Science of the Total Environment 751 (2021) 142161

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Science of the Total Environment

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

Microbial removal of vanadium (V) from groundwater by sawdust used

as a sole carbon source
Liting Hao a,b, Yongjie Liu a, Nan Chen a, Xiaodi Hao b, Baogang Zhang a, Chuanping Feng a,⁎
a
School of Water Resources and Environment, MOE Key Laboratory of Groundwater Circulation and Environmental Evolution, China University of Geosciences (Beijing), Beijing 100083, PR China
b
Sino-Dutch R&D Centre for Future Wastewater Treatment Technologies, Beijing Advanced Innovation Center for Future Urban Design, Beijing University of Civil Engineering and Architecture,
Beijing 100044, 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

• V(V) is effectively reduced by microbes

using sawdust as solid carbon sources.
• Medical stone and phosphate rock en-
hance V(V) bioremediation in actual
groundwater.
• Accumulation of microbes such as
Thauera could contribute to V
(V) reduction.
• Sawdust supports effective V(V) removal
in column bioreactors with various
inocula.

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

Article history: Bioremediation of vanadium (V) (V(V)) for polluted groundwater is an emerging topic globally. With this study,
Received 20 May 2020 microbial removal of V(V) was investigated by sawdust of pine used as a sole carbon source. The removal effi-
Received in revised form 23 July 2020 ciency of V(V) reached up to 90.3% with anaerobic sludge as inocula and sawdust as the carbon source in nutrient
Accepted 1 September 2020
solution. Microbial removal of V(V) could be enhanced by adding medical stone and phosphate rock, from 53.2%
Available online 02 September 2020
up to 82.6% in real groundwater. Microbiological analysis revealed such microbes as Thauera accumulated, which
Editor: Deyi Hou could contribute to V(V) reduction. Such functional species as Bacteroidetes vadinHA17 norank and
Anaerolineaceae norank helped degradation of sawdust. In column experiments with domesticated sludge or in-
Keywords: digenous microbes from soils, microbial V(V) removal efficiencies (on 26 d) with sawdust were around 58.7%
Vanadium (V) (BS), 54.8% (BP) and 38.4% (BU), respectively. The study can offer a potential approach to microbially removing
Sawdust V(V) for contaminated groundwater and even for disposal of agricultural and forestry wastes.
Groundwater © 2020 Elsevier B.V. All rights reserved.
Bioremediation
Microbial community

1. Introduction 2018). 176–268 μg/L V in groundwater was detected in Panzhihua

Excessive amounts of Vanadium (V) could be released by intensive
mining of V-containing minerals and other anthropogenic activities,
causing severe public health issues (Chen and Liu, 2017; Jiang et al.,
⁎ Corresponding author.
E-mail address: fengcp@cugb.edu.cn (C. Feng).
(China) (Zhong et al., 2015). Up to 0.77 mg/L V in groundwater were re-
ported in Rifle, Colo. (Ortiz-Bernad et al., 2004). 0.05–2.47 mg/L V was
found in groundwater samples of Argentina (Fiorentino et al., 2007).
For the drinking water, the limit value of V in China is 0.05 mg/L (GB
3838-2002, 2002), and California proposed 15 μg/L as a notification
level of V (OEHHA, 2019). V(V) as vanadate is the most toxic because
the toxicity of V would increase with solubility and valence state

https://doi.org/10.1016/j.scitotenv.2020.142161
0048-9697/© 2020 Elsevier B.V. All rights reserved.
L. Hao, Y. Liu, N. Chen et al. Science of the Total Environment 751 (2021) 142161

