JOURNAL OF BIOSCIENCE AND BIOENGINEERING
©2005, The Society for Biotechnology, Japan
Vol. 100, No. 3, 260–265. 2005
Hydrogen and Ethanol Production from Glycerol-Containing
Wastes Discharged after Biodiesel Manufacturing Process
Takeshi Ito,1Yutaka Nakashimada,1Koichiro Senba,1
Tomoaki Matsui,1and Naomichi Nishio1*
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan1
Received 20 December 2004/Accepted 13 May 2005
H2 and ethanol production from glycerol-containing wastes discharged after a manufacturing
process for biodiesel fuel (biodiesel wastes) using Enterobacter aerogenes HU-101 was evaluated.
The biodiesel wastes should be diluted with a synthetic medium to increase the rate of glycerol uti-
lization and the addition of yeast extract and tryptone to the synthetic medium accelerated the
production of H2 and ethanol. The yields of H2 and ethanol decreased with an increase in the con-
centrations of biodiesel wastes and commercially available glycerol (pure glycerol). Furthermore,
the rates of H2 and ethanol production from biodiesel wastes were much lower than those at the
same concentration of pure glycerol, partially due to a high salt content in the wastes. In continu-
ous culture with a packed-bed reactor using self-immobilized cells, the maximum rate of H2 pro-
duction from pure glycerol was 80 mmol/l/h yielding ethanol at 0.8mol/mol-glycerol, while that
from biodiesel wastes was only 30 mmol/l/h. However, using porous ceramics as a support mate-
rial to fix cells in the reactor, the maximum H2 production rate from biodiesel wastes reached
63mmol/l/h obtaining an ethanol yield of 0.85mol/mol-glycerol.
[Key words: hydrogen, ethanol, Enterobacter aerogenes, glycerol, biodiesel]
Biodiesel fuels are defined as fatty acid methyl or ethyl
esters from vegetable oils or animal fats and they are used
as fuel in diesel engines and heating systems (1, 2). Since
biodiesel fuels have various advantages such as an alter-
native to petroleum-based fuel, renewable fuel, a favorable
energy balance, lower harmful emissions and nontoxic fuel,
they have drawn much attention recently. Although biodie-
sel fuels are produced chemically and enzymatically, glyc-
erol is essentially generated as the by-product (3, 4). Glyc-
erol generated is presently applied, for example, as a ingre-
dient of cosmetics, but a further increase in the production
of biodiesel fuels would raise the problem of efficiently
treating wastes containing glycerol.
The microbial conversion of glycerol to various com-
pounds has been investigated recently with particular focus
on the production of 1,3-propanediol, which can be applied
as a basic ingredient of polyesters (5–7). The fermentation
of glycerol to 1,3-propanediol has been studied using micro-
organisms such as Klebsiella pneumoniae (8–10), Citro-
bacter freundii (11, 12), Clostridium butyricum (13, 14) and
Enterobacter agglomerans (15). However, the biological pro-
duction of H2 and ethanol from glycerol is also attractive be-
cause H2 is expected to be a future clean energy source and
ethanol can be used as a raw material and a supplement to
Enterobacter aerogenes HU-101, isolated as a high-rate
H2 producer from methanogenic sludge (16, 17), can con-
vert various carbohydrates, such as sugars and sugar alco-
hols, to H2, ethanol, 2,3-butanediol, lactate and acetate. H2
can be biologically produced either by photosynthetic micro-
organisms (18, 19) or fermentative anaerobes (20). Among
the latter, Clostridium species have received much attention
for their ability to produce either solvents (butanol and ace-
tone) or acids (butyrate and acetate) as well as H2 (21). We
have studied H2 production using E. aerogenes (22, 23) be-
cause E. aerogenes, unlike clostridia, exhibits uninhibited
growth in an atmosphere of 100% H2. During the course of
these studies, we found that E. aerogenes HU-101 mainly
produces H2 and ethanol with a minimal production of other
by-products when glycerol was used as the substrate. Thus,
the microorganism can be utilized for the high-yield produc-
tion of H2 and ethanol from biodiesel wastes containing
In this study, we evaluated the culture conditions of E.
aerogenes HU-101 for the efficient production of H2 and
ethanol from biodiesel wastes. We also demonstrated the con-
tinuous production in a packed-bed reactor with and without
a support material.
