Chemical Engineering Journal 263 (2015) 461–470

Contents lists available at ScienceDirect

Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

Influence of zero valent scrap iron (ZVSI) supply on methane production

from waste activated sludge
Guangyin Zhen a,b,⇑, Xueqin Lu a, Yu-You Li a,⇑, Yuan Liu c, Youcai Zhao d
a
Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan
b
National Institute for Environmental Studies, Onogawa 16-2, Tsukuba, Ibaraki 305-0053, Japan
c
Graduate School of Environmental Studies, Tohoku University, Sendai, Miyagi 980-8579, Japan
d
The State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Zero valent scrap iron (ZVSI) was used

to stimulate the anaerobic digestion
of sludge.
 The highest methane yield was
174.9 ± 1.5 mL/g VSSfed with ZVSI
1.0 g/g VSS, increasing by 38.3%.
 ZVSI enhanced the methanogenesis
via acting as electron donors and
creating favorable environment.
 Responsible mechanisms for
enhancing methane formation in the
presence of ZVSI were proposed.

a r t i c l e i n f o a b s t r a c t

Article history: The use potential of zero valent scrap iron (ZVSI) for promoting anaerobic production of waste activated
Received 27 August 2014 sludge (WAS) was assessed. The results indicated that the presence of ZVSI effectively enhanced WAS
Received in revised form 19 October 2014 methane production. Methane yield reached the maximum of 174.9 ± 1.5 mL/g VSSfed with addition of
Accepted 1 November 2014
1.0 g-ZVSI/g VSS, increasing by 38.3%. The corresponding values for sludge hydrolysis (%H), acidogenesis
Available online 7 November 2014
(%A), and methanogenesis rate (%M) increased into 52.6 ± 0.4%, 44.5 ± 2.9% and 40.8 ± 0.3%, respectively,
versus 45.2 ± 0.5%, 40.9 ± 2.0% and 29.5 ± 0.2% in the test without ZVSI. Further investigations confirmed
Keywords:
that ZVSI could offer electrons (H2/[H]), promote H2 consumption and afford beneficial pH environment
Waste activated sludge
Zero valent scrap iron (ZVSI)
for WAS digestion, which stimulated the metabolisms of critical microbes involved in the process and,
Anaerobic digestion subsequently accelerated the hydrolysis–acidification–methanation steps of WAS. As a result of this,
Methane production methane production was upgraded. The superior ability of ZVSI to serve as a source of electron donors
will open a new door for WAS treatment. Moreover, ZVSI is less expensive, and convenient to use,
providing a cost-efficient alternative for simultaneous energy recovery along with waste iron scrap
conservation.
Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction society [1]. WAS anaerobic digestion (AD) has garnered much
attention recently [2–4] as it allows sludge stabilization, odor
Huge production of waste activated sludge (WAS) is becoming control [5] and bioenergy recovery [6]. AD usually involves four
one of the most critical environmental challenges facing modern steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis.

⇑ Corresponding authors at: Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba-ku,
Sendai, Miyagi 980-8579, Japan. Tel.: +81 22 795 4867.
E-mail addresses: zhenguangyin@163.com (G. Zhen), yyli@epl1.civil.tohoku.ac.jp (Y.-Y. Li).

http://dx.doi.org/10.1016/j.cej.2014.11.003
1385-8947/Ó 2014 Elsevier B.V. All rights reserved.
462 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470

Hydrolysis of particulate organics associated with microbial cells Japan. The sludge, pre-screened through a 2.0 mm mesh to remove
and extracellular polymeric substances (EPS) has been proven as any large particles before use, was concentrated by settling at 4 °C
the rate-limiting step [7–10] for the startup of the anaerobic sys- for 4 h and its main characteristics after settlement are given in
tems while methanogenesis is considered rate-limiting for further Table 1. The pre-screened sludge was then kept at 4 °C to maintain
fermentation of the released soluble substrates [11]. WAS is a com- its freshness.
plex multiphase medium, composed of a diversity of microbes as
well as other organic and inorganic particles [12,13]. EPS, located 2.2. Biochemical methane potential (BMP) assay
at the cell surface, constitute 50–60% of the organic fraction of
sludge [14] and glue sludge particles together firmly. WAS with Batch-scale experiments of influence of ZVSI (approx. 2.0 mm in
high contents of microbial cells and EPS thus has the sturdy struc- width and 0.35 mm in thickness) (Shanghai CNPC powder material
ture against hydrolytic enzyme and is more difficult to hydrolyze. Co., China) on the hydrolysis, acidogenesis and methanogenesis of
If without a pretreatment step, a very long solids retention time sludge were carried out in series of 120 mL serum bottles. Each
(20–50 days) will be necessary to obtain a 20–50% degradation of bottle was fed with 90 mL sludge suspensions. The pH of the liquor
organics [2,15]. Hence, to boost the hydrolysis and subsequent in each bottle was then checked carefully and neutralized to
AD, different pretreatment options, including mechanical [16], around 7.0. Afterwards, ZVSI was added with its dosage being 0,
ultrasonic [10,17], chemical treatments [7] or the combination of 0.05, 0.1, 0.25, 0.5 and 1.0 g/g VSS, respectively. After sealed with
two of them [15,18] have been developed. rubber stoppers, all bottles were flushed with nitrogen gas to
Zero valent iron (ZVI), a strong reducing reagent remove oxygen and then incubated in a water bath (20 ± 0.5 °C)
(E0 = 0.447 V), has been universally used in contaminated site (BT 100, Yamato, Japan) under continuous shaking (100 ± 1 rpm)
remediation [19], decolorization of Rhodamine B [20], degradation for digestion. After 12 days of operation, temperature was
of azo dye [21] and microbial removal of perchlorate [22]. Most increased and maintained at 35 ± 0.5 °C to examine the effect of
recently, the promotion for AD using ZVI has also been docu- operating temperatures. During the running test, 1.0 mL of liquid
mented [1,23,24]. The preliminary idea of using ZVI is based on sample was manually withdrawn at a given time interval for the
the release of hydrogen through electron transfer during ZVI corro- analysis of pH, SCOD, protein, carbohydrate and VFAs. All the fer-
sion/oxidation [23], which can serve as an electron-donor source mentation experiments were conducted in duplicate to ensure
for methanogens [25–28], sulfate reducing bacteria (SRB) [21] or the reproducibility of results. The biogas production was measured
dechlorinating bacteria [22,23]. For instance, the activities of key periodically by releasing the pressure in bottles. At each sample
enzymes in hydrolysis-acidification were reported to increase event, the compositions of biogas were measured and CH4 yield
0.6–1 time through adding ZVI and methane yield raised 43.5% [1]. in the course of time was calculated as the values under standard
Unlike the exciting results achieved before, slight stimulation conditions (101.325 kPa, 273.15 K).
and even potential inhibition of ZVI on methanogens [29] and
SRB [30] have been observed. In a study nano ZVI at 1 mM and 2.3. Analytical methods
above reduced methane production by higher than 20%; at
30 mM, nano ZVI greatly increased soluble COD and volatile fatty 2.3.1. Biodegradability of sludge
acids (VFAs) along with an accumulation of H2, leading to a reduc- The effect of ZVSI on the hydrolysis (%H), acidogenesis (%A),
tion of methane by 69% [29]. The toxicity principles of nano ZVI methanogenesis (%M) and biodegradability (%B) were calculated
may include: (i) fast H2 production and accumulation, which according to Field et al. [35] and Astals et al. [36] (Eqs. (1)–(4)):
impedes the thermodynamical conversions of propionic and buty-
ðSCOD  SCOD0 þ CODCH4 Þ
ric acids into acetic acid [31] and methanogenesis (H2 threshold of %H ¼  100 ð1Þ
TCOD0  SCOD0
23–75 nM for methanogens [32]); and (ii) disruption of cell mem-
branes via reductive decomposition of ZVI [29,33], which inhibits
methanogenesis. Clearly, up to date, the behaviors of ZVI in anaer-
obic systems are still inconsistent and sometimes contradictory.
The questions therefore remains as to whether and how ZVI will Table 1
affect hydrolysis, acidogenesis and methanogenesis in anaerobic Characteristics of WAS used in this study.
process. Besides, most previous studies were undertaken mainly Parameter Unit Valuea SDb
with ZVI, and very limited research referred to zero valent scrap pH 6.2 0.0
iron (ZVSI) has been conducted [1,34]. ZVSI is a byproduct pro- TS (total solids) g/L 27.3 0.6
duced from machinery industries and easily available at local level VS (volatile solids) g/L 22.4 0.4
[21]; moreover it has excellent reactivity similar to ZVI but very TSS (total suspended solids) g/L 26.5 0.3
VSS (volatile suspended solids) g/L 21.9 0.2
low price. Therefore, provided that ZVSI can substitute ZVI, this
TCOD (total chemical oxygen demand) mg/L 30141 1003
will not only lower the overall project costs but also stimulate SCOD (soluble chemical oxygen demand) mg/L 86 2
the waste scrap conservation. Owing to the above factors, a series Total protein mg/L 8881.1 563.9
of batch experiments with six different dosages of ZVSI were thus Total carbohydrate mg/L 2292.9 42.0
performed herein to exploit the exact roles of ZVSI in WAS diges- Soluble protein mg/L 11.7 0.7
Soluble carbohydrate mg/L 12.3 0.6
tion, with the primary aim of developing a sustainable strategy Ammonia nitrogen (NH+4-N) mg/L 13.2 0.2
to drive sludge stabilization while accomplishing bioenergy Sulfate (SO2
4 ) mg/L 2.6 0.2
recovery. Acetic acid mg/L – –
Propionic acid mg/L – –
Iso-butyric acid mg/L – –
N-butyric acid mg/L – –
2. Materials and methods Iso-valeric acid mg/L – –
N-valeric acid mg/L – –
2.1. Source of WAS Total VFAs mg/L – –

