Nanoscale zero-valent iron/persulfate enhanced upflow anaerobic sludge blanket reactor for dye removal: Insight into microbial metabolism and microbial community
Nanoscale zero-valent iron/ persulfate enhanced upflow anaerobic sludge blanket reactor for dye removal: Insight into microbial metabolism and microbial community
Wen Liu 4
Fei Pan 0 1
Xiaohan Zhong 0
Dongsheng Xia 0
Fan Li 3
Dongye Zhao 3
Haodong Ji 3
0 School of Environmental Engineering, Wuhan Textile University , Wuhan, 430073 , P.R. China
1 Engineering Research Centre for Clean Production of Textile Dyeing and Printing, Ministry of Education , Wuhan, 430073 , P.R. China
2 School of Materials Science and Engineering, Wuhan Textile University , Wuhan 430073 , P.R. China
3 Environmental Engineering Program, Department of Civil Engineering, Auburn University , Auburn, AL 36849 , USA
4 School of Civil
OPEN This study investigated the efficiency of nanoscale zero-valent iron combined with persulfate (NZVI/ PS) for enhanced degradation of brilliant red X-3B in an upflow anaerobic sludge blanket (UASB) reactor, and examined the effects of NZVI/PS on anaerobic microbial communities during the treatment process. The addition of NZVI (0.5 g/L) greatly enhanced the decolourization rate of X-3B from 63.8% to 98.4%. The Biolog EcoPlateTM technique was utilized to examine microbial metabolism in the reactor, and the Illumina MiSeq high-throughput sequencing revealed 22 phyla and 88 genera of the bacteria. The largest genera (Lactococcus) decreased from 33.03% to 7.94%, while the Akkermansia genera increased from 1.69% to 20.23% according to the abundance in the presence of 0.2 g/L NZVI during the biological treatment process. Meanwhile, three strains were isolated from the sludge in the UASB reactors and identified by 16S rRNA analysis. The distribution of three strains was consistent with the results from the Illumina MiSeq high throughput sequencing. The X-ray photoelectron spectroscopy results indicated that Fe(0) was transformed into Fe(II)/Fe(III) during the treatment process, which are beneficial for the microorganism growth, and thus promoting their metabolic processes and microbial community.
improve anaerobic digestion performance considering its low price and conductive properties10?12. Zhang et al.13
found that ZVI could promote the performance of UASB reactor on degradation of azo dye at low hydraulic
retention time (12 h) and low temperature (25 ?C). Some reports also proved that nanoscale zero-valent iron (NZVI)
can accelerate the fermentation and hydrolysis stages for its action as electron donor14,15.
Nanoparticles (NPs) can interact with biological systems and humans, when released into the environment16.
Many studies have reported that NPs could accumulate in aquatic organisms and cells, which would lead to stress
or even mortality17?19. Metal nanoparticles, a major type of NPs, have shown strong antimicrobial effects for
microorganisms20. For example, the antimicrobial activity of iron-based nanoparticles against bacteriophage21
and Escherichia coli (E. coli)22,23 has been reported. NZVI could cause serious damage to the respiratory activity
and to the integrity of the cell membrane of E. coli in the absence of oxygen22?24. Therefore, more attention should
be paid to the application and disposal of manufactured NPs, so as to avoid unintended environmental harms.
Although previous works studied removal of contaminants in the ZVI/PS system, little information is known
about the effects of NZVI/PS on bio-communities and organisms, and the underlying mechanism also should be
figured out. Moreover, there is limited information on the environmental fate of most concerned NPs (e.g. NZVI)
in WWTPs (e.g. UASB or SBR reactor).
Biolog EcoPlatesTM has been used to analyze the bacterial communities in various environments25,26, and the
Illumina MiSeq sequencing technique has been widely used for analyzing the microbial functional and species
in many environments samples27. By means of these molecular biological methods, we can better understand the
bacterial communities in various environments.