MATERIALS AND METHODS
Microorganism and culture conditions
used in this study was E. aerogenes HU-101 isolated from a meth-
anogenic sludge developed in our laboratory (24). Cultures were
* Corresponding author. e-mail: firstname.lastname@example.org
phone: +81-(0)82-424-7760 fax: +81-(0)82-424-7046
H2 AND ETHANOL PRODUCTION FROM BIODIESEL WASTEVOL. 100, 2005 261
maintained at −80°C with 15% glycerol. A synthetic medium used
in this study contained (per liter) 7.0g of K2HPO4, 5.5g of KH2PO4,
1.0g of (NH4)2SO4, 0.25g of MgSO4⋅7H2O, 0.021g of CaCl2⋅2H2O,
0.12 g of Na2MoO4⋅2H2O, 2.0mg of nicotinic acid, 0.172mg of
Na2SeO3, 0.02 mg of NiCl2 and 10 ml of trace element solution
containing 0.5g of MnCl2⋅4H2O, 0.1 g of H3BO4, 0.01g of
AlK(SO4)2⋅H2O, 0.001 g of CuCl2⋅2H2O and 0.5 g of Na2EDTA
per liter. A complex medium was prepared by adding the desired
concentrations of yeast extract and tryptone to the synthetic
medium. The biodiesel wastes containing glycerol were supplied
from a biodiesel manufacturing factory in Hiroshima prefecture,
Japan. The biodiesel fuel was chemically produced with potassium
hydroxide as the alkali catalyst. The wastes contained 41% (w/w)
glycerol. The amount of total organic carbon (TOC) in the wastes
was 540 g/l, of which 524 g was soluble. The impurities were main-
ly composed of ash (8%, w/v) and methanol (25%, w/w). Although
0.04% (w/w) diacylglycerol and 0.01% (w/w) monoacylglycerol
were contained in the wastes, triacylglycerol was not detected.
A modified Hungate technique in combination with the serum
bottle technique (25) was used to culture the bacterium anaerobi-
cally. The medium without glycerol and phosphate buffer was
boiled for 20 min, cooled on ice with continuous bubbling of N2
gas, dispensed into serum bottles sealed with black butyl rubber
stoppers, and then sterilized (18 min, 121°C). Concentrated aque-
ous solutions of glycerol and phosphate buffer autoclaved sepa-
rately were then injected into the medium using a hypodermic
syringe. After the inoculation of 2 ml of seed culture into serum
bottles (approximately 125ml bottles containing 50 ml of the cul-
ture medium) and adjustment of the pH to 6.8, the bottles were in-
cubated at 37°C with agitation (120 rpm) (16).
A cylindrical glass column reactor
(φ2.7×17 cm height) with a working volume of 60 ml was used for
the continuous culture. Fresh medium was supplied from the
bottom by a peristaltic pump (Decarf N-10; Taiyo, Tokyo) and
evolved gas and effluent liquid were discharged from the top of the
reactor (22). Two ml of the seed culture was transferred into the re-
actor. After 12 h of incubation in the batch mode, continuous culti-
vation was initiated by feeding the sterilized medium at a dilution
rate of 0.1 h–1 with the peristaltic pump. The cells were cultivated
anaerobically at 37°C without controlling pH. After the accumula-
tion of cell flocs was observed at the bottom of the reactor, the vol-
ume and the content of gas produced were measured periodically.
A quasi-steady state was confirmed, except for the cell mass in the
reactor, on the basis of a constant H2 evolution rate, remaining
glycerol concentration and pH of the effluent. These values were
measured at least twice per day. Dilution rate was increased step-
Nagao Porcell (diatomaceous clay; particle size, 4 to 10 mm
[diameter]; apparent density, 0.38 g/ml; true density, 2.17 g/ml;
porosity, 81%; average pore diameter, 128 µm; Nagao & Co.,
Okayama) was used as a support material to increase the number
of cells retained in the reactor for continuous culture with biodiesel
Gas production was measured periodically by the
displacement of saturated aqueous sodium chloride in a graduate
cylinder. The concentrations of CO2 and H2 were determined by
gas chromatography (GC 8A; Shimadzu, Kyoto) with a thermal
conductivity detector (27). Lactate, acetate, ethanol, and 1,3-pro-
panediol were measured using an HPLC system as previously de-
scribed (28). Glycerol and formate were determined by enzymatic
analysis using F-kit glycerol and F-kit formate (Roche Diagnostics
K. K., Tokyo), respectively. The cell concentration was not mea-
sured because the medium containing biodiesel wastes was turbid.
