenic archaea and sulfate-reducing bacteria. There-
lower than those that were theoretically estimated, indicating
fore, it is probable that an anaerobic homoacetogenic micro-
the presence of stagnant backwater, mainly in the entrance of
organism is involved in the uptake of H2 and CO2.
the reactor.
While operating their reactor under conditions similar to
To evaluate the flow patterns in the reactor, the tanks-in-
those that were applied in this study, Fernandes et al. [14] and
series model was adjusted to the response curves and the
Peixoto et al. [15] concluded that the microorganisms involved
number of completely mixed reactors in series (N ) was
in hydrogen production in a fixed-bed reactor belong to the
obtained (Fig. 2). The recycle ratio that was applied in these
genus Clostridium. However, some microorganisms related to
experiments was chosen by trying to use values that would
hydrogen production can also use WoodeLjungdahl pathway
cover the hydrodynamic behavior from plug flow (high values
to reduce CO2to acetate. Tanner et al. [16] described a species
of Clostridium, Clostridium ljungdahlii, an anaerobic homo-
acetogenable to ferment sugars, other organic compounds, or
CO2/H2 and synthesis gas (CO/H2). According to Köpke et al.
[17], C. ljungdahlii grows heterotrophically in sugars (hexoses
or pentoses), or autotrophically on gases (CO or CO2 þ H2).
Acetate and methanol are the main end-products from the
autotrophic pathway. In fact, the data for the soluble products
of the operation with R equal to 0.5, which showed the best
hydrogen production results, indicated that the decrease in
biogas production coincided with a simultaneous increase in
acetic acid production. When comparing the maximum point
reached and the last day of operation, hydrogen showed
Fig. 2 e The relationship between the number of mixed a decrease of about 10.2 mmol L
1 and carbon dioxide showed
reactors in series (N ) and the recycle ratio (R). a decrease of about 6.5 mmol L
1. In turn, acetic acid showed
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 9 6 3 0 e9 6 3 5 9633

Fig. 3 e The (A) sucrose conversion, (B) biogas production (Qg), (C) Hydrogen molar flow, and (D) Hydrogen yield (YH2 ). The
dashed line divides the transient and steady-state operations with respect to the sucrose conversion.

an increase in production of approximately 3.0 mmol L
1 in recycle rate on hydrogen generation can clearly be observed in
the same period. These values are based on the stoichiometry this table.
of the metabolic pathway used by C. ljungdahlii: Statistical test ANOVA (ɑ ¼ 0.05) was performed for yield
and hydrogen production data. For the hydrogen yield, the F
4H2 þ 2CO2 / CH3COOH þ 2H2O (1) value found was 3.72 while the critical F was 2.70, thus
showing that there was a significant difference between the
The average (avg.) and maximum (max.) results obtained recycle rates tested. This difference was also found for the
for the biogas flow rate, the percentage of hydrogen and hydrogen production rate, which showed an F value of 4.38
carbon dioxide in the biogas, the hydrogen flow rate, the and a critical F of 2.70.
hydrogen yield and the hydrogen produced are shown in The hypothesis of an optimum operating point for
Table 1. The average values were calculated from the values hydrogen production with respect to the degree of back-
recorded during 60 days of operation. The influence of the mixing could be confirmed by correlating the hydrogen yield

Table 1 e Values of biogas flow rate (Qg), composition of biogas (XH2 and XCO2 ), hydrogen molar flow rate (wH2 ), hydrogen
yield (YH2 ) and hydrogen production rate (HPR) under all the recycle ratios tested.
R ¼ 0.25 R ¼ 0.5 R ¼ 1.0 R ¼ 2.0

Avg. Max. Avg. Max. Avg. Max. Avg. Max.

