ARTICLE

pubs.acs.org/est

Nano-TiO2 Enhanced Photofermentative Hydrogen Produced from

the Dark Fermentation Liquid of Waste Activated Sludge
Yuxiao Zhao and Yinguang Chen*
State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University,
1239 Siping Road, Shanghai 200092, China
bS Supporting Information
ABSTRACT: After anaerobic dark fermentation of waste activated sludge
Downloaded from pubs.acs.org by OPEN UNIV OF HONG KONG on 01/23/19. For personal use only.

(WAS) for hydrogen production, there are a large number of organic com-
pounds including protein, polysaccharide, and volatile fatty acids left in the dark
fermentation liquid, which can be further bioconverted to hydrogen by
photofermentation techniquea. In this study, the enhancement of photofer-
mentative hydrogen produced from WAS dark fermentation liquid by using
nano-TiO2 is reported. First, high concentration of NH4+-N in the dark
fermentation liquid was observed to inhibit the photofermentative hydrogen
production, and its removal was essential. Then the effect of nano-TiO2 on
photofermentative hydrogen generation was investigated, and the addition of
Environ. Sci. Technol. 2011.45:8589-8595.

100 mg/L nano-TiO2 increased hydrogen by 46.1%. Finally, the mechanisms for
nano-TiO2 improving hydrogen production were investigated. It was found that
nano-TiO2 improved the decomposition of protein and polysaccharide to small-
molecule organic compounds and promoted the growth of photosynthetic bacteria and the activity of nitrogenase but decreased the
H2-uptake hydrogenase activity.

’ INTRODUCTION mineralized via photofermentation with photosynthetic bacteria

Waste activated sludge (WAS) is a byproduct of biological
municipal wastewater treatment plant. It contains significant
amounts of protein and polysaccharide. Recently, much atten-
tion has been paid to sustainable hydrogen biological produc-
tion from WAS, by which human resources, such as fossil fuels,
are saved, and sludge is reused.1
4 The most common method
for biological hydrogen production from WAS is anaerobic dark
fermentation.
Hydrogen production by anaerobic dark fermentation in-
cludes several stages, such as solubilization of sludge particulate
organic matter, hydrolysis of solubilized sludge organic materi-
als, acidification of hydrolyzed products with hydrogen genera-
tion, and hydrogen consumption. The hydrogen production of
anaerobic dark fermentation is rather low (0.08 mmol H2/g
dried sludge), which has been widely believed to be caused by
the low WAS hydrolysis efficiency and the rapid consumption
of generated H2 by hydrogen consumers.3 Thus, most of the
strategies reported in the literature for improving biohydrogen
production are from the aspects of increasing sludge hydrolysis
and/or killing hydrogen consumers by pretreating sludge with
ultrasonic, acidic, alkaline, heat-shocking, or freezing
thawing
methods.5
8
It is well-known that anaerobic dark fermentation of organic
matter produces not only hydrogen but volatile fatty acids
(VFAs), which results in a low observed hydrogen production.3
Some of the soluble intermediate metabolites left in the
anaerobic dark fermentation liquid can be further degraded or
(PSB) to produce hydrogen.9,10 The stoichiometries of photo-
fermentative hydrogen production from acetic, propionic, and
butyric acids can be described by the following equations:11
CH3 COOH þ 2H2 O f 4H2 þ 2CO2 ð1Þ
CH3 CH2 COOH þ 2H2 O f 3H2 þ CH3 COOH þ CO2
ð2Þ
CH3 CH2 CH2 COOH þ 2H2 O f 2H2 þ 2CH3 COOH
ð3Þ
It seems that when biohydrogen is recovered from waste
activated sludge, a higher hydrogen production can be achieved
through the combination of dark fermentation and photofer-
mentation. Figure 1 illustrates the proposed schematic diagram
of two-step fermentation for hydrogen production from WAS
(Figure 1). Our previous work showed that by controlling the
anaerobic dark fermentation of WAS at constant pH 10 (i.e.,
step 1 in Figure 1), a remarkably higher hydrogen production
of 0.87 mmol H2/g dried sludge was achieved due to the
improvement of the anaerobic process (sludge solubilization,
Received: May 12, 2011
Accepted: August 18, 2011
Revised: August 10, 2011
Published: August 18, 2011

