Journal of Environmental Biotechnology
Vol. 12, No. 2, 141–146, 2012

Original paper (regular paper)

Anaerobic Bioconversion to Value-Added Byproducts from Two Promising
Biomass in the Future, Crude Glycerol and Sewage Sludge
Hayato Tokumoto* and Chie Kimura
Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531* E-mail: tokumoto@chemeng.osakafu-u.ac.jp* TEL & FAX: +81–72–254–9301(Received; 2 July, 2012/Accepted; 20 August, 2012)
Sewage sludge generates an enormous amount of new biomass in urban cities. As new biomass, crude glycerol is
co-produced from biodiesel production and its generation is expected to increase in the future. Sewage sludge containing
0.01% (v/v) crude glycerol showed the highest methane production rate of 10.82 mL/d/g-TS on day 7, which was 1.52 times
higher than the control sludge without supplementation of crude glycerol. Addition of crude glycerol at 0.4% (v/v) resulted
in production of valuable resources such as hydrogen and 1,3-propanediol (1,3-PDO). A strongly alkaline solution of crude
glycerol enhanced the solubilization of organic matter to various organic acids from the solid phase of the sewage sludge,
which resulted in a significant decrease in total solid (TS) content in the sludge compared with the control, an estimated
60% decrease from the first day. Anaerobic co-digestion of sewage sludge and crude glycerol as a co-substrate is considered
to be a cost eﬀective method for incineration of sewage sludge (with high water content) and should help to reduce landfill
capacity. The proposed process promotes the possibility of biodiesel production by anaerobic bioconversion of crude glycerol.
Key words: Sewage sludge, Crude glycerol, Hydrogen, methane, 1,3-Propanediol

1. Introduction
The production of sewage sludge is increasing in accordance with the extension of sewer distribution networks andnew installation of wastewater treatment plants. The cost of
the treatment and disposal of sewage sludge is estimated tocomprise half of the total cost of wastewater treatment27).
Therefore, minimizing the negative impact of sewage sludge
on the environment is a major challenge. Anaerobic digestion is an appropriate technique for reducing the volume
and weight of excess sludge before final disposal, and it isemployed worldwide as the oldest and most important process
for sludge stabilization. Anaerobic sewage sludge digestion
can remove pathogens and thereby help to protect humans
and the environment9). Nordberg et al.18) reported that highly
concentrated methane gas is obtained from sewage sludge
during anaerobic digestion which is useful as a way to partly
recover the bioenergy of sludge8,20). However, conventional
anaerobic digestion can be ineﬃcient because of the loworganic content in a combined sewer system. Co-digestionin the presence of higher organic content such as food waste
could be a reliable option to enhance the activity of anaerobic
microorganisms. A proper mixture provides synergistic andcomplementary advantages that oﬀset the lack of carbon
sources in the sewage sludge and dilute harmful or excessive substances which may inhibit the action of anaerobes
in food waste14). A review of the literature suggests that forco-digestion of sewage sludge in the presence of organic
waste, inclusion of crude glycerol with a large amount oforganic matter derived from waste food oil makes the ideal
substrate mixture for anaerobic digestion of sewage sludge.
Biodiesel is a major biofuel that is used in place of fossil
fuels for mitigating greenhouse gas emissions17). Biodiesel ismainly produced from the transesterification of vegetableoils with alcohol21), the main byproduct of which is glycerol.
Crude glycerol is a major byproduct of biodiesel production.
It is estimated that for every 9 kg of biodiesel produced,
about 1 kg of crude glycerol is formed7). The significant
increase of late in the production of biodiesel has createda glycerol surplus that has resulted in a dramatic 10-folddecrease in crude glycerol prices30). In the past, the biodiesel
industry considered glycerol a desirable co-product that could
contribute to the economic viability of biodiesel production; nowadays, glycerol is often regarded as a waste stream
with an associated disposal cost. Tokumoto and Tanaka
reported that glycerol was readily decomposed when sewage
sludge, acting as a fermentation promoter, was added to theanaerobic reactor26). Overall, the fermentation resulted in
the generation of hydrogen, 1,3-propanediol (1,3-PDO) andvarious organic acids.
The main objective of this work was to evaluate the use of
crude glycerol as a co-substrate to enhance the productivityof the anaerobic digestion of sewage sludge. The eﬀect of thetotal solid (TS) content in sewage sludge on the productivity

