See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/229357669

Enhancement of Hydrogen Production from Glucose by Nitrogen Gas Sparging

Article in Bioresource Technology · May 2000

DOI: 10.1016/S0960-8524(99)00130-3

CITATIONS READS

544 311

5 authors, including:

Richard M Dinsdale
University of South Wales
167 PUBLICATIONS 11,404 CITATIONS

SEE PROFILE

All content following this page was uploaded by Richard M Dinsdale on 25 May 2022.

The user has requested enhancement of the downloaded file.

 Bioresource Technology 73 (2000) 59±65

Enhancement of hydrogen production from glucose by nitrogen gas

sparging
Osamu Mizunoa, Richard Dinsdaleb, Freda R. Hawkesc, Dennis L. Hawkesb,*,
Tatsuya Noikea
a
Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Sendai, Aoba 980-8597, Japan
b
School of Design and Advanced Technology, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UK
c
School of Applied Sciences, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UK
Received 2 July 1999; received in revised form 13 August 1999; accepted 18 August 1999

Abstract
The e€ect on hydrogen yield of N2 sparging was investigated in non-sterile conditions using a hydrogen-producing mixed culture
previously enriched from soya bean meal. A continuous stirred-tank reactor (CSTR) at 35°C and pH 6.0 was operated on a mineral
salts-glucose (10 g lÿ1 ) medium at a hydraulic retention time (HRT) of 8.5 h, and organic loading rate of 27.02 g glucose litre
reactorÿ1 dayÿ1 . Results are reported from an 8 week period of continuous operation, and the enrichment culture gave stable results
over an extended period. A hydrogen yield of 0.85 moles H2 /mole glucose consumed was obtained after 5 HRT, the gas produced
being 53.4% H2 . With N2 sparging at a ¯ow rate approximately 15 times the hydrogen production rate, the hydrogen yield was 1.43
moles H2 /mole glucose consumed. The speci®c hydrogen production rate increased from 1.446 ml hydrogen minÿ1 gÿ1 biomass to
3.131 ml hydrogen minÿ1 gÿ1 biomass under sparging conditions. It is suggested that hydrogen partial pressure in the liquid phase
was an important factor a€ecting hydrogen yield. Energy could be recovered as hydrogen from processes generating volatile fatty
acids for ®ne chemicals and liquid bio-fuels or from acidi®cation reactors preceding normal anaerobic biological treatment of sugary
wastewaters. Ó 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Hydrogen partial pressure; Biological hydrogen production; Hydrogen yield; N2 sparging

1. Introduction of hydrogen however is potentially more attractive, es-

Hydrogen gas is a clean fuel, producing H2 O as its
only by-product as it burns, not contributing CO2 , NOx ,
sulphur or particulates to global atmospheric pollution.
It has a high energy content per unit weight (122 kJ gÿ1 )
and thus would have considerable possibilities as a fuel
if the cost were low enough. With the development of
storage technologies (e.g. as metal hydrides) it can be a
multipurpose fuel. For nations such as Japan which
import petroleum-based fuels, research on hydrogen
production is particularly signi®cant, and has become a
focus of governmental support (Benemann, 1996; Ueno
et al., 1996; Lay et al., 1999).
Hydrogen can be generated in a number of ways, for
example through fossil fuel processing, or by electrolysis
using solar power. However, these processes are energy
intensive and therefore expensive. Biological production
*
Corresponding author.
pecially if wastewater or other biomass could be used as
the raw material. The two biological routes to hydrogen
production, photosynthetic and fermentative, have been
extensively reviewed by Nandi and Sengupta (1998) and
Benemann (1996). These and other workers (e.g. Yokoi
et al., 1995) point to the advantages of the anaerobic
fermentative route since H2 can be generated using less
complex plant from a large number of sources such as
refuse or waste products. Glucose or other carbohy-
drates are the preferred carbon source for the fermen-
tations, which give rise to acetic and butyric acids
together with hydrogen gas:
C6 H12 O6 ‡ 2H2 O ! 2CH3 COOH ‡ 2CO2 ‡ 4H2 1†
C6 H12 O6 ! CH3 CH2 CH2 COOH ‡ 2CO2 ‡ 2H2 2†
The e‚uent from fermentative hydrogen generation,
rich in organic acids, could be further exploited; e.g. by
methanogenesis.