r 2011 American Chemical Society 8589 dx.doi.org/10.1021/es2016186 | Environ. Sci. Technol. 2011, 45, 8589–8595
Environmental Science & Technology
Figure 1. Two-step fermentation for biohydrogen production from waste activated sludge.
hydrolysis, and acidification) and the inhibition of hydrogen
consumption.12 Nevertheless, it is unclear whether the dark
fermentation liquid can be used as the substrate of PSB for
efficient photofermentative hydrogen production (i.e., step 2 in
Figure 1).
The purpose of this study was to investigate the feasibility of
photofermentative hydrogen production from WAS anaerobic
dark fermentation liquid. First, the influence of high concentra-
tions of NH4+-N in the dark fermentation liquid on photofer-
mentative hydrogen production was studied, and its removal was
found to be essential for hydrogen generation by PSB. Then the
enhancement of photofermentative hydrogen production by the
use of nano-TiO2 was reported. As there were no references
available on the mechanisms of nano-TiO2 improving photo-
fermentative hydrogen production, the role of nano-TiO2 was
finally investigated in this paper.
’ MATERIALS AND METHODS
Enrichment of PSB and Preparation of WAS Anaerobic
Dark Fermentation Liquid. The photosynthetic bacterium used
in this study was isolated from waste activated sludge and was
identified as Rhodopseudomonas palustris after analysis of its 16S
rDNA sequence. The isolation method was as follows: 5 g of
sludge and 50 mL of deionized water were put in a conical flask,
which was then oscillated for 20 min at 120 rpm. After settling for
1 h, 5 mL of the suspension was transferred to a 600 mL serum
bottle and mixed with a common phototrophic medium 27 (see
Supporting Information). The serum bottle was purged with
argon gas for 10 min to remove oxygen and then sealed with
rubber stopper. The bacteria were enriched at 30 ( 1 °C by use
of halogen tungsten lamps as a light source at a light intensity of
200 W/m2 (6000
7000 lx, 350
820 nm, unless otherwise
stated) for 7 days until small quantities of red bacteria were
observed on the bottle wall. Then the red bacteria were cultured
again for 3 days by the same method, and the same procedure was
repeated 3 times. The bacteria were finally purified by plate
isolation, and the isolated PSB were cultured at 30 ( 1 °C and a
light intensity of 200 W/m2 under sterilized conditions with
phototrophic medium 27 for 7 days for the photofermentative
hydrogen production experiments.
The preparation of WAS anaerobic dark fermentation liquid
and its characteristics are shown in Supporting Information All
the following experiments were conducted in triplicate, and one-
way analysis of variance (ANOVA) at 0.05 level was used to
analyze the data.
TiO2 Nanoparticles. Nano-TiO2 (<25 nm, anatase) was
purchased from Sigma
Aldrich. Its stock dispersion was pro-
duced by adding 2 g of nano-TiO2 to 1000 mL of distilled water,
8590
ARTICLE
vigorously stirring for 10 min, and sonicating for 30 min
according to ref 13. While the advertised size of nano-TiO2
was below 25 nm, the particles almost immediately aggregated
in distilled water. Dynamic light scattering (DLS) analysis
indicated that the particles of nano-TiO2 in the stock dispersion
aggregated to a size distribution of 80
260 nm with an average
particle size of 185 nm, which was in accordance with the
observation made by Ge et al.13 that TiO2 nanoparticles were
prone to aggregate to a size distribution of 190
230 nm even in
nanopure water.
Effect of NH4+-N Removal from Sludge Dark Fermentation
Liquid on Photofermentative H2 Production. The average
NH4+-N concentration in the liquid of WAS dark fermentation
was 164 mg/L. It was removed by adding MgCl2 and KH2PO4
to form struvite according to the optimal conditions reported in
our previous publication.14 The characteristics of sludge dark
fermentation liquid before and after NH4+-N removal are
shown in Table 1. Two groups of 600 mL serum bottles were
used in this test with three parallels each. Dark fermentation
liquid before NH4+-N removal (300 mL) was added to each
bottle of group 1, and dark fermentation liquid after NH4+-N
removal (300 mL) was added to each bottle of group 2. The
initial pH in each serum bottle was adjusted to 8.0 by adding 4
M hydrochloric acid (HCl), and the PSB were added to achieve
a biomass concentration of approximately 400 mg/L. The