–: below the limits of detection.

WAS sample was obtained from the secondary sedimentation a
All values are expressed in average data.
b
tank of a municipal wastewater treatment plant (WWTP) in Sendai, SD: standard deviation.

ðCODVFA  CODVFA0 þ CODCH4 Þ
%A ¼  100
TCOD0
CODCH4
%M ¼  100
TCOD0
 
YA CODVFA0 YM
%R ¼ %A þ  %A  100  þ  %M
1  YA TCOD0 1  YM
where YA is the yield of acidogenic microorganisms (0.050 g
COD g1 COD) and YM is the yield of methanogenic archaea
(0.029 g COD g1 COD).
2.3.2. Sludge fractioning protocol
Sludge was separated into four different fractions, i.e., slime
EPS, loosely bound EPS (LB-EPS), tightly bound EPS (TB-EPS) and
pellet based on the previous method [37,38]. Protein and carbohy-
drate in different fractions were then analyzed.
2.4. Other analysis methods
The pH, TSS, VSS and COD were analyzed following the Standard
Methods [39]. Particulate COD (PCOD) was calculated as follows:
PCOD = TCOD  SCOD [40]. Protein was determined by the
modified Lowery procedure [14]. Carbohydrate was stained with
the phenol sulfuric acid method [41]. CH4 and CO2 were analyzed
using a gas chromatograph (SHIMADZU GC-8A) equipped with a
thermal conductivity detector (TCD, 80 mA) and an 2 m stainless
steel column packed with Porapak Q. Helium was the carrier gas
at a flow rate of 30 mL/min. The column temperature was 70 °C,
and the injector and detector temperatures were both 100 °C. H2
was determined by means of a gas chromatograph (SHIMADZU
GC-8A) equipped with a thermal conductivity detector (TCD,
80 mA) and an 2 m stainless steel column packed with Unibeads
C. Nitrogen was the carrier gas at 46 mL/min. The operating tem-
perature of injector, column and detector was 100 °C, 70 °C, and
100 °C, respectively. The concentrations of VFAs including acetic,
propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids
were determined by an Agilent 6890 gas chromatography
equipped with a flame ionization detector (FID) using helium as
carrier gas (38.6 kPa, 5.5 mL/min) in a DB-WAXetr capillary
column (30 m  0.53 mm I.D., 1 lm). The temperature of detector
was 280 °C. The equivalent relationships between COD and
substrates were as follows: 1.5 g-COD/g protein, 1.06 g-COD/g car-
bohydrate, 1.07 g-COD/g acetic acid, 1.51 g-COD/g propionic acid,
1.82 g-COD/g butyric acid and 2.04 g-COD/g valeric acid [42].
NH+4-N was measured by the indophenol blue method; SO2 4 was
measured using an ion chromatography (DIONEX, DX-120). Scan-
ning electron microscopy (SEM) images of sludge and ZVSI were
180
Cumulative CH4 yield (mL/g VSS)
160
140
120
100
80
60
40
20
0 0
0 10
Fig. 1. Effect of ZVSI dosages on CH4 production: (a) CH4 yield and (b) CH4 content.
G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470 463
collected using a JSM-6500F integrating energy dispersive X-ray
ð2Þ
analyzer (EDX) (Japan Electron Optics Laboratory Co., Japan).
ð3Þ 2.5. Statistical analysis
Statistical significance of the results obtained during different
experiments was conducted by means of one-way variance analy-
ð4Þ
sis (ANOVA) using a 95% confidence interval (p < 0.05).
3. Results and discussion
3.1. Effects of ZVSI on biogas production
Fig. 1 illustrates cumulative methane yield profiles under differ-
ent temperatures and ZVSI dosages. As can be seen, temperature, as
a key operational parameter, played a decisive role in methane
production and controlled the overall biokinetics of digestion
process. During the first stage (20 °C), methane yield showed negli-
gible discrepancy irrespective of ZVSI dosages (p > 0.05), and the
average values were lower than 1.5 ± 0.1 mL/g VSSfed attributable
to the relatively low anaerobes activities [43]. In contrast, increas-
ing temperature to 35 °C could profoundly promote methane pro-
duction, consistent with observations by Fey and Conrad [44].
Moreover, the higher the ZVSI was added the more the methane
generated. For example, with an increase of ZVSI from 0 (ZVSI-free)
to 0.05 g/g VSS, methane yield exhibited a progressive climb
from126.4 ± 1.0 to 132.4 ± 3.7 mL/g VSSfed (p = 0.16), which was
further enhanced (p < 0.05) as ZVSI increased to 0.1 g/g VSS and
above. The highest methane production of 174.9 ± 1.5 mL/g VSSfed
was obtained at 1.0 g-ZVSI/g VSS, 38.3% higher relative to the
ZVSI-free (Fobserved = 1489.5, Fcrit = 18.5, p = 0.0007 < 0.05). It sug-
gested that mesophilic condition (35 °C) is always required for sim-
ulating the function of ZVSI and maintaining the superior stability
of AD process. As with the methane content, the distinct difference
was also observed under mesophilic condition. From Fig. 1b, meth-
ane content at 0 g-ZVSI/g VSS was quite low and stable in 62.9–
63.2% during 27–52 days, which further rose to 65.1–68.3% and
then to 72.6–75.5% (p < 0.05) as ZVSI elevated to 0.5 and 1.0 g/g VSS.
Methane content achieved at the optimum ZVSI of 1.0 g/g VSS in
this experiment was comparably higher than 60.9 ± 0.8% of raw
sludge, 60.2 ± 2.6% of acid pretreated sludge [7], 64–70% of alka-
line-ultrasound pretreated sludge [45], 62–69% of sono-alkalization
pretreated sludge [46] or 68.9% of ZVI-sludge [1].
The commonly accepted mechanism of electron transfer from
ZVSI to microorganisms is via the microbial corrosion and surface
oxidation of ZVSI. Under anaerobic conditions, ZVSI is oxidized to
ferrous iron (Fe2+), producing cathodic hydrogen (H2/[H]) via Eq.
(5) [47]. Hence, H2 production can be used as a direct measure to
20 OC 35 OC 20 OC 35 OC
100
0 g/g VSS
(a) 0.05 g/g VSS
(b)
0.10 g/g VSS 80
0.25 g/g VSS
CH4 content (%)
0.50 g/g VSS
1.00 g/g VSS 60
40
20
20 30 40 50 0 10 20 30 40 50
Duration (days) Duration (days)
464 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470