In this study, we evaluated the efficiency of NZVI/PS enhanced degradation of brilliant red X-3B in a UASB
reactor, and determined the effects of NZVI/PS on anaerobic microbial communities in the reactor system. The
main objectives were to: (
) test degradation efficiency of brilliant red X-3B by NZVI activated PS, (
the performance of UASB combined with NZVI/PS for degradation of brilliant red X-3B and the chemical oxygen
demand (COD) removal, (
) determine the temporal and spatial changes in microbial metabolism of anaerobic
microorganisms in UASB through Biolog EcoPlateTM test28 following the addition of NZVI/PS; and (
analyse the change of microbial communities of the anaerobic microorganisms in UASB using the Illumina MiSeq
sequencing technique in the presence of NZVI/PS. The results may facilitate improved degradation efficacy of
persistent organic substances such as dyestuffs by incorporating nanotechnologies into biological treatment
processes in WWTPs.
Results and Discussion
Degradation of reactive brilliant red X-3B by NZVI/PS enhanced UASB reactor. Figure?1A shows
the schematic diagram of the UASB reactor, and Fig.?1B,C show the SEM images of NZVI. Fresh NZVI appeared
as aggregated nanospheres with individual sizes of 50?100 nm (Fig.?1B). After reaction in the UASB reactor for
30 days (Fig.?1C), the nanospheres were transformed into larger bulk aggregates, with some rod-like
microorganisms attached on the materials. The EDS line-scanning indicated that Fe was the main element for fresh NZVI
(through sections 1?1?) (Figure?S1A), while O accounted for a large percentage for used NZVI (Figure?S1B)
(through sections 2?2?), suggesting oxidation of Fe(0) during the reaction. In addition, the presence of C
indicated the attachment of microorganisms.
Table?1 gives the COD removal efficiency, decolorization ratio and COD in influent and effluent concentration
in stable operation stage of UASB reactor. The addition of NZVI into the sludge greatly affected the UASB reactor
process. Low dosages (0.1 g/L, 0.2 g/L and 0.5 g/L) of NZVI remarkably promoted the removal of COD and X-3B.
COD removal Decolorization
efficiency (%) ratio (%)
COD in effluent
G2 (0.1 g/L)
G3 (0.2 g/L)
G4 (0.5 g/L)
G5 (1.0 g/L)
However, an excessive dosage (1.0 g/L) of NZVI would pose a negative impact on the UASB reactor performance.
Figure?2A shows the decolourization rates in the UASB systems with different NZVI/PS dosages. At an NZVI
dosage of 0.1 g/L, the decolorization extent was increased from 63.8% to 93.9% at 60 min. Further increasing the
NZVI dosage to 0.5 g/L enhanced the decolorization rate to 98.4%, and the decolourization was decreased to
90.6% at a higher dose of 1.0 g/L NZVI. As shown in Fig.?2B, the COD removal was presented distinct effects after
addition of NZVI. When compared with reactor G1 which was operated without NZVI, all of reactors (G2-G5)
showed better COD removal (88.57% ? 93.53%) during the stage of stable operation. Moreover, Fig.?2B showed
that the maximum COD removal occurred at a concentration of 0.5 g/L and that the minimum COD removal
occurred at a concentration of 1.0 g/L with NZVI range (from 0.1 g/L to 1.0 g/L).
