1 s2.0 S0304389420324201 Main

Journal of Hazardous Materials 408 (2021) 124430
Contents lists available at ScienceDirect
Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat
Research paper
Response of carbon and microbial properties to risk elements pollution in

arctic soils
Xiaowen Ji a, b, c, Evgeny Abakumov b, Svetlana Chigray b, Sheker Saparova b,
Vyacheslav Polyakov b, d, e, Wenjuan Wang b, Daishe Wu a, Chunlan Li f, g, Yu Huang h,
Xianchuan Xie a, i, *
a
Key Laboratory of Poyang Lake Environment and Resource Utilization, Ministry of Education, School of Resources Environmental & Chemical Engineering, Nanchang
University, Nanchang 330031, PR China
b
Department of Applied Ecology, Saint Petersburg State University, Saint Petersburg 199178, Russian Federation
c
School of Environment and Sustainability, University of Saskatchewan, Saskatoon SK, S7N 5B3, Canada
d
Arctic and Antarctic Research Institute, Saint Petersburg, 199397, Russian Federation
e
Department of Soil Science and Agrochemistry, Saint-Petersburg State Agrarian University, Pushkin, Saint Petersburg 19660, Russian Federation
f
Institute for Global Innovation and Development, East China Normal University, Shanghai 200062, PR China
g
School of Urban and Regional Sciences, East China Normal University, Shanghai 200241, PR China
h
Center for Eco-Environment Research, Nanjing Hydraulic Research Institute, Nanjing 210098, PR China
i
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, PR China
A R T I C L E I N F O A B S T R A C T
Editor: Dr. R. Debora A 180-day incubation study was conducted to evaluate the effects of risk elements (REs) on organic carbon use
and microbial activities in organic soils in the Arctic during the summer snowmelt period. Soils were artificially
Keywords: spiked with Cd, Pb, Cr, Ni, Cu, As, and a combination of these REs according to the levels measured in Arctic soils
Arctic soil from REs-polluted industrial sites. During the incubation period, microbial activities and microbial biomass
Microbial biomass carbon
carbon (MBC) formation were inhibited, and microbial quotient (qCO2) values were relatively high in the spiked
Microbial community
soils indicating that more energy was used by microbes for maintenance under REs stress. Meanwhile, microbial
Microbial carbon decomposition
Risk elements metabolism was significantly restrained. Microbial Specific phospholipid fatty acids (PLFAs) were reduced in RE
spiked soils relative to the control, especially in the As- and multi-RE-spiked soils. The abundance of both fungi
and bacteria was reduced in response to RE amendments by 14–24% and 1–55%, respectively. PLFA biomarkers
indicated a shift in soil microbial community structure and activities influenced by REs, consequently having a
negative effect on soil organic carbon degradation. This study addresses the knowledge gap regarding the
alternation of biochemical reactions in Arctic soils under anthropogenic REs with relevant contamination levels.
1. Introduction communities (Tipayno et al., 2018; Azarbad et al., 2013; Beattie et al.,
2018; Xu et al., 2019); some studies have used full-length 16S rRNA gene
Microorganisms play important roles in biogeochemical cycles in sequences as an indicative of general microbial community (Hur and
healthy ecosystems (Madsen, 2011). Microbial processing of soil is Park, 2019; Sobolev and Begonia, 2008).
susceptible to changes in response to environmental conditions or In the Arctic, microorganisms are vital for biogeochemical cycling,
available substrates through alterations in the microbial composition despite low temperatures, resulting in low activity and slow growth
and relative abundance for metabolism (Kallenbach et al., 2016; Gmach (Neufeld and Mohn, 2005). During the summer months, the annually
et al., 2020). Risk elements (REs) contamination, such as enhanced thawed active layer results in a great increase in microbial activity and
levels of heavy metals and metalloids in soils, can alter microbial com nutrient cycling (Malard and Pearce, 2018). Arctic soils store large
munity structure by reducing diversity and biomass. These influences quantities of organic carbon (C), over twice the amount stored in the
have been previously reported for aerobic and anaerobic soil microbial atmosphere (Loveland, 2011; Hugelius et al., 2014). Active soil
* Corresponding author at: Key Laboratory of Poyang Lake Environment and Resource Utilization, Ministry of Education, School of Resources Environmental &
Chemical Engineering, Nanchang University, Nanchang 330031, PR China.
E-mail addresses: [email protected], [email protected] (X. Xie).
https://doi.org/10.1016/j.jhazmat.2020.124430
Received 5 August 2020; Received in revised form 27 October 2020; Accepted 28 October 2020
Available online 31 October 2020
0304-3894/© 2020 Elsevier B.V. All rights reserved.
X. Ji et al. Journal of Hazardous Materials 408 (2021) 124430
microorganisms are an essential driving force in the regulation of C and 2. Materials and methods
nutrient cycling (Mooshammer et al., 2014; Buchkowski et al., 2019).