0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 3 0 - 3
60 O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
From the ratios of acetic and butyric acids often
formed, a hydrogen yield of approximately 2.5 mol H2 /
mol hexose degraded can be expected, or approximately
0.3 m3 kgÿ1 carbohydrate utilised, with about 60% H2 in
the o€-gas. However it is dicult to establish a high
hydrogen yield because the amount of fermentation
products is signi®cantly in¯uenced by various factors
such as nutrient levels (Bahl and Gottschalk, 1984;
Dabrock et al., 1992), stirring (Lamed et al., 1988), and
levels of carbon dioxide (Tanisho et al., 1998).
Hydrogen production by a wide range of bacterial
species, both pure and mixed, de®ned, cultures grown on
sterile medium, and unde®ned enrichment cultures
grown in non-sterile conditions, has been reviewed by
Nandi and Sengupta (1998). From an engineering point
of view a process using a stable enrichment culture
yielding hydrogen from non-sterile organic wastes is
required. Roychowdhury et al. (1988) demonstrated
hydrogen production by enrichment cultures from cane
juice, corn pulp and sacchari®ed cellulose, but not in
continuous culture. Mizuno et al. (1997) reported hy-
drogen production from tofu manufacturing waste in
batch culture from the culture used in the experiment
reported here. Kalia et al. (1994) studied hydrogen
production from damaged wheat grains by Bacillus li-
cheniformis in continuous culture over a 40 day period,
though low yields were reported The experiments of
Ueno et al. (1996) on hydrogen production from sugar
factory wastewater by a mixed micro¯ora in chemostat
culture are the most successful known to the authors.
Operation at a hydraulic retention time (HRT) of 0.5
days for 20 days is reported, giving a good hydrogen
yield.
Hydrogen partial pressure in the liquid phase is one
of the key factors a€ecting hydrogen production. Many
controversial observations regarding the in¯uence of
hydrogen gas on the anaerobic breakdown of saccha-
rides have been reported (Ruzicka, 1996). Tanisho et al.
(1998) on sparging with argon obtained an increase in
residual NADH, which might be expected to give an
increased hydrogen production, although the hydrogen
production was not actually measured. These authors
found the same e€ect on NADH when sparging with
hydrogen, and attributed this to CO2 removal.
In this study we examined the e€ect of nitrogen
sparging on hydrogen yield in a continuous culture of
mixed anaerobic micro¯ora operating on a glucose-
mineral salts non-sterile medium.
2. Methods
2.1. Inoculum
The anaerobic micro¯ora (predominantly Clostridium
sp.) was obtained from fermented soybean-meal
(ESPRIT, 1989; Lay et al., 1999) and maintained in the
laboratories of Tohoku University on a sucrose mineral
salts medium in continuous culture at 35°C and a 10 h
HRT at an uncontrolled pH of between 4.7 and 5.0,
stirred by gas recirculation. After one week in transit to
the UK at ambient temperature, the 50 ml culture was
used to inoculate 500 ml glucose-mineral salts medium
in a stoppered 1 l ¯ask at 35°C without pH control, with
a gas outlet under water. An addition of 2 g lÿ1 of
Na2 CO3 was made to bu€er the medium. This culture
produced hydrogen after 3 days incubation, and the pH
dropped by the fourth day to pH 4.5. The 500 ml culture
was used to inoculate 1 l of growth medium and after 1