reflect the reactivity and corrosion extent of ZVSI. Fig. 2 shows the CH3 COO þ Hþ ! CH4 þ CO2 DG00 ¼ 75:7 kJ=mol ð8Þ
H2 evolution in ZVSI-sludge system. As can be seen, fast H2 produc-
Except for methane production, the prompt consumption of H2
tion and accumulation took place during the initial 2 days at 20 °C.
also is the prerequisite of continuous microbial-assisted corrosion
With the increase of ZVSI, the yield of H2 increased and reached the 0
of ZVSI. Because of high Gibbs free energy (DG0 = 5.02 kJ/mol Fe0
greatest of 12.3 ± 1.5 mL at 1.0 g-ZVSI/g VSS. It was reported previ-
(Eq. (5)), the formation of cathodic hydrogen through ZVSI
ously that high H2 content (or partial pressure) was detrimental to
corrosion is thermodynamic slow [47]. The accumulation of H2 is
methane production [29] because of the associated CO production
unfavorable for the corrosion reaction, and even halts the genera-
[48] and/or the impeded thermodynamical conversions of propio-
tion of cathodic hydrogen. H2 removal by hydrogen-utilizing
nate and butyrate into acetate [31], which inhibited acetoclastic
microorganisms via Eqs. (6)–(7) can effectively decline the amount
methanogenesis (Methanosaeta and Methanosarcina [29]). Notably,
of H2 and drive the ZVSI bio-corrosion [52] (Fig. S1, Supporting
H2 did not accumulate continuously herein even at the highest
Information and Fig. 8). Thus the otherwise unfavorable ZVSI
dosage of ZVSI 1.0 g/g VSS, but was quickly consumed during the
bio-corrosion by cathodic depolarization occurred by continual
following biological process (especially at 35 °C), during which
microbial conversion of H2 so that its pressure was kept low
no H2 was detected. The results confirmed that H2 produced during
[52]. In this case, methane formation was enhanced. Besides,
both ZVSI corrosion could be effectively utilized by hydrogeno-
another evidence, as hypothesized by Karri et al. [47], also noted
trophic methanogens (e.g., Methanobacterials, Methanococci and
that methanogenesis can convert CO2 to methane with ZVSI as
Methanomicrobials) [29] via Eq. (6) to produce CH4 [23,29,43,49].
the direct electron donor (Eq. (9)), which was necessarily beneficial
The addition of ZVSI contributed to the production of more H2,
for accelerating ZVSI corrosion. The above-named results clearly
and thereby methane yield was also expected to increase
demonstrated that ZVSI can serve as an electron donor for
(Fig. 1a). Meanwhile the autotrophic methanogenesis via using
methanogenesis (i.e., methanogenic archaea) and favored the
H2 as energy source could not only form extra CH4 but also con-
improvement of methane production.
sume part of CO2 (Eq. (6)) and, as a consequence, the percentage
of methane increased with the applied ZVSI dosages (Fig. 1b). Like-
8Hþ þ 4Fe0 þ CO2 ! CH4 þ 4Fe2þ þ 2H2 O
wise homoacetogenic bacteria, as indicated by Thauer et al. [49]
and Rajagopal and LeGall [50], could also use CO2/H2 as sole energy DG00 ¼ 150:5 kJ=mol CH4 ð9Þ
source for the formation of methanogenic precursor acetate (Eq.
(7)), which further reduced CO2/H2 contents and raised methane
percentage (Eq. (8)) [44]. Other authors similarly noted that iron 3.2. Effects of ZVSI on sludge solubilization
could be involved in energy metabolism as a cytochrome and fer-
redoxin in methylotrophic methanogens [51]. The solubilization of sludge is a key step before hydrolysis. The
dynamic changes of SCOD, protein and carbohydrate during AD
Fe0 þ 2H2 O ! Fe2þ þ H2 þ 2OH DG00 ¼ 5:02 kJ=mol Fe0 process were thus determined (Fig. 3). On the overall, the average
ð5Þ SCOD, protein and carbohydrate concentrations in different
sludges rapidly rose during the initial 12 days (20 °C) regardless
4H2 þ CO2 ! CH4 þ 2H2 O DG00 ¼ 131 kJ=mol ð6Þ of ZVSI, presumably due to the continued endogenous biomass
decay and slow rate of methanogenesis (Fig. 1a). The former made
a large amount of soluble compounds release while the later
4H2 þ 2CO2 ! CH3 COO þ Hþ þ 2H2 O DG00 ¼ 95 kJ=mol
retarded the further conversion of these organics into methane.
ð7Þ Also, as for ZVSI, at a certain time, SCOD, protein and carbohydrate

Fig. 2. Production of H2 during ZVSI corrosion/oxidation: (a) 0, (b) 0.05, (c) 0.10, (d) 0.25, (e) 0.50 and (f) 1.00 g-ZVSI/g VSS.
 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470 465

20 OC 35 OC Unequivocal evidence from Etchebehere et al. [54] and Pervin

et al. [55] has accumulated that Coprothermobacter, having prefer-
(a) ence for fermentation of protein and amino acids, survive more
SCOD (mg/g VSS)

300 easily in mesophilic digestion while Thermotogae, which excrete

hydrolytic enzymes to catalyze a wide range of polysaccharides,
200 0 g/g VSS are in thermophilic condition. In the view of this, the abundant
0.05 g/g VSS presence of protein-dependent microorganisms in this study might
0.10 g/g VSS be the core reason for much quicker consumption of protein versus
100 0.25 g/g VSS
0.50 g/g VSS carbohydrate during mesophilic process (12–52 days). The
1.00 g/g VSS enhanced solubilization of particulate organics and following
0 hydrolysis, particularly in the presence of higher ZVSI, provided
(b) the bountiful substrates for subsequent acidogenesis and methan-
Protein (mg/g VSS)

