Enhancing hydrogen production of Clostridium butyricum using a column reactor with square-structured ceramic fittings

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 3 ( 2 0 0 8 ) 6 5 4 9 – 6 5 5 7

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Enhancing hydrogen production of Clostridium butyricumusing a column reactor with square-structuredceramic fittings

Markus Fritscha,**, Winfried Hartmeiera, Jo-Shu Changb,c,*aDepartment of Biotechnology, RWTH Aachen University, Sammelbau Biologie, Worringer Weg 1, D-52074 Aachen, GermanybDepartment of Chemical Engineering, National Cheng Kung University, No. 1 University Road, Tainan 701, TaiwancSustainable Environment Research Center, National Cheng Kung University, Tainan, Taiwan

a r t i c l e i n f o

Article history:

Received 27 April 2008

Received in revised form

13 July 2008

Accepted 14 July 2008

Available online 16 October 2008

Keywords:

Dark fermentation

Clostridium butyricum

Biohydrogen production

Monolith

* Corresponding author. Department of CheTaiwan. Tel.: þ886 62757575x62651; fax: þ88** Corresponding author. Tel.: þ49 2418027578

E-mail addresses: [email protected]/$ – see front matter ª 2008 Interndoi:10.1016/j.ijhydene.2008.07.070

a b s t r a c t

A new approach in bioreactor development for fermentative hydrogen production was

undertaken using monoliths (square-structured ceramic fittings) as seeds to enhance

bubble formation in the fermentation broth. Clostridium butyricum CGS2 was used

throughout the batch and continuous experiments on sucrose substrate. In a batchwise

column reactor with a COD of 20 g/L, the hydrogen-production performance increased and

caused a shift in the metabolism when the ceramic fittings were installed. Compared to the

stirred tank the H2 yield and the volumetric production rate were lower, but the specific

production rate turned out to be maximized using three monoliths. Continuous runs with

and without monoliths were carried out with various sucrose concentrations leading to

a COD loading of 10–40 g/L at an HRT of 5.5 h. Compared to the standard column reactor

(SCR), the novel monolith column reactor (MCR) showed a 35% increase in hydrogen-

production rate to 305 mL/L/h and a 30% increase in hydrogen yield to 0.92 mol per mol

hexose consumed. Most interestingly, the specific hydrogen-production rate increased by

about 60% to 292 mL/h/g CDW at a COD of 20 g/L. It is concluded that ceramic fittings in

fermentative biohydrogen production offer a great potential for further improvements

without the drawbacks of traditional methods to increase hydrogen-production

performance.

ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights

reserved.

1. Introduction been applied to maximize H2 production. Especially high-cell-

In recent years several dark fermentative processes of

hydrogen production from sugars or waste materials have

been developed in labscale [1,2]. Different reactor configura-

tions ranging from stirred tanks [3–5] to reactors with artifi-

cially immobilized [6–8] and self-immobilized cells [9–11] have

mical Engineering, Natio6 62357146.; fax: þ49 2418022387.aachen.de (M. Fritsch), chational Association for H

density processes led to the highest hydrogen-production

rates [10–12].

Most of the microorganisms in dark fermentative

systems – either in pure or mixed cultures – belong to the

genera Clostridium and Enterobacter, and the fermentation

patterns are often similar. That is why those processes also

nal Cheng Kung University, No. 1 University Road, Tainan 701,

[email protected] (J.-S. Chang).ydrogen Energy. Published by Elsevier Ltd. All rights reserved.

mailto:[email protected]

mailto:[email protected]

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often share the same limitations and problems: Clostridia –

produce hydrogen from anaerobic conversion of sugars to

organic acids, e.g., butyric and acetic acid. Other by-products –

depending on the strains, substrates and fermentation

conditions – could be other organic acids, lower alcohols or

solvents lowering the hydrogen yield [3,13–15]. While dark

fermentations are superior compared to other biological ways

in terms of hydrogen-production rate they suffer in low

hydrogen yield as defined by the amount of hydrogen

produced per substrate consumed [16,17].