Tokumoto and Kimura142

was examined. There was also an attempt to investigate theTS removal eﬃciency during the anaerobic digestion.
2. Materials and methods
2.1. Seed sludge
The seed sludge used for the anaerobic co-digestion test
was sampled from the Senboku sewage disposal plant (Sakai,
Japan). The seed sludge was collected on the same day as theanaerobic co-digestion test because otherwise it underwent
decay within a few days. The collected sludge contained alarge amount of water (about 99.5%), thus, the TS concentration in the sludge was only 4.0 g-dry/L. Therefore, the sludge
was centrifuged to condense it and the TS concentration was
adjusted to 20.4 g-dry/L and 42.8 g-dry/L as needed.
2.2. Co-substrate
Crude glycerol was derived from biodiesel fuel producedby the transesterification reaction of waste cooking oil fromthe cafeteria at our university. The crude glycerol contained
554 g/L of glycerol and 482 g/L of total organic carbon.
2.3. Anaerobic digestion test of sewage sludge using crude
glycerol as the co-substrate
All anaerobic digestion tests were performed in auto
sampler vials (PerkinElmer 20-CV) with butyl rubber andaluminum seals at 310 K without shaking. Glass vials witha capacity of 21.6 mL were filled with 5 mL of a mixtureof 4 mL of sewage sludge (TS concentration was set at
4.0 g-dry/L, 20.4 g-dry/L or 42.8 g-dry/L) and 1 mL ofcrude glycerol (glycerol concentration was adjusted to thedesired level). The head space of the vials was replaced with
100% nitrogen under 1.2 atm.
2.4. The production of methane and hydrogen from theanaerobic digestion
During the anaerobic co-digestion, gas in the headspace ofthe vials was removed in 0.5 ml samples using a 2.5 ml gastight syringe (Ito Co., Shizuoka, Japan). The gas composition(H2, N2, CH4, CO2) was analyzed by gas chromatography(GC-8APT; Shimadzu, Kyoto, Japan) using a stainless-steel
80/100 mesh Porapak Q column (3 m×3.0 mm) and athermal conductivity detector. Argon was used as the carrier
gas at a rate of 20 mL/min. The injector, oven, and detector
temperatures were 373 K, 343 K, and 373 K, respectively. A
80% N2/20% CO2 gas mixture and CH4 gas were used as
the standard gases. There were three independent samples
measured for each condition.
2.5. The concentrations of organic acid, glycerol and 1,3PDO in the fermentation broth from the anaerobic
digestion
The concentrations of several organic acids were determined by high-performance liquid chromatography (HPLC)
using a Shimadzu LC-10AD VP pump equipped with two
ion-exclusion chromatography columns (Shim-pack SCR-