dayÕs growth this was used to inoculate the continuous
stirred tank reactor (CSTR).
2.2. Glucose-mineral salts de®ned medium
The medium contained the following ingredients in 1 l
of tap water (modi®ed from Mizuno et al., 1998):
NH4 Cl, 2600 mg; K2 HPO4 , 250 mg; MgCl2 á 6H2 O, 125
mg; FeSO4 á 7H2 O, 5 mg; CoCl2 á 6H2 O, 2.5 mg; MnCl2 á
4H2 O, 2.5 mg; KI, 2.5 mg; Na2 MoO4 á 2H2 O, 0.5 mg;
H3 BO4 , 0.5 mg; NiCl2 á 6H2 O, 0.5 mg; ZnCl2 , 0.5 mg.
Glucose was added to a ®nal concentration of 10 g lÿ1 .
To prevent excessive foaming, anti-foam agent (Dehy-
san Z 2111, Henkel KGaA, Dusseldorf, Germany) was
added to the nutrient reservoir to a ®nal concentration
of 0.3 ml lÿ1 . Medium was made up freshly every day
(weekdays) and 2.5 days (weekends) at 13 times the ®nal
concentration, and diluted by tap water at the point of
delivery to the reactor. The concentrated mineral salts
nutrient solution was adjusted to below pH 2 with HCl.
The 130 g lÿ1 glucose solution and the mineral salts
medium were not sterilised or refrigerated, and micro-
bial degradation did not take place under the conditions
described here.
2.3. Operation of CSTR
An anaerobic CSTR of 2.5 l capacity with 2.3 l
working volume and a 8.5 h retention time, mixed by
mechanical stirring at 100 rpm, was used. A schematic
of the experimental apparatus is given in Fig. 1. The
reactor was ®tted with a di€user for use in sparging
experiments. Circulation through the water jacket was
by a Grant FH15 ¯ow heater, (Cambridge Instruments,
Cambridge, UK) adjusted to maintain the reactor tem-
perature at 35°C ‹ 1°C. The pH value was kept constant
at 6.0 by automatic titration (Kent EIL 9142 pH con-
troller (ABB Kent-Taylor Ltd., Cambridge, England))
with a Watson-Marlow 101U pump (Falmouth,
England) using 6M sodium hydroxide. The culture was
maintained in these steady state conditions for 50 days
before the experiments reported here. Gas production
rate was measured continuously by a low ¯ow gas meter
 O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
Fig. 1. Anaerobic continuously stirred tank reactor.
developed in this laboratory (Guwy et al., 1995) cali-
brated using a bubble ¯ow meter (Phase Separations,
UK). The data were logged to a Viglen 486DX 33
computer ®tted with a RTI data acquisition board
down-loading ®les to Excel for data processing.
In duplicate experiments, normal conditions were
followed by gas sparging. Nitrogen (GC grade, Distillers
Company, UK) containing less than 2 ppm O2 was used
as sparging gas at a ¯ow rate of 110 ‹ 2.6 ml minÿ1
measured using a bubble ¯ow meter (Phase Separations,
UK). After 50 days of normal operation, nitrogen was
sparged for a 49 h period, followed by a return to nor-
mal conditions for 5 days. A second period of sparging
lasted for 4.5 days.
2.4. Chemical analysis
Hydrogen was determined with a gas chromatograph
(Varian 6500) equipped with a thermal conductivity
detector and a 2.0 m (1/4 in. inside diameter) steel col-
umn ®lled with Porapak Q (50/80 mesh). Nitrogen was
used as the carrier gas at a ¯ow rate of 40 ml minÿ1 . A
1 ml sample was injected except during calibration. A
three-point calibration was performed daily for analysis
61
of the gas produced in normal operation, when 1.0, 0.5
and 0.1 ml of 100% H2 were used. For sparging condi-
tions, 0.1 and 0.05 ml of 100% hydrogen and 5 ml of
0.2117% hydrogen in nitrogen (BOC Gases, Guildford,