Several carbon sources can be glycolytically degraded to

pyruvate and subsequently to acetyl-CoA. Thereby CO2,

reduced ferredoxin, NADH, and ATP are generated. Two

molecules of acetyl-CoA yield in butyric acid while consuming

NADH and thus regenerating NAD for glycolysis. Molecular

hydrogen is mainly produced by oxidation of the reduced

ferredoxin with ferredoxin:Hþ-oxidoreductase (hydrogenase).

No reoxidation of NADH to NAD occurs if acetic acid is the

only by-product. Therefore the redox state is not in balance.

Excess NADH has to be removed by oxidation using NADH:-

ferredoxin-oxidoreductase. Electrons are transferred to

ferredoxin which otherwise gets oxidized by hydrogenase

releasing additional molecular hydrogen [18]. But the electron

transfer from NADH to ferredoxin is only thermodynamically

feasible if the dissolved hydrogen concentration is below

a critical level. Angenent et al. [19] stated that, under standard

reaction conditions and gas-saturated liquid phase, the

hydrogen partial pressure in the reactor headspace must be

lower than 60 Pa. Then hydrogen formation can occur via the

NADH:ferredoxin-oxidoreductase pathway, and it can be

lower than 30 kPa, so that hydrogen is released via the pyr-

uvate:ferredoxin-oxidoreductase pathway. Higher yields and/

or production rates according to the law of Le Chatelier should

also be favoured at lower dissolved hydrogen concentrations

[4,20–23].

Several researchers found hydrogen supersaturations in

the liquid of biogas and biohydrogen fermentations. The

degree of gas saturation was higher than expected from offgas

analytics: Pauss et al. [24] reported 35–70 times and Frigon and

Guiot [25] reported up to 13 times higher hydrogen concen-

trations in a methane reactor. Kraemer and Bagley [20] still

measured 11-fold H2 supersaturations in hydrogen dark

fermentations although nitrogen sparging was applied. Hence

it is essential to discriminate between dissolved hydrogen

concentrations and hydrogen partial pressures in the reactor

headspace and to point out that only the dissolved gas

concentrations are of importance for the microbial behaviour.

Different approaches have been tried in dark fermenta-

tions to reduce dissolved hydrogen concentrations, e.g.,

intensive stirring [26], stripping with inert or recycle gases

[4,27–30], headspace pressure reduction [22] and gas removal

via silicon hollow fibres in the reactor [31]. Although some of

those approaches showed significant increases in hydrogen

production, the most successful cases required considerable

energy or even gas input into the reactor and led to an increase

in production costs or a decrease in product concentrations.

None of those studies focussed directly on bubble forma-

tion in the liquid as a (or maybe the) crucial step in mass

transfer of gases out of the liquid. Pauss and Guiot [32] stated

that the only way to increase the mass transfer of hydrogen

out of supersaturated liquids is to increase the interphasic

specific area. Thus, gas supersaturations can be reduced by

nucleation (commonly known as bubble formation) at pre-

existing gas cavities or nucleation sites on surfaces. That

means if you have supersaturated solutions you can improve

bubbling with seeds in the liquid whereas there are several

possible mechanisms of nucleation [33]. Monoliths provide

very high specific surfaces with its uneven structure and thus

can be suitable for that purpose. Despite the fact that those

ceramic carriers have been originally used in catalytic reac-

tions only some reports on their application in bioprocesses

exist [34–36]. However, there is only one study using mono-

liths in anaerobic systems available [37]. No applications in

biohydrogen production have been reported.

Due to their properties monoliths should be ideal to opti-

mize hydrogen mass transfer by enhancing nucleation, even

without diluting the product or the necessity of an intensive

energy input, making them subject to our study on their effect

and potential application in a fermentative biohydrogen

process.

2. Materials and methods

2.1. Strain, medium and culture conditions

Clostridium butyricum CGS2, isolated from a high-rate anaer-

obic hydrogen-producing reactor as previously described [38]

and characterized by Lo et al. [39], was used for H2 production.