20
ogenisis, and undoubtedly methane yield rose.
15

10 3.3. Effects of ZVSI on pH, and VFAs production and composition

5 Fig. 4 shows the variations of pH and VFAs under different dos-

ages of ZVSI. It reveals that the trend of pH highly agreed with the
0 VFAs accumulation [2,29]. All pH values dropped below 6.0
Carbohydrate (mg/g VSS)

20 (c) throughout the overall low-temperature digestion and the first

3 days of mesophilic process, owing to the accumulation of VFAs.
15 After a distinct drop, a quick pH recovery was detected; moreover
it increased more at higher ZVSI. The pH, for example on day 30,
10 increased from 6.3 ± 0.4 to 6.4 ± 0.0, 6.3 ± 0.1, 6.5 ± 0.4, and
6.7 ± 0.1 with increasing ZVSI from 0 to 0.05, 0.10, 0.25, and
5 0.50 g-ZVSI/g VSS, and it further increased to 7.3 ± 0.1 as ZVSI
was 1.00 g-ZVSI/g VSS likely due to the continued dissolution of
0 ZVSI via Eq. (5), and consequently enhanced VFAs consumption.
0 10 20 30 40 50 These observations evidenced the advantageous buffering capacity
Duration (days) of ZVSI, which could prevent pH drop and induce a stable anaerobic
Fig. 3. Variations of SCOD (a), protein (b) and carbohydrate (c) during the overall 20 oC 35 oC
digestion process. 8.0
(a)
had an ascending trend with ZVSI dosages (Fig. 3). As an example, 7.5
with the fermentation time of 12 days at 20 °C, SCOD in the groups
7.0
of 0, 0.05, 0.10, 0.25, 0.50 and 1.00 g-ZVSI/g VSS were 178.2 ± 3.5,
178.0 ± 12.3, 181.2 ± 6.4, 179.4 ± 12.9, 184.1 ± 2.4 and 199.2 ± 6.5
19.1 mg/g VSS, respectively. The distinct increment of SCOD, par-
pH

ticularly as exposed to 1.00 g-ZVSI/g VSS, in part hinted the physi- 6.0 0 g/g VSS
cal disruption of cell membranes and leakage of intracellular 0.05 g/g VSS
substances [33]. The decomposition of bound EPS and glycan cross 5.5 0.10 g/g VSS
0.25 g/g VSS
linked by peptide chains of the external membranes caused the
5.0 0.50 g/g VSS
rupture of partial cells and damage of sludge integrity. In this 1.00 g/g VSS
sense, the solubilization of WAS was advanced. 4.5
The disintegration of sludge caused by ZVSI might be effectively 0 10 20 30 40 50
upgraded as the operational temperature increased to 35 °C. The
Duration (days)
elevated temperature accelerated ZVSI corrosion and thus greatly
promoted sludge solubilization. It can be seen from Fig. 3 that 20oC 35oC
4500
the soluble organics in the fermentation liquor increased fleetly acetic
with the maximum SCOD and protein in the groups of 0, 0.05,
(b) propionic
4000
iso-butyric
0.10, 0.25, 0.50 and 1.00 g-ZVSI/g VSS ranging from 306.9 ± 1.6 to n-butyric
3500
VFA concentration(mg/L)

351.3 ± 16.3 mg/g VSS (p  0.05) and 19.6 ± 0.6 to 22.8 ± 0.4 mg/ iso-valeric
n-valeric
g VSS (p = 0.03) only after 3 days of mesophilic digestion. As 3000
■ 0 g/g VSS ▲ 0.05 g/g VSS
expected, after a transient rise, these organics, mainly protein ● 0.10 g/g VSS Ⴗ 0.25 g/g VSS
2500 ღ 0.50 g/g VSS Ⴟ 1.0 g/g VSS
(Fig. 3b), fast dropped suggesting the rapid start-up of acidogenesis
and/or methanogenesis, as indicated by increased methane yield at 2000
35 °C. Whereas, in comparison with protein, the progressive rise in
1500
carbohydrate still persisted until the day 30, then followed by a
marked decrease. Similar finding was also documented by Ahn 1000
et al. [53], who reported the enhanced reduction of nitrate by 500 ■ ▲ ●Ⴗ ღ Ⴟ
zero-valent iron at elevated temperatures, and they attributed this
to the overcome energy barrier by temperature increase. Besides, 0
0 2 4 8 12 15 30 52
temperature influenced not only the ZVSI corrosion but also the Duration (days)
composition of microbial community [44], and even the metabo-
lism pathway of organics during methanogenic degradation. Fig. 4. Variations of pH (a) and VFAs (b) during the overall digestion process.
466 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470

system. What is more, during the fermentation of soluble organics,
or more exactly protein, some by-products such as CO2, NH+4 and
HCO 3 are generated that can also to some extent alleviate liquor
pH.
The results of VFAs components are presented in Fig. 4b. Consis-
tent with SCOD, protein and carbohydrate profiles, total VFAs and
acetate showed a gradual accumulation with the operating time
and ZVSI dosages. On day 12, for instance, VFAs at 0, 0.05, 0.10,
0.25, 0.50 and 1.00 g-ZVSI/g VSS was 2059.9 ± 110.8, 2114.7 ±
40.1, 2121.5 ± 35.0, 2156.0 ± 11.2, 2195.9 ± 10.8 and 2340.4 ±
73.0 mg/L; the corresponding acetate increased from 779.6 ± 18.2
to 783.6 ± 12.2, 790.3 ± 9.4, 807.3 ± 12.0, 820.2 ± 8.0 and then
865.5 ± 24.8 mg/L, respectively. It suggested that ZVSI provoked
VFAs, typically acetate production. This result is supported by Yang
et al. [29] who obtained the maximum VFAs and acetate of 49.3 ±
6.2 and 24.9 ± 4.0 mg/L, respectively, with the addition of 30 mM
nano ZVI, as opposed to the negligible VFAs/acetate in ZVI-free
one. It is well known that only acetate in VFAs, together with for-
mic acid, hydrogen, methanol and methylamine can be utilized as
the direct substrates for methane production. Therefore, it can be
speculated that the enhanced acetate production in the presence
of ZVSI would be thermodynamically favorable for methanogenisis.
Furthermore, as operation time elapsed, VFAs and acetate
increased drastically and peaked on day 15 (i.e., 3 days after the
start of mesophilic digestion). Afterwards, a significant decline of
VFAs, especially acetate, was observed on day 30 and nearly no
sign of acetate was detected, indicating its complete degradation.
On the contrary, propionate, at this moment, showed a maximum
ranging from 1242.4 ± 47.4 to 412.4 ± 47.1 mg/L depending on
ZVSI dosages, which might derive from conversion of other VFAs.
The buildup of propionate is considered to be unfavorable in any
anaerobic process unless it is decomposed by b-oxidation into
acetate and H2. However, the conversion is frequently difficult to
the functional integrity, strength and dewaterability of sludge
[38,63–65] as well as biodegradability. Fig. 5 compares different
EPS fractions during AD processes. It is apparent that EPS in raw
sludge were mainly distributed in pellet and TB-EPS, and very
few in S-EPS and LB-EPS. As been well known, S-EPS are well-
distributed in aqueous phase and LB-EPS, a highly porous slime
layer, extend from TB-EPS to the liquid. TB-EPS, in contrast,
accumulate both on the outside of cells and in the interior of
microbial aggregates (i.e., pellet) [66] and strongly support the
mechanical stability of sludge. The sludge with high TB-EPS and
pellet contents is thus much stiffer, and has more difficulties to
be hydrolyzed. Therefore, the transfer of TB-EPS and pellet into
S-EPS and LB-EPS after even 12 days (20 °C) was still extremely
limited (Fig. 5b). Comparatively, the transfer under the help of ZVSI
appeared to be enhanced. Of course, owing to the slow dissolution
of ZVSI, the promotion looked somewhat slight.
EPS not only act as a protective net-like layer against stressful
external disturbance, which are also a carbon/energy reservoir
[64] during biological process. Alone with operating time, the sol-
ubilization of EPS from TB-EPS and pellet into LB-EPS, and typically
into S-EPS increased (Fig. 5c), which further were used for methane
formation. Therefore, the appropriate rupture of EPS will facilitate
the release of more substrates and subsequent methanogenesis.
Simultaneously, it is worth recalling that on day 52 (Fig. 5c), the
sludge treated by ZVSI displayed a higher level of TB-EPS, and
particularly pellet as compared to the ZVSI-free. Microbial cells
comprise the sludge pellet, and the increased pellet indicated ZVSI
has a positive effect on the growth of microbes, as a result of
20 oC
500
(a) ▲ (b) ● S-EPS Ⴗ LB-EPS ■ TB-EPS ▲ Pellet
Different EPS fractions (mg/g VSS)