serum bottles were purged with argon for 10 min and then
sealed with rubber stoppers. All serum bottles were maintained
at 30 ( 1 °C with halogen tungsten lamps as the light source at a
light intensity of 200 W/m2.
Effect of Nano-TiO2 Concentration on Photofermentative
H2 Production from Sludge Dark Fermentation Liquid.
Sludge dark fermentation liquid with NH4+-N removed (1500 mL)
was divided equally into five serum bottles. The nano-TiO2
concentrations in these five serum bottles were adjusted to 0, 50,
100, 150, and 200 mg/L by adding stock dispersions of nano-TiO2.
The PSB were added to each serum bottle to achieve a biomass of
approximately 400 mg/L. All other operations were the same as
described above.
Effect of Nano-TiO2 on Hydrogen Production in the
Absence of PSB. The following test was arranged to examine
whether the presence of nano-TiO2 could cause the production
of hydrogen when there were no PSB. Dark fermentation liquid
with NH4+-N removed (300 mL) was put into a 600 mL serum
bottle, and then nano-TiO2 was added to a concentration of 100
mg/L. All other operations were the same as described above.
Effect of Nano-TiO2 on Degradation of Protein, Polysac-
charide, and Acetic Acid in the Absence of PSB. Protein, poly-
saccharide, and acetate were the main organic compounds in sludge
dark fermentation liquid. To examine the effects of nano-TiO2
dx.doi.org/10.1021/es2016186 |Environ. Sci. Technol. 2011, 45, 8589–8595
Environmental Science & Technology
Table 1. Compositions of Sludge Dark Fermentation Liquid before and after NH4+-N Removal
SCOD (mg-COD/L) VFAs (mg-COD/L) protein (mg-COD/L) polysaccharide (mg-COD/L) NH4+-N (mg/L) SOP (mg/L)
before NH4+-N removal 5988 ( 300 2381 ( 119a
after NH4+-N removal 5412 ( 270 2296 ( 114b
avg removal efficiency (%) 9.6 3.6
a
VFAs were composed of the following acids: acetic 1540 ( 77, propionic 342 ( 17, n-butyric 95 ( 5, isobutyric 169 ( 8, and isovaleric 235 ( 12 mg-
COD/L. b VFAs were composed of the following acids: acetic 1492 ( 75, propionic 337 ( 16, n-butyric 84 ( 4, isobutyric 152 ( 7, and isovaleric 231 (
12 mg-COD/L.
on the degradation of these organic compounds in the absence
of PSB, the following synthetic wastewater tests of bovine
serum albumin (BSA, average molecular weight 670 000,
model protein compound used in this study) or dextran
(Mw ∼ 42 000, model polysaccharide compound) or sodium
acetate were conducted. Distilled water (600 mL) was divided
equally into two serum bottles. BSA (0.51 g) (or 0.18 g of dextran
or 0.48 g of sodium acetate) was added to both bottles. Then
nano-TiO2 was added to one bottle to achieve a nano-TiO2
concentration of 100 mg/L. All other operations were the same
as described above.
Effect of Nano-TiO2 on the Activity of PSB. To investigate
the effect of nano-TiO2 on the activity of PSB, the following
synthetic wastewater fermentation tests were conducted. Syn-
thetic wastewater (600 mL) containing (mg/L of distilled water)
1640 sodium acetate (sole carbon source), 500 sodium glutamate
(sole nitrogen source), 500 KH2PO4, 50 CaCl2, 400 MgSO4 3 7
H2O, and 400 NaCl, with 0.4 mL of vitamin B12 solution and
1 mL of trace element solution (Supporting Information), was
divided equally into two serum bottles, and nano-TiO2 was added
to one bottle to achieve a concentration of 100 mg/L. Then each
serum bottle was inoculated with PSB (approximately 400 mg/
L). The biomass concentration in the nano-TiO2 addition bottle
was measured by subtracting the concentration of nano-TiO2. All
other operations were the same as described above.
Analytical Methods. Ethylene was analyzed on a GC 6890 gas
chromatograph with a flame ionization detector, and nitrogen
was used as a carrier. Determinations of hydrogen, VFAs, protein,
polysaccharide, and biomass were the same as described in our
previous publications.12,15 The pH was measured by a pH meter.
Molecular weight (Mw) distributions of protein and dextran
were determined by gel-filtration chromatography (GFC) ana-
lyzer (Shimadzu Co., Japan). A TSK G4000SW type gel column
(Tosoh Co., Japan) was used in this work with Milli-Q water as
mobile phase at a flow rate of 0.5 mL 3 min
1. The samples were
filtered through a 0.45 μm hydrophilic filtration membrane and
diluted to an approximate concentration before being analyzed