However, increasing industrial activities in the Arctic, such as metal 2.1. Soil sampling, spiking levels, and analysis
lurgy and mining, have led to different soil contamination levels in
recent years. Our previous investigations and other studies have docu The sampling sites for determining spiked-REs levels in the incuba
mented high heavy-metal pollution levels in soils near industrial areas tion experiment were selected near areas of industrial activity in the
compared to background areas or government-permissible concentra Russian subarctic regions in the north of Western Siberia, including
tions in Arctic regions (Ji et al., 2019a, 2019b; Abakumov et al., 2015, seven settlements: Aksarka (A), Salekhard (S), Labytnangi (L), Kharsaim
2017; Launer et al., 1993; Ryaboshapko et al., 1998; Walker et al., (K), Nadym (N), Novy Urengoy (NU), and Gaz-Sale (GS). Numerous
2003). The soil micro-population is highly sensitive to alterations in industries have been established in these settlements since the 1970s,
environmental conditions stressing the utilization of microbial C asso such as metallurgical and mining factories (Ji et al., 2019b; Moskov
ciated with changes in soil parameters (Frey et al., 2001). Certain ele chenko et al., 2017). During REs investigation in topsoil in these set
ments are essential as micronutrients for the survival and growth of soil tlements, concentrations of six REs (Pb, Cr, Cu, Cd, Ni and As) were
microbes (Alloway, 2013), while excess REs in soils may adversely affect
microbial activities and community composition (Zhang et al., 2016;
Shentu et al., 2014; Xu et al., 2012; Lenart-Boroń and Wolny-Koładka, Table 1
2015). Microbial activity changes both physical and biochemical prop Concentrations (mean ± standard deviation) of risk elements (Res; mg kg− 1) in
erties of soils that affect the bioavailability of REs and the affinity of soils from polluted areas in Western Siberia (n = 3) and selected soil materials (n
= 3) from background areas for the incubation experiment, and spiked dose of
ligand ions for elemental cations (Seshadri et al., 2015; Abdu et al.,
individual RE for the incubation experiment.
2017). Despite previously reported interactions between REs and soil
physicochemical components, e.g., basal respiration, organic C, and clay Sampling Pb Cr Cu Cd Ni As
sites
minerals (Romero-Freire et al., 2016; Enya et al., 2020), little is known
regarding the effects of REs on microbial C changes and soil microbes in A 15.85 9.77 ± 15.45 0.33 ± 74.16 5.30 ±
± 0.57 0.56 ± 0.47 0.02 ± 0.26 0.02
Arctic soil during the annual permafrost thawing.
S 22.10 16.06 26.76 0.35 ± 75.12 5.54 ±
The identification of phospholipid fatty acids (PLFAs) serves as a ± 0.64 ± 1.14 ± 1.28 0.02 ± 0.46 0.04
nonselective approach to detect possible changes in soil microbial L 17.60 10.00 24.13 0.33 ± 82.89 6.45 ±
communities and has been applied to REs toxicity to microbes in soils ± 0.37 ± 0.27 ± 0.27 0.01 ± 0.13 0.03
(Xu et al., 2019; Frostegård et al., 1993). Additionally, different REs can K 19.35 10.44 23.80 0.32 ± 78.62 7.42 ±
± 0.34 ± 0.46 ± 0.15 0.02 ± 0.33 0.04
have different effects on the processes of soil organic C (Enya et al.,
N 18.44 10.33 23.70 1.11 ± 93.04 9.21 ±
2020). The effects of REs contamination on soil microorganisms can be ± 0.10 ± 0.37 ± 0.26 0.02 ± 0.25 0.02
highly variable, which may cause uncertainty in the use of respirometry NU 22.66 23.67 18.41 0.71 ± 84.28 10.42
responses as a reliable indicator to assess organic matter degradation. ± 0.17 ± 2.66 ± 0.29 0.02 ± 0.17 ± 0.06
GS 22.83 10.87 19.74 0.33 ± 94.17 6.32 ±
The microbial metabolic quotient (qCO2), defined as microbial respira
± 0.15 ± 0.87 ± 0.36 0.02 ± 0.14 0.04
tion per unit of biomass, can be applied to assess the effect of changes in NP 0.22 ± 1.20 ± 1.15 ± 0.03 ± 0.27 ± 0.13 ±
environmental conditions on soil microbial biomass (Anderson and 0.02 0.07 0.15 0.002 0.02 0.01
Domsch, 1993). Soil microbial functional groups are intricately involved GBC a
11.50 4.67 10.76 0.13 51.84 2.00
in soil biochemical reactions. Previous studies have used the contami Spiked dose added to soils for incubation experiment
Low dose 16 10 15 0.3 74 5
nation REs levels at government-permissible concentrations (Romero-
High dose 23 24 27 1.0 93 10
Freire et al., 2016; Enya et al., 2020). However, these permissible con Final metal concentrations measured in spiked soils