To provide a viable culture over a prolonged period cell

suspension was transferred monthly in sterile 100 mL argon

flushed serum vials containing fresh medium with the

following components (given in g/L): sucrose (15), (NH4)2SO4

(3), Na2HPO4 (5), KH2PO4 (1), NaCl (2), MgSO4$9H2O (0.1), agar

(2), Na2S (0.5), Resazurin (0.001). The transfer ratio was 1:25.

The preculture for the experiments was obtained by

transferring 1.2 mL of the culture into 30 mL of a modified

agar-free medium as described before, containing 17.81 g/L of

sucrose being equivalent to a COD of 20 g/L. Incubation

temperature was 37 �C for 48 h. For the inoculation of batch

and continuous experiments 30 mL of the preculture were

given into 270 mL of anaerobic production medium as

described by Wu et al. [11]. Sucrose concentration (as the sole

carbon source) varied in COD between 10 and 40 g/L in

continuous experiments and was set to COD-value of 20 g/L

for batch experiments. The fermentations were not pH

controlled.

2.2. Reactor setup

Two different reactors were used. For batch and continuous

experiments a column type reactor system as shown in Fig. 1

consisting of a glass column (22.5 cm� 4.5 cm) with

a maximum volume of 360 mL without any internals was

established. In continuous mode liquid left the reactor via an

overflow pipe which accounted for additional 130 mL of liquid

volume. The gas from the column and the overflow was

collected at the top of the reactor and went through a Ritter

TG01 gas meter (Bochum, Germany). The volumes were cali-

brated at 25 �C under normal atmospheric pressure.

Fig. 1 – Scheme of the continuous monolith column reactor

(MCR). The total volume of the reactor including the volume

of the overflow varied between 460 and 490 mL, depending

on the number of monoliths applied to the column. For

batch experiments overflow and recycle connections were

closed and the initial liquid volume was set to 300 mL.

Fig. 2 – Top-view on a cylindrical monolith.

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 3 ( 2 0 0 8 ) 6 5 4 9 – 6 5 5 7 6551

In continuous operation the feed entered the reactor in the

middle of the column, while liquid was recycled from the top

part of the column into the bottom part. Samples for optical

density (OD) measurement were taken out of the recycle loop

to prevent errors from cell sedimentation while samples for

HPLC were taken from the overflow outlet. In batch mode the

overflow, feed and recycle outlets were closed. At the top of

the reactor a pH probe (Mettler Toledo, Giessen, Germany) was

installed and connected to the Suntex pH-Controller PC3200

(Kunshan, Taiwan). Various numbers of cylindrical-shaped

monoliths kindly provided by Rauschert Kloster Veilsdorf

GmbH (Veilsdorf, Germany) made of highly porous cordierite

with a height of 35 mm and a diameter of 25 mm were put on

each other on a metal support at the bottom center of the

column. Each of them accounted for approximately 10 mL of

loss in liquid working volume. The capillary cells of the

monoliths were quadratic with a side length of 1.53 mm each

and walls of 0.26 mm thickness resulting in 200 cells per

square inch. A picture of the ceramic fitting is shown in Fig. 2.

Due to the different configurations we distinguish between

the standard column reactor (SCR) without monoliths and the

monolith column reactor (MCR). For comparable studies

a stirred batch reactor consisting of a standard 1 L bottle from

Schott (Mainz, Germany) was used. Adequate stirring was

accomplished with a magnetic stir bar at the bottom

(3.0 cm� 0.6 cm) rotating at 750 rpm. Both reactors were set

into a temperature controlled water bath.

2.3. Batch experiments

For column and stirred tank experiments the reactors

(including the monoliths if necessary) were autoclaved at

121 �C for 20 min. Two hundred seventy milliliters of sterile

production medium as described above was given into the

reactor. The initial pH value of the medium was adjusted to 7.1

using 2.55 mL of 5 N HCl. Thirty milliliters of inoculum were

given into the reactor followed by 5 min of argon flushing to

ensure anaerobic conditions in the reactor. After that the

reactor was put into a temperature controlled water bath at

37 �C. The initial batch reaction volume was 300 mL. Samples

of gas and liquid products were taken over the course of

fermentation.