increase (Hao et al., 2015; Zhang et al., 2010). V(V) contaminated In present study, the feasibility of sawdust as solid organic carbon
groundwater requires efficient remediation in order to prevent adverse sources for microbial V(V) remediation was investigated. The enhance-
environmental impacts. ment of microbial V(V) removal in actual groundwater was also evalu-
As the promising, environmentally-friendly and efficient technol- ated with medical stone and phosphate rock. Microbial analyses were
ogy, microbial V(V) removal has attracted the great attention (Hao performed for better insights into mechanisms of microbial V
et al., 2018; Yelton et al., 2013). V(V) could be microbiologically reduced (V) reduction. Furthermore, continuous flow column bioreactors were
and immobilized (Huang et al., 2015). Currently, the majority of micro- also employed to explore the uninterrupted bioremediation of real V
bial V(V) removal treatments rely on heterotrophic bacteria with or- (V) contaminated groundwater. This study is expected to provide theo-
ganic carbon substrate required. Lots of heterotrophic microorganisms retical and technical supports for the practical application of microbial V
capable of reducing V(V) have been reported. For example, Shewanella (V) remediation in groundwater with agricultural and forestry wastes.
oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate
to the reduction of V(V) (Carpentier et al., 2003). However, organic car- 2. Materials and methods
bon availability decreases with depth below the ground surface (Starr
and Gillham, 1993). Groundwater in Panzhihua region (China) with V 2.1. Preparation of materials and cultivation of microorganisms
mining and smelting activities was reported to show oligotrophic char-
acteristics with less total organic carbon (TOC) compared to surface The sawdust of pine was obtained from Tai'an of Shandong Province
water (Zhang et al., 2019). Hence, challenges still exist regarding the in China (Jin et al., 2019). Sawdust (1–4 mm) was prepared via sample
lack of carbon sources in the V contaminated groundwater. It was re- sieves screened. The C, H and N contents of sawdust were 47.67% ±
ported that the organics (glucose) addition was effective for microbial 1.38%, 5.73% ± 0.17%, and 0.20% ± 0.01%, determined by an elemental
V(V) reduction enhancement (Zhang et al., 2015a). To biostimulate con- analyzer (Flash 2000, Thermo Fisher, Italy). The content of organic car-
taminated sediments, acetate was directly injected into the aquifer, re- bon in sawdust was 46.49% ± 0.97% (CS230, LECO, USA). Natural phos-
sulted in prolonged removal of aqueous V for a period of at least two phate rock was obtained from Jinan of Shandong Province in China, and
years (Yelton et al., 2013). During 12 h, acetate was reported to effec- it was crushed by manual hammering and screened to obtain 1–4 mm
tively support microbial V(V) removal with its efficiency of 75.6% (Liu particle sizes. Medical stone (1–4 mm) was from Tongliao of Inner
et al., 2016). Therefore, appropriate amount of carbon sources should Mongolia in China. The obtained sawdust, phosphate rock and medical
be provided during the application process to promote V stone were rinsed three times with deionized water and allowed to air
(V) bioremediation. However, it must be pointed out that methanol, dry naturally.
ethanol, acetic acid and other traditional liquid carbon sources are al- Nutrient solution and actual groundwater were used in this study.
ways expensive, with risk of overdosing causing secondary pollution Actual groundwater was taken from a well located at China University
(Shao et al., 2009). Furthermore, it is not easy for liquid carbon sources of Geosciences (Beijing) in February–May 2019. The concentrations of
to build reactors or in situ repair process. main cations in the raw actual groundwater (Table 1a) were analyzed
Compared with liquid carbon sources, solid carbon sources could act by ICP-OES (OPTIMA 8000, PE, USA). Except for Na, Fe, Cu and other reg-
as both the carbon sources and carriers for microbial growth. The ular indices, some other indices such as Co and Ni were also included in
immobilized anaerobic biofilm was reported to perform better for mi- the raw groundwater samples. Nutrient solution contained the follow-
crobial V(V) reduction than free suspended culture (Zhang et al., ing components (per liter): NH4Cl (0.1557 g), CaCl2 (0.2464 g),
2015a). Saliling et al. (2007) reported that wood chips were degraded MgCl2`6H2O (1.0572 g), NaCl (0.4459 g), KCl (0.0283 g), NaHCO3
in the lower sections of reactors to remove nitrate, however, it could re- (0.8082 g) and KH2PO4 (0.0299 g). V(V) was added into above solution
main sturdy enough to support media in the upper sections. Solid car- in the form of NaVO3 at the designated concentration. The glucose or
bon sources with high-quality slow-release ability have already sawdust were added as liquid or solid organic carbon sources in the
captured people's attention in denitrification systems (Zhao et al., study.
2018). Solid carbon sources were initially hydrolyzed by extracellular en- Jars with silica gel stoppers were employed for the cultivation of in-
zymes secreted by the microbes, decomposed into soluble and small mol- ocula. Sludge was obtained from Qinghe Wastewater Treatment Plant in
ecule organic matter, and then, most of the organic matter could be
utilized by microbes to reduce nitrate (Si et al., 2018). Sawdust and
Table 1
other wood by-products have been successfully tested in bioreactors de- (a) The concentrations of main cations in the raw actual groundwater; (b) Alpha-diversity
signed for nitrate treatment (Robertson, 2010; Zhao et al., 2018). Sawdust of microbial communities in the inoculated sludge (Sludge) and batch reactor samples
was already reported to be successfully applied in field-scale projects for (NSS, GSS and GSMPS).
nitrate removal (Schmidt and Clark, 2012). Using sawdust and other solid (a) The concentrations of main cations in the raw actual groundwater
carbon sources could better avoid the risks of overdosing or insufficient Name of cations Concentration (mg/L)
dose, and the control and supervision of the process were simple, which
K 1.738
were already applied for in-situ groundwater remediation (Wang and Ca 63.066
Chu, 2016). However, few attempts have been made to remove V(V) by Na 18.087
microbes with solid carbon sources, with advantages of low processing Mg 34.492
Mn 0.014
cost and low risk of secondary pollution.
Ni 0.012
It has been reported that there would be a risk of clogging when re- Fe 0.043
actors were built with pine sawdust only (Jin et al., 2019). Thus, suitable Cu 0.013
auxiliary materials could also be screened, which might be also helpful Co 0.024
for V(V) bioremediation enhancement in real contaminated groundwa-
(b) Alpha-diversity of microbial communities in the inoculated sludge (Sludge)
ter. Medical stone was reported to be widely used in wastewater treat-
and batch reactor samples (NSS, GSS and GSMPS).
ment (Wang et al., 2016b), which could leach some trace elements to Name Ace Chao Coverage Shannon Simpson Sobs
promote the growth of bacteria (Hong et al., 2012). Phosphate rock
Sludge 1463.6845 1447.3367 0.9975 5.0367 0.0216 1376
could meet microbial growth needs of biological phosphorus (Jin et al., NSS 1162.0486 1008.5385 0.9943 3.4773 0.1345 696
2019), already successfully applicated for remediation of nitrate pol- GSS 1433.1003 1417.3989 0.9960 4.3997 0.0562 1216
luted groundwater (Zhang et al., 2015c). Both of them were reported GSMPS 1374.0786 1367.8182 0.9939 4.1822 0.0868 1096
to successfully improve the pH condition for bioreaction and enhance (NSS: nutrient solution+sawdust+sludge; GSS: groundwater+sawdust+sludge;
microbial activities (Liu et al., 2020). GSMPS: groundwater+sawdust+medical stone and phosphate rock+sludge)