by a refractive index detector (RID-10A). The Mw was calculated
with class VP 5.03 software (Shimadzu Co., Japan).
Hydroxyl radical was detected by the salicylic acid method with
a few modifications.16 The hydroxyl radical (•OH) is extremely
reactive and reacts with salicylic acid to generate hydroxylated
salicylate complex. Two serum bottles were used to assay hydro-
xyl radical. The salicylate concentration was 1 mmol/L in each
bottle. One serum bottle was used as control with no additions,
but nano-TiO2 was added to the other bottle to achieve a
concentration of 100 mg/L. Both bottles were treated for 1 h at
30 ( 1 °C with halogen tungsten lamps as the light source at a
light intensity of 200 W/m2. Samples (5 mL) from the reaction
system was collected by centrifugation for 30 min at 4800 rpm,
ARTICLE
2123 ( 106 770 ( 38 164 ( 9 108 ( 5
1747 ( 87 601 ( 29 15 ( 1 12 ( 1
17.7 21.9 90.9 88.9
0.25 mL of HCl and 0.5 g of NaCl were added to each solution,
and 4 mL of ether was used as the extractant. After the extraction,
3 mL of ether in the supernatant was put in a 10 mL tube, which
was evaporated to dryness at 40 °C. Trichloroacetic acid (10%)
(0.15 mL), 0.25 mL of sodium tungstate (10%), and 0.25 mL of
sodium nitrite (0.5%) were added to the tube. After 5 min,
0.25 mL of NaOH (1M) and 3.10 mL of distilled water were
added to the tube. Hydroxylated salicylate complex was measured
at 562 nm.
Nitrogenase activity was assayed by the acetylene reduction
method of Zhang et al.17 H2-uptake hydrogenase was measured
according to Goodman and Hoffman.18 An aliquot (1.5 mL) of
anaerobically grown cells was centrifuged and dissolved in 1 mL
of 20 mM phosphate buffer (pH = 7.0). Phosphate buffer (1 mL)
was added, together with 100 mL of 40 mM benzyl viologen. The
mixture was first flushed with nitrogen gas to make an anaerobic
environment and then with hydrogen gas as a substrate for H2-
uptake hydrogenase for 5 min. This mixture was prepared in an
anaerobic cuvette and the kinetics of the reaction was measured
by the spectrophotometer. The color change due to reduction of
benzyl viologen was recorded for a certain time at 600 nm.
Bacterial strain identification was carried out by 16S rDNA
sequence analysis. Colonies of photosynthetic bacteria underwent
DNA amplification by PCR using the bacterial universal primers
27f and 1492r. The PSB were determined to be Rhodopseudomo-
nas palustris on the basis of16S rDNA sequence analysis.
’ RESULTS
Removal of NH4+-N from WAS Dark Fermentation Liquid
Affects Photofermentative Hydrogen Production. In the
literature, it was found that the presence of NH4+-N at a
concentration of greater than 28 mg/L inhibited photofermen-
tative hydrogen production by PSB.19 In this study the average
concentration of NH4+-N in the WAS dark fermentation liquid
was 164 mg/L. It seems that it is necessary to remove the NH4+-
N before photofermentative hydrogen is recovered from the dark
fermentation liquid. The formation of struvite (magnesium
ammonium phosphate) was reported to be an efficient method
to remove NH4+-N from sludge dark fermentation liquid.14 The
main compositions of fermentation liquid before and after NH4+-
N removal are summarized in Table 1. There was only 15 mg/L
NH4+-N left in the fermentation liquid after NH4+-N removal,
and only a little soluble chemical oxygen demand (SCOD),
protein, and polysaccharide were removed from the liquid. The
VFAs before and after NH4+-N removal were 2381 and 2296 mg-
COD/L, respectively, indicating that NH4+-N removal had little
effect on the VFAs concentration. After NH4+-N removal, the
dark fermentation liquid had a soluble COD of 5412 mg/L,
which was composed of 42.4% VFAs, 32.3% protein, 11.1%
8591 dx.doi.org/10.1021/es2016186 |Environ. Sci. Technol. 2011, 45, 8589–8595
Environmental Science & Technology
Figure 2. Effect of nano-TiO2 concentration on photofermentative
hydrogen produced from dark fermentation liquid with NH4+-N
removed. Error bars represent standard deviations of triplicate tests.
polysaccharide, and 14.2% remaining unknown. The VFAs
consisted of 65.0% acetic acid, 14.7% propionic acid, 10.1%
isovaleric acid, 6.6% isobutyric acid, and 3.6% n-butyric acid.
The influence of NH4+-N removal on photofermentative
hydrogen production is shown in Figure S1 (Supporting
Information). At 7 and 10 days, the accumulated hydrogen
was only 6.6 and 7 mL, respectively, when NH4+-N was not