2.4. Continuous experiments

Continuous studies were performed using the column reactor

only. The start-up was accomplished with an initial batch

phase as described before. After reaching a pH of 5.5 the

continuous feed was set to a dilution rate of 0.1 h�1 until

reaching steady-state conditions. After that the hydraulic

retention time (HRT) was set to 4 h, while varying the recycle

ratio (as defined by the ratio of the recycle flow rate divided by

the feed rate) between 3 and 10 and the sucrose concentra-

tions between 10 and 40 g COD/L. The pH of the feed was

adjusted to 9.3. Samples were taken under steady-state

conditions, meaning that gas production was nearly constant.

If not noted differently at least four samples in equilibrium

were taken until the next experiment was started.

2.5. Analytical methods

Gas composition (H2 and CO2) was measured with a gas

chromatograph (GC) equipped with a thermal conductivity

detector as described elsewhere [40]. Concentrations of

sucrose and organic acids in the filtered supernatant (0.2 mm)

of the fermentation broth were analyzed using HPLC (RID-10A,

ro

du

ctio

n [m

L]

150

200

250

300

350


Shimadzu, Tokyo, Japan). Detailed procedures and equipment

are reported in our recent work [39]. Optical density (OD) was

measured at 600 nm using a Hitachi U-2100 spectrophotom-

eter (Tokyo, Japan). Cell suspensions from steady-state

continuous cultures of a known OD and volume were centri-

fuged (3690 g, 10 min) and washed twice with tab water. The

pellet was dried at 105 �C for 48 h. The cell dry weight (CDW)

was calculated and resulted in 0.22 g/L per unit OD600.

H2 p

0

50

100 0 monoliths1 monolith2 monoliths3 monolithsstirred

Bio

mass co

ncen

tratio

n

[g

C

DW

/L

]

0,0

0,5

1,0

1,5

2,0 0 monoliths1 monolith2 monoliths3 monolithsstirred

Time [h]

10 15 20 25 30 35

Sp

ecific H

2 p

ro

du

ctio

n rate

[m

L/h

/g

C

DW

]

0

100

200

300

0 monoliths1 monolith2 monoliths3 monolithsstirred

Fig. 3 – Comparison of different column reactor designs

with various numbers of monoliths and a stirred reactor in

batch experiments regarding hydrogen production,

biomass concentration and specific hydrogen-production

rate. The initial pH of the culture was set to 7.1. The

temperature was controlled at 37 8C. The initial working

volume was 300 mL.

3. Results and discussion

3.1. The effect of monolith on batch H2 production

Fig. 3 compares the H2-production performance of column

batch experiments using various numbers of monoliths with

the stirred fermenter. Lag time varied between 12 and 18 h

until rapid gas production occurred, consisting only of H2 and

CO2. The control experiment without any monoliths or stir-

ring shows the lowest hydrogen-production rate and H2 yield.

Table 1 summarizes the production parameters estimated by

a modified Gompertz regression as described elsewhere [41].

While the stirred experiment still showed the highest

hydrogen-production rate and yield, the application of

monoliths in the column reactor increased the H2 yield about

50% from 0.55 to 0.82 mol per mol hexose consumed, whereas

the application of two and three monoliths gained similar

results. Sucrose conversion was incomplete, varying from 50.6

to 57.3%. Hence, the H2 yield from total sugar is slightly better

in the three-monolith experiment (0.46 mol per mol total

hexose) compared to the two-monolith experiment. Incom-

plete sucrose conversion seems to be due to a decreasing pH to

values around pH 5 and low contact between substrate and

cells or a substrate inhibition, since the medium composition

proved to be sufficient for total sugar conversion in various

studies before [6,11,39,42]. It is often reported that Clostridia

neither grow nor produce hydrogen at pH values around 5

[2,38,43,44].