2
L. Hao, Y. Liu, N. Chen et al.
Beijing (China). The initial total solids (TS) of the sludge was 16.2 g/L
with the volatile solids (VS) of 9.9 g/L. The sludge above was domesti-
cated for approximately 30 d with refreshing the fresh nutrient solution
once every 3 d. The glucose (C6H12O6, 0.75 g/L) was used as the liquid
organic carbon source in the solution containing V(V) (75 mg/L). Soil
samples were collected from V ore mining area (petroleum associated
minerals, Hubei Province, China) and farmland near V ore mining area
(uvanite, Guizhou Province, China). Jars filled with sampled soils
(100 g) were also refreshed once every 3 d with fresh nutrient solution
above containing V(V) and glucose for incubation.
2.2. Experimental setup and operation
2.2.1. Batch tests
Batch experiments were conducted to verify sawdust could serve as
the solid carbon sources for the bioremediation of V(V) contaminated
groundwater. Six 250 mL serum bottles were employed as reactors
and divided evenly into three groups (NSS, NS and NGS). Each reactor
was filled with 200 mL nutrient solution. V(V) was added in the form
of NaVO3 with the concentration of 75 mg/L. The sawdust (1.5 g) was
added into the two reactors (NSS) as carbon sources. And incubated
sludge (20 mL) was centrifuged at 3000 rpm for 20 min, with solid res-
idue used as inocula in each reactor. The left bottles were control sets. To
determine the influence of materials themselves on the V(V) in experi-
ments, adsorption experiments without sludge inoculated (NS) were
conducted. The reactors of NGS were also inoculated with 20 mL sludge
as NSS, but glucose was added as the liquid carbon source instead of
sawdust.
Another eight 250 mL serum bottles were employed as reactors and
2 serum bottles for each group (GSS, GSMPS, GS and GSMP) to investi-
gate the V(V) removal with sawdust in real groundwater. Each reactor
was filled with 200 mL actual groundwater with NaVO3 added for the
V(V) concentration of 75 mg/L. The sawdust (1.5 g) was added into all
the 8 reactors, with residue of 20 mL incubated and centrifuged sludge
as inocula in GSS and GSMPS. The left reactors were control sets and
without inoculation (GS and GSMP). The enhancement of microbial V
(V) removal in actual groundwater was investigated with sterilized
phosphate rock (4.0 g) and medical stone (4.0 g) added into GSMPS
and GSMP.
The serum bottles, sawdust, phosphate rock and medical stone used
above were sterilized in the clean bench with UV for 12 h before use.
After the serum bottles were filled with materials and nutrient solution
or groundwater, the headspace of bottles was flushed with N2 for 5 min
to maintain anaerobic environment. All the reactors were operated at
room temperature. Samples were collected and then filtered through
0.45 μm membrane prior to analysis. Each sample from each condition
was tested for three times and average results were used.
2.2.2. Experimental column apparatus and procedure
Sawdust as carbon sources and biofilm carriers for the biological V
(V) removal were also investigated in continuous flow column bioreac-
tors. Four acrylic glass columns with same dimensions (inner diame-
ter = 30 mm, height = 300 mm) were constructed as the
experimental bioreactors (BC, BS, BP and BU). The top and bottom of
columns were filled with 2.0–4.0 mm glass beads (filling height of
15 mm) as the support and buffer layer to ensure influent distributed
evenly and sufficient water level. The height of working layer (between
the support and buffer layer) was 270 mm. The sawdust, phosphate
rock and medical stone were completely mixed in a volume ratio of
4:1:1 (18.0 g sawdust, 52.0 g phosphate rock and 48.0 g medical
stone) and filled in the working layer of reactors. The media porosity
(ne) of the whole bioreactors was 57.3%.
Among them, BC column was the blank control, packed with filter
only (without inoculation) to investigate the adsorption performance
of V(V) on packed substances. For BS column, the domesticated anaero-
bic sludge as inocula was completely mixed with the filling materials.
3
Science of the Total Environment 751 (2021) 142161
The domesticated samples of soil from V ore mining area (petroleum as-
sociated minerals) were inoculated in BP. And for BU, the domesticated
samples of surface farmland soil near V ore mining area (uvanite) were
employed as inocula. Working layer materials were immersed with var-
ious kinds of inocula (100 mL), mixed thoroughly and sealed for 1 d at
room temperature (about 25 °C). And then, V(V) (75 mg/L) contami-
nated groundwater was fed to the bottom of columns by using a peri-
staltic pump (BT100-1F, Longer Precision Pump Co., Ltd., China) at the
flow rate of 1 mL/min. The effluent was re-pumped into reactor devices
as the influent using the pump, with the internal circulation performed.
The circulating biofilm acclimatization period before formal operation
was approximately 40 d.
2.3. Chemical and biological analyses
Soluble V(V) concentration was measured using a spectrophotomet-
ric method (Zhang et al., 2018). The dissolved organic carbon (DOC) and
dissolved inorganic carbon (DIC) were measured by a TOC analysis
meter (multi N/C 2100S, Analytik Jena, Germany). The morphology of
the samples was observed using an environmental scanning electron
microscope (ESEM, Quanta 200, Netherlands). Precipitates were charac-
terized by X-ray photoelectron spectroscopy (XPS) (Thermo Fisher Sci-
entific, ESCALAB 250Xi, USA).
Microbial samples were collected from the inoculated sludge as well
as the batch reactor samples after operation to characterize the micro-
bial community. DNA of samples was extracted using the FastDNA
SPIN Kit (Qiagen, CA, USA). The extracted DNA was pooled, amplified
by PCR with primers 515FmodF (5′-GTGYCAGCMGCCGCGGTAA-3′)
and 806RmodR (5′-GGACTACNVGGGTWTCTAAT-3′) for bacteria and
archaea. The samples were sent to Majorbio Technology (Shanghai,
China) for high-throughput Illumina MiSeq sequencing analysis.
2.4. Data analysis
The operational taxonomic units (OTUs) were clustered from se-
quences at 97% similarity and assigned taxonomy based on the SILVA
database. Rarefaction curves and alpha-diversity were obtained by
using Mothur (version v.1.30.1). Rank-Abundance curves, Venn dia-
gram, Bar graph and Hierarchical cluster analysis were performed by
using R, FastTree (version 2.1.3) and FastUniFrac. Circos diagram was
conducted by Circos-0.67-7. Networks analysis was performed based
on Networkx.
3. Results and discussion
3.1. Bioremediation of V(V) in nutrient solution with sawdust
3.1.1. The variation of V(V) concentrations
With the sludge as inocula and the sawdust used as solid organic car-
bon sources, the variation of V(V) concentration in nutrient solution
was shown in Fig. 1a. The TS of the inoculated sludge was 12.9 g/L,
with the VS of 8.5 g/L. In the batch reactors of NSS, the concentration
of V(V) decreased significantly, with the removal efficiency of around
90.3% (10 d). At the beginning of the reaction (0–4 h), the average V
(V) removal rate was up to about 42.1 mg V(V)/(L·d). Within 0–2 d,
the average V(V) removal rate showed a rapidly and continuously de-
creasing trend. During 2–10 d, the average V(V) removal rate fluctuated
and then slowly decreased. At the end of the experimental period (10
d), the average V(V) removal rate was around 6.5 mg V(V)/(L·d). The
precipitates in NSS (10 d) were collected and analyzed by XPS
(Fig. 1b). The peaks at around 516.4 and 523.6 eV in the V 2p spectrum
corresponded to V(IV) (Fan et al., 2020), providing direct evidence that
V(V) was bio-reduced to V(IV). Less toxic V(IV) was the main reduction
product of V(V) through microbial transformation, which could precip-
itate naturally in near-neutral conditions in situ, consistent with previ-
ous findings (Qiu et al., 2017; Zhang et al., 2018; Zhang et al., 2019).
L. Hao, Y. Liu, N. Chen et al. Science of the Total Environment 751 (2021) 142161