centrations are not appropriate for boreal / Arctic soils due to high Pb-spiked 14.60 – – – – –
active layer dynamics influenced by permafrost and heterogeneous soil low ± 1.02
Pb-spiked 21.59
properties (Beer, 2016; Yi et al., 2018). Therefore, the relationship be – – – – –
high ± 2.16
tween RE contamination levels from polluted sites and soil biochemical Cr-spiked – 10.30 – – – –
reactions in Arctic soils remains to be identified. low ± 0.72
Comparing to soils in temperate zone, Arctic soils store the one of the Cr-spiked – 23.69 – – – –
largest terrestrial C due to low rate of organic matter decomposition high ± 1.90
Cu-spiked 15.34
(Loya and Grogan, 2004; Marion and Oechel, 1993). Decomposition of
– – – – –
low ± 1.53
organic matter in the Arctic soils is restricted by low temperature, Cu-spiked – – 25.90 – – –
nutrient limitation of microbial heterotrophic activity, and anaerobic high ± 2.07
conditions as poor drainage linked to underlying permafrost. Although Cd-spiked – – – 0.30 ± – –
low 0.02
harsh environmental conditions, soil microbial communities (response
Cd-spiked – – – 0.96 ± – –
to soil organic matter decomposition) in Arctic are as diverse as those high 0.08
observed in other biomes (Chu et al., 2010). Based on this, we hypoth Ni-spiked – – – – 72.04 –
esize that soil microbial diversity and activity are likely to be influenced low ± 6.48
by REs which indirectly influenced dynamics of soil microbial C during Ni-spiked – – – – 87.67 –
high ± 7.01
annual permafrost thawing when the microbial activity is the most
As-spiked – – – – – 4.98 ±
active. The purpose of this study is to explore how REs pollutions in low 0.50
fluence soil microbial C usage and microbial activities during snow As-spiked – – – – – 9.52 ±
melting through a long-term (180 days) incubation of soil from the high 0.95
Russian Arctic spiked lead (Pb), chromium (Cr), copper (Cu), cadmium Multi- 14.60 10.42 15.50 0.31 ± 68.33 4.62 ±
spiked ± 1.02 ± 0.73 ± 1.55 0.02 ± 6.83 0.42
(Cd), nickel (Ni), and arsenic (As), based on the relevant REs levels in low
polluted soils. Multi- 21.36 22.68 26.74 0.94 ± 86.74 9.12 ±
spiked ± 1.72 ± 2.27 ± 1.60 0.05 ± 4.34 0.55
high
a
Geochemical baseline values for surf ace soils (O/A horizon) of the Russian
Arctic (Ji et al., 2020).
2
observed to be significantly higher than the geochemical baseline we ray (EDX) analysis (JSM-6390LA, EX-2300, JEOL, Tokyo, Japan). EDX
previously established for the Russian Arctic (Table 1) (Ji et al., 2020). can qualitatively and quantitatively determine chemical components
One non-polluted (NP) site from the natural tundra terraces far from through analyzing characteristic radiations of solid materials without
anthropogenic activities (>50 km) was sampled (Fig. 1a). The concen digestion (Sitko et al., 2004; Fiamegos and de la Calle Guntiñas, 2018).
trations of the same six REs were much lower than the geochemical The emission by characteristic radiation of each element is based on all
baseline, and therefore, this soil was considered clean for the incubation forms of chemical bonds. The characteristic peak for every element is
experiment. accurate as atomic level precision and directly transferred as mg kg− 1
Full-profile pits were dug until the frozen bottom for diagnosis of the for elemental concentration. The dataset was generated using EDX AZF
soil horizon and soil classification. These soils were classified as Turbic quantification. All EDX analysis for each soil sample were conducted in
Histic Cryosols and Histic Stagnic Podzols according to the World triplicate. The final data presented here were obtained from the average
Reference Base for Soil Resources (WRB) (I. World Reference Base for of three different points from each sample. Soil certificated reference
Soil Resources (WRB), World Reference Base for Soil Resources, 2014). material SQC001 (Sigma-Aldrich, Saint Louis, MO, US) was used to
The diagnosed organic layers (O and Ah horizon above B horizon as check analytical accuracy. The analytes (six REs) in SQC001 was all at
shown in Fig. 1b, <10 cm together) were sampled with a scoop. The 40 mg kg− 1. The standard deviation of running three samples was
organic layer can be seen without cryogenic influences and has clear 1.70 mg kg− 1 for Pb, 1.53 mg kg− 1 for Cr, 2.70 mg kg− 1 for Cu,
humus accumulation with boundary with B horizon. The selection of this 1.72 mg kg− 1 for Cd, 0.81 mg kg− 1 for Ni and 1.50 mg kg− 1 for As.