1
Qg (mL h ) 225.5  119.1 480.5 244.1  162.2 513.6 217.1  106.9 482.3 176.4  141.5 485.4
XH2 (%) 49.8  6.7 68.4 55.9  10.6 72.5 56.2  14.8 75.6 45.7  11.1 61.9
XCO2 (%) 36.1  5.3 50.6 39.0  6.7 52.7 34.4  9.2 52.5 37.0  5.2 48.1
wH2 (mmol H2 h
1) 4.6  2.4 9.3 5.9  3.4 13.8 5.1  2.4 8.5 3.6  2.9 9.4
YH2 (mol H2 mol
1 sucrose) 1.1  0.6 2.3 1.4  0.9 4.0 1.3  0.8 3.4 0.9  0.8 2.6
HPR (mL H2 h
1 L
1) 74.4  36.9 145.2 124.8  71.8 289.8 96.6  51.0 168.0 73.5  60.5 193.7
9634 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 9 6 3 0 e9 6 3 5

Fig. 4 e (A) The relationship between the maximum YH2 and N; (B) The relationship between the average HPR and N.

(YH2 ) and the hydrogen production rate (HPR) with the number transfer and the kinetic behavior of the irreversible reactions
of continuously stirred tanks in series (N ), which is defined as in series. The last factor is influenced by the flow pattern,
a function of recycle rate (Fig. 2). The HPR was obtained by mainly when an intermediate is the desirable product, as
dividing the hydrogen flow by the useful volume of the previously discussed.
reactor, which was calculated using a flow rate of Although the quantitative influence of each interfering
21 mL min
1. This should be applied to the useful volume of factor was impossible to obtain, the sum of all of these kinetic
the reactor (2.55 L) to obtain the theoretical HRT of 2 h and the and transport phenomena optimized the process by only
real HRT that is obtained in hydrodynamic assays. manipulating a simple operating parameter, the recycle ratio.
Fig. 4 depicts the behavior of the maximum values of YH2 The distribution of the intermediate products did not
(4A) and the average value of the HPR (4B) as a function of N. A clearly differentiate among the various operation phases.
polynomial function was fitted to the experimental data to Acetic acid, butyric acid and ethanol were the main interme-
obtain an N value that provides the optimum values of YH2 and diate products under all the recycle ratios tested, and the first
HPR. The polynomial functions were derived to search for the two are indicators of maximum hydrogen production [18]
maximum point that could be obtained from an N value of 8, (Table 2).
given optimum values of 4.22 mol H2 mol
1 sucrose and
128.13 mL H2 h
1 L
1. This value of N is related to a recycle
ratio of 0.6, which was obtained from the relationship
between N and R (Fig. 2). Such optimum values correspond to 4. Conclusions
53% of the theoretical values predicted by the stoichiometric
relationships that would be obtained if sucrose was only This paper demonstrates the relevant role of the back-mixing
converted into acetic acid. on hydrogen production in anaerobic fixed-bed reactors. A
The optimum operating point was obtained as a conjuga- value for the degree of back-mixing that optimizes hydrogen
tion of three factors related to the various degrees of back- production in such reactor was found. There was a tendency
mixing to which the reactor was subjected: mass transfer to increase the production of hydrogen by increasing the
from the bulk liquid to the biocatalyst, liquid-to-gas mass recirculation until a peak was reached, after which the
production decreased.
The optimum hydrogen yield (4.22 mol H2 mol
1 sucrose)
was obtained for an R of 0.6 when the reactor behaved as 8
Table 2 e The average concentrations of the organic acids continuous stirred-tank reactors in series. This point was
and alcohols produced under all of the experimental inferred from an empirical mathematical model as a result of
conditions. mass transfer and kinetic effects due to the application of
Average concentrations of the organic various degrees of back-mixing.
acids and alcohols (mg L
1) The reduction in biogas production observed in all of the
operation phases may have been caused by the presence of
Recycle ratio 0.25 0.5 1.0 2.0
anaerobic, homoacetogenic microorganisms, which are
Acetic acid 241.28 265.61 239.09 265.00 involved in the uptake of H2 and CO2.
Propionic acid 9.00 20.01 20.61 21.50
Isobutyric acid 0.45 8.40 34.50 0.69
Butyric acid 135.37 100.00 116.46 168.90
Isovaleric acid 0.50 3.00 4.44 0.00
Valeric acid 0.00 8.00 10.10 0.00 Acknowledgements
Caproic acid 0.00 5.02 8.65 0.00
Acetone 14.11 4.01 4.68 9.26 This work was funded by the Fundação de Amparo à Pesquisa
Methanol 0.00 0.49 0.00 0.00
do Estado de São Paulo (FAPESP e Process number 2009/15984-
Ethanol 116.70 191.87 160.24 123.00
0) and the Conselho Nacional de Desenvolvimento Cientı́fico e
n-Butanol 0.30 0.00 0.30 0.00
Tecnológico (CNPq), Brazil.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 9 6 3 0 e9 6 3 5 9635