Generally biomass concentrations were low. None of our

experiments showed the formation of granules, only sus-

pended cells were observed. The batch growth curves in Fig. 3

show the highest biomass concentrations during stirred and

non-stirred column fermentations whereas the monolith

experiments only reach lower values. Subsequently the

specific H2-production rates calculated from these numbers

show the highest value for the three-monolith experiment,

while the two-monolith experiment shows a comparable

range as the stirred reactor. Increasing the number of mono-

liths applied increased the specific hydrogen-production

rates. This indicates that the presence of monoliths improves

environmental conditions. Most likely the liquid concentra-

tions of hydrogen (and carbon dioxide) were lowered due to

the increased bubble formation. Thus enhancing the mass

transfer of the gaseous products out of the liquid lowers H2

inhibitions of the enzymatic hydrogen evolving steps in the

broth [23,26]. Although a better H2-production performance

was achieved in the stirred experiments, the monolith

experiments gained remarkable results without any addi-

tional energy input. That is why this configuration was chosen

for the continuous experiments. Given the fact that no stirring

or pH control was applied to the system the monolith exper-

iments led to noteworthy results in terms of specific

hydrogen-production rates [38,45].

3.2. Soluble metabolites in batch H2 fermentation

The final soluble metabolite concentrations of the performed

batch experiments are listed in Table 2. The supernatant only

contained lactate, formate, acetate, and butyrate. It can be

seen that butyrate was the main by-product followed by

formate and lactate in nearly equal amounts. Acetate turned

Table 1 – Patterns of H2 production in various batch reactors using 20 g COD/L

Monoliths vH2 max

[mL/L/h]x

H2

production [mL]YH2 [mol H2 per molhexose consumed]

Sucroseconversion [%]

Final pH

Column

reactor

0 140 173 0.55 52.0 4.99

1 195 217 0.70 50.6 4.93

2 230 278 0.82 53.4 5.06

3 210 280 0.81 56.8 4.94

Stirred tank 0 383 328 0.95 57.3 5.03


out to be the least produced metabolite. Whereas the

production of acetate or butyrate ideally lead to the formation

of 4 or 2 mol H2 per mol acid formed [46], respectively, other

metabolites as lactate and biomass as well consume hydrogen

or are unfavourable to H2 production when produced [46,47].

Thus, it is consistent that the experiments with the highest

hydrogen yields also show the highest butyrate production.

No clear trend can be seen in acetate production since its

concentration scatters around 0.82 g/L regardless of the

numbers of applied monoliths. It is also noted that for the

stirred reactor the acetate concentration was considerably

higher than for the unstirred ones.

Increased hydrogen yields can either be obtained by

a higher acetate and butyrate production or a smaller hydrogen

consumption through the production of less lactate. It could be

seen from our experiments that lactate concentrations also

gradually decreased up to 17% when more monoliths were

applied in the column. Similar observations were seen for

formate with an exception for the two-monolith batch.

Increased mass transfer by intensive stirring was found to

change the metabolic pattern of Clostridium batch fermenta-

tions elsewhere before [26]. Assuming that monoliths increase

bubble formation and decrease dissolved gas concentrations

in the liquid, our findings of lower lactate concentrations with

increasing number of monoliths applied can be interpreted as

the result of a lower tendency of C. butyricum CGS2 to regen-

erate NAD for glycolysis through electron transfer from NADH

on pyruvate. In addition, the lower dissolved hydrogen

concentrations shift the metabolism to a more butyrate-type

fermentation in batch experiments.