Le et al.29 found that 100% color removal efficiency and 54% TOC removal efficiency were achieved in
45 min with an initial dye Reactive Blue 19 concentration of 0.1 mM under typical conditions (pH 7.0, 0.8 g?L?1
ZVI, 10 mM persulfate and 30 ?C). Rodriguez et al.30 found that complete azo dye Orange G (OG) removal was
achieved within the first 30 min when PS was activated by Fe(II), but the longer reaction time required when
ZVI was employed. In this study, NZVI and PS were firstly added into the feed tanks of the reactors full of
dyeing wastewater and the following stoichiometric reactions between NZVI and PS were initiated immediately in
SO4?? + H2O ? SO24? + ?OH + H+
X ? 3B + SO4??/?OH ? intermediates ? products
The formed sulfate radicals (SO4??) and hydroxyl radicals (?OH) are strong reactive oxygen species (ROS),
which can efficiently degrade X-3B. The dyeing wastewater with NZVI and PS in the five feed tanks was
continuously agitated by a constant stirrer with a 200 rpm of mixing for 60 min, while the degradation of organic
pollutants using NZVI/PS may take only a few minutes14. Moreover, it was reported that the formed sulfate radicals
(SO4??) could react with S2O82? to form SO42? and S2O8?? under acidic conditions36, and SO4?? only existed in
aqueous-phase atmosphere for instance in cloud droplets37. In addition, iron could be initiated in anoxic
environments38. Therefore, the reaction of PS and NZVI occurred in the feed tank, while the biological degradation
process on X-3B and the reaction of NZVI with sludge mainly occurred in UASB reactors. It is a self-consumption
reaction for ZVI with regarding to the reactions 1?5, and transformation of Fe(0) was later confirmed by XPS
in the later Section. Therefore, reuse of reacted material for continuous cycle of reaction should not be available,
while re-addition of ZVI and PS after initialization of UASB should be a better choice in real application for
economic consideration. Fe-based materials including NZVI can also enhance the biological process in UASB due
to the release of Fe2+/Fe3+, which are essential elements and beneficial for the microbial growth39. Furthermore,
NZVI may affect the microbial metabolism and communities, and that the microbial degradation effectiveness
(to be discussed in detail in the following sections). However, high dosages of NZVI may cause a potential
toxicity to bacteria40, the addition of NZVI in anammox UASB reactors favored the growth of anammox bacteria and
promoted their aggregation in flocs41.
Microbial metabolism. The community-level physiological profiling (CLPP) of microbial
communities could be represented by the average well color development (AWCD) of Biolog EcoPlateTM technique25,42.
Figure?3A shows the AWCD changes of the 5 groups of UASB reactors. The results indicate that the CLPP, which
reflects the substrate utilization patterns by microbial communities, varied significantly in the UASB reactors
in response to the NZVI additions. In contrast, the AWCD curves of 5 UASB reactors showed similar patterns.
The microbial activities of G2, G3 and G4 were higher than those of G1and G5 in the initial 24 h, with G1 (the
control) showing the lowest microbial activity. In all cases, the microbial activity increased sharply at 96 h, and
then reached a moderately stable stage after 120 h. Among the 5 UASB reactors, the much greater microbial
activities of G2-G5 over G1 (Control) clearly indicates that NZVI could greatly improve CLPP, which also explains
the improved COD removal by the addition of NZVI in the UASB reactors (Table?1). Among the UASB reactors
which received the different doses of NZVI, the CLPP of G2-G4 increased slightly with increasing NZVI dose,
while that of G5 was lowered. Figure?3A shows that the maximum CLPP occurred at 0.5 g/L of NZVI while
the minimum CLPP occurred at 1.0 g/L in the different NZVI ranges (from 0.1 g/L to 1.0 g/L). Although
addition of NZVI with all dosages promoted the CLPP, the CLPP was firstly increased from 0.1 g/L to 0.5 g/L, while
then decreased with NZVI further increasing from 0.5 g/L to 1.0 g/L. The optical density (OD) data, which were
obtained from Biolog EcoPlateTM assay, were normalized and subjected to PCA to study the impact of NZVI on
the CLPP of microbial communities25. Figure?3B?F show the ordination diagrams of CLPPs from the principal
component analysis (PC1 and PC2) of 31 carbon source utilization profiles in five UASB reactors, which reflects
the change in the utilization pattern of carbon sources substrates of the microbial communities in response to
after the different NZVI doses. It is evident from the PC1-PC2 diagrams that the carbon source substrates are
mostly found in the lower right and upper left regions for the control reactor G1 (without NZVI), while the are
mainly located in the lower left and the upper right areas for G2-G5. These observations indicate that the addition
of NZVI notably affects the utilization patterns of carbon source substrates.