0
occur in thermodynamics (DG0 =+76.1 kJ/mol) (Eq. (10)) [49]
because of the strict requirements for H2 partial pressure
400
(3  104 – 106 atm) [56], acetate concentration and oxidation–
reduction potential (ORP) (<278 mV) [57]. Interestingly, these
Protein
intrinsic defects appeared to be overcome herein in the presence
300 Carbohydrate
of ZVSI (Fig. 4b). Propionate decreased at a considerable level on 50 ■
day 52 while accompanied by the formation of acetate, especially
for the sludges treated with 0.50 and 1.00 g-ZVSI/g VSS, with
respect to the continuous buildup in ZVSI-free one. 25
Based upon this finding, it is obvious that ZVSI might have
reduced DG in the decomposition of propionate (Eq. (10)) through
consuming either acetate or H2, or both together; this provided a ●Ⴗ
0
favorable free-energy change for propionate b-oxidation [58], facil- Initial 0 0.05 0.10 0.25 0.50 1.00
itating the final methanogenesis [26]. The reason for this might be ZVSI dosage (mg/g VSS)
principally ascribed to the intrinsic reducing properties of ZVSI,
35 oC
and thus its addition could decrease ORP and create a more reduc-
500
tive atmosphere for AD process [1,59]. In addition, according to the (b)
450 (c)
Different EPS fractions (mg/g VSS)

infinitesimal H2 content under mesophilic conditions as set forth, 400

we reasonably assumed that the enhanced hydrogen-utilizing 350
methanogenesis and homoacetogensis by ZVSI depleted H2 and 300
250
reduced its partial pressure, making propionate degradation more
200
thermodynamically possible. So, acetogenesis and methanogenisis 150
were intensified.
50
CH3 CH2 COO þ 3H2 O ! HCO3 þ CH3 COO þ Hþ þ 3H2
Protein
DG00 ¼ þ76:1 kJ=mol ð10Þ
25 Carbohydrate

3.4. Variations of different EPS fractions

0
0 0.05 0.10 0.25 0.50 1.00
EPS, i.e., a three-dimensional, gel-like and negative-charged ZVSI dosage (mg/g VSS)
matrix [60,61], take a critical part in cells adhesion and cohesion
of sludge [59,62]. Its presence is of great importance in controlling Fig. 5. Effects of ZVSI on EPS: raw (a) and digested sludges on day 12 (b) and 52 (c).
 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470
which, EPS formation was provoked. The finding was in line with
those of Liu et al. [59] who demonstrated the useful impact of
ZVI on EPS production and sludge granulation. Other works by
Shen et al. [51] also observed the helpful influence of iron on the
amount of carbohydrates extracted. EPS could provide some pro-
tective shielding, helping create a safe environment for bacteria
and methanogenic archaea to survive. In this case, AD performance
maintained stable, and methane production was reinforced.
3.5. Effects of ZVSI on the hydrolysis, acidification and methanogenesis
of sludge
To further clarify the exact impacts of ZVSI on methane
production, each step (hydrolysis, %H; acidogenesis, %A; and
methanogenesis, %M) involved in the anaerobic process as well
as biodegradability (%B) were calculated after 52 days of digestion.
As can be seen from Fig. 6a, the influence of ZVSI on hydrolysis of
solubilized products (i.e., soluble protein and carbohydrate) was
dosage-dependent. At 0.05–0.25 g/g VSS, the influence of ZVSI
was inappreciable (p > 0.05), but the degradation efficiency of sol-
ubilized products at 0.50 and 1.00 g/g VSS significantly increased in
comparison with the ZVSI-free (p = 0.019 and 0.003, respectively),
and reached up to 48.3 ± 0.3% and 52.6 ± 0.4% (versus 45.2 ± 0.5%
in the ZVSI-free) at the end of test, respectively. Evidently, ZVSI
could considerably accelerate the hydrolysis involved in sludge
digestion.
The data in Fig. 6b relation to acidogenesis suggested that no
significant difference in VFAs production existed among the ZVSI-
free and others. Despite this, with increasing ZVSI the acidogenesis
was promoted. Acidogenesis rate was 40.9 ± 2.0%, 39.1 ± 2.0%,
40.8 ± 0.2% and 39.2 ± 1.1% for the reactors of ZVSI-free, 0.05,
0.10 and 0.25 g/g VSS, respectively, which was further raised into
43.1 ± 0.7% and 44.5 ± 2.9% as ZVSI was 0.50 and 1.00 g/g VSS,
respectively. The above observation manifested the previous find-
ing that ZVSI could effectively accelerate the hydrolysis-acidificat-
ion of sludge [1]. This might be one of the pivotal reasons for the
increased methane yield treated by higher ZVSI.
For the effects of ZVSI on methanation step, the results from
Fig. 6c hinted that ZVSI contributed more to the methanogenesis.
Methanogenesis for those exposed to 0.05, 0.10 and 0.25 g/g VSS
increased to 30.9 ± 0.9%, 31.7 ± 0.3% and 31.2 ± 1.9%, respectively,
even not very significantly versus 29.5 ± 0.2% of the ZVSI-free
(p > 0.05). The remarkable progress was obtained at 0.50 and
50 (a)
Hydrolysis (%H)
40
30
20
10
0 0
(c)
Methanogenesis (%M)
40
30
20
10
0 0
0 0.05 0.10 0.25 0.50 1.00
ZVSI dosage (mg/g VSS)
Fig. 6. Effects of ZVSI on hydrolysis (a), acidogenesis (b), methanogenesis (c) and biodegradability (d) of WAS at 52 days. Asterisks indicate statistical significance (p < 0.05)
from the ZVSI-free.
467
1.00 g-ZVSI/g VSS, in which methanogenesis ran up to 36.1 ± 0.7%
(p = 0.006) and 40.8 ± 0.3% (p = 0.001), respectively, suggesting that
ZVSI was beneficial to the growth of methanogens and enhanced
the bio-conversion of acetate to methane, corresponding well with
methane profiles (Fig. 1) and COD balance (Fig. S2, Supporting
Information). The earlier work performed by Belay and Daniels
[67] also confirmed the positive effect of Fe0 on methanogenesis.
In contrast, the effect of ZVSI on biodegradability was insignificant
(p > 0.05) (Fig. 6d). The responsible reasons are still not well known
and more research is required. Whatever, by comparing the data in
Fig. 6a–d, we still can conclude that the accelerated hydrolysis and
methanation steps might be the fundamental mechanisms behind
the enhanced methane formation by ZVSI.
3.6. Evidence for dissolution of ZVSI and its stimulation on anaerobic
process
SEM-EDX analysis were conducted to support our findings that
ZVSI could stimulate the methane production of WAS. SEM images
in Fig. 7 revealed the surface of un-used ZVSI was flat with
high-purity iron detected by EDX examination (Fig. 7a), while it
became scraggy and rough at the end of digestion (Fig. 7b). A
higher magnification of SEM revealed that many ‘‘flower-shaped’’
crystals comprised of prismatic iron (hydr)oxides clusters (e.g.,
FeCO3, Fe3O4, Fe2O3, Fe(OH)3 and FeOOH) [21,68] prevailed the sur-
face of used-ZVSI, showcasing the microbe-driven dissolution of
ZVSI throughout digestion process.
To comprehensively verify the underlying enhancement mech-
anisms, the morphology of digested sludge and distribution of iron
in sludge were characterized (Fig. 8). The group of ZVSI-free had
relatively low background iron and sludge flocs displayed a com-
pact and rigid microstructure (Fig. 8a). Whereas, with the increase
of ZVSI the level of iron in sludge elevated, agreeing well with the
results of iron EDX mapping (Fig. S3, Supporting Information).
Correspondingly, the sludge became well-regulated, distinct and
stable, and the amount of microbial cells increased as well, in
accordance with the increasing trends of methane (Fig. 1a), EPS
(Fig. 5) and methanogenesis rate (Fig. 6c). From this point of view,
the microbes observed in sludge matrix were presumably related
to methanogens and hydrogen-utilizing microbes. Based on a
higher magnification of SEM for the sludge treated with 1.00 g-
ZVSI/g VSS (Fig. S4, Supporting Information), some crystalline dots
on the surface of cells were identified, possibly attributed to
50
* * (b)
Acidogenesis (%A)
40
30
20
10
* 50 (d)
Biodegradability (%B)
*
40
30
20
10
0 0.05 0.10 0.25 0.50 1.00
ZVSI dosage (mg/g VSS)
468 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470