102H; 8×300 mm; Shimadzu) with post column pH-buﬀered
conductivity detection (Shimadzu CDD-6A). The mobile
phase consisted of a 5 mM p-toluenesulfonic acid (PTSA)
solution at a flow rate of 0.8 mL/min. Mixtures of 5 mM
PTSA and 100 mM ethylenediaminetetraacetic acid wereused as post-column reagents, both at flow rates of 0.8 mL/
min. The column temperature was maintained at 313 K.
The concentrations of glycerol and 1,3-PDO were
measured by HPLC using a HSS-1500 system (Jasco International Company, Limited, Tokyo, Japan) equipped with asize-exclusion column (Shodex SUGAR KS-801, 8×300 mm;
SHOWA DENKO Company Limited, Tokyo, Japan) and adiﬀerential refractometer (RI-2031; Jasco). The mobile phase
was ultrapure water at a flow rate of 0.4 mL/min. The column
temperature was maintained at 343 K. The retention time ofglycerol and 1,3-PDO was confirmed by injecting a sample
containing a known amount of glycerol and 1,3-PDO.
3. Result and discussion
3.1. The eﬀect of using crude glycerol as a co-substrateon the productivity of the anaerobic digestion of
sewage sludge
Crude glycerol is strongly alkaline with a pH of 12.6.
Therefore, a preparatory experiment was conducted toinvestigate the relationship between the crude glycerol
concentration in the reactor and the pH of a mixture of the
sewage sludge (4 mL) and the crude glycerol (1 mL). The pH
increased moderately in accordance with increasing crude
glycerol concentrations. Generally, the sewage sludge has
the capability to provide buﬀering action because it contains
a large amount of salt (e.g. potassium, sodium). When the
crude glycerol concentration was over 0.4% (v/v), the pH
rapidly increased to 8.93. Thus, the minimum concentrationof crude glycerol used as a co-substrate was set at 0.01%(v/v) (pH value: 6.59) and the maximum concentration was
set at 0.4% (v/v).
Figure 1 shows the biogas production from the anaerobic
co-digestion of sewage sludge with diﬀerent concentrationsof crude glycerol. The control sludge without crude glycerol
as a co-substrate produced methane over time. Glycerol
degraded rapidly on day 1 in the sludge supplemented with
0.01% (v/v) crude glycerol. The highest methane production
and rate occurred on day 7 and was estimated at 1.58 mL
and 10.82 mLCH4/d/g-TS respectively (Fig. 1a). Other
researchers have come to similar conclusions, but the supplemented crude glycerol concentration varies greatly across thereported studies. Fountoulakis et al.10) found that digestion of
sewage sludge produced 10.4±0.97 mL CH4/d/g-TS before
the addition of crude glycerol and 16.4±1.20 mL CH4/d/g-TS
after the addition of crude glycerol (1% (v/v) in the feed). It
would appear that the large amount of organic materials (e.g.
methanol, organic acids, amino acids and free fatty acids)in the crude glycerol enhanced the activity of the methanogenic bacteria in the sewage sludge. A significant decrease of
methane production in the digester fed with sewage sludge

Anaerobic co-digestion of sewage sludge and crude glycerol

143

Fig. 1. (a) Biogas production, pH value and glycerol concentration in the anaerobic co-digestion of sewage sludge with 0.01% (v/v) crude
glycerol. (b) Biogas production, pH value and glycerol concentration in the anaerobic co-digestion of sewage sludge with 0.4% (v/v) crude
glycerol. ( ● ) Glycerol concentration, ( □ ) Methane, ( △ ) Hydrogen. Sewage sludge contained 4.0 g/L TS. The co-digestion was incubated at 310 K for 7 days. Data are means±standard deviations of results from three independent samples.