RESULTS AND DISCUSSION
Medium composition for treatment of biodiesel wastes
To ferment biodiesel wastes to H2 and ethanol using E.
aerogenes, it would be desirable not to add any supplements
that support cell growth to reduce the cost of fermentation
and wastewater treatment after fermentation. Therefore,
batch fermentation was first carried out with biodiesel wastes
diluted with deionized water. When biodiesel wastes were
diluted to 80 mM glycerol with deionized water, glycerol was
not completely consumed even after 48h and no growth
was observed after 48h. This indicated that some nutrients
should be added to ferment glycerol in biodiesel wastes.
Therefore, the synthetic medium was used for dilution of
biodiesel wastes. The rate of glycerol utilization further in-
creased using the synthetic medium. When biodiesel wastes
were diluted to 80mM glycerol with the synthetic medium,
glycerol was completely utilized after 24 h, yielding H2 at
0.89 mol/mol-glycerol and ethanol at 1.0 mol/mol-glycerol
(data not shown), respectively. The addition of both yeast
extract and tryptone to the synthetic medium was effective
in increasing the rates of H2 and ethanol production (Fig. 1).
Even in the medium containing 0.5 g/l yeast extract and
0.5g/l tryptone, ethanol and H2 production levels markedly
increased after 12h compared with those of the synthetic
medium. The addition of 5 g/l yeast extract or tryptone was
effective in increasing the rate of glycerol consumption as in
the case of adding both (data not shown), suggesting that
FIG. 1. Typical time courses of H2 and ethanol production in batch
culture using biodiesel wastes diluted with deionized water (closed tri-
angles), synthetic medium (open circles) and complex medium con-
taining 0.5 (closed squares), 1 (open triangles), 2.5 (closed circles),
and 5 g/l (open squares) each of yeast extract and tryptone. Culture
conditions: initial pH, 6.8; glycerol, 80mM. Experimental values rep-
resent averages of at least duplicate cultures.
ITO ET AL.J. BIOSCI. BIOENG., 262
some nutrients such as specific amino acids and vitamins
that are still unknown are needed for the better growth of E.
Effect of concentration of biodiesel wastes on fermen-
To minimize the reactor size and running cost, it
is desirable that the concentration of biodiesel wastes is as
high as possible. Therefore, batch fermentation was carried
out with biodiesel wastes diluted with the complex medium,
which consisted of the synthetic medium containing 5 g/l
yeast extract and 5g/l tryptone to 1.7, 3.3, 10 and 25 g/l as
glycerol concentrations. The culture times needed for the
complete utilization of glycerol and yields of end-products
at the indicated times are shown in Table 1. The yield of H2
from glycerol (1.12 mol/mol) exceeded the theoretical max-
imum yield of H2 (1.0 mol/mol) with 1.7g/l glycerol. This
suggested that unknown carbon sources or electron sources
in the wastes contributed to H2 production. The yield of eth-
anol was almost the same as the theoretical maximum yield
from glycerol and small amounts of acetate and 1,3-pro-
panediol were detected in the case of using 1.7g/l glycerol.
However, the yields of H2, ethanol and acetate decreased
whereas the yield of lactate increased with the increase in
the concentration of biodiesel wastes. Furthermore, when
the glycerol concentration was 25g/l, glycerol was not com-
pletely consumed even after 48 h and a decreased H2 pro-
duction was observed.
To determine the sole effect of glycerol concentration on
substrate utilization and product formation, the complex me-
dium supplemented with commercially available glycerol
(pure glycerol) was applied to batch cultures. The micro-
organism completely consumed 5 g/l or 10 g/l pure glycerol
within 6 h and 25g/l after 12h (Table 2). Although the
yields of H2 and ethanol were 1 mol/mol-glycerol using 5 g/l
glycerol, they decreased with the increase in glycerol con-
centration, as observed in biodiesel wastes. The result indi-
cated that a higher concentration of glycerol decreased the
yields of H2 and ethanol.
Effect of impurities on H2 and ethanol production by
As shown in Tables 1 and 2, the rates of
glycerol utilization in the medium with biodiesel wastes
supplemented to provide 10 and 25g/l glycerol were much
lower than that in the medium with pure glycerol. Biodiesel
fuel is currently produced using an alkali-catalyzed technol-
ogy. The most commonly used alkali catalysts are sodium
hydroxide, sodium methoxide and potassium hydroxide (3).
Since an alkali is neutralized with an acid after esterifica-
tion, biodiesel wastes may contain high concentrations of
salts such as sodium chloride, which might inhibit cell
growth. Figure 2 shows the effect of the concentration of
added sodium chloride on the rate of glycerol utilization.