Fig. 7. SEM-EDX analysis (+) of ZVSI before (a) and after (b, 1.00 g-ZVSI/g VSS) WAS anaerobic digestion.

Fig. 8. SEM-EDX analysis (+) of sludge exposed to 0 (a), 0.25 (b), and 1.00 g-ZVSI/g VSS (c).

iron-bearing compounds according to EDX (Fig. 8c). Unlike the
commonly observed inhibition from zero valent iron due to the for-
mation of iron precipitates (e.g., iron-phosphorous complexes [29])
or physical disruption to cell membrane [33], the bactericidal
effects did not occur in our study. By contrast, the released iron
ions could penetrate into the inner part of cells and stimulate the
synthesis of key enzymes and growth of microbes, particularly
methanogens. These results were confirmed by those of Liu et al.
[59] who reported that the relative abundance of methanogens
in the presence of ZVI was 86.25% while 74.81% and 63.31% in
two controls, respectively. Dinh et al. [69] found that a newly
isolated Methanobacterium-like archaeon produced methane with
Fe0 faster than do known hydrogen-using methanogens, again
suggesting the simulating effects of Fe0. It is still unclear whether
iron ions binding specific DNA may directly upgrade the enzymatic
functions of methanogens, and speed up cell replication. Neverthe-
less, based on the above results, several possible pathways of ZVSI
prompting methane production could still be proposed (Fig. 9).
These biotic/abiotic factors all eventually led to the observed
increase in methane production. Our work here builds solid
 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470
Fig. 9. Proposed enhancement mechanisms for methane production from WAS in the presence of ZVSI.
support for the potential applicability of ZVSI for WAS digestion
and provides an additional route for enhancing methane produc-
tion accompanied with waste iron scrap reuse.
4. Conclusion
The above studies confirmed that ZVSI could be used as a source
of electron donors for the anaerobic digestion of waste activated
sludge. The highest methane yield of 174.9 ± 1.5 mL/g VSSfed was
obtained with ZVSI 1.0 g/g VSS by the end of mesophilic operation,
increasing by 38.3% relative to the ZVSI-free one. Use of ZVSI could
stimulate the metabolism and growth of critical microbes involved
in anaerobic process through serving as direct or indirect (H2/[H])
electron donors, as well as affording a low H2 concentration (or
partial pressure) and beneficial pH environment, etc. which
enhanced sludge hydrolysis, acidogenesis, methanogenesis rate,
and final methane production. The potential ability to offer elec-
trons will open many new doors for our better understanding
and application of ZVSI in sludge anaerobic digestion and sustain-
able management.
Acknowledgements
The authors wish to thank the Japan Society for Promotion of
Science (JSPS, ID No. PU 14016) and China Scholarship Council
(CSC, File No. 201306890003) for partial support of this study.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cej.2014.11.003.
References
[1] Y. Feng, Y. Zhang, X. Quan, S. Chen, Enhanced anaerobic digestion of waste
activated sludge digestion by the addition of zero valent iron, Water Res. 52
(2014) 242–250.
[2] H.M. Jang, H.W. Cho, S.K. Park, J.H. Ha, J.M. Park, Influence of thermophilic
aerobic digestion as a sludge pre-treatment and solids retention time of
mesophilic anaerobic digestion on the methane production, sludge digestion
and microbial communities in a sequential digestion process, Water Res. 48
(2014) 1–14.
[3] G. Zhen, X. Lu, J. Niu, L. Su, X. Chai, Y. Zhao, Y.Y. Li, Y. Song, D.J. Niu, Inhibitory
effects of a shock load of Fe(II)-mediated persulfate oxidation on waste
activated sludge anaerobic digestion, Chem. Eng. J. 233 (2013) 274–281.
[4] G. Zhen, X. Lu, Y.Y. Li, Y. Zhao, Combined electrical-alkali pretreatment to
increase the anaerobic hydrolysis rate of waste activated sludge during
anaerobic digestion, Appl. Energy 128 (2014) 93–102.
[5] L. Appels, J. Baeyens, J. Degreve, R. Dewil, Principles and potential of the
anaerobic digestion of waste-activated sludge, Prog. Energy Combust. 34
(2008) 755–781.
[6] S. Luostarinen, S. Luste, M. Sillanpää, Increased biogas production at
wastewater treatment plants through co-digestion of sewage sludge with
grease trap sludge from a meat processing plant, Bioresour. Technol. 100
(2009) 79–85.
[7] D.C. Devlin, S.R.R. Esteves, R.M. Dinsdale, A.J. Guwy, The effect of acid
pretreatment on the anaerobic digestion and dewatering of waste activated
sludge, Bioresour. Technol. 102 (2011) 4076–4082.
469
[8] I. Dogan, F.D. Sanin, Alkaline solubilization and microwave irradiation as a
combined sludge disintegration and minimization method, Water Res. 43
(2009) 2139–2148.
[9] R. Uma Rani, S. Adish Kumar, S. Kaliappan, I.T. Yeom, J. Rajesh Banu, Low
temperature thermo-chemical pretreatment of dairy waste activated sludge
for anaerobic digestion process, Bioresour. Technol. 103 (2012) 415–424.
[10] A. Tiehm, K. Nickel, M. Zellhorn, U. Neis, Ultrasonic waste activated sludge
disintegration for improving anaerobic stabilization, Water Res. 35 (2001)
2003–2009.
[11] S. Ponsa, I. Ferrer, F. Vazquez, X. Font, Optimization of the hydrolytic-
acidogenic anaerobic digestion stage (55 °C) of sewage sludge: influence of pH
and solid content, Water Res. 42 (2008) 3972–3980.
[12] S. Bala Subramanian, S. Yan, R.D. Tyagi, R.Y. Surampalli, Extracellular