layer considers neither strong natural geochemical weathering in Soil samples from NP site were individually spiked using solutions of
fluences nor cryoturbation. Oil sampling and site remediation was in soluble salts from the abundant RE species that exist in the soils, namely
accordance with previously described sampling protocols for Pb (II) [Pb(NO3)2], Cr (VI) [K2CrO4], Cu (II) [Cu(NO3)20.3 H2O], Cd (II)
permafrost-affected soils (Ping et al., 2013), while soil sampling was [Cd(NO3)2], Ni (II) [NiCl20.6 H2O], and As (V) [Na2HAsO4⋅H2O]. Seven
conducted during July 2018. The topsoil (<15 cm) was under water- groups of individually spiked soils were prepared: (1) Pb-spiked soils,
saturated status and the temperature of surface in daytime was 16 ◦ C (2) Cr-spiked soils, (3) Cu-spiked soils, (4) Cd-spiked soils, (5) Ni-spiked
and 30 ◦ C for minimum and maximum temperatures, respectively. The soils, (6) As-spiked soils, and (7) Multi-spiked soils (combination of six
moss (Calliergon giganteum) and sedges (Carex sp., Carex aquatilis, Poa REs). Each RE-spiked group of soil incubation were carried out in trip
arctica, and Salix sp.) were sampled by a knife from the soil pit after soil licate. For every artificially polluted soil group, two pollution doses, low
sampling. dose (LD) and high dose (HD), were set (Table 1), which were deter
All soil samples were transported to the laboratory of the Applied mined by the REs concentrations in field-polluted soils (L, S, K, A, N, NU
Ecology Department of St. Petersburg State University for processing. and GS). The highest concentration of each RE was labeled as the HD,
Soil and vegetation (moss and sedge) samples were kept in polyethylene and the lowest concentration was selected as the LD. The final measured
bags and stored in refrigerator at 4 ◦ C prior to experiment. Fine roots concentrations of REs in each spiked group are shown in Table 1 with the
and debris were removed from the samples, which were then air-dried at recovery rate of 90–95%. 500 mL glass beakers were used to load the
room temperature (~23 ◦ C) until the weight was stable and milled using soils. In order to obtain homogenization, soils were mixed and stirred by
an electric soil grinder to manually pass through a 2-mm sieve. A soil: a rolling shaker for 12 h. Approximately 50 g fully mixed with 10 g
water (1:5) extract was prepared to determine redox potential (Eh) and clipped mosses/sedges were added with the proper amount of the in
electrical conductivity (EC), and a soil: 1 M KCl (1:2.5) extract was dividual chemical reagent to reach the set spiked levels. These sedges
prepared to determine pH. Eh, EC, and pH were measured using a and mosses originated from the sampling sites. The purpose to add these
HI9813–6 portable pH/EC meter (Hanna Instruments, Smithfield, RI, sedges and mosses is to simulate bigger sedges/mosses tissues in this soil
US). Soil organic C (%) and nitrogen content (%) was determined using organic layer as part of carbon sources. This incubation was conducted
LECO Tru-SPEC elemental analyzer (Leco Corp., St. Joseph, MI, US). Soil for 180 days from May 1 to November 27, 2019 at a constant temper
particle size distribution were determined using ASTM hydrometers ature of 25 ◦ C. To simulate the water-saturation status found in soils
(Fisher Scientific, Pittsburgh, PA, US) (Gee and Bauder, 1979). The saturated with smelting water, the water layer was 1 cm higher than the
cation-exchange capacity was determined by the ammonium acetate soils. Water layer level was maintained by an assembled horizontal line
extraction method at pH 7 (Sumner and Miller, 1996). The major detector controlled by an Arduino (micro controller) (Fig. S1). When the
characteristics of the sampled soils are presented in Table 2. water level was lower than the position that we set for horizontal line
REs were determined in field-sampled soils with energy dispersive x- detector, Arduino would turn on the value connected to the water tap,
Fig. 1. Map of sampling sites in Yamal-Nets autonomous area (a). A, Aksarka; S, Salekhard; L, Labytnangi; K, Kharsaim; N, Nadym; NU, Novy Urengoy; GS, Gaz-Sale;
NP, non-polluted site. (b) Soil profile of non-polluted sampling site.
3
Table 2
Physicochemical properties (mean ± standard deviation) of soils sampled (n = 3) from polluted areas in west Siberia and selected soil materials (n = 3) from back
ground areas for the incubation experiment.
Sampling sites A S L K N NU GS B
Depth (cm) 0–6 0–9 0–16 0–16 0–16 0–10 0–7 0–12
pH-KCl 4.2 ± 0.6 4.6 ± 0.7 5.7 ± 0.7 5.7 ± 0.7 5.8 ± 0.9 5.7 ± 0.9 5.5 ± 0.6 5.5 ± 0.7
SOC (%) 5.47 ± 0.6 26.62 ± 3.5 5.23 ± 0.6 6.23 ± 0.7 5.16 ± 0.7 4.09 ± 0.6 4.16 ± 0.5 6.03 ± 0.8
TN (%) 0.22 ± 0.03 1.18 ± 0.17 0.02 ± 0.003 0.02 ± 0.002 0.02 ± 0.002 0.02 ± 0.003 0.03 ± 0.004 0.28 ± 0.03
Clay (%) 8.9 ± 0.9 0.15 ± 0.02 20.0 ± 2.1 10.0 ± 1.6 19.9 ± 2.7 10.0 ± 1.2 14.9 ± 1.6 10.2 ± 1.4
Silt (%) 8.9 ± 1.0 29.6 ± 3.1 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 14.6 ± 1.7 8.2 ± 0.9 3.2 ± 0.4
Sand (%) 82.1 ± 9.7 70.3 ± 8.9 79.6 ± 10.5 89.6 ± 13.6 79.7 ± 10.4 75.4 ± 9.5 76.9 ± 11.4 86.6 ± 10.3
Eh (mV)a − 24.7 ± 3.8 − 21.6 ± 2.7 − 28.8 ± 3.6 − 26.9 ± 3.6 − 25.0 ± 3.2 − 21.4 ± 2.9 − 24.0 ± 3.1 − 24.3 ± 3.3
EC (mS/cm) 2.4 ± 0.3 2.3 ± 0.3 3.7 ± 0.4 4.1 ± 0.6 3.1 ± 0.4 2.0 ± 0.2 4.4 ± 0.5 3.1 ± 0.5
CEC (cmol + kg− 1) 22.3 ± 2.6 21.1 ± 2.7 20.9 ± 2.4 20.9 ± 2.5 23.7 ± 3.6 23.2 ± 3.2 21.1 ± 2.8 21.6 ± 2.2
CEC, cation-exchange capacity; SOC, soil organic carbon; TN, total nitrogen.