3.3. The effect of monoliths on continuous H2 production

Two separate sets of continuous hydrogen fermentations in

the standard column (SCR) and monolith column reactor

(MCR) were accomplished at different sucrose feed concen-

trations. Starting with a COD of 20 g/L the feed concentration

Table 2 – Final concentrations of soluble metabolites inbatch experiments

Monoliths Lactate[g/L]

Formate[g/L]

Acetate[g/L]

Butyrate[g/L]

Column

reactor

0 1.11 1.30 0.83 2.17

1 1.05 1.05 0.73 2.15

2 0.92 1.16 0.91 2.29

3 0.92 1.01 0.82 2.42

Stirred tank 0 0.93 1.03 1.02 2.45

was stepwise shifted to a COD of 40 g/L and then lowered to

10 g/L. Each shift was done after the previous experiment has

been measured under steady-state conditions. At a COD of

40 g/L the recycle ratio in the column was kept at 3 and 10 to

further investigate for mass transfer limitations. The

hydraulic retention time for all investigations was set to 5.5 h.

Table 3 summarizes the hydrogen-production character-

istics. The steady-state pH and optical density can be found in

Table 4. It turned out that the sugar conversion was incom-

plete in both investigated reactor configurations although the

pH value was ranging well above 5.5 in all experiments. The

sucrose conversion degree generally ranged between 43 and

99% and decreased with increasing substrate concentration.

Conversely pH rather increased with decreasing sucrose feed

concentration. Compared to the corresponding batch experi-

ments the hydrogen-production rates increased about 60 and

45% at a COD of 20 g/L without and with monoliths in the

continuous fermentations, respectively. Parallel the yields

increased about 29 and 13% which is due to the increased

levels of the pH in the continuous fermentations.

The maximum hydrogen-production rates reached

237 mL/L/h at a COD of 30 g/L for the SCR and 305 mL/L/h at

a COD of 20 g/L for the MCR. Generally the production rates for

hydrogen were in a narrow range for COD of 20–40 g/L, but

significantly above experiments with a COD of 10 g/L only.

Since the hydrogen yield and the sucrose conversion reach

maximum values at a COD of 20 g/L for both reactor configu-

rations, a substrate inhibition above a COD-value of 20 g/L is

seen to be the reason for the behaviour of C. butyricum CGS2.

Similar reports are available for the related strain CGS5 [38].

An insufficient contact between substrate and cells in the

column reactor cannot be the reason, since the increase in the

recycle ratio of reactor broth from 3 to 10 at a COD of 40 g/L

only showed a minor impact in the hydrogen-production

performance in the SCR.

Our investigations show that the MCR increased the

hydrogen-production performance compared to the SCR.

Hydrogen-production rates at optimum substrate concentra-

tion increased about 35% and the hydrogen yield about 30% to

0.92 mol hydrogen per mol of hexose consumed. Most inter-

estingly, as can be seen in Fig. 4, the specific hydrogen-

production rate at a COD of 20 g/L increased about 60% to

292 mL/h/g CDW which is a remarkable value for a non-stirred

and non-pH-controlled dark fermentation [45]. However, since

the optical density was rather low, the volumetric hydrogen-

production rate only increased from 225 mL/L/h to 305 mL/L/h.

The offgas only contained H2 and CO2 whereas the hydrogen

concentration varied between 48 and 58%, with similar

patterns in both reactor configurations. This implies that the

Table 3 – Hydrogen production in continuous dark fermentations under various sucrose influent concentrations anddifferent column reactor configurations

Reactor SucroseCOD [g/L]

Recycleratio

vH2

[mL/L/h]cH2 [%] Specific H2

production rateCDW [mL/h/g]

YH2 [mol H2 per molhexose consumed]

Sucroseconversion [%]

SCR 20 3 225� 7.2 52� 0.42 182� 4.0 0.71� 0.02 80� 0.46

30 3 237� 5.7 52� 0.27 206� 5.0 0.63� 0.02 58� 2.51

40 3 212� 6.8 52� 0.17 187� 4.1 0.62� 0.05 43� 2.16

10 3 61� 0.2 55� 0.98 63� 0.8 0.31� 0.01 99� 0.32

MCR 20 3 305� 8.6 52� 0.28 292� 25.7 0.92� 0.02 84� 0.96

30 3 300� 9.8 51� 0.48 278� 2.8 0.82� 0.02 61� 0.66

40 3 270� 7.5 51� 0.3 250� 10.2 0.65� 0.04 52� 2.91

10 3 88� 2.2 58� 1.2 121� 3.3 0.44� 0.02 90� 1.31

SCR 40 10 247� 6.8 48� 0.71 202� 3.4 0.64� 0.07 49� 5.00

MCRa 40 10 255� 7.8 52� 0.23 221� 6.1 0.59� 0.03 54� 1.25

a Deviation from two measurements.