polymeric substances (EPS) producing bacterial strains of municipal
wastewater sludge: isolation, molecular identification, EPS characterization
and performance for sludge settling and dewatering, Water Res. 44 (2010)
2253–2266.
[13] B.M. Wilén, B. Jin, P. Lant, The influence of key chemical constituents in
activated sludge on surface and flocculating properties, Water Res. 37 (2003)
2127–2139.
[14] B. Frolund, R. Palmgren, K. Keiding, P.H. Nielsen, Extraction of extracellular
polymers from activated sludge using a cation exchange resin, Water Res. 30
(1996) 1749–1758.
[15] D.H. Kim, E. Jeong, S.E. Oh, H.S. Shin, Combined (alkaline + ultrasonic)
pretreatment effect on sewage sludge disintegration, Water Res. 44 (2010)
3093–3100.
[16] K.Y. Hwang, E.B. Shin, H.B. Choi, A mechanical pretreatment of waste activated
sludge for improvement of anaerobic digestion system, Water Sci. Technol. 36
(1997) 111–116.
[17] A. Gallipoli, A. Gianico, M.C. Gagliano, C.M. Braguglia, Potential of high-
frequency ultrasounds to improve sludge anaerobic conversion and
surfactants removal at different food/inoculum ratio, Bioresour. Technol. 159
(2014) 207–214.
[18] J. Ariunbaatar, A. Panico, G. Esposito, F. Pirozzi, P.N.L. Lens, Pretreatment
methods to enhance anaerobic digestion of organic solid waste, Appl. Energy
123 (2014) 143–156.
[19] D. OCarroll, B. Sleep, M. Krol, H. Boparai, C. Kocur, Nanoscale zero valent iron
and bimetallic particles for contaminated site remediation, Adv. Water Resour.
51 (2013) 104–122.
[20] J.B. Parsa, S.A.E. Zonouzian, Optimization of a heterogeneous catalytic
hydrodynamic cavitation reactor performance in decolorization of
Rhodamine B: application of scrap iron sheets, Ultrason. Sonochem. 20
(2013) 1442–1449.
[21] A.K. Yadav, S. Jena, B.C. Acharya, B.K. Mishra, Removal of azo dye in innovative
constructed wetlands: influence of iron scrap and sulfate reducing bacterial
enrichment, Ecol. Eng. 49 (2012) 53–58.
[22] A. Son, J. Lee, P.C. Chiu, B.J. Kim, D.K. Cha, Microbial reduction of perchlorate
with zero-valent iron, Water Res. 40 (2006) 2027–2032.
[23] L. Zhu, H.Z. Lin, J.Q. Qi, X.Y. Xu, H.Y. Qi, Effect of H2 on reductive transformation
of p-ClNB in a combined ZVI-anaerobic sludge system, Water Res. 46 (2012)
6291–6299.
[24] X. Xiao, G.P. Sheng, Y. Mu, H.Q. Yu, A modeling approach to describe ZVI-based
anaerobic system, Water Res. 47 (2013) 6007–6013.
[25] J. Zhang, Y. Zhang, X. Quan, Y. Liu, X. An, S. Chen, H. Zhao, Bioaugmentation and
functional partitioning in a zero valent iron-anaerobic reactor for sulfate-
containing wastewater treatment, Chem. Eng. J. 174 (2011) 159–165.
[26] X. Meng, Y. Zhang, Q. Li, X. Quan, Adding Fe0 powder to enhance the anaerobic
conversion of propionate to acetate, Biochem. Eng. J. 73 (2013) 80–85.
[27] B.K. Chastain, T.A. Kral, Zero-valent iron on Mars: an alternative energy source
for methanogens, Icarus 208 (2010) 198–201.
[28] Y. Liu, Y. Zhang, Z. Zhao, Y. Li, X. Quan, S. Chen, Enhanced azo dye wastewater
treatment in a two-stage anaerobic system with Fe0 dosing, Bioresour.
Technol. 121 (2012) 148–153.
[29] Y. Yang, J. Guo, Z. Hu, Impact of nano zero valent iron (NZVI) on methanogenic
activity and population dynamics in anaerobic digestion, Water Res. 47 (2013)
6790–6800.
[30] N. Kumar, E.O. Omoregie, J. Rose, A. Masion, J.R. Lloyd, L. Diels, L. Bastiaens,
Inhibition of sulfate reducing bacteria in aquifer sediment by iron
nanoparticles, Water Res. 51 (2014) 64–72.
470 G. Zhen et al. / Chemical Engineering Journal 263 (2015) 461–470
[31] Y.P. Hou, D.C. Peng, X.D. Xue, H.Y. Wang, L.Y. Pei, Hydrogen utilization rate: a
crucial indicator for anaerobic digestion process evaluation and monitoring, J.
Biosci. Bioeng. 117 (2014) 519–523.
[32] C.R. Smatlak, J.M. Gossett, Comparative kinetics of hydrogen utilization for
reductive dechlorination of tetrachloroethene and methanogenesis in an
anaerobic enrichment culture, Environ. Sci. Technol. 30 (1996) 2850–2858.
[33] C. Lee, J.Y. Kim, W.I. Lee, K.L. Nelson, J. Yoon, D.L. Sedlak, Bactericidal effect of
zero-valent iron nanoparticles on Escherichia coli, Environ. Sci. Technol. 42
(2008) 4927–4933.
[34] Y. Zhang, Y. Feng, Q. Yu, Z. Xu, X. Quan, Enhanced high-solids anaerobic
digestion of waste activated sludge by the addition of scrap iron, Bioresour.
Technol. 159 (2014) 297–304.
[35] J. Field, R. Sierra-Alvarez, G. Lettinga, Ensayos anaerobios, in, Proceeding of the
4th Symposium on Wastewater Anaerobic Treatment, Valladolid, Spain, 1988.
[36] S. Astals, M. Esteban-Gutiérrez, T. Fernández-Arévalo, E. Aymerich, J.L. García-
Heras, J. Mata-Alvarez, Anaerobic digestion of seven different sewage sludges:
a biodegradability and modelling study, Water Res. 47 (2013) 6033–6043.
[37] X.Y. Li, S.F. Yang, Influence of loosely bound extracellular polymeric substances
(EPS) on the flocculation, sedimentation and dewaterability of activated
sludge, Water Res. 41 (2007) 1022–1030.
[38] G. Zhen, X. Lu, Y.Y. Li, Y. Zhao, Innovative combination of electrolysis and
Fe(II)-activated persulfate oxidation for improving the dewaterability of waste
activated sludge, Bioresour. Technol. 136 (2013) 654–663.
[39] APHA, Standard Methods for the Examination of Water and Wastewater, 20th
ed., A.P.H. Association, Washington, DC, USA, 1998.