A, Aksarka; S, Salekhard; L, Labytnangi; K, Kharsaim; N, Nadym; NU, Novy Urengoy; GS, Gaz-Sale; B, background site for incubation experiment.
a
The reference electrode: 245 mV.
allowing water to enter the breaker thereby maintaining constant levels. fumigated by chloroform) to MBC (Wu et al., 1990).
During all incubation, the breakers were open. Soil PLFAs were used to evaluate the microbial community of all
tested soils. The PLFA extraction procedure followed the method de
2.2. Potentially bioavailable REs and analysis scried by Frostegård et al., (1991) with minor modifications for humus/
organic sandy-loam soils. Briefly, total lipids were extracted from 1 g
A previous study concluded that the diffusive gradients in thin-films moist soil by a one-phase mixture (1:2:0.8 v/v/v) of chloroform,
(DGT) technique is the most applicable method for measuring metal methanol, and citrate buffer (0.15 M, pH 4.0). Then the extracts were
bioavailability in sandy-clay-organic soils, similar to our soils (Soriano- split into two phases by adding chloroform and buffer, and the phase
Disla et al., 2010). Commercial DGT devices were purchased from DGT containing lipid was dried by a mild stream of nitrogen and stored at
Research Ltd. (Lancaster, UK). The assembled DGT device had a diffu − 15 ◦ C. Polar lipids were fractioned from neutral and glycolipids by
sive gel layer with thickness of 0.78 and exposure window of 2.54 cm2. solid phase extraction cartridges (normal phase silica, 40–75 µm, 70 Å,
Three types of resin gel, NMDG for Cd (VI), Chelex for Pb, Cr, Cu, and Ni, 100 mg/3 mL, Hawach Scientific, Xi’an, China). Polar lipids were con
and Zr-oxide for As, were separately contained in individual DGT de verted to fatty acid methyl esters by following a mild alkaline meth
vices. The devices were stored in a well-sealed Minigrip bag containing a anolysis (Dowling et al., 1986), and the resulting fatty acid methyl esters
small amount of 0.1 M NaCl solution at 4 ◦ C prior to deployment. DGT were separated using a gas chromatograph coupled to a flame ionization
devices for measuring bioavailable RE concentration were deployed on detector (Agilent 7890, Santa Clara, CA, US). Hydrogen was used as a
days 5 and 180. The DGT devices were gently pressed into soil slurry to carrier gas and a spitless mode was used for injections. The temperature
maintain physical contact between the diffusive gel and soil for 24 h. setting of the gas chromatograph followed that of Frostegård, et al.
The binding gel was removed from the device and eluted with 1 mL of Frostegård et al., (1993). PLFA peaks were identified using MIDI peak
1 M HNO3, and the solutions were centrifuged at 1008×g for 15 min at identification (Sherlock microbial identification system, MIDI Inc.
room temperature (~23 ◦ C). Finally, the solutions were filtered through Newark, DE, US). The nonanoic methyl ester 9:0 and nonadecanoic
a 0.45 µm membrane filter and analyzed using ICP− MS (NexION® methyl ester 19:0 were added to each sample as internal standards. The
2000, PerkinElmer, Waltham, MA, US). DGT-derived REs concentrations abundance of different microbial groups was quantified as nmol signa
were calculated as described by Zhang et al., (2001). ture PLFA g − 1 soil. According to previously reported PLFA biomarker
data, PLFA biomarkers were considered for Gram-positive (G+) bacte
2.3. Microbial parameters ria: 14:0iso, 15:0anteiso, 150iso, 16:0iso, 17:0anteiao, and 17:0iso.
Gram-negative (G− ) bacteria were 16:1ω7c, 16:1ω9c, 17:0cyclo,
On the days 1, 7, 30, 40, 60, 100, 135, and 180 during incubation, 17:1ω8c, 19:0cyclo, and 18:1ω7c (Frostegård and Bååth, 1996). The
soil was determined using a µTrac 4200 analyzer (SY-LAB, Madrid, biomarkers for actinomycete were 10Me18:0, 10Me17:0, and 10Me16:0
Spain). Briefly, approximately 5 g soil was mixed with 50 mg talc: (Zelles, 1997). The biomarkers to represent fungal PLFAs were 18:2ω6
glucose at a ratio of 10:1 and moved into a PTFE Teflon tube that was and 18:1ω9c (Bååth and Anderson, 2003). Total PLFAs were determined
introduced into a measuring cell with 0.2% KOH. The equipment was as the sum of all PLFAs.
operated at a constant temperature of 30 ºC for 36 h and the evolved
CO2 was monitored every 10 min by measuring the decrease in solution 2.4. Statistical analysis
impedance.
Soil microbial biomass C (MBC) was determined following the After verifying the normal distribution of data using the
fumigation–extraction method (Vance et al., 1987). At the beginning Kolmogorov-Smirnov test, significance between the control soils and
and end of the experiment, approximately 8 g of well-mixed moist soil different RE-contaminated soils was determined using one-way ANOVA.
was extracted with 32 mL of 0.05 M K2SO4 and shaken at 4×g for 2 h. Multiple comparisons among different RE treatments were tested using a
Another 8 g of moist soil was first fumigated with alcohol-free chloro post hoc Duncan Multiple Range test. The least significant difference test
form in a dark chamber under vacuum for 24 h and extracted in the same was used to test for significant differences (p < 0.05). Spearman corre
way. Extracts were filtered through filter paper (Grade 40, Whatman, lation coefficient was used to determine correlation among soil and
Maidstone, UK). The extracts were then analyzed for total C concen microbial properties. Principal components analysis (PCA) was applied
tration using a TOC/TIC analyzer (Multi N/C 2100S, Analytik Jena AG, for microbial PLFA values to interpret variation and covariation in the
Jena, Germany). Total MBC concentration was reported as the difference PLFA biomarkers and microbial groups using factor analysis of varimax
between extracted C from fumigated and non-fumigated soils, using a rotation. All statistical analyses were run in IBM SPSS 25 version (Chi
correction factor of 0.45 converting chloroform C (C from soils cago, IL, US).