UK) were used.
Carbon dioxide and methane were determined with a
gas chromatograph (Varian 3400cx) equipped with a
thermal conductivity detector and a 2 m (1/8 inch inside
diameter) steel column ®lled with Poropak T (80±100
mesh). The temperatures at the injection port, column
and TCD were 110°C, 60°C and 200°C, respectively.
Helium was used as the carrier gas at a ¯ow rate of 30 ml
minÿ1 . A single point calibration was performed with
41.52% CO2 in methane, supplied by BOC Gases,
Guildford UK. The limit of detection was approxi-
mately 1%.
Volatile fatty acids (VFA, C2 ±C5 ) were determined by
gas chromatography as in Peck et al. (1986). Other
fermentation end-products were determined by gas
chromatography (Pye Unicam 104, Cambridge) ®tted
with FID and a Perkin Elmer LCI-100 Integrator
(Perkin Elmer, Beacons®eld). Centrifuged aqueous
samples were injected on to a 2 m long (2 mm id) glass
column packed with 80/120 Carbopack BAW/6.6% PEG
62 O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
20M (Supelco/Sigma Aldrich UK). Helium was used as
the carrier gas at a ¯ow of 30 ml minÿ1 and with an oven
temperature ramp of 80°C to 200°C at 4°C minÿ1 . The
glucose concentration was measured colourimetrically
(Dubois et al., 1956). Biomass concentration was de-
termined according to Standard Methods (APHA,
1992).
3. Results and discussion
The culture obtained from Japan was easily grown up
to form an inoculum despite 7 days in transit at ambient
temperature. It recovered from a period of accidental
washout in continuous culture, after which the reactor
was re-inoculated from growth deposited in the exit U
bend tube, recovering steady state biomass levels within
4 days. The culture operated at an 8 h retention time and
was white in colour. Where biomass was stagnant an-
aerobically, a black colour appeared. While the culture
is predominantly Clostridium sp., studies at Tohoku
University have shown the presence of both sulphate-
reducing and methanogenic bacteria in this enrichment
culture, and growth at longer HRTs allows these to
develop.
The biomass concentration in the reactor was for
non-sparging conditions on average 1.45 ‹ 0.25 g lÿ1 ,
and for sparging conditions after 5 HRTs 1.06 ‹ 0.08 g
lÿ1 . A change in the state of the biomass was visible after
several days sparging, from a ¯occulating appearance
throughout the reactor to a homogeneous culture. Pos-
sibly the increased mixing on sparging disrupted the
¯ocs. The decrease in biomass on sparging might pos-
sibly suggest the ¯ocs were incompletely suspended by
the stirrer device and some biomass retention was al-
lowed to occur which was not possible in the sparging
Table 1
Liquid phase parameters
ÿ1 a
Residual glucose (mg l )
Glucose removal %a
Biomass (g lÿ1 )a
Speci®c loading rate (g glucose gÿ1 biomass dayÿ1 )
a
Four samples measured.
Table 2
Gas phase parameters
ÿ1 ÿ1
Biogas production rate (ml min l )
Hydrogen %a
Hydrogen production rate (ml minÿ1 lÿ1 )
Hydrogen yield (mol molÿ1 glucose)
Speci®c hydrogen production rate (ml minÿ1 gÿ1 biomass)
a
Six samples measured.
situation. Alternatively, physiological changes could
lead to a lower growth yield in spaxrging conditions. It
should be noted that the nitrogen gas used was 99.998%
pure, and that no pretreatment was used to remove the
O2 which could have been present. Possibly these traces