supplemented with 0.4% (v/v) crude glycerol was observed.
This occurred due to low pH (5.33) and the build-up ofvolatile fatty acids (VFA). This is strongly supported by theprevious work by Holm-Nielsen et al.13), who found that withlow concentrations (<5 g/L) of glycerol, VFA and individual
fatty acids showed no signs of organic loading; however,when the glycerol content was increased, organic overloading
tended to occur. The optimal crude glycerol concentration
for methane production may depend on the properties ofthe seed sludge. In this study, a supplement of 0.01% (v/v)
crude glycerol had a positive eﬀect on methane production
from the anaerobic digestion of the sludge.
Figure 1b shows the hydrogen production from the anaerobic
co-digestion of sewage sludge with crude glycerol. Hydrogen
was only detected from the sludge supplemented with 0.4%(v/v) crude glycerol. The production reached a maximumof 3.38 mL on day 1 when the glycerol was completely
decomposed. When the anaerobic digestion of 6.0% (v/v)
glycerol occurred in the presence of fermentation promoter,hydrogen generation reached 9.85 mL after 7 days of incubation26). Although the hydrogen decomposed over time,
the rate of hydrogen production was independent of themethane concentration. Generally hydrogen is rapidly taken
up and converted to other products by hydrogen-consuming
microorganisms during the methanogenic stage of anaerobic
digestion12,20). To harvest hydrogen, the anaerobic digestionof glycerol must be blocked at the hydrogen and acetic acidformation stage, namely, the acidogenic stage of anaerobic
digestion. In a previous study, it was found that sterilized
sludge produced hydrogen from the anaerobic digestion of
sewage sludge because sterilization of the sewage sludge
screened hydrogen-producing spore-forming microorganisms(against heat shock) from the sludge microflora28). However,this process required high temperature, which is unfavorable formaintaining an economical bioprocess. Another study showedthat the photosynthetic bacterium Rhodopseudomonas
palustris is capable of the photofermentative conversion ofglycerol, both pure and crude, to hydrogen23). However, theusefulness of this process is limited because of diﬃculties
with light penetration and uniform light distribution15). The
process proposed herein can achieve hydrogen production
through the simple addition of 0.4% (v/v) crude glycerol
to sewage sludge. Compared with the studies describedabove, our experimental protocol is more advantageous forhydrogen production from anaerobic digestion.
Figure 2a shows the 1,3-PDO concentration in the fermentation broth from the anaerobic co-digestion of sewage
sludge supplemented with diﬀerent concentrations of crude
glycerol. An increase of crude glycerol concentration to 0.4%(v/v) resulted in a significant production of 1,3-PDO, which
achieved a high concentration and yield on day 2, estimated at 38.4 mM and 0.57 mol/mol-glycerol respectively.
Even after reaching the maximum, the 1,3-PDO was notdecomposed. These results suggested that the productivityof the anaerobic digestion of sewage sludge was primarily
related to the supplemented crude glycerol concentration. In
fact, a supplement of 0.01% (v/v) crude glycerol improved
the methane productivity whereas 0.4% (v/v) crude glycerol
promoted both hydrogen and 1,3-PDO productivity.
Figure 2b shows the composition of the organic acids inthe fermentation broth from the anaerobic co-digestion of
sewage sludge with two diﬀerent concentrations of crude glycerol. Total production of organic acids increased as the crude
glycerol concentration increased. The sludge supplemented
with 0.4% (v/v) crude glycerol produced a significant amountof total organic acids and attained a maximum concentrationof 31.7 mM on day 6. After that, the concentration becamealmost constant. Propionic acid (26%) and acetic acid (38%)
were the major organic acids followed by butyric acid (14%).
It appears that a significant amount of organic acid lead toa pH (5.33) decrease in the digester, which likely slowed
methanogenic activity. In other words, a significant amountof organic acids may prevent the conversion of intermediates
such as hydrogen and 1,3-PDO into methane.
Although there is not a general consensus, many studies
have demonstrated that anaerobic fermentation under alka-

144

Tokumoto and Kimura

Fig. 2. (a) 1,3-PDO concentration in the fermentation broth from the anaerobic co-digestion of sewage sludge with diﬀerent concentrations ofcrude glycerol.
Sewage sludge contained 4.0 g/L TS. Crude glycerol concentration was set at ( ○ ) 0 (control), ( ● ) 0.01% (v/v), ( △ ) 0.4% (v/v). The
co-digestion was incubated at 310 K for 6 days. Data are means of the results from three independent samples. (b) Total organic acidconcentration and composition in the fermentation broth after 6 days incubation. Data are means of the results from three independent
samples.