removed. However, after NH4+-N removal, the produced
hydrogen was respectively 94.8 and 101.1 mL. The observation
of hydrogen production significantly enhanced by NH4+-N
removal could be made at any other time. It is well-known that
hydrogen production with PSB is catalyzed by nitrogenase,
which enables them to reduce H+ to H2 under light-anaerobic
conditions. As seen in Table S1 (Supporting Information), the
nitrogenase activity of PSB was remarkably increased after
NH4+-N removal. It has been reported that the photofermen-
tative hydrogen production of PSB is also affected by the
activity of H2-uptake hydrogenase, which catalyzes the conver-
sion of molecular hydrogen to electrons and protons and
decreases hydrogen production.17,18 The data in Table S1
(Supporting Information) show that the activity of H2-uptake
hydrogenase was not affected by NH4+-N removal. Further
investigation in Table S1 indicated that the removal of NH4+-N
did not significantly influence the growth of PSB. According to
data in Figure S1 and Table S1 (Supporting Information), it can
be concluded that the removal of NH4+-N is necessary for
photofermentative hydrogen produced from sludge dark fer-
mentation liquid because higher concentrations of NH4+-N
inhibited the activity of nitrogenase.
Effect of Nano-TiO2 Concentration on Photofermentative
Hydrogen Produced from Sludge Dark Fermentation Liquid.
As seen in Figure 2, the accumulated hydrogen in all tests
increased with time during the initial 7 days, but further
increasing the time to 8 days did not result in significant
improvement in hydrogen production (see Table S2, Support-
ing Information, for statistical analysis). At fermentation time of
7 days, the accumulated hydrogen was 116.6, 138.5, 109.8, and
103.4 mL at nano-TiO2 concentrations of 50, 100, 150, and 200
mg/L, respectively, which was 22.9%, 46.1%, 15.8%, and 9.1%
higher than without nano-TiO2 addition (94.8 mL). The
addition of nano-TiO2 to the PSB system improved hydrogen
production, and the maximal hydrogen was achieved at nano-
TiO2 concentration of 100 mg/L. The reasons for hydrogen
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Figure 3. Effect of nano-TiO2 on the changes of main organic com-
pound concentration in dark fermentation liquid. Error bars represent
standard deviations of triplicate tests.
production increased by nano-TiO2 were investigated and are
discussed below.
Effect of Nano-TiO2 on Hydrogen Production in the
Absence of PSB. When there were no PSB in the reaction
system, the influence of TiO2 on hydrogen production via
photocatalytic decomposition of water or organic compounds
was first examined, and the results showed that no detectable
hydrogen was produced. Apparently, nano-TiO2 improving the
photofermentative hydrogen production, which was observed in
Figure 2, was not caused by its photocatalytic decomposition of
water or organic compounds. Several researchers reported that
after nano-TiO2 was modified by doping metal or nonmetal
materials, it exhibited the ability of decomposing water to
produce hydrogen.20,21 Nevertheless, the present study revealed
that no hydrogen gas was produced when pure anatase nano-
TiO2 was investigated with the halogen tungsten lamps as the
light source at a light intensity of 200 W/m2 (6000
7000 lx,
350
820 nm) and temperature 30 ( 1 °C.
Variations of Main Organic Compounds during Photo-
fermentative Hydrogen Production from Sludge Dark Fer-
mentation Liquid. Protein, polysaccharide, and VFAs are the
main organic components of WAS dark fermentation liquid. As
shown in Figure 3, the concentrations of these three organic
compounds decreased with fermentation time in all tests. Never-
theless, the concentrations of protein, polysaccharide, and VFAs
in the presence of nano-TiO2 were lower than those in the
control at any time. For example, at 7 days the average soluble
protein, soluble polysaccharide, and VFAs in the presence and
absence of nano-TiO2 were respectively 648 and 1005, 197 and
291, and 386 and 827 mg-COD/L. There are two possible
dx.doi.org/10.1021/es2016186 |Environ. Sci. Technol. 2011, 45, 8589–8595
Environmental Science & Technology
Figure 4. Effect of 100 mg/L nano-TiO2 on the Mw distributions of protein and dextran in the synthetic wastewater experiments in the absence of PSB.