of O2 may have a€ected the growth of the anaerobic
biomass. Low biomass yields may be advantageous in
the production of VFA for chemical re®ning as the
feedstock may be directed to volatile products.
The organic loading rate to the reactor was 27.02 g
glucose lÿ1 dayÿ1 . E‚uent glucose levels during the
whole period of operation reported here were low
(Table 1), averaging 76.9 mg lÿ1 in non-sparging and
45.4 mg lÿ1 in sparging conditions. Thus over 99% of the
added glucose was converted to fermentation end
products. The speci®c organic load to the biomass was
18.63 g glucose gÿ1 biomass dayÿ1 for sparging condi-
tions after 5 HRT, and 25.37 g glucose gÿ1 biomass
dayÿ1 in sparging conditions.
Average gas production rate from the reactor during
non-sparging operation after 5 HRT was 8.9 ml minÿ1 .
Average hydrogen measured in the gas was 53.4% dur-
ing this period of non-sparging operation, and 5.3%
measured after 5 HRT during sparging (Table 2).
Measurements of the rate of ¯ow of the nitrogen
sparging gas showed that this averaged 110.0 ‹ 2.6 ml
minÿ1 . Allowing for dilution by this background level of
nitrogen sparging gas, the rate of gas production during
sparging by the whole reactor was on average 33.2 ml
minÿ1 . From these measurements the hydrogen con-
centration in the biogas produced during sparging was
22.9%.
The hydrogen yield during steady-state operation,
based on the average gas ¯ow and average hydrogen
concentration, was 0.85 moles hydrogen/mole glucose
utilised. Similar calculations for the hydrogen yield
Non-sparging Sparging
76.9 ‹ 35.4 45.4 ‹ 13.6
99.2 ‹ 0.3 99.5 ‹ 0.1
1.45 ‹ 0.25 1.06 ‹ 0.08
18.38 25.49
Non-sparging Sparging
3.895 ‹ 1.15 14.499 ‹ 3.035
53.4 ‹ 1.7 5.3 ‹ 0.2
2.08 3.31
0.85 ‹ 0.32 1.43 ‹ 0.12
1.434 3.126
 O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
under sparging conditions gave 1.43 moles hydrogen/
mole glucose utilised. The hydrogen partial pressure in
the reactor liquid during sparging conditions might have
been an order of magnitude lower.
Traces of methane were found in the biogas, 2%
methane with 41% carbon dioxide in non-sparging
conditions. As the amount of CO2 in the biogas in-
creased from 46% to 77% during sparging (by di€erence,
assuming the biogas was chie¯y H2 and CO2 ), changes
in fermentation end-products might have been expected.
The levels of volatile metabolic products in the reactor
e‚uent are shown in Table 3.
From the distribution of products in Table 3 it can be
seen that the products were principally volatile fatty
acids, with virtually no solvent production: i.e. the cul-
ture was acidogenic in nature under the conditions de-
scribed. The principal VFA was n-butyric, as also found
by Zoetemeyer et al. (1982) and Cohen et al. (1979) in
anaerobic acidogenic reactors. The main di€erence be-
tween the non-sparging conditions and the sparging
conditions was a small increase in n-butyric concentra-
tion in the sparging conditions from an average of 1742
mg lÿ1 to 1929 mg lÿ1 . No major shift in metabolic
pathways, i.e. from acids to acetone-butanol produc-
tion, was seen.
In hydrogen production, conditions are sought max-
imising acetic acid production as this gives the maxi-
mum hydrogen yield (Eq. (1)). The concept of
fermentative hydrogen production is contrary to the
more well-studied solvent producing acetone-butanol
fermentation in which the production of molecular hy-