line conditions can significantly improve VFA production
from sewage sludge3,31). It has been observed that alkali
improves the solubilization of organic matter from the solid
phase of the sludge and then increases its bioavailability foracidogens in anaerobic reactors. Therefore, it seemed thatthe strong alkaline nature of the crude glycerol resulted in thesolubilization of organic matter from the solid phase of thesludge which in turn, enhanced the activity of the acetogenic
bacteria.
3.2. The eﬀect of TS content in sewage sludge on the
productivity of the anaerobic digestion
Figure 3 and Table 1 shows the 1,3-PDO concentrationand yields in the fermentation broth from the anaerobic
co-digestion of diﬀerent TS contents of sewage sludge inthe presence of 0.4% (v/v) crude glycerol. An increase of
TS content to 20.4 g/L resulted in a higher production of
1,3-PDO. The concentrated sludge (de-watered by centrifugation) showed a maximum production and yield of 45.7 mM
and 0.64 mol/mol-glycerol on day 2, which was 1.13 times
higher than from the non-concentrated sludge containing
4.0 g/L TS. However, a further increase of TS content to42.8 g/L had a negative eﬀect on 1,3-PDO production. The
anaerobic digestion of 6.0% (v/v) glycerol in the presence offermentation promoter yielded a conversion of 0.424 mol/
mol-glycerol after 7 days of incubation27). In a previous study,
the production of 1,3-PDO by a newly isolated C. butyricum
strain was investigated19). The highest 1,3-PDO concentration
obtained from a continuous culture was 407–631 mM, witha conversion yield of 0.67 mol/mol-glycerol. Another studyof a fed-batch fermentation with sucrose as the co-substrateyielded 1.10 M 1,3-PDO and 0.62 mol/mol-glycerol witha productivity of 1.61 g/L/h in the presence of Klebsiella
oxytoca M5a1, a mutant strain deficient in lactic acidbiosynthesis29). The first report of a pilot-scale production

Fig. 3. 1,3-PDO concentration in the fermentation broth from theanaerobic co-digestion of diﬀerent TS concentrations of sewage sludge with 0.4% (v/v) crude glycerol.
TS concentration in sewage sludge was set at ( ○ ) 4.0 g/L, ( ● )20.4 g/L, ( △ ) 42.8 g/L. The co-digestion was incubated at
310 K for 6 days. Data are means of the results from three independent samples.

of 1,3-PDO using Klebsiella pneumoniae M5a1 obtained
773 mM with a yield of 0.52 mol/mol-glycerol5). Although
the maximum concentration and yield of 1,3-PDO in thisstudy is not the highest among the above described studies,
there are some advantages to this process for producing
1,3-PDO. First, there is no requirement for a single purebacterial strain that can produce 1,3-PDO from glycerol andsecond, the instruments necessary to carry out the digestion
can be directly installed at the sewage disposal plant so thatthe high concentration of sewage sludge can be remediatedimmediately. Additionally, the sludge concentration favors
not only 1,3-PDO production but also the sludge treatment,
which allows more sludge to be treated at a time than using
an anaerobic digestion process to treat the non-concentrated
sludge.

Anaerobic co-digestion of sewage sludge and crude glycerol

145

Table 1. Comparison of the yields of major digestion products(1,3-PDO, acetic acid, and propionic acid) from the anaerobic co-digestion of sewage sludge with diﬀerent concentrationsof crude glycerol on day 2 when glycerol was decomposedcompletely. Sewage sludge contained 20.4 g/L TS. Crude
glycerol concentration was set at (a) 0 (control), (b) 0.01%(v/v), (c) 0.4% (v/v). The co-digestion was incubated at 310
K for 6 days. Data are means of the results from three independent samples.

(a) Control(b) 0.01% (v/v)
crude glycerol(c) 0.4%(v/v)
crude glycerol

Yield of digestion products (g/g-TS)
1,3-PDO*1 Acetic acid Propionic acid
0
0
0
0.313
0.0526
0.03480.529

0.07470.0406*1 1,3-PDO yield is the ratio of 1,3-PDO production to digested
glycerol.