(a1) GFC chromatograms of protein wastewater, (a2) percentage of Mw < 1000 in protein wastewater, (c) GFC chromatograms of dextran wastewater,
and (d) percentage of Mw < 1000 in dextran wastewater.
reasons for this difference. One is that degradation of protein and
polysaccharide was enhanced by the addition of nano-TiO2, and
the other is that activity of PSB was increased by nano-TiO2,
which resulted in improved utilization of VFAs. The detailed
mechanisms were investigated by the following synthetic waste-
water experiments.
Effect of Nano-TiO2 on Degradation of Protein, Polysac-
charide, and Acetic Acid. When protein or dextran was used as
the sole substrate of synthetic wastewater photodegradation
tests (with light and 100 mg/L TiO2 but without PSB inocu-
lation), after 7 days of reaction time the degradation efficiencies
of protein and dextran were respectively 23.5% and 16% in the
case of TiO2 added (Table S3, Supporting Information).
Nevertheless, degradation of protein or dextran was not ob-
served in the control tests (no TiO2 addition). Also, there were
no significant changes in the concentration of SCOD even in
the presence of TiO2 (data not shown), and no CO2 generated
(Table S3, Supporting Information). The molecular weight
(Mw) distributions of protein and dextran in the synthetic
wastewater experiments in the absence of PSB were further
studied and the results are shown in Figure 4. The Mw of protein
was between 0 and 1 200 000, and that of dextran was between 0
and 90 000. It was observed that only one peak (at around
22 min) appeared in the range of Mw 0
1000 in both protein
and dextran systems (Figure 4a1,b1), and the percentage of Mw <
1000 increased from 13.1% to 29.6% (protein wastewater,
Figure 4a2) and from 3.2% to 14.2% (dextran wastewater,
Figure 4b2) after 7 d reaction time. Apparently nano-TiO2
enhanced the photodecompositions of protein and polysac-
charide to small-molecule compounds other than CO2, which
might be more easily utilized by PSB. The photofermentative
hydrogen produced by PSB was therefore improved at nano-
TiO2 concentration of 100 mg/L (Figure 2). Although it was
noted that, in the acetic acid synthetic wastewater tests, the
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ARTICLE
presence of light and TiO2 (without PSB) caused the decom-
position of acetic acid (the preferred substrate for PSB producing
hydrogen) and the formation of CO2, the degradation efficiency
of acetic acid was only 8.5% (Table S3, Supporting Information).
Generation of hydroxyl radical was thought to be an important
reason for the decomposition of some organic substrates caused
by nano-TiO2.22 The hydroxyl radical is extremely reactive and
can react with salicylic acid to generate hydroxylated salicylate
complex, which absorbs at 562 nm.16 Thus the absorbance at
562 nm can represent the concentration of hydroxyl radical.
Without addition of nano-TiO2, there was no hydroxyl radical
detectable. However, the measured hydroxyl radical was 0.046 at
nano-TiO2 of 100 mg/L. It seems that, in the presence of nano-
TiO2, the observed degradation of main organic compounds of
sludge dark fermentation liquid was due to the generation of
hydroxyl radical.
Effects of Nano-TiO2 on Growth of PSB and Activities of
Hydrogen Production-Related Enzymes. With acetic acid as
the sole carbon source of PSB, the effects of nano-TiO2 (100 mg/L)
on hydrogen production, PSB growth, and activity of key
enzymes (nitrogenase and H2-uptake hydrogenase) in the syn-
thetic wastewater tests are illustrated in Figure 5. As seen in
Figure 5a,b, both hydrogen production and nitrogenase activity in
the presence of nano-TiO2 were significantly higher than those in
the control at any fermentation time investigated. Nevertheless,
the data in Figure 5c show that the H2-uptake hydrogenase activity
of PSB decreased with addition of nano-TiO2 at any time,
suggesting that less hydrogen was consumed and more hydrogen
could be accumulated. From Figure 5d it can be seen that, with the
addition of nano-TiO2, there were greater biomass concentrations
at any fermentation time, which indicated that nano-TiO2 pro-
moted the growth of PSB. The same observations were made
when the sludge dark fermentation liquid was utilized to produce
dx.doi.org/10.1021/es2016186 |Environ. Sci. Technol. 2011, 45, 8589–8595
Environmental Science & Technology ARTICLE