drogen and acetate is unnecessary and decreases solvent
recovery. End products such as H2 , CO2 , acetate and
butyrate are the result of side reactions in the acetone-
butanol fermentation process (Kim et al., 1984). Thus a
study of the conditions detrimental to solvent produc-
tion will give information on those conditions favouring
hydrogen and acetate production.
Glucose is the fundamental resource for hydrogen
production. Glucose is fermented via the EMP pathway
to pyruvate. Pyruvate oxidation to acetyl coenzyme A
requires ferredoxin (Fd) reduction. Reduced Fd is oxi-
dized by hydrogenase, which generates Fd and releases
Table 3
Volatile metabolic products in the liquid phasea
Product Non-Sparging (mg lÿ1 )
Acetic 773 ‹ 48 (3)
Propionic 114 ‹ 6 (3)
i-butyric 29 ‹ 15 (3)
n-butyric 1742 ‹ 31 (3)
i-valeric ND (3)
n-valeric 10 ‹ 9 (3)
Acetone ND (3)
Ethanol 58 ‹ 20 (3)
Butanol 4 ‹ 1 (3)
a
ND ˆ Not Detectable; ( ) ˆ number of samples.
63
electrons as molecular hydrogen. Therefore hydrogen
production is the means by which bacteria lose excess
electrons. The reaction is reversible and depends on
hydrogen partial pressure (pH2 ), suggesting that hydro-
gen yield is signi®cantly in¯uenced by pH2 . Therefore it is
important to control pH2 in the liquid phase for en-
hancement of hydrogen yield. Gas sparging is a useful
method for decreasing pH2 although industrially a
method of hydrogen utilisation, e.g. a fuel cell or metal
hydrides, would be employed (Levy et al., 1981).
The e€ects of hydrogen on the metabolism and the
fermentative pattern of the anaerobic bacteria have been
demonstrated in previous studies. Clostridium cello-
bioparum produces more hydrogen when it is removed
by hydrogen-consuming methanogens (Chung, 1976).
The quantitative composition of the fermentation
products depends on the pH2 . Van Andel et al. (1985)
demonstrated that sparging a pure culture of Clostridi-
um butyricum with nitrogen increased the rate of acetate
production both absolutely and relative to the rate of
butyrate production. The production of acetate and
hydrogen by Clostridium thermocellum has been con-
sidered an obstacle to the use of this organism in ethanol
production (Lamed et al., 1988) and stirring the batch
cultures favoured hydrogen and acetate production.
This was attributed to accumulation of hydrogen at
supersaturated concentrations in unstirred conditions
inhibiting acetate production. The CSTR reactor used
here was already well stirred.
Other workers who have grown enrichment cultures
on glucose-mineral salts medium in continuous culture
include Nakamura et al. (1993) who studied the e€ect of
HRT from 2±10 h on an enrichment from sewage
sludge. These workers do not report the e‚uent glucose
concentrations, but assuming these were low, a hydro-
gen yield of 0.07 moles H2 /mole glucose consumed was
achieved at a 2 h HRT, which gave the highest hydrogen
% and total gas production. Ueno et al. (1996) also used
a sewage sludge enrichment grown in continuous culture
on a sugary industrial wastewater. The maximum hy-
drogen yield reported was 2.52 moles H2 /mole carbo-
hydrate consumed (assuming carbohydrate has the
formula C6 H12 O6 ) at a 12 h HRT and pH 6.8. Kumar et
Sparging (mg lÿ1 )
785 ‹ 79 (3)
74 ‹ 1 (3)
13 ‹ 2 (3)
1929 ‹ 170 (3)
2 ‹ 2 (3)
13 ‹ 2 (3)
1 ‹ 1 (3)
30 ‹ 17 (3)
6 ‹ 2 (3)
64 O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65