In general, a low utilization of carbon source substrates has a negative loading on PC1, while a high
utilization of carbon source substrates would present a high loading on PC1 (Fig.?3B?F).The variability on PC1 can
be explained by the change in the substrate utilization patterns by the microbial communities, such as
carboxylic acids, carbohydrates, phenolic compounds, polymers and amino acids25. The microbial communities in G1
favored utilization of carbohydrates (C2, D2, and E2), and amino acids (A4, D4, and F4). in contrast, the addition
of NZVI (0.1 g/L, 0.2 g/L and 0.5 g/L) showed relatively high utilization of carboxylic acids (A3, B3, D3, E3, F3,
F2, and H3), polymers (C1, D1, and E1) and amines/amides(G4 and H4), indicating that NZVI could promote
the metabolism of the microbial communities. The favorable impact of NZVI on the microbial communities in
the UASB reactors could be due to improved sulfate reduction and methane production43. Indeed, it was reported
that the microbial communities, particularly homoacetogenic bacteria and propionate-utilizing bacteria, could
be enhanced with the addition of ZVI44. Moreover, in the presence of low concentrations of NZVI, the microbial
communities are able to mitigates to toxic effects and overcome the oxidative stress associated with the ROS
through mechanisms such as the production of extracellular or intracellular substances45, and/or some kind of
cell repair mechanisms25. However, further investigations are needed to confirm these assertions.
In contrast, at 1.0 g/L NZVI, the utilizations of amino acids (B4, C4, and F4), amines/amides (G4 and H4),
carboxylic acids (C3 and H3), and carbohydrates (B2 and C2) were all decreased. This observation explains the
inhibitive effect of NZVI at elevated doses on the microbial communities. The results agree with the reported data
that 1.0 g/L NZVI may pose toxic effects on indigenous bacteria in bioremediation processes46,47.
MiSeq-pyrosequencing results and microbial community structures. Figure?2A shows that the
decolorization ratio was significantly changed upon the addition of different doses of NZVI. The decolorization
ratios of G2 and G5 were similar, and those of G3 and G4 were comparable. According to the decolorization ratios
and the NZVI dosages, samples collected G1, G3 and G5 were subjected to the Illumina pyrosequencing.
The total of 223288 effective 16S rRNA sequence reads (51836, 101098, and 70354 for G1, G3 and G5,
respectively) were generated by the Illumina MiSeq high-throughput sequencing of the samples from the corresponding
UASB reactors. Table?2 lists the sequence information on the 3 samples. The Simpson/Shannon diversity level and
Chao1/Ace estimator are used to reflect the microbial communities? phylotype richness levels. Table?2 shows that
the Chao1/Ace values at low NZVI doses (0.2 g/L) (G3) and high NZVI doses (1.0 g/L) (G5) were both higher
than that for the control (G1). Rarefaction analyses were then performed to compare and standardize the detected
taxon richness among the samples48. Figure?S2 in the Supporting?Information (SI) presents the rarefaction curves.
The rank abundance curves in Figure?S3 in SI show that G1, G3 and G5 all had extremely abundant microbes,
though G3 and G5 exceeded G1 in both microbial diversity and richness. As indicated in the Venn diagram in
Figure?S4 in SI, 1,690 OTU species occupied group G1, 2,678 OUT species occupied group G3, and 1,982 OTU
species occupied group G5. However, the Venn diagram indicated that only 7.83% (497 OTUs) OUT species of
the total OTUs were common for the three samples, and the shared OTUs between G1 and G3, G1and G5, and
G3 and G5 were 11.07%, 10.27% and 13.07%, respectively, indicating that the microbial compositions among G1,
G3 and G5 were quite different.