Fig. 1. Profiles of V(V) concentration (a); XPS spectra of V 2p for the generated precipitates (NSS) (b); DOC/DIC concentration (c); ESEM images of the sawdust before (d) and after (e) the
experimental operation (NSS). (NSS: nutrient solution+sawdust+sludge; NS: nutrient solution+sawdust; NGS: nutrient solution+glucose+sludge)

This observation better suggested that V(V) pollution could be miti-
gated through microbial reduction with sawdust served as carbon
sources.
In the control set of NS (without inoculation), V(V) concentration
decreased slightly along with time (Fig. 1a), with the removal efficiency
of approximately 19.0% (10 d). One possible reason might be the occur-
rence of abiotic reactions such as the adsorption of V(V) by sawdust. It
was reported that sawdust was one effective adsorbent of heavy metals
in solutions (Li et al., 2005). Sawdust was also reported to be a promis-
ing adsorbent for the removal of nickel from untreated industrial
wastewater (Shukla et al., 2005). Compared with NSS, results reflected
that the sawdust could be used as carbon sources to support the micro-
bial removal of V(V) in nutrient solution, which could provide a basis for
bioremediation of V(V) pollution with sawdust as solid organic carbon
sources in real groundwater.
In NGS (with glucose as the carbon sources), the V(V) decreased sig-
nificantly along with time (Fig. 1a). The removal efficiency of V(V) was
about 98.7% (10 d), with the average removal rate of around 7.1 mg V
(V)/(L·d). By comparison with NSS, the average V(V) removal rate in
NGS was significantly higher during the first 5 d. However, during the