ratio of produced H2 and CO2 was not affected, thus the slightly

higher pH values in the MCR fermentations were not due to

reactions between the CO2 and the bicarbonate buffer.

The enhanced specific hydrogen-production rate in the

MCR implies optimized fermentation conditions for C. butyr-

icum CGS2. We believe that the high specific surfaces of the

monoliths increased bubble formation in the liquid super-

saturated with hydrogen and thus decreased hydrogen

inhibitions.

3.4. Soluble metabolites in continuous H2 fermentation

Since the hydrogen-production characteristics in continuous

experiments differed from batch experiments it was likely to

detect changes in the concentrations of metabolic products. It

can be seen from Table 4 that the predominant soluble prod-

ucts were lactate followed by butyrate. While lactate

concentrations nearly quadrupled compared to batch experi-

ments at a COD of 20 g/L, butyrate concentrations only

increased about 24 and 52% with and without the use of

monoliths, respectively. Less formate was detected while the

concentration of acetate was in a comparable range as in

Table 4 – Soluble metabolites and growth parameters from conconcentrations and different column reactor configurations

Reactor SucroseCOD [g/L]

Recycleratio

Lactate[g/L]

Forma[g/L]

SCR 20 3 4.1� 0.09 0.7� 0

30 3 4.3� 0.20 0.7� 0

40 3 4.5� 0.24 0.6� 0.

10 3 3.4� 0.18 1.0� 0.

MCR 20 3 4.2� 0.13 0.4� 0.

30 3 4.7� 0.07 0.5� 0.

40 3 4.8� 0.19 0.4� 0.

10 3 5.2� 0.28 0.3� 0.

SCR 40 10 4.7� 0.30 0.6� 0

MCRa 40 10 5.1� 0.30 0.2� 0

a Deviation from two measurements.

batch experiments. The MCR showed higher acetate produc-

tion than the column reactor without monoliths. The strong

accumulation of organic acids is seen to be the major reason

for the low hydrogen yields.

Overall, more soluble metabolites were produced since

more sucrose was converted in the continuous runs than in the

batchwise reactions corresponding to the enhanced hydrogen-

production performance in the continuous system. This could

be due to the increased pH value in the reactor. Maybe the

higher pH also caused the shift to lactate production.

Each set of experiments with COD of 20–40 g/L shows

comparable metabolic profiles which also correlates to their

similar hydrogen-production profiles as reported in Section

3.3. Not only the runs with a COD of 10 g/L show much lower

concentrations of lactate and butyrate but also a much lower

gas production. In general, the continuous MCR shows higher

acetate and lactate concentrations while the concentrations

of formate were lower compared to the runs without mono-

liths. Since the lactate and butyrate levels are generally high it

is also possible that not a substrate but a product inhibition

caused by organic acids contributed for low conversions and

hydrogen production [4,48].

tinuous dark fermentations under various sucrose influent

te Acetate[g/L]

Butyrate[g/L]

Opticaldensity

pH

.03 0.7� 0.03 3.3� 0.12 5.6� 0.25 5.9� 0.02

.05 0.9� 0.04 3.5� 0.11 5.2� 0.21 5.9� 0.03

06 0.8� 0.07 3.1� 0.20 5.1� 0.20 5.8� 0.04

07 0.5� 0.05 1.8� 0.03 4.4� 0.09 6.3� 0.01

04 1.0� 0.05 3.3� 0.15 4.5� 0.19 6.1� 0.03

02 1.0� 0.07 3.4� 0.12 4.9� 0.10 6.1� 0.04

09 0.9� 0.03 3.4� 0.15 5.0� 0.23 6.1� 0.01

02 0.8� 0.03 2.9� 0.13 3.9� 0.17 6.7� 0.03

.07 1.0� 0.03 3.7� 0.19 5.6� 0.20 5.9� 0.01

.02 0.9� 0.05 3.1� 0.20 5.3� 0.14 6.0� 0.02

Fig. 4 – Specific production rates in continuous column

dark fermentations with and without the use of monoliths.