Taxonomic complexities of bacterial communities. The functions and behaviors of bacteria in
microbial communities influence the biomass and microbial activity49, The bacteria from G1, G3 and G5 displayed
greatly different abundance levels. Figure?4A illustrates the bacterial community abundance levels of G1, G3 and
G5 at the phylum level. A total of 22 phyla were identified in the bacteria from the 3 samples. The classified phyla
could be separated into five branches based on the species distribution in the 3 samples: Phy01-Phy14 (present in
all 3 samples), Phy15-Phy19 (only in G1), and Phy20 (in G3 and G5), Phy21 (only in G5) and Phy22 (in G1and
Phy03-Bacteroidetes, Phy06-Firmicutes, Phy10-Proteobacteria and Phy13-Verrucomicrobia were the
dominant phyla in the bacteria communities from the 3 samples, and the 4 dominant phyla differed distinctively in
the 3 samples. The percentages of these phyla in G1, G3 and G5 were measured at: Phy03-Bacteroidetes = 9.3%,
15.8% and 13.7%, Phy06-Firmicutes 46.7%, 37.3% and 32.5%, Phy10-Proteobacteria = 29.6%, 24.4% and 33.3%,
and Phy13-Verrucomicrobia = 1.9%, 19.2% and 14.9%, respectively. Thus, Phy06-Firmicutes were weakened
while Phy03-Bacteroidetes and Phy13-Verrucomicrobia were enriched with the NZVI treatments. However,
Phy10-Proteobacteria was weakened at the low dose of NZVI (G3) while enriched at the high dose of NZVI (G5).
The Bacteroidetes phylum is a large group of bacteria, which are found in many aquatic systems, revealing a high
level of metabolic and phenotypic diversity50. The enrichment of Bacteroidetes phylum is likely to play a role in
improving the anaerobic degradation of the dye stuff in the UASB reactors.
The microbial community?s abundance was further investigated at the genera level (Fig.?4B). A total of 88
genera were identified in the bacteria from the 3 samples. The classified genera are separated into six branches
based on the species distribution in the 3 samples: Gen01-Gen48 (existed in all 3 samples), Gen49-Gen56 (only in
G1), Gen57-Gen63 (in G3 and G5), Gen64-Gen71 (only in G3), Gen72-Gen83 (in G1 and G5) and Gen84-Gen88
(in G1 and G3). Gen05-S24-7 (Family), Gen14-Lactococcus , Gen32-Sphingomonas, Gen34-Comamonas and
Gen48-Akkermansia were the dominant genera in the bacteria from the 3 samples, in which the dominant genera
The percentages of these genera in G1, G3 and G5 were: Gen14-Lactococcus = 33.03%, 7.94% and 16.49%,
Gen48-Akkermansia = 1.69%, 20.23% and 15.74%, Gen34-Comamonas = 10.15%, 1.91% and 4.39%,
Gen32-Sphingomonas = 0.11%, 1.48% and 14.88%, S24-7 (Family) = 7.55%, 13.88% and 13.38%,
respectively. Evidently, Gen14-Lactococcus and Gen34-Comamonas were weakened with the NZVI treatments,
while Gen48-Akkermansia, Gen05-S24-7 (Family) and Gen32-Sphingomonas were enriched. Akkermansia are
well known to utilize mucins (sulfate groups and complex glycoproteins) as nitrogen, sole carbon and energy
sources51,52, and were found to exist in greater abundance with higher metagenome richness53. The increase in
Akkermansia abundance by NZVI benefited to the metabolic profile of the microbial community54. The increase
in S24-7 (Family) abundance favored the metabolism of microbial community. Sphingomonas was found having
great exuberant vitality and intense ability to fit for the environment55.
This bacterial community was further examined with the heat-map to recognize differences and similarities
among the 3 samples. As presented in Fig.?4c, 86 bacterial genera in the 3 samples were recognized at the 0.2%
abundance level, indicating the remarkably different microbial community structures in G1, G3 and G5.
The genera of the 3 samples in Fig.?4C were divided into four branches based on the species richness. From
the top to the bottom of Fig.?4c, the first branch covers Ruminococcaceae to Acinetobacter with 20 genera, the
second covers Rummeliibacillus to Peptostreptococcaceae with 66 genera, the third covers Ruminococcaceae to
Aneurinibacillus with 25 genera, and the fourth covers Victivallaceae to Peptostreptococcaceae with 61 genera.