Both H2 and ethanol productions at 1% sodium chloride
were almost the same as those without sodium chloride in
the medium with pure glycerol. On the other hand, when
sodium chloride was added to the medium with biodiesel
wastes, H2 and ethanol productions significantly decreased
even at 1% sodium chloride. Indeed, when biodiesel wastes
were diluted to 10 or 25g/l glycerol, the solution should
contain about 0.2% or 0.5% ash, respectively (see Materials
and Methods). If the resulting ash is considered to be mostly
sodium chloride, the decrease in the rate of product forma-
tion caused by the presence of the ash in biodiesel wastes
seemed to be excessive compared with that caused by the
TABLE 1. Yields of end products for glycerol in biodiesel wastes di-
luted with complex medium
Glycerol concentration (g/l)
Time required for complete
glycerol consumption (h)
Ethanol 0.96 0.83 0.670.56
LactateND 0.050.11 0.17
Formate0.140.2 0.19 ND
Batch cultures were carried out using the complex medium contain-
ing various concentrations of biodiesel wastes and 80mM (1.7–3.3g/l
glycerol) or 160 mM (10–25g/l glycerol) phosphate buffer at an initial
pH of 6.8. Experimental values represent averages of at least duplicate
cultures. ND, Not detected.
a Glycerol was not completely consumed within the time shown in
TABLE 2. Yields of end products for commercially
Glycerol concentration (g/l)
Time required for complete
glycerol consumption (h)
Culture conditions: initial pH, 6.8; pure glycerol; complex medium;
phosphate buffer, 80mM. Experimental values represent averages of
at least duplicate cultures.
a When glycerol concentration was 25g/l, phosphate buffer was
used at 160mM.
FIG. 2. Effect of sodium chloride concentration on H2 (circles) and
ethanol (squares) production by E. aerogenes HU-101 from biodiesel
wastes (closed symbols) or pure glycerol (open symbols) in synthetic
medium. Culture conditions: culture time, 24h; initial pH, 6.8; glycerol,
10g/l. Experimental values represent averages of at least duplicate
H2 AND ETHANOL PRODUCTION FROM BIODIESEL WASTEVOL. 100, 2005263
presence of sodium chloride in the medium with pure glyc-
erol as shown in Fig. 2. A high salinity of the medium with
biodiesel wastes would be one of the causal factors for the
inhibition of product formation.
Since about 25% (w/w) methanol was also contained in
biodiesel wastes used in this study, about 1.5% methanol
should be contained in biodiesel wastes diluted to 25 g/l
glycerol, the concentration at which glycerol consumption
significantly decreased. Therefore, the effect of methanol
concentration on the growth of E. aerogenes was investi-
gated using the complex medium containing 10g/l pure glyc-
erol. However, even when methanol was added up to 3%, no
inhibition of the cell growth and glycerol consumption was
observed, suggesting that methanol was not an inhibitory
factor for this microorganism at the range of dilutions tested.
Continuous cultures using packed-bed reactor with
To elucidate the production rate
of H2 and ethanol from biodiesel wastes, we carried out con-
tinuous culture using immobilized-cell systems, which have
become common alternatives to suspended-cell systems in
continuous operations because they are more efficient in
solid/liquid separation and can be operated at high dilution
rates without encountering washout of cells. Recent studies
have clearly demonstrated that immobilized-cell systems
using various support matrices are suitable for continuous
hydrogen fermentation (29–32). In particular, since E. aero-
genes flocculates, a continuous culture system using a fixed-
bed reactor with self-immobilized cells have been developed
as reported previously (22). Therefore, in this study, this
continuous culture system was attempted to increase the
productivity of H2 and ethanol.
Figure 3 shows the production rates of H2 and the concen-
trations of ethanol and other metabolites in the effluent from
110 mM pure glycerol and biodiesel wastes in the complex
medium (5.0g/l yeast extract, 5.0 g/l tryptone) in continu-
ous culture with self-immobilized cells. The volumetric max-
imum H2 production rate reached 80mmol/l/h and glycerol
was consumed completely at dilution rates up to 1.3 h–1
(Fig. 3A). This rate of H2 production was 2.6-fold and 1.3-
fold higher than that from glucose using the same strain of
E. aerogenes and the high-H2-producing mutant AY-2 using
the same culture system, respectively (22). This result indi-
cates that glycerol is a very suitable substrate for producing
H2 as suggested previously (17). The yield of ethanol was
maintained at more than 0.9 mol/mol-glycerol during the cul-
ture although the production of 1,3-propanediol increased at
a higher dilution rate.