Although the batch monolith experiments showed

decreasing lactate concentrations with increasing numbers of

monoliths, in the continuous system no lactate decrease

could be detected. However, the application of monoliths still

increased the hydrogen production. Other possible reasons

therefore include favourable H2 production due to less

biomass growth, higher acetate and lower formate produc-

tion, and a better contact between cells and substrate. Further

investigations are necessary to understand those patterns.

3.5. Practical and future aspects for using monoliths indark H2 fermentation

Since we believe that the surface of the monoliths is impor-

tant for enhancing the nucleation of gaseous products in the

liquid, biofilm formation on the monoliths would limit this

effect. After 3 weeks of continuous fermentation we did not

observe any visible biofilms except only a staining of the

monoliths due to the presence of Na2S in the medium. This

finding has to be confirmed in future long-term studies. The

fact that we did not observe any biofilms also underlined that

our calculations of the specific hydrogen-production rates

based on the suspended cells was appropriate.

Although our batch studies using monoliths did not ach-

ieve a higher hydrogen-production performance than that of

the stirred reactor, we still believe that the application of

monoliths in dark fermentations can be of future impact since

no additional energy input in the reactor is necessary to gain

mass transfer enhancement. This and the fact of not diluting

the product gas are the major advantages of our system

compared to other methods used to increase hydrogen-

production yield like gas stripping or vacuum conditions

[4,20,22,27]. Since the stirred batch reactor showed the best

performance in this study, a combination of monoliths and

stirring might be a promising approach in the future. This

study was only preliminary and we expect big potential in

optimized reactor designs like stirred monolith reactors or

monoliths in the recycle loops of high-density column reac-

tors, which may lead to marked improvement in hydrogen

production.

4. Conclusions

This study was the first reported investigation of using

monoliths in dark hydrogen fermentation. Since those

ceramics have very high specific surfaces we wanted to see if

its tendency of enhancing bubble formation in supersaturated

liquids could be a benefit for bacterial hydrogen production in

a column reactor without the disadvantages of other mass

transfer enhancing strategies. C. butyricum CGS2 was chosen

for cultivation on sucrose and showed a mixed acid fermen-

tation pattern.

First we studied the effect of monoliths in batchwise

column reactions and compared them to a stirred fermenta-

tion. Although the stirred reaction showed slightly better

H2-producing performance, the application of monoliths

stepwise increased the hydrogen production in rate and yield

without any additional energy input. Also changes in the

metabolic pattern were observed when monoliths were

introduced. After that continuous column reactions with and

without monoliths were accomplished. This further enhanced

hydrogen production. Especially the specific hydrogen-

production rate was significantly enhanced through monolith

application. Using different sucrose feed concentrations it

turned out that the investigated strain is inhibited by sucrose

concentrations equivalent to a COD above 20 g/L.

Our study showed that monoliths are a novel promising

approach to enhance hydrogen production in dark fermenta-

tions without the drawbacks of energy or gas input and should

be further investigated in future studies of this context.

Acknowledgements

The authors appreciated the support of National Cheng Kung

University (Project No. A029), National Science Council of

Taiwan (Grant Nos. 96-2628-E-006-004-MY3, 96-2218-E-006-

295 and 95-2221-E-006-164-MY3) and the German Academic

Exchange Service (Grant No. 332 404 106). Also we like to thank

Mr. Yung-Chung Lo and Mrs. Yi-Chen Su for their assistance

with the experiments.

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Enhancing hydrogen production of Clostridium butyricum using a column reactor with square-structured ceramic fittings