According to Fig.?4c, the first branch was a high abundance branch in G1 and G5, while the second branch was a
low abundance branch in G1 and G5, the third branch was a high abundance branch in G3, and the fourth branch
was a low abundance branch in G3.
Functional microbial strain analysis. Two strains were isolated from G1, one strain was isolated from G2,
two strains were isolated from G3, one strain was isolated from G4 and one strain was isolated from G5. Some of
the seven strains were identified by 16S rRNA as the same strain and three different strains were finally identified.
Then the three different strains were denoted as FP-A1, FP-B1 and FP-C1. FP-A1 was only present in G1; FP-B1
was present in all of 5 samples, whereas FP-C1 was only present in G3. All the three strains are able to use reactive
brilliant red X-3B as their sole carbon and energy source, thereby bio-catalyzing the degradation of dye X-3B.
Figure?5A?C shows the growth rates and the biological degradation rates for the three strains.
The three strains were further identified as Bacillus cereus (FP-A1), Escherichia fergusonii (FP-B1) and
Rummeliibacillus pycnus (FP-C1) by 16S rRNA analysis and the taxon of three strains were shown in
Fig.?5A-1?C1. The NCBI Numbers of three strains were KX421197 (FP-A1), KX421198 (FP-B1) and KX421199 (FP-C1); the
phylogenetic trees of three strains are presented in Figures?S5?S7 in SI, and the morphologies of the three strains,
which were observed under FE-SEM, are given in Fig.?5A-2?C-2. Moreover, the results from high-throughput
sequencing also showed the genera of three strains as dye-degrading bacterial genera. Bacillus cereus belongs
to Bacillus (genus) and it was found only in G1 (Fig.?4B); Escherichia fergusonii belongs to Enterobacteriaceae
(Family) and it existed in all the three samples (Fig.?4B); Rummeliibacillus pycnus belongs to Rummeliibacillus
(genus) and it was observed in G3 only (Fig.?4B). The distribution of three strains was consistent with that of
genera in Fig.?4B. As a result, the high-throughput sequencing results were consistent with those from the bacterial
Nowadays, the microbiome studies by high-throughput sequencing of 16S rRNA gene, e.g., Polony
sequencing technology, 454 pyrosequencing technology and Illumina pyrosequencing technology, have attracted great
interests56. However, the reported results are usually difficult to reproduce across investigations which are caused
by the large variation in the complex multistep process of 16S rRNA-based microbiome analysis, such as sample
collection, DNA extraction, and purification, Polymerase chain reaction (PCR) process, sequencing and
bioinformatics. In this study, we presented the conventional microbial isolation and pure culture, which should be a useful
tool to confirm the results of high-throughput 16S rRNA gene sequencing.
Mechanisms for effects of NZVI/PS on UASB efficiency. Based on the above results, the addition of
NZVI/PS can affect the performance of UASB reactors, leading to much enhanced X-3B decolourization rate
(Fig.?2A). Following the addition of NZVI/PS, remarkable changes in the microbial community in the UASB
reactors were observed (Figs?3 and 4), i.e. a higher CLPP of microbial communities (Fig.?3) and much enhanced
microbial communities, particularly homoacetogenic bacteria, and propionate-utilizing bacteria44. Moreover, the
predominant microorganisms in the UASB reactors were changed. Specifically, (
) the competing bacteria (e.g.,
Phy06-Firmicutes) were inhibited, leading to enhanced microbial diversity; (
) Phy03-Bacteroidetes and
Phy13Verrucomicrobia in the UASB reactors were enriched, leading to a higher level of metabolic and phenotypic
diversity50; and (
) the increases of Gen48- Akkermansia, Gen05-S24-7 (Family) and Gen32-Sphingomonas bacteria
with the addition of NZVI in UASB reactor are also beneficial to the biological degradation process.