On the other hand, when biodiesel wastes were applied to
the same culture system, the number of cells remaining in
the reactor was much lower than in the reactor with pure
glycerol. The flocs formed in biodiesel wastes were very
downy and fragile, and easily washed out from the reactor
with the increase in dilution rate. Some components in
biodiesel wastes such as salts and/or oils that remained after
the biodiesel manufacturing process might disturb the for-
mation of rigid flocs. Residual glycerol was observed even
at a low dilution rate (Fig. 3B), possibly, due to the low cell
density in the reactor. The maximum rate of H2 production
with biodiesel wastes was 30mmol/l/h at a dilution rate of
0.8h–1, which was much lower than that with pure glycerol.
Indeed, the lower rate of glycerol consumption resulted in
the lower rate of H2 production. Furthermore, the accumula-
tion of formate could also explain why H2 production rate
decreased in the case of using biodiesel wastes because for-
mate is generally converted to H2 and CO2 as catalyzed by
formate hydrogen lyase, although the reason for formate ac-
cumulation is still unclarified.
Continuous culture using packed-bed reactor with
The results obtained from the analysis
using the packed-bed reactor system with self-immobiliza-
FIG. 3. H2 production rates and end production concentrations during continuous culture by self-immobilization. (A) Cultivation on pure glyc-
erol. (B) Cultivation on glycerol waste discharged from biodiesel process. Symbols: closed circles, H2; open circles, glycerol; open squares, etha-
nol; closed triangles, formate; open triangles, 1,3-propanediol; closed squares, lactate; open diamonds, acetate. Culture conditions: complex me-
dium (yeast extract, 5g/l; tryptone, 5g/l; glycerol, 110mM) at 37°C.
ITO ET AL.J. BIOSCI. BIOENG., 264
tion demonstrate that it is difficult to retain a sufficient num-
ber of cells at a higher dilution rate when biodiesel wastes
are applied. To increase the production of H2 and ethanol
more, a packed-bed reactor with a carrier matrix seemed to
be an attractive system for preventing the efflux of cell flocs
from the reactor. Thus, this system was used and the results
of culture using the carrier matrix with the complex medium
containing biodiesel wastes are presented in Fig. 4. The cells
were successfully retained in the reactor throughout the cul-
ture period unlike the culture with self-immobilized cells
(data not shown), resulting in an almost complete consump-
tion of glycerol at dilution rates up to 1.2 h–1 at which the
volumetric maximum H2 production rate increased to 63
mmol/l/h with an ethanol yield of 0.85mol/mol-glycerol.
The main by-products were 1,3-propanediol, lactate and for-
mate. This volumetric rate of H2 production from biodiesel
wastes by E. aerogenes HU-101 was almost the same as that
from glucose with the mutant AY-2 of E. aerogenes HU-101
(22). This result demonstrates that the use of the carrier ma-
trix was very effective for H2 and ethanol production from
biodiesel wastes although the hydrogen production was lower
than that in the cultivation using pure glycerol.
In this study, it was shown that H2 and ethanol were pro-
duced by E. aerogenes HU-101 with a high yield and a high
production rate from biodiesel wastes containing glycerol.
These findings may lead to a decrease in the use of fossil
fuel that may in turn lead to the alleviation of global warm-
ing because H2 can be applied to fuel cells to generate elec-
tricity and heat without the emission of carbon dioxide, and
ethanol can be supplemented to gasoline or used as a re-
source for biodiesel production instead of methanol which
is usually produced from natural gas. Indeed, there are some
problems that need to be solved before this technology can
have practical applications. For example, it is necessary to
increase glycerol concentration used in the production of
H2 and ethanol because an excessive dilution of biodiesel
wastes using the medium increases the cost for the recovery
of ethanol and wastewater treatment. Although H2 and etha-
nol production from biodiesel wastes was demonstrated us-
ing the wild strain of E. aerogenes HU-101 in this study, it
is necessary to further optimize culture conditions and to
breed mutants with a high tolerance to a high concentration
of glycerol or salts by conventional breeding methods or
genetic engineering. Since 90 g/l raw glycerol derived from
biodiesel wastes was completely consumed and 47.1 g/l
1,3-propanediol was produced by Clostridium butyricum
F2b after 35h (33), further explorations of H2 and ethanol-
producing microorganisms with such useful properties in
nature are also required for this technology to develop.
This study was partially supported by the Industrial Technology
Research Grant Program in 2004 from New Energy and Industrial
Technology Development Organization (NEDO) in Japan.
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