4
3. Results from 418 µg kg− 1 to 543 µg kg− 1 from 30 to 180 days for the HD, while
the difference in As was only 9 µg kg− 1 for the LD. For the increasing
3.1. Soil parameters and REs pattern of other REs, the difference in Cu and Pb for the LD was
32 µg kg− 1 and 62 µg kg− 1, respectively, while the difference in Cu, Pb,
3.1.1. Changes in total organic C, and nitrogen and Ni for the HD was 66 µg kg− 1, 6 µg kg− 1, and 63 µg kg− 1,
Soil organic C (SOC) and soil nitrogen (SN) were determined after respectively.
180 days of incubation as shown in Table 3. The majority of SOC and SN
in different REs treatments ranged similarly to those in the control.
There was no significant difference (p > 0.05) between the control and 3.2. Influence of REs on microbial C utilization
REs treatments in SOC (except for LD of Pb and Ni spiked soils) and SN
(except for HD of multi-REs-spiked soils). The LD of Ni-spiked soils had 3.2.1. Influence of REs on soil respiration
the lowest SOC (6%) and C/N (20). However, the largest SN was The soil microbial respiration rate and cumulative evolved CO2 in
observed in the HD of multi-RE-spiked soils (SN = 0.28%). The C/N the soil were determined to understand the effects of REs on soil mi
values in all treatments were significantly different (p < 0.05) among crobial activity (Fig. 3). Soil respiration rates reached the maximum rate
the control and treatments except for between the control and the LD of in all treatments on the first day of incubation and gradually decreased
As-spiked soils. Significant differences in the ratio of MBC to SOC (MBC/ with incubation time (Fig. 3a). The control soil had significantly higher
SOC) were observed between the control and all treatments except for respiration rates than the soil in other treatments over the whole incu
LD and HD of As-spiked and multi-REs-spiked soils. The lowest MBC/ bation period. The order of respiration rate (µg CO2-C day− 1 g− 1 soil)
SOC was 0.04 in the LD of multi-RE-spiked soils, and the largest MBC/ under REs-spiked soils was: multi-HD (5.8) < multi-LD (7.8) < As-HD
SOC was in the control (0.49). (8.1) < Cu-HD (11.3) < As-LD (13.7) < Cu-LD (14.1) < Pb-HD (17.62)
< Cr-HD (18.0) < Cd-HD (19.0) < Ni-HD (20.1) < Cd-LD (21.1) < Ni-LD
3.1.2. Potentially bioavailable concentrations of REs (21.5) < Cr-LD (21.9) < Pb-LD (22.2). After 180 days of incubation, the
For single RE treatment, the bioavailable concentrations (extracted total released CO2-C differed under different RE treatments (Fig. 3b).
by DGT devices) of Pb-, Cr-, and Ni-spiked soils did not change at 30 and The largest total respired CO2− C was observed in uncontaminated soil
180 d of incubation in both the LD and HD amendments (Fig. 2). A 33% (570 µg CO2− C g− 1 soil). For the LD, compared with the control, the
and 40% reduction was observed in Cd-spiked soils for the LD and HD, cumulative respiration declined by 24%, 26%, 33%, 61%, 68%, 79%,
respectively. At 180 days of incubation, the bioavailable Cr concentra and 83% in Cd-, Pb-, Cr-, Ni-, Cu-, As, and multi-RE-spiked soils,
tion slightly increased from 151 µg kg− 1 to 158 µg kg− 1 for the LD, and a respectively. For the HD, the decrease was by 29%, 32%, 40%, 72%,
similarly slight increase was observed in As for the LD (from 91 µg kg− 1 82%, 89%, and 94% in Cd, Pb, Cr, Ni, Cu, As, and multi-RE-spiked soils
to 98 µg kg− 1), whereas there was an increase in As for the HD from compared with the control. The lowest cumulative soil respiration of
291 µg kg− 1 to 376 µg kg− 1. Bioconcentrations of Cu increased by 9% CO2-C was observed for the HD of multi-RE amendments (37 µg CO2-C
and 12% for LD and HD at the end of incubation without significant g− 1 soil), followed by 61 µg CO2-C g− 1 soil for the HD of As amendment.
differences (p > 0.05).