references Dissertação (mestrado), Escola de Engenharia de São Carlos,

USP; 1987 [in portuguese].
[10] Rojas MPA. Influência da relação C/N na produção de
hidrogênio em reator anaeróbio de leito fixo [Influence of C/N
[1] Veziroglu TN, Sahin S. 21st century’s energy: hydrogen
ratio on hydrogen production in anaerobic fixed-bed reactor].
energy system. Energy Convers Manag 2008;49:1820e31.
Dissertação (mestrado), Escola de Engenharia de São Carlos,
[2] Veziroglu TN, Barbir F. Hydrogen: the wonder fuel. Int J
USP; 2010 [in portuguese].
Hydrogen Energy 1992;17(6):391e404.
[11] Leite JAC, Fernandes BS, Pozzi E, Barboza M, Zaiat M.
[3] Ren NQ, Xing DF, Rittmann BE, Zhao LH, Xie TH, Zhao X.
Application of an anaerobic packed-bed bioreactor for the
Microbial community structure of ethanol type fermentation
production of hydrogen and organic acids. Int J Hydrogen
in bio-hydrogen production. Appl Environ Microbiol 2007;9:
Energy 2008;33:579e86.
1112e25.
[12] Dubois SM, Gilles KA, Hamilton JL, Rebers PA, Smith F.
[4] Han SK, Shin HS. Biohydrogen production by anaerobic
Colorimetric methods for determination of sugar and related
fermentation of food waste. Int J Hydrogen Energy 2004;29:
substance. Anal Chem 1956;228:13e21.
569e77.
[13] APHA. Standard methods for the examination of water and
[5] Wang J, Wan W. Factors influencing fermentative hydrogen
wastewater. New York: American Public Health Association;
production: a review. Int J Hydrogen Energy 2009;34:799e811.
1998.
[6] Levenspiel O. Chemical reaction engineering. 3rd ed. New
[14] Fernandes BS, Peixoto G, Albrecht FR, Saavedra NK, Zaiat M.
York: John Wiley & Sons Inc; 2000.
Potential to produce biohydrogen from various wastewaters.
[7] Pauss A, Andre G, Perrier M, Guiot SR. Liquid-to-gas mass
Energy Sustain Dev 2010;14:143e8.
transfer in anaerobic processes: inevitable transfer
[15] Peixoto G, Saavedra NK, Varesche MBA, Zaiat M. Hydrogen
limitations of methane and hydrogen in the
production from soft-drink wastewater in an upflow anaerobic
biomethanation process. Appl Environ Microbiol 1990;
packed-bed reactor. Int J Hydrogen Energy 2011;36:8953e66.
56(6):1636e44.
[16] Tanner RS, Miller LM, Yang D. Clostridium ljungdahlii sp. nov.,
[8] Camargo EFM, Ratusznei SM, Rodrigues JAD, Zaiat M,
an acetogenic species in clostridial rRNA homology group I.
Borzani W. Treatment of low-strength wastewater using
Int J Syst Bacteriol 1993;43:232e6.
immobilized biomass in a sequencing batch external loop
[17] Köpkea M, Heldb C, Hujer S, Liesegangb H, Wiezer A,
reactor: influence of the medium superficial velocity on the
Wollherr A, et al. Clostridium ljungdahlii represents
stability and performance. Braz J Chem Eng 2002;19(3):
a microbial production platform based on syngas. PNAS
267e75.
2010;107(34):13087e92.
[9] Del Nery V. Utilização de lodo anaeróbio imobilizado em gel
[18] Levin DB, Pitt L, Love M. Biohydrogen production: prospects
no estudo de partida de reatores de fluxo ascendente com
and limitations to practical application. Int J Hydrogen
manta de lodo [Use of anaerobic sludge immobilized in gel in
Energy 2004;29:173e85.
the start study of upflow anaerobic sludge blanket reactor].