Table 4
Hydrogen yields reported in the literature
Condition Substrate H2 yield (mol/mol-glucose)
Mixed culture this study N2 sparging, continuous glucose 1.43
Mixed culture this study continuous glucose 0.85
Enterobacter aerogenes (Tanisho et al., 1998) Ar sparging, batch molasses 1.58
Enterobacter aerogenes (Tanisho et al., 1998) batch molasses 0.52
Enterobacter aerogenes (Yokoi et al., 1995) continuous glucose 1.00
Clostridium butyricum (Van Andel et al., 1985) continuous glucose 1.60
Clostridium sp. strain No.2 (Taguchi et al., 1995) continuous glucose 1.61±2.36
Citrobacter intermedius (Brosseau and Zajic, 1982) batch glucose 1.00
Clostridium pasteurianum (Brosseau and Zajic, 1982) batch glucose 1.50
Clostridium beijerinckii AM21B (Taguchi et al., 1995) batch glucose 1.3±2.0

al. (1995) using a strain of Bacillus licheniformis isolated 4. Conclusions

from cattle dung report hydrogen yields in semi-con-
tinuous culture over 60 days of 0.37 moles/mole glucose
utilised, though yields of up to 1.1 moles H2 /mole glu-
cose were reported in shorter term batch-type experi-
ments. It should be noted that in each case the gas
volume was measured by the water displacement meth-
od, which may be less accurate than the gas meter
measurements available in the present study. Hydrogen
yields from pure culture studies reviewed by Nandi and
Sengupta (1998) vary from 0.7 to 2.4 moles H2 /mole
glucose, and a summary of yields from pure culture
work is given in Table 4.
The speci®c hydrogen production rate found here of
3.84 mmol hÿ1 gÿ1 dry weight of biomass (Table 2)
compares well with rates for Clostridium pasteurianum
of 2.5 mmol hÿ1 gÿ1 biomass in log growth phase in
batch culture and 1.2 mmol hÿ1 gÿ1 biomass in sta-
tionary phase (Brosseau and Zajic, 1982). However,
Taguchi et al. (1995) obtained speci®c hydrogen pro-
duction rates with Clostridium sp. strain No. 2 grown on
glucose in continuous culture of between 7.8 and 32.2
mmol hÿ1 gÿ1 dry weight of biomass depending on the
retention time.
It appears possible that at an industrial scale, energy
could be recovered as hydrogen from an acidogenic re-
actor as part of a typical biological treatment for car-
bohydrate-containing wastewaters or from an
acidogenic stage used to produce volatile fatty acids for
chemical or liquid bio-fuel production (Levy et al.,
1981). The e‚uent from the hydrogen producing stage,
already at 35°C, could then pass to a methanogenic
stage for which the fermentation end products are ideal
substrates, or the volatile products could be extracted to
supply chemicals or be used as liquid bio-fuels (Levy et
al., 1981; Brosseau and Zajic, 1982). The mixed culture
used here grows also on sucrose and starch, tofu man-
ufacturing waste, wheat bran and rice bran, in batch
culture (Mizuno et al., 1997) so that this process could
be of wide application to food industry wastewaters,
which would then be seen as a resource for high grade
renewable energy production.
The hydrogen-producing enrichment culture was ex-
tremely stable when grown at pH 6.0 and a HRT of
8.5 h, re-growing well after dormancy and accidental
washout. A ¯occulant biomass of concentration 1.5 g lÿ1
dry weight was obtained in the CSTR without sparging,
a non-¯occulant biomass of a lower concentration re-
sulting during sparging.
Hydrogen yields of 0.85 and 1.43 mol/mol glucose
were obtained under non-sparging and sparging condi-
tions, respectively; sparging with nitrogen resulted in a
68% increase in hydrogen yield. No signi®cant change in
fermentation end products was observed.
Acknowledgements
The authors wish to thank Dr. A.J. Guwy and Miss
H. Forsey for their expert technical assistance, and the
UK EPSRC for an equipment grant to DLH and FRH
(GR/M38346).
References
Andel, J.G., Zoutberg, G.R., Crabbendam, P.M., Breure, A.M., 1985.
Glucose fermentation by Clostridium butyricum grown under a self
generated gas atmosphere in chemostat culture. Applied Microbi-
ology and Biotechnology 23, 21±26.
APHA, 1992. Standard Methods for the Examination of Waste and
Wastewater, 18th ed. American Public Health Association, Wash-
ington, USA.
Bahl, H., Gottschalk, G., 1984. Parameters a€ecting solvent produc-
tion by Clostridium acetobutylicum in continuous culture. Biotech-
nology and Bioengineering Symposium 14, 215±223.
Benemann, J., 1996. Hydrogen biotechnology: progress and prospects.
Nature Biotechnology 14, 1101±1103.
Brosseau, J.D., Zajic, J.E., 1982. Hydrogen-gas production with