4
L. Hao, Y. Liu, N. Chen et al.
6–9 d, the average removal rate was lower than that in NSS
(7.5–9.2 mg V(V)/(L·d)). Results suggested that microbes could stably
and continuously take organic matter from the sawdust to maintain V
(V) reduction performance during the long-term operation.
3.1.2. Concentration profiles of DOC/DIC
The changes of DOC and DIC concentrations in reactors were moni-
tored (Fig. 1c) to investigate the carbon release of sawdust during the
microbial V(V) removal processes. The initial concentration of DOC in
the fresh nutrient solution was below the detection limit (1.19 mg/L).
In the initial stage, the DOC concentration in NSS was relatively high,
with around 178.1 mg/L after 4 h. The DOC accumulated to
208.2 mg/L after 1.5 d, and then it decreased rapidly with the passing
time. At the end of the experimental period (10 d), the DOC concentra-
tion decreased to approximately 40.2 mg/L. The easily soluble fraction of
carbon could cause rapid microbial adaptation growth. Therefore, better
average V(V) removal rates during the initial stage of NSS was observed.
While, with the consumption of easy dissolution carbon, degradation-
resistant was slowly replacing easy dissolution parts of sawdust as the
main using form of carbon sources. Some insoluble macromolecular or-
ganic matter of sawdust could be slowly degraded by microbes into
small molecular substances, which could be used by microorganisms
during the process. The continuous release of TOC from woodchips
may be controlled by intraparticle diffusion and the microbial degrada-
tion rate (Abusallout and Hua, 2017). Correspondingly, the average V
(V) removal rates were gradually reduced compared to the initial
stage. ESEM images (Fig. 1d) revealed that the sawdust before use ex-
hibited rich in pores and large specific surface area with rigid and highly
ordered fibrils, conducive to microbial attachment. However, after the
sawdust was used as solid organic carbon sources and carriers by mi-
crobes, it showed less order (Fig. 1e), with still many fibrous substances
difficult to be degraded.
DOC concentration in NS had shown a slowly decrease trend along
with time, probably due to the incomplete sterilization causing the ex-
istence of residual indigenous microbes. Therefore, some organic carbon
sources were used with slight DOC concentration decreasing (Fig. 1c),
which was also consistent with the slight V(V) removal in NS (Fig. 1a).
The initial concentration of DIC in the fresh nutrient solution was
118.2 mg/L (Fig. 1c). By comparing the results of NSS and NS, it could
be found that the DIC in NSS was slightly higher than that in NS. The
concentration of DIC in NSS was about 1.2 times of the DIC in NS at
Fig. 2. The variation of V(V) concentrations with sawdust in groundwater during the 10 d operation. (GSS: groundwater+sawdust+sludge; GSMPS: groundwater+sawdust+medical
stone and phosphate rock+sludge; GS: groundwater+sawdust; GSMP: groundwater+sawdust+medical stone and phosphate rock)
5
Science of the Total Environment 751 (2021) 142161
the end of whole period (10 d). The nutrient solution was used in the
study, and the fresh solution itself contained a certain amount of DIC.
Moreover, DOC in NSS originated from the sawdust and it could be fi-
nally converted into DIC in the reactors. Thus, the rest of the DIC in
the reactors was presumed to be generated by the respiration of micro-
organisms. Two ways of V(V) bio-reduction were already reported, res-
piration by microbes via electron transfer, for detoxifying or V binding
to reductases of other electron acceptors (Yelton et al., 2013).
3.2. V(V) removal with sawdust and its enhancement in real groundwater
Fig. 2 presents the variation of V(V) concentration with sludge as in-
ocula and sawdust as solid carbon sources in actual groundwater. The TS
of the inoculated sludge was 11.8 g/L, with the VS of 7.8 g/L. After 10 d,
the V(V) removal efficiency of GSS was approximately 53.2%. In the con-
trol sets of GS (no inocula), V(V) concentration decreased slightly and
fluctuated, with removal efficiency of about 18.2% (10 d). The V
(V) removal in GS might be also due to the non-biological reactions
such as adsorption or residual microbial V(V) removal caused by incom-
plete inactivation. Results of GSS and GS reflected that some agricultural
and forestry waste such as sawdust could be used as solid carbon
sources for V(V) bioremediation in the real groundwater. While, com-
pared with NSS (nutrient solution), the removal efficiency of GSS (ac-
tual groundwater) was obviously lower.
Thence, the medical stone (4.0 g) and phosphate rock (4.0 g) were
added in GSMPS in order to enhance the performance of microbial V
(V) removal with sawdust in actual groundwater. The medical stone
could leach some trace elements to promote the growth of bacteria
(Hong et al., 2012). The phosphate rock was also reported to have the
potential to enhance biological denitrification (Zhang et al., 2015c). Re-
sults confirmed that the V(V) removal efficiency was around 82.6% (10
d), higher than GSS (53.2%). Moreover, the average V(V) removal rate in
GSMPS was around 5.7 mg V(V)/(L·d) (10 d), more than 3.7 mg V(V)/
(L·d) in GSS. In the control sets of GSMP (without inoculation) with
medical stone and phosphate rock, V(V) concentrations fluctuated
along with time, with removal efficiency of about 21.8%, slightly high
than 18.2% (10 d) in GS. It was probably due to the V(V) adsorption abil-
ities of medical stone and phosphate rock themselves. The medical
stone and phosphate rock added in the study could serve as the carriers