3.3. The TS removal eﬃciency during the anaerobic
digestion of sewage sludge using crude glycerol as aco-substrate
Recently, the disposal of sewage sludge to sanitary landfills
has gradually decreased, whereas the desire to re-use treatedsludge as a soil amendment or as a biofuel has increased.
The trends in sewage sludge management in many European
countries between 1992 and 2005 showed that although landfilling of sludge has not been significantly reduced since 1992,
incineration and recycling have increased steadily. Incineration aims to reduce the volume of sludge and render it inert(biologically and chemically) before landfilling. However, thisprocess requires significant amounts of energy and is therefore
more costly compared with conventional treatment options.
In Europe, one of the most desirable current uses of treatedsludge is in agriculture as a soil amendment. The anaerobic
digestion process is a well-known technology that improves
sewage sludge quality for agricultural use24), while at the sametime reducing the volume and weight of the excess sludge. In
this study, the anaerobic digestion of sewage sludge in theabsence of crude glycerol as a co-substrate resulted in a slightdecrease in the TS content of the sludge in 6 days, which was a
30% decrease from the first day (Figure 4a). Surprisingly, thesludge supplemented with 0.4% (v/v) crude glycerol showeda significant decrease in the TS content of the sludge in 6
days, calculated as a 59% decrease from the first day (Figure4b). At the same time, the total organic acid concentration
reached 8.68 g/L on day 6, which was 3.81 times higher
than the yield from the control sludge. The results provedthat the use of a strongly alkaline crude glycerol solution
resulted in the solubilization of organic material to variousorganic acids from the solid phase of the sludge. Some
researchers have found that depending on the concentration,
VFA may have a stimulating or inhibiting eﬀect on bacterial growth. Sometimes stimulation (at low concentration)and inhibition (at high concentration) have been identified
for the same bacteria22,25). Amon et al.1) showed that when
the VFA concentration significantly exceeded 5.0 g/L, theFig. 4. (a) TS content in sewage sludge from the anaerobic digestion of sewage sludge after 6 days of incubation. Sewage
sludge contained 20.4 g/L TS.
The digestion was incubated at 310 K for 6 days. Data aremeans of the results from three independent samples. (b) TS
content in sewage sludge from the anaerobic co-digestion of
sewage sludge with 0.4% (v/v) crude glycerol after 6 days of
incubation. Except for the addition of crude glycerol, the codigestion was performed under the same conditions. Data aremeans of the results from three independent samples.

anaerobic digestion process was no longer stable and organic
overloading was likely. Ginkel and Logan11) found that anincrease of VFA, particularly acetic acid, could inhibit further
growth of hydrogen-producing microorganisms, resulting in adecrease in the hydrogen production rate. From the research
reported herein, supplementation of 0.4% (v/v) crude glycerol enhanced the activity of acetogenic bacteria in the sludge
but the resulting metabolic products limited their growth. The
growth inhibition of the acetogenic bacteria, which appeared
to be a drawback of the anaerobic digestion process, would
result in a significant decrease in the excess sludge. Moreover,excessive cell growth could be unfavorable to the 1,3-PDO
synthesis because increased carbon flow could contribute tobiomass synthesis rather than 1,3-PDO production2,4,6,16). In
summary, sludge supplemented with 0.4% (v/v) crude glycerol results in both 1,3-PDO formation in good yield fromglycerol and a significant decrease in the excess sludge.

4. Conclusion
A supplement of crude glycerol had a positive eﬀect on the
productivity of the anaerobic digestion of sewage sludge. The
eﬀect depended on the crude glycerol concentration. Crude
glycerol supplementation at 0.01% (v/v) increased methane
production from the anaerobic digestion. An increase of
crude glycerol concentration to 0.4% (v/v) resulted in the
production of valuable resources such as hydrogen and
1,3-PDO. The use of concentrated sludge improved the
1,3-PDO yield and the sludge disposal capacity more thanTokumoto and Kimura146

the non-concentrated sludge. A strongly alkaline crude
glycerol supplement enhanced the solubilization of organic
material to various organic acids from the solid phase of thesludge, which resulted in a significant decrease of TS content
in the sludge compared with the control sludge without crude
glycerol supplementation.

Acknowledgments
This research was supported by a Grant-in-Aid for challenging Exploratory Research (19656243) and Grant-in-Aid
for Scientific Research (A) (21241022) from the Japan Society
for the Promotion of Science.

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