By comparing with the control without the addition of NZVI, it is evident that the change in microbial
community is attributed to the NZVI effects and may result from the modulation of NZVI. Figure?6 shows XPS
spectra of spent NZVI after 30 days? reactions in the UASB reactors. The main elements of used NZVI included:
Fe (55.4%), O (35.2%) and C (9.4%). The much elevated O percentage indicates oxidation of Fe(0) during the
reaction, while the presence of C on the materials manifests interactions of the microorganisms with NZVI. The
two major peaks at 724.4 ? 0.2 eV and 710.8 ? 0.2 eV are assigned to Fe 2p1/2 and Fe 2p3/2, respectively. The three
peaks at 706.5, 710.5 and 712.4 eV with a peak area ratio of 2.9:24.1:73.0 are attributed to Fe(0), Fe(II) and Fe(III)
oxidation states57,58, respectively (Fig.?6B). Therefore, most of Fe(0) was transformed into Fe(II) and Fe(III), which
are beneficial species for the microorganism growth. Therefore, the production of Fe(II)/Fe(III) and the
associated effects on the biological growth and microbial community can be also responsible for the enhanced
biodegradation of the dyestuff. In addition, the formed Fe(II) plays an important role for the formation of sulfate radicals
)), thus promoting the degradation of X-3B. Moreover, the dissolve irons (Fe(II) and Fe(III)) may
also catalyze the decomposition rate of X-3B. The roles of different iron ions should be further investigated in the
Material and Methods
Experimental methods. Five UASB reactors (G1-G5) were employed. PS was added into the UASB reactors
(G1-G5) at 0.125 g/L. The G1 reactor was used as the control without addition of NZVI, and the G2-G5 reactors
were subjected to various NZVI dosages (0.1, 0.2. 0.5 and 1.0 g/L). Figure?1A shows the schematic diagram of the
UASB reactors and the detailed information of the reactors is provided in Text S1 in SI.
All reagents were obtained in high-purity and were used as received, including NZVI (? 99.9%, Aladdin
Industrial Inc. Shanghai, China), Potassium persulfate (?99.0%, Aladdin Industrial Inc. Shanghai, China),
Reactive Brilliant Red X-3B (Sinapharm Chemical reagent Co., Ltd. Shanghai, China).
Inoculated sludge and synthetic dyeing wastewater. Inoculated sludge was taken from anaerobic
digesters of the sewage treatment plant based at Wuhan Textile University (Wuhan, China). The floc sizes of the
inoculated sludge ranged from ~1.0 to ~3.0 mm (Figure?S8, SI) and pH from 6.5 to 7.2. The detailed information
of the inoculated sludge and the influent dyeing wastewater are provided in Text S2 in SI.
Degradation of reactive brilliant red X-3B by NZVI/PS enhanced UASB system. In this study, the
dyeing wastewater was firstly purged with high-purity nitrogen gas at a constant flow rate of 0.5 L/min for 5 min
to remove oxygen and then was pumped into UASB reactor. The UASB reactors were first operated with a influent
COD from 100 to 4,000 g/(m3?d) for two months for the pre-acclimation of the anaerobic microbial communities.
COD levels in both influent and effluent were measured according to the standard method. Afterward, the UASB
reactors were operated over four stages, i.e., start-up (0-62 d), X-3B loading increase (63-112 d), recovery
(113131 d) and stable operation (132-208 d). High-purity nitrogen gas was purged into the UASB reactor at a constant
flow rate of 0.5 L/min for 20 min to maintain the DO concentration every 24 h during the reaction processes. And
the DO concentration in UASB reactor was ranged from 0.18 to 0.20 mg/L. On Day 63, PS was firstly added into
the five feed tanks of the reactors (G1-G5) which were full of dyeing wastewaters with the same concentration
of 0.125 g/L. Afterwards, NZVI was added at dosage of 0.1, 0.2, 0.5 and 1.0 g/L into the four tanks of the G2-G5
reactors. During the reaction, the dyeing wastewater with NZVI and PS in the five feed tanks were continuously
agitated by a constant stirrer with a 200 rpm of mixing for 60 min. Consequently, the dyeing wastewaters were
pumped into UASB reactors by a peristaltic pump after purging with high-purity nitrogen gas at a constant flow
rate of 0.5 L/min for 5 min to remove oxygen. On the other hand, the remaining NZVI was firstly took out from
the feed tank and then putted into the reaction zone of UASB reactor, which was then fixed with anaerobic sludge.