for microbial growth, dissolve some trace elements and facilitate the en-
richment of microorganisms. Furthermore, the self-metabolism, growth
L. Hao, Y. Liu, N. Chen et al.
and reproduction of microbes might also promote the dissolution of
trace elements (Liu et al., 2020), thereby improving the activity of mi-
croorganisms. Results reflected that the addition of medical stone and
phosphate rock could be helpful for the enhancement of microbial V
(V) removal in real groundwater.
3.3. Microbiological analysis
3.3.1. Richness and diversity
The microbial ecology of NSS, GSS and GSMPS samples collected at
the end of batch experimental operation and the original sample (inoc-
ula) named Sludge were analyzed by high-throughput 16S rRNA gene
sequencing. High-quality reads of 67,529, 41,992, 48,786 and 67,623
were obtained for the samples of Sludge, NSS, GSS and GSMPS. Shown
by the Rank-Abundance curves (Fig. 3a), the relative abundance of
Sludge was the highest among the 4 samples. The relative abundance
of NSS was lower than that of GSS, which reflected that the complex
composition of actual groundwater was helpful for the growth and re-
production of microbes. While, compared with GSS, GSMPS showed a
higher abundance, which might be owing to the obvious effect of the ac-
tive minerals on the microbial community structure changes.
The rarefaction curves for all the samples displayed reasonable de-
grees of saturation (Fig. 3b). The Coverage showed that the sequencing
results represented an almost overall microbial diversity in these
(a)
(c)
GSS NSS
Sludge GSMPS
Sludge GSS NSS GSMPS
Fig. 3. Rank-Abundance curves (a), rarefaction curves (b), Venn diagram of the samples at OTUs level (c) and microbial community compositions at phylum level (d) based on
pyrosequencing of microbial communities in the inoculated sludge (Sludge) and batch reactor samples (NSS, GSS and GSMPS). (NSS: nutrient solution+sawdust+sludge; GSS:
groundwater+sawdust+sludge; GSMPS: groundwater+sawdust+medical stone and phosphate rock+sludge)
Science of the Total Environment 751 (2021) 142161
samples (>99.39%) (Table 1b). From the values of Shannon and
Simpson index in the Table 1b, the microbial community of Sludge
was more diverse than that in other samples. Furthermore, more diver-
sity of GSS and GSMPS with actual groundwater were observed than
NSS (with nutrient solution). The value of Ace, Chao and Sobs
(Table 1b) also indicated that the microbial community richness of the
original sample was more than that of the others, implying the gradual
formation of unique functional microbial groups with the domestication
of specific conditions.
The Venn diagrams showed that there were totally 528 OTUs de-
tected in all the 4 samples, accounting for 38.4%, 75.9%, 48.2%, and
43.4% of the total OTUs in Sludge, NSS, GSS and GSMPS, respectively
(Fig. 3c). The existence of V(V) could be one possible reason for the
shared OTUs. The phyla of microbial communities were similar in each
sample, while the proportion of each phylum varied greatly among
them (Fig. 3d). Proteobacteria was the highest proportion of both Sludge
(41.3%) and GSMPS (40.4%). With sawdust as solid carbon sources, the
phylum of Bacteroidetes was detected most both in NSS (54.5%) and
GSS (32.5%). In addition to Proteobacteria and Bacteroidetes, the top 6
relative abundance of phyla in each sample included Chloroflexi,
Firmicutes, BRC1 and Actinobacteria. The Proteobacteria, Bacteroidetes,
Chloroflexi and Firmicutes accounted for 27.1%, 32.5%, 26.7% and 4.5%
of GSS, with the sum more than 90%. For GSMPS, the relative abundance
of the 4 phyla was 40.4%, 28.0%, 15.8% and 4.5%, respectively. The
(b)
(d)
6
L. Hao, Y. Liu, N. Chen et al.
selective enrichment of Bacteroidetes, Chloroflexi and Firmicutes were
detected in GSS and GSMPS compared with Sludge, possibly due to
the fact that all of them were the known phyla related to cellulose deg-
radation (Rastogi et al., 2010), heavy metal tolerance (Legg et al., 2012)
or reduction (Dong et al., 2016).
3.3.2. Identification of the involved microbes
The most dominant genera (relative abundance in the top 5) in NSS
were Bacteroidetes (35.0%), Paludibacter (7.3%), Erythrobacteraceae un-
classified (4.8%), Thauera (3.5%) and Draconibacteriaceae norank (3.2%)
(Fig. 4a). While in GSS, they were BSV13 (28.0%), Anaerolineaceae
norank (9.3%), Chloroflexi 1–2 norank (4.9%), Anaerolineaceae unclassified
(3.4%) and Bacteroidetes vadinHA17 norank (3.1%). Furthermore, the top
5 relative abundance genera of GSMPS were BSV13 (21.0%), Zoogloea
(5.5%), Chloroflexi 1–2 norank (4.8%), Novosphingobium (4.7%) and
Bacteroidetes vadinHA17 norank (3.9%). Many of the dominant bacteria
in NSS, GSS, and GSMPS (Fig. 4b) were reported to be able to utilize
(a)
(c)
(b)
(d)
Fig. 4. Circos diagram of the samples and species (Genus) (a); Hierarchical cluster analysis of microbial communities at genus level (b); Networks analysis of co-occurrence patterns among
genera in microbial communities (c); Bar graph of clusters of orthologous groups (COG) function classification (d). (NSS: nutrient solution+sawdust+sludge; GSS:
groundwater+sawdust+sludge; GSMPS: groundwater+sawdust+medical stone and phosphate rock+sludge)
7
Science of the Total Environment 751 (2021) 142161
the organics difficult to degrade. For example, Bacteroidetes organisms
were considered to be highly correlated with the degradation of com-
plex polysaccharides (Zhang et al., 2015b). Paludibacter jiangxiensis sp.
nov., was a strictly anaerobic, propionate-producing bacterium isolated