Except for Cr and Cd in the multi-REs treatment, bioavailable con 3.2.2. Pattern of soil microbial biomass C
centrations of other REs (Pb, Cu, Ni, and As) increased during the in Soil MBC in the control increased from 324 to 563 mg kg− 1 soil from
cubation period (Fig. 2). The bioconcentration of Cr in the HD was 30 to 180 d of incubation, whereas the MBC in soils spiked with REs only
observed to slightly decrease from 30 days (521 µg kg− 1) to 180 days slightly increased compared with that in control soils (Fig. 4a), and was
(501 µg kg− 1). The bioavailable concentration of Cd showed a higher significantly suppressed by REs addition compared to control. MBC
decrease from 6 µg kg− 1 to 4 µg kg− 1 for LD, and from 7 µg kg− 1 to values in REs-spiked soils at 180 days were slightly higher than those at
5 µg kg− 1 for HD. Compared with single-RE treatment, the bioavailable 30 days of incubation were. After 30 d of incubation, the MBC values in
concentrations of multi-REs were higher. By days 30 and 180 in the Cd-spiked soils with the LD were 12% higher than those with the HD;
multi-REs amendments, the increase in available concentrations (LD and those in Pb-, Cr-, and Ni-spiked soils with the LD were 5% higher than
HD), compared with single-RE amendments, were by 42% for Pb, 46% those with the HD; those in Cu-spiked soils with the LD were 6% higher
and 44% for Cr, 50% and 43% for Cu, 41% and 49% for Cd, 31% and than those with the HD; those in As-spiked soils with the LD were 14%
36% for Ni, and 44% and 44% for As, respectively. Although the input higher than those with the HD; those in multi-RE-spiked soils with the
rate was similar, the significantly higher bioavailability of As increased LD were only 2% higher than those with the HD. The lowest MBC values
were found in multi-REs-spiked soils with the LD (58 mg kg− 1 soil) and
Table 3
Content of soil organic carbon (SOC), soil nitrogen (SN), and microbial biomass carbon (MBC) in different RE-spiked doses after 180 days of incubation. Results are
means ± standard deviation (n = 3).
RE Sample SOC (%) SN (%) C/N MBC/SOC
Control 8.56 ± 1.23a 0.32 ± 0.02a 27.03 ± 0.06a 0.49 ± 0.06a

Pb Low dose 6.92 ± 0.45a 0.34 ± 0.03a 20.35 ± 0.01b 0.19 ± 0.01a
High dose 8.74 ± 1.00a 0.35 ± 0.02a 24.96 ± 0.05b 0.18 ± 0.01a
Cr Low dose 9.44 ± 1.00a 0.31 ± 0.02a 30.45 ± 0.06b 0.16 ± 0.01a
High dose 9.26 ± 1.05a 0.33 ± 0.02a 28.06 ± 0.06b 0.13 ± 0.02a
Cu Low dose 8.22 ± 1.06a 0.33 ± 0.02a 24.91 ± 0.05b 0.15 ± 0.01a
High dose 8.49 ± 0.92a 0.36 ± 0.03a 23.58 ± 0.03b 0.14 ± 0.02a
Cd Low dose 9.48 ± 0.68a 0.31 ± 0.02a 30.58 ± 0.03b 0.14 ± 0.01a
High dose 11.24 ± 1.43a 0.30 ± 0.02a 37.47 ± 0.08b 0.12 ± 0.01a
Ni Low dose 6.31 ± 0.38b 0.32 ± 0.02a 19.72 ± 0.02b 0.14 ± 0.01a
High dose 8.36 ± 1.32a 0.31 ± 0.02a 26.97 ± 0.09b 0.13 ± 0.01a
As Low dose 8.33 ± ± 1.32a 0.34 ± 0.03a 24.50 ± 0.04b 0.08 ± 0.01b
High dose 9.32 ± 1.18a 0.36 ± 0.04a 25.19 ± 0.03b 0.07 ± 0.01b
Multi Low dose 8.36 ± 0.89a 0.37 ± 0.03a 22.59 ± 0.03b 0.06 ± 0.003b
High dose 8.12 ± 0.52a 0.28 ± 0.03a 29.00 ± 0.02b 0.04 ± 0.003b
Letters a and b in individual treatments indicate significant differences (one-way ANOVA, p < 0.05) between the control and different RE treatments.
5
Fig. 2. Bioavailable concentrations (extracted by diffusive gradients in thin-films devices) of REs under single-RE and multi-REs treatments with the low dose (LD)
and high dose (HD) amendments (n = 3). Data are shown as means with standard deviation.
HD (59 mg kg− 1 soil), followed by As-spiked soils with the HD under REs stress. There was no significant difference between the con
(74 mg kg− 1 soil). By the end of the incubation period, compared with trol soil (24 nmol g− 1 soil) and the spiked soils (25 and 24 nmol g− 1 soil,
the control, the order of decrease in MBC values in contaminated soils 24 and 26 nmol g− 1 soil, 28 and 28 nmol g− 1 soil, 24 and 21 nmol g− 1
was: Pb- (67% and 71% for LD and HD) < Cr- (69% and 71% for LD and soil, 24 and 22 nmol g− 1 soil, 23 and 21 nmol g− 1 soil, and 21 and
HD) < Cd- (66% and 68% for LD and HD) < Ni- (75% and 80% for LD 20 nmol g− 1 soil for the LD and HD, respectively, in Pb-, Cr-, Cu-, Cd-,
and HD) < Cu- (81% and 82% for LD and HD) < As- (84% and 86% for Ni-, Ni-, As-, and multi-REs-spiked soils, respectively) after 30 days of
LD and HD) < multi-REs-contaminated (89% and 90% for LD and HD) incubation (Fig. 5). The PLFA values in the control soil at the end of
soils. However, the MBC values in the control significantly increased incubation were significantly higher than those in the spiked soils were.