Citrobacter intermedius and Clostridium pasteurianum. Journal
Chemical Technology and Biotechnology 32, 496±502.
Chung, K.T., 1976. Inhibitory e€ects of H2 on growth of Clostridium
cellobioparum. Applied and Environmental Microbiology 31, 342±348.
Cohen, A., Zoetemeyer, R.J., Van Deursen, A., Van Andel, J.G., 1979.
Anaerobic digestion of glucose with separated acid production and
methane formation. Water Research 13, 571±580.
 O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
Dabrock, B., Bahl, H., Gottschalk, G., 1992. Parameters a€ecting
solvent production by Clostridium pasteurianum. Applied and
Environmental Microbiology 58, 1233±1239.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F.,
1956. Colorimetric method for determination of sugars and related
substances. Analytical Chemistry 28, 350±356.
ESPRIT, 1989. Research Report of the explosion of soybean-meal in a
silo. ESPRIT Co. Ltd. (In Japanese).
Guwy, A.J., Hawkes, D.L., Hawkes, F.R., 1995. On-line low ¯ow
high-precision gas metering systems. Water Research 29, 977±979.
Kalia, V.C., Jain, S.R., Kumar, A., Joshi, A.P., 1994. Fermentation of
bio-waste to H2 by Bacillus licheniformis. World Journal of
Microbiology and Biotechnology 10, 224±227.
Kim, B.H., Bellows, P., Datta, R., Zeikus, J.G., 1984. Control of
carbon and electron ¯ow in Clostridium acetobutylicum fermenta-
tions: utilization of carbon monoxide to inhibit hydrogen produc-
tion and to enhance butanol yields. Applied and Environmental
Microbiology 48, 764±770.
Kumar, A., Jain, S.R., Sharma, C.B., Joshi, A.P., Kalia, V.C., 1995.
Increased H2 production by immobilized microorganisms. World
Journal of Microbiology and Biotechnology 11, 156±159.
Lamed, R.J., Lobos, J.H., Su, T.M., 1988. E€ect of stirring and
hydrogen on fermentation products of Clostridium thermocellum.
Applied and Environmental Microbiology 54, 1216±1221.
Lay, J.J., Lee, Y.J., Noike, T., 1999. Feasibility of biological hydrogen
production from organic fraction of municipal solid waste. Water
Research 33 (11), 2579±2586.
Levy, P.F., Sanderson, J.E., Kispert, R.G., Wise, D.L., 1981.
Biore®ning of biomass to liquid fuels and organic chemicals.
Enzyme Microbiol Technology 3, 207±215.
Mizuno, O., Li, Y.Y., Noike, T., 1998. The behavior of sulfate-
reducing bacteria in acidogenic phase of anaerobic digestion. Water
Research 32, 1626±1634.
Mizuno, O., Ohara, T., Noike, T., 1997. Hydrogen Production from
Food Processing Waste by Anaerobic Bacteria. Journal of Envi-
View publication stats
65
ronmental Systems and Engineering, JSCE (573/VII-5), 111±118 (in
Japanese).
Nakamura, M., Kanabe, H., Matsumoto, J., 1993. Fundamental
studies on hydrogen production in the acid-forming phase and its
bacteria in anaerobic treatment processes-the e€ects of solids
retention time. Water Science Technology 28 (7), 81±88.
Nandi, R., Sengupta, S., 1998. Microbial production of hydrogen: an
overview. Critical Reviews in Microbiology. 24 (1), 61±84.
Peck, M.W., Skilton, J.M., Hawkes, F.R., Hawkes, D.L., 1986. E€ects
of temperature shock treatments on the stability of anaerobic
digesters operated on separated cattle slurry. Water Research 20
(4), 453±462.
Roychowdhury, S., Cox, D., Levandowsky, M., 1988. Production of
hydrogen by microbial fermentation. International Journal of
Hydrogen Energy 13, 407±410.
Ruzicka, M., 1996. The e€ect of hydrogen on acidogenic glucose
cleavage. Water Research 30, 2447±2451.
Taguchi, F., Mizukami, N., Saito-Taki, T., Hasegawa, K., 1995.
Hydrogen production from continuous fermentation of xylose
during growth of Clostridium sp. strain No.2.. Canadian Journal of
Microbiology 41, 536±540.
Tanisho, S., Kuromoto, M., Kadokura, N., 1998. E€ect of CO2
removal on hydrogen production by fermentation. International
Journal of Hydrogen Energy 23, 559±563.
Ueno, Y., Otauka, S., Morimoto, M., 1996. Hydrogen production
from industrial wastewater by anaerobic micro¯ora in chemostat
culture. Journal of Fermentation and Bioengineering 82, 194±197.
Yokoi, H., Ohkawa, T., Hirosse, J., Hayashi, S., Takasaki, Y., 1995.
Characteristics of hydrogen production by aciduric Enterobacter
aerogen strain HO-39. Journal of Fermentation and Bioengineering
80, 571±574.
Zoetemeyer, R.J., Arnoldy, P., Cohen, A., Boelhouwer, C., 1982.
In¯uence of temperature on the anaerobic acidi®cation of glucose
in a mixed culture forming part of a two stage digestion process.
Water Research 16, 313±321.