from rice paddy field (Qiu et al., 2014). Paludibacter was reported to be
one of the dominant species in the anoxic system for enhanced pyridine
biodegradation (Shen et al., 2015). Thauera was reported to be one of
the dominant lignin-degrading strains (Wang et al., 2016a).
Draconibacteriaceae was reported to be fermentative bacteria (Cheng
et al., 2018). It was reported that Anaerolineaceae could play an impor-
tant role in the process of fermentation (Liang et al., 2015). Members
of the Bacteroidetes environmental groups VadinHA17, WCHB1-69, and
U29-B03 were reported to be found in environments undergoing com-
plex carbon degradation, and clone VadinHA17 came from anaerobic di-
gester treating winery wastewater (Baldwin et al., 2015). Members of
the vadinHA17 were found to be more important during the late phases
of progressing rice straw decomposition, implying that it was an
L. Hao, Y. Liu, N. Chen et al.
important bacterial group during rice straw degradation especially for
the recalcitrant part decomposition (Ji et al., 2018). These genera
above could contribute to decomposing macromolecular organic to
small molecule, easily utilized by microbes and helpful for V
(V) removal.
In addition, some enhanced species in batch reactors could also con-
tribute to V(V) reduction. For instance, enriched Thauera was reported
to bio-reduce V(V) to V(IV) (Zhang et al., 2019). It was reported that
Thauera selenatis could obtain energy by respiring anaerobically with
selenate as the terminal electron acceptor (Schröder et al., 1997). The
biosorption of Fe(III), Cr(VI), Pb(II), Cu(II) and Ni(II) ions on Zoogloea
ramigera and Rhizopus arrhizus was studied as a function of initial
metal ion concentration and temperature (Saǧ and Kutsal, 2000). More-
over, some strains belonged to genus Novosphingobium could grow at
500 mg Cd/L, 1000 mg Zn/L and 500 mg As/L (Pereira et al., 2015).
The similarities and differences of microbial communities for the 4
samples were visualized by hierarchical cluster analysis at genus level
(Fig. 4b). The community structures of GSS and GSMPS were more sim-
ilar, with large differences among NSS and the other three groups. The
co-occurrence patterns among genera were investigated using network
analysis as shown in Fig. 4c. Results showed that the genera of BSV13,
Bacteroidetes vadinHA17 norank, Anaerolineaceae norank, Chloroflexi
1–2 norank, Xanthomonadales Incertae Sedis norank, Thauera,
Fig. 5. V(V) (a) and DOC/DIC concentrations (b) in the effluents of column bioreactors. (BC: bioreactor without inoculation; BS: bioreactor inoculated with sludge; BP: bioreactor
inoculated with domesticated soil samples from V ore mining area (petroleum associated minerals); BU: bioreactor inoculated with domesticated farmland soil samples near V ore
mining area (uvanite)).
8
Science of the Total Environment 751 (2021) 142161
Denitratisoma, BRC1 norank and Hydrogenophaga were the important
nodes.
The Cluster of Orthologous Groups (COG) microbial functional fea-
tures were examined for the samples to explore the mechanisms of V
(V) reduction with sawdust degradation. As shown in Fig. 4d, the pre-
diction results revealed that among the 25 COG categories, some gene
abundance were higher of NSS, GSS and GSMPS than that of Sludge,
such as Categories G (Carbohydrate transport and metabolism), D
(Cell cycle control, cell division, chromosome partitioning), and P (Inor-
ganic ion transport and metabolism). The results could suggest that the
addition of sawdust strengthened the microbial activities and functions,
with a positive impact on V(V) bio-reduction.
3.4. Column reactors repairing V(V) contaminated actual groundwater
3.4.1. V(V) removal performance
The hydraulic retention time (HRT) was set as 1 d for the formal col-
umn operation, with flow rate of around 84.4 μL/min. The V(V) profiles
in the effluents of BC, BS, BP and BU were shown in the Fig. 5a. The efflu-
ent V(V) concentration of BC (without inoculation) during 0–5 d grad-
ually increased to 60.1 mg/L. From 6 d to 26 d, V(V) in the effluents
stabilized in the range of 60.0–66.0 mg/L, with relevant V(V) removal
efficiency of 15.5% on 26 d. The effluent V(V) concentration of BS
L. Hao, Y. Liu, N. Chen et al.
(inoculated with domesticated sludge) continued increasing slowly
during 0–11 d. Correspondingly, the V(V) removal efficiency also de-
creased slowly, from 91.5% to 51.1%. After that, V(V) in the effluents
gradually flattened and showed a slight downward trend, and its re-
moval efficiency showed a steady and slowly increasing trend accord-
ingly. On 26 d, V(V) concentration in outlet was 30.9 mg/L, with
removal efficiency of around 58.7%, and the corresponding V
(V) average removal rate of about 44.1 mg V(V)/(L·d). Results showed
that sawdust could efficiently remove V(V) from contaminated ground-
water with sludge as inocula in the column bioreactor. While, more suit-
able operating conditions with better removal ability need to be
explored in future studies.
The domesticated samples of soil from V ore mining area (petroleum
associated minerals) and farmland soil near V ore mining area (uvanite)
were inoculated in BP and BU, separately. In the first 5 d of the formal
operation, almost no V(V) in the effluents were detected, indicating
that the inoculated microbes quickly adapted to both systems. The V
(V) concentration in the outlet began to slightly rise from the 6 d, al-
though both of their V(V) removal efficiencies still exceeded 99.2%. Sub-
sequently, removal effect in BP and BU gradually deteriorated and the V