Figure 5. Effect of nano-TiO2 on (a) accumulated hydrogen production, (b) nitrogenase activity, (c) H2-uptake hydrogenase activity, and (d) biomass
concentration in the acetic acid synthetic wastewater tests. Error bars represent standard deviations of triplicate tests.

According to the above investigations, it can be seen that

removal of NH4+ removal was necessary for photofermentative
H2 produced from sludge dark fermentation liquid, and H2
production could be significantly increased by the addition of
100 mg/L TiO2 to the PSB fermentation system due to the
improvement of protein and polysaccharide decomposition,
PSB growth and nitrogenase activity, and the inhibition of H2-
uptake hydrogenase activity. The hydrogen production was
0.87 mmol H2/g dried sludge in the dark fermentation stage12
and 1.01 mmol H2/g dried sludge in the photofermenta-
tion step, and the total hydrogen in these two steps reached
1.88 mmol H2/g dried sludge. In the literature, the reported
maximal hydrogen productions with raw and pretreated WAS
self-fermentation were respectively 0.08 and 0.74 mmol H2/g
dried sludge.3,6 It seems that the hydrogen recovery efficiency
from waste sludge can be remarkably increased by the two-step
process proposed in this study.

’ ASSOCIATED CONTENT

bS Supporting Information. Additional materials and

methods, three tables, and one figure as described in the text.
This material is available free of charge via the Internet at http://
pubs.acs.org/.

’ AUTHOR INFORMATION

Figure 6. Effect of 100 mg/L nano-TiO2 on the activity of (a) Corresponding Author
nitrogenase and (b) H2-uptake hydrogenase and (c) the concentration *E-mail: yg2chen@yahoo.com; tel: 86-21-65981263; fax: 86-21-
of biomass during photofermentation of sludge dark fermentation liquid 65986313.
for hydrogen production. Error bars represent standard deviations of
triplicate tests.
’ ACKNOWLEDGMENT
hydrogen (Figure 6). As a result, a higher photofermentative This work was financially supported by the Foundation of
hydrogen production was observed by the use of nano-TiO2. State Key Laboratory of Pollution Control and Resource Reuse.
8594 dx.doi.org/10.1021/es2016186 |Environ. Sci. Technol. 2011, 45, 8589–8595
Environmental Science & Technology ARTICLE

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