Table?S1 in SI provides the experimental schedule for influent COD and X-3B loading. The pH of dyeing
wastewater in the UASB reactors was adjusted by 1.0 mol/L NaOH and 1.0 mol/L H2SO4, and pH was kept from
6.5 to 7.2. The operation temperature of the UASB reactors was maintained at 30? 2 ?C, the hydraulic retention
time (HRT) was kept at 18 ? 2 h, and the average cell residence time ranged from 20 to 40 d. On Day 198, samples
were collected from each UASB reactors for further analyses.
The morphology and elemental percentages of NZVI in the UASB reactors was obtained on an SU8020
Ultra-High-Resolution field emission scanning electron microscope (FE-SEM) (Hitachi, Tokyo, Japan), and
energy dispersive spectra were collected at the same time. X-ray photoelectron spectroscopy (XPS) of NZVI
was conducted using a Thermo ESCALAB 250XI Multifunctional imaging electron spectrometer (Thermo
Fisher Scientific, Waltham, USA). The microbial community-level physiological profiling was performed
through the Biolog EcoPlateTM test26. The anaerobic microorganism genera were identified via Illumina MiSeq
Microbial community-level physiological profile (CLPP) determination. The Biolog EcoPlateTM
technique was employed to evaluate the microbial community physiological metabolic characteristics by
assessing utilization of separate sole carbon source substrates distributed in a 96-well plate during 7 days incubation28.
The detailed information of Biolog EcoPlateTM is provided in Table?S2 of SI. And the procedure of the Biolog
EcoPlateTM assay is described in Text S3 of SI.
Illumina high-throughput sequencing. The microbial communities in the UASB reactors were identified
by Illumina MiSeq high-throughput sequencing. The detailed information on DNA extraction, PCR
amplification, the multiplexed DNA libraries construction and sequences fragments analysis were described in Text S4
of SI following Pan et al.26, and the Illumina MiSeq high-throughput sequencing was conducted by Personal
Biotechnology Co., Ltd. (Shanghai, China).
Functional microbial strain analysis. Functional strain analysis of the bacteria with X-3B degrading
ability was carried out from the MLSS collected from G1-G5. And the method of bacterial isolation by Hamaki59 was
used with minor modification. The strains were subjected to decolorization ability assessment by measuring the
decolorization and growth curves, and the morphology of strains was analysed on a ZEISS ULTRA 55 FE-SEM
(ZEISS, Oberkochen, Germany). In the end, the phylogenetic characterization of the strains was performed by
16S rRNA gene sequencing. The detailed information is shown in Text S5 of SI.
The statistical and bioinformatics analysis were also performed according to procedures described in our prior
work26. All the assays were conducted in triplicate.
The authors thank the Collaborative Innovation Plan of Hubei Province for Key Technologies in the Eco-Ramie
Industry. This work was financially supported by the National Key Technology R&D Program on ?research and
demonstrations of key technologies for clean production in printing and dyeing? (2014BAC13B02) and the
National Natural Science Foundation of China (Grant 41230638).
F.P. and W.L. conceived the experiments, F.P., X.Z., F.L., and H.J. conducted the experiments, F.P., X.Y. D.X. and
D.Z. analysed the data. All authors participated in writing the paper.
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing Interests: The authors declare no competing financial interests.
How to cite this article: Pan, F. et al. Nanoscale zero-valent iron/persulfate enhanced upflow anaerobic sludge
blanket reactor for dye removal: Insight into microbial metabolism and microbial community. Sci. Rep. 7, 44626;
doi: 10.1038/srep44626 (2017).
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