(V) concentrations in the effluents did not stabilize until 20 d. On 26 d,
the V(V) removal efficiencies of effluents from BP and BU reactors were
54.8% and 38.4%, with the average removal rates of around 41.1 and
28.8 mg V(V)/(L·d), respectively. Overall, BS, BP and BU with sawdust
could all remediate V(V) contaminated groundwater. The indigenous
microbes of domesticated soils from petroleum associated minerals
ore mining area and farmland near the uvanite ore mining area could
quickly exert sustained V(V) remediation abilities in BP and BU. While
the bioreactor (BS) with domesticated anaerobic sludge as inocula
could remove V(V) in real groundwater more effectively for a longer
time.
3.4.2. DOC/DIC changes
DOC was also a key parameter in wastewater treatment and the ad-
dition of organics was effective for microbial V(V) reduction enhance-
ment (Zhang et al., 2015a). DOC concentration of influent (real
groundwater) was below the detection limit. Nevertheless, after the cir-
culating biofilm acclimatization period (40 d), DOC in the outlets (BC,
BS, BP and BU) were 800.2, 76.6, 108.9 and 81.4 mg/L, with DIC of
44.2, 79.8, 47.5 and 43.8 mg/L. The variation of DOC and DIC in the efflu-
ents of bioreactors were shown in the Fig. 5b. During the formal opera-
tion, the highest DOC in outlet of BC was found on 1 d (around
120.6 mg/L), possibly related to the residual DOC of the internal circula-
tion. From 1 d, the DOC tended to be stable after declining, with about
6.0 mg/L (26 d) detected.
In BS, BP and BU, DOC continued declining and fluctuating over time.
After 6 d, the DOC concentration in the effluents remained basically sta-
ble. Furthermore, the DOC (from 6 d to 26 d) decreased slowly, which
almost associated with the V(V) variation in the bioreactors. The outlet
DOC concentrations in the BS, BP and BU were finally maintained ap-
proximately at 5.8–11.4 mg/L (26 d). In the initial stage of the formal op-
eration, easily soluble or utilized carbon released by sawdust remained
in the column bioreactors (Jin et al., 2019), which could facilitate the mi-
crobial growth, with better V(V) removal performance observed. How-
ever, with gradually consumption of easy utilized organic above,
degradation-resistant parts gradually became the main using form,
with V(V) removal observed slightly worse than before accordingly. Ex-
cept for the carbon limitation above, a short HRT (1 d) might be another
related reason, which might lead to some loss of organics and microbes.
With insufficient contact time, DOC was also utilized and decomposed
deficiently. Results reflected that the DOC release capability was vital
in achieving sustainable bioremediation of V(V) contaminated ground-
water with sawdust, which could be enhanced in future studies.
The organic carbon in bioreactors originated from sawdust, which
was eventually converted to inorganic carbon in the systems. Obviously,
the DIC concentrations in the effluents of BS, BP and BU were higher
9
Science of the Total Environment 751 (2021) 142161
than that of BC. The experimental influent was actual groundwater, con-
taining a certain amount of DIC (about 50.6 mg/L). It was inferred that
the remaining DIC in the bioreactors were generated by microbial respi-
ration. Microbial activity could be inferred by the TIC produced rate
(Zhao et al., 2018). The DIC in the effluents of BS and BP were signifi-
cantly higher than that of BU and BC, consistent with the better V
(V) removal in BS and BP.
In summary, microbial removal of V(V) could be achieved by saw-
dust used as a sole carbon source. Furthermore, microbially removing
V(V) was also efficient with sawdust during long-term operation with
domesticated sludge or indigenous microbes from soils, emphasizing
necessities of this study. To our best knowledge, this potential way to
microbially remove V(V) for contaminated groundwater and simulta-
neously dispose of agricultural and forestry wastes has been reported
for the first time. These findings should have vital implications for the
bioremediation of actual V(V) contaminated groundwater.
4. Conclusions
Sawdust of pine as the solid carbon source could efficiently support
the microbial V(V) removal and its removal efficiency reached up to
90.3% in nutrient solution. In actual groundwater, medical stone and
phosphate rock were helpful for bioremediation enhancement of V
(V) contaminated groundwater, with the V(V) removal efficiency
from 53.2% up to 82.6% obtained. Microbiological analysis indicated
that V(V) reduction was mediated through synergistic interactions of
microbes. Sawdust with domesticated sludge or indigenous microbes
from soils as inocula supported effective V(V) removal in column biore-
actors. This study has important implications for developing new pro-
cesses for remediation of V(V) contaminated groundwater.
CRediT authorship contribution statement
Liting Hao: Conceptualization, Methodology, Formal analysis, Inves-
tigation, Data curation, Writing - original draft, Writing - review &
editing. Yongjie Liu: Investigation, Validation, Formal analysis, Writing
- review & editing. Nan Chen: Resources, Funding acquisition. Xiaodi
Hao: Writing - review & editing. Baogang Zhang: Resources.
Chuanping Feng: Conceptualization, Resources, Writing - review &
editing, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors acknowledge financial support from the Major Science
and Technology Program for Water Pollution Control and Treatment
(No. 2017ZX07202002) and the Fundamental Research Funds for the
Central Universities (No. 2652019284).
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