[40] N.M.G. Coelho, R.L. Droste, K.J. Kennedy, Evaluation of continuous mesophilic,
thermophilic and temperature phased anaerobic digestion of microwaved
activated sludge, Water Res. 45 (2011) 2822–2834.
[41] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method
for determination of sugars and related substances, Anal. Chem. 28 (1956)
350–356.
[42] C.P.L. Grady, G.T. Daigger, H.C. Lim, Biological wastewater treatment, Marcel
Dekker, Inc., New York, 1999.
[43] J.J. Chen, Biomass to renewable energy processes, CRC Press, Taylor & Francis
Group, Boca Raton London, New York, 2010.
[44] A. Fey, R. Conrad, Effect of temperature on carbon and electron flow and on the
archaeal community in methanogenic rice field soil, Appl. Environ. Microbiol.
66 (2000) 4790–4797.
[45] N.D. Park, S.S. Helle, R.W. Thring, Combined alkaline and ultrasound pre-
treatment of thickened pulp mill waste activated sludge for improved
anaerobic digestion, Biomass Bioenergy 46 (2012) 750–756.
[46] R. Uma Rani, S. Adish Kumar, S. Kaliappan, I.T. Yeom, J.R. Banu, Enhancing the
anaerobic digestion potential of dairy waste activated sludge by two step
sono-alkalization pretreatment, Ultrason. Sonochem. 21 (2014) 1065–1074.
[47] S. Karri, R. Sierra-Alvarez, J.A. Field, Zero valent iron as an electron-donor for
methanogenesis and sulfate reduction in anaerobic sludge, Biotechnol. Bioeng.
92 (2005) 810–819.
[48] S.H. Zinder, T. Anguish, Carbon monoxide, hydrogen, and formate metabolism
during methanogenesis from acetate by thermophilic cultures of
Methanosarcina and Methanothrix strains, Appl. Environ. Mircobiol. 58 (1992)
3323–3329.
[49] R.K. Thauer, K. Jungermann, K. Decker, Energy conservation in chemotrophic
anaerobic bacteria, Bacteriol. Rev. 41 (1977) 100–180.
[50] B.S. Rajagopal, J. LeGall, Utilization of cathodic hydrogen by hydrogen-
oxidizing bacteria, Appl. Microbiol. Biotechnol. 31 (1989) 406–412.
[51] C.F. Shen, N. Kosaric, R. Blaszczyk, The effect of selected heavy metals (Ni, Co
and Fe) on anaerobic granules and their extracellular polymeric substances
(EPS), Water Res. 27 (1993) 25–33.
[52] L. Daniels, N. Belay, B.S. Rajagopal, P.J. Weimer, Bacterial methanogenesis and
growth from CO2 with elemental iron as the sole source of electrons, Nature
237 (1987) 509–511.
[53] S.C. Ahn, S.Y. Oh, D.K. Cha, Enhanced reduction of nitrate by zero-valent iron at
elevated temperatures, J. Hazard. Mater. 156 (2008) 17–22.
[54] C. Etchebehere, M.E. Pavan, J. Zorzopulos, M. Soubes, L. Muxi,
Coprothermobacter platensis sp. nov., a new anaerobic proteolytic
thermophilic bacterium isolated from an anaerobic mesophilic sludge, Int. J.
Syst. Bacteriol. 48 (1998) 1297–1304.
[55] H.M. Pervin, P.G. Dennis, H.J. Lim, G.W. Tyson, D.J. Batstone, P.L. Bond, Drivers
of microbial community composition in mesophilic and thermophilic
temperature-phased anaerobic digestion pre-treatment reactors, Water Res.
47 (2013) 7098–7108.
[56] F. Kus, U. Wiesmann, Degradation kinetics of acetate and propionate by
immobilized anaerobic mixed cultures, Water Res. 29 (1994) 1437–1443.
[57] N.Q. Ren, H. Chua, S.Y. Chan, Y.F. Tsang, Y.J. Wang, N. Sin, Assessing optimal
fermentation type for bio-hydrogen production in continuous-flow acidogenic
reactors, Bioresour. Technol. 98 (2007) 1774–1780.
[58] P.L. McCarty, D.P. Smith, Anaerobic wastewater treatment, Environ. Sci.
Technol. 20 (1986) 1200–1206.
[59] Y. Liu, Y. Zhang, X. Quan, S. Chen, H. Zhao, Applying an electric field in a built-
in zero valent iron-anaerobic reactor for enhancement of sludge granulation,
Water Res. 45 (2011) 1258–1266.
[60] E. Neyens, J. Baeyens, A review of thermal sludge pre-treatment processes to
improve dewaterability, J. Hazard. Mater. B98 (2003) 51–67.
[61] S. Tsuneda, H. Aikawa, H. Hayashi, A. Yuasa, A. Hirata, Extracellular polymeric
substances responsible for bacterial adhesion onto solid surface, FEMS
Microbiol. Lett. 223 (2003) 287–292.
[62] E. Neyens, J. Baeyens, R. Dewil, B. De heyder, Advanced sludge treatment
affects extracellular polymeric substances to improve activated sludge
dewatering, J. Hazard. Mater. 106 (2004) 83–92.
[63] S.S. Adav, D.J. Lee, J.H. Tay, Extracellular polymeric substances and structural
stability of aerobic granule, Water Res. 42 (2008) 1644–1650.
[64] J. Abelleira, S.I. Pérez-Elvira, J. Sánchez-Oneto, J.R. Portela, E. Nebot, Advanced
thermal hydrolysis of secondary sewage sludge: a novel process combining
thermal hydrolysis and hydrogen peroxide addition, Resour. Conserv. Recy. 59
(2012) 52–57.
[65] T.L. Poxon, J.L. Darby, Extracellular polyanions in digested sludge:
measurement and relationship to sludge dewaterability, Water Res. 31
(1997) 749–758.
[66] X.M. Liu, G.P. Sheng, H.W. Luo, F. Zhang, S.J. Yuan, J. Xu, R.J. Zeng, J.G. Wu, H.Q.
Yu, Contribution of extracellular polymeric substances (EPS) to the sludge
aggregation, Environ. Sci. Technol. 44 (2010) 4355–4360.
[67] N. Belay, L. Daniels, Elemental metals as electron sources for biological
methane formation from CO2, Antonie Van Leeuwenhoek 57 (1990) 1–7.
[68] T. Uchiyama, K. Ito, K. Mori, H. Tsurumaru, S. Harayama, Iron-corroding
methanogen isolated from a crude-oil storage tank, Appl. Environ. Microbiol.
76 (2010) 1783–1788.
[69] H.T. Dinh, J. Kuever, M. Mußmann, A.W. Hassel, M. Stratmann, F. Widdel, Iron
corrosion by novel anaerobic microorganisms, Nature 427 (2004) 829–832.