from 324 mg kg− 1 soil to 563 mg kg− 1 soil at the end of the incubation In comparison to the control soil after 180 days, PLFA values dropped by
period. Thus, microbial qCO2 was calculated for C use by microbes. 59% and 60%, 57% and 58%, 56% and 56%, 55% and 59%, 59% and
Compared with 30 days, qCO2 in the control and all treatments 60%, 60% and 61%, 62% and 62% for the LD and HD in Pb-, Cr-, Cu-, Cd-
decreased at the end of incubation (Fig. 4b). The largest qCO2 values , Ni-, Ni-, As-, and multi-REs-spiked soils, respectively. There was no
were found in the LD and HD of Ni-spiked soils, whereas the control soils significant difference among PLFA values in all treatment soils
had the lowest qCO2 values. Despite the largest values being shown with (p > 0.05). However, there was no significant correlation observed be
the LD and HD of Ni-spiked soils at 30 days, the values significantly tween SOC and different microbial functional groups (Table S1). MBC
declined after 180 days. was positively significantly correlated with G+ bacteria, PLFAs, and
fungi; G+ bacteria were also positively significantly correlated with
3.3. Microbial activity PLFAs.
3.3.1. Microbial PLFAs 3.3.2. Microbial group structure

Total microbial PLFAs were quantitatively determined for different The contents of the individual microbial functional groups are shown
microbial functional groups and for microbial community composition in Table 4. At the end of the incubation period, the highest biomarker
6
Fig. 3. Soil respiration (a) and cumulative evolved CO2− C from soils under
different RE amendments (b) during 180 days of incubation (n = 3). Control Fig. 4. Microbial biomass carbon (a) and microbial quotient (b) in different
represents background soils; HD and LD represent high and low doses of RE treatment soils (n = 3). Control represents background soils; HD and LD
amendment, respectively. Data are shown as means with standard deviation. represent high and low doses of RE amendment, respectively. Data are shown as
Different letters in individual treatments indicate significant differences means with standard deviation. Different letters in individual treatments indi
(p < 0.05) among the control and different treatments. cate significant differences (p < 0.05) among the control and
different treatments.
values were found in the control soil as G+ bacteria, 94 nmol g− 1 soil;

G− bacteria, 2 nmol g− 1 soil; actinomycetes, 124 nmol g− 1 soil; fungi, were distributed in G+/G− bacteria and actinomycetes (10Me18:0 and
4 nmol g− 1 soil. The lowest PLFA content after 180 d was found in the 10Me17:0) (Fig. 6a). The most evident discrepancy was revealed in G+
HD of multi-REs-spiked soils (G+ bacteria, 30 nmol g− 1 soil; actino biosignature 17:0anteiao, 14:0iso, and 150iso, G– biosignature 18:1ω7c,
mycetes, 41 nmol g− 1 soil) and in the LD for fungi with 0.4 nmol g− 1 and fungi biosignature 18:2ω6 and 18:1ω9c. These values were posi
soil. Actinomycetes in the control soils were 64% higher than those in tively positioned on PC1 and negatively on PC2 at 180 days of incuba
the HD of multi-REs-spiked soils. Microbial community population on tion, whereas they were positive on PC2 at 30 d. This result indicated
30 d in the control soils had the biggest value on either 30 days or 180 that REs negatively influenced microbial populations. However, the
days incubation. At 30 days, the microbial biomarker contents were 43, least shift in microbial populations was found in the G− biosignature.
0.6, 54, and 3 nmol g− 1 soil for G+ bacteria, G– bacteria, actinomycetes, Generally, the decrease in G+, actinomycetes, and fungi in RE-spiked
and fungi, respectively. The lowest concentrations of G+ and G– bac soils resulted in a shift in the microbial community.
teria (21 and 0.3 nmol g− 1 soil), actinomycetes (25 nmol g− 1 soil), and Compared with the PCA results at 30 d, different profiles of microbial
fungi (2 nmol g− 1 soil) were observed in the HD of multi-REs-spiked populations were observed at 180 d. After 180 d of incubation, PLFA
soils. biomarkers for G+ and G– bacteria (14:0iso, 15:0anteiso, 150iso,
G+/G– bacteria declined at the end of incubation compared with 16:0iso, 17:0anteiao, 17:0iso, 16:1ω7c, 16:1ω9c, 17:0cyclo, 17:1ω8c,
those at 30 d of incubation, whereas bacteria/fungi (B/F) ratios 19:0cyclo, and 18:1ω7c) and fungi (18:2ω6 and 18:1ω9c) were all pos
increased except for those in the control soils, LD in Cd-spiked soils, and itive on PC1, whereas biomarkers for G+ and G– bacteria except for
HD in multi-REs-spiked soils. Although all microbial functional groups 16:1ω7c and 18:1ω7c were negatively positioned on PC2. Biomarkers for
in the control soil were higher than those in REs-contaminated soils fungi were positive on PC2 and those for actinomycetes (10Me18:0,
were, the microbial population varied greatly in the ratio of G+/G– 10Me17:0, and 10Me16:0) were negative on PC1 (the most negative
bacteria and B/F. At 180 d of incubation, the highest G+/G– bacterial actinomycete was 10Me18:0), whereas the values were all positive on
ratio was found in the LD of RE-spiked soils (78), followed by the LD and PC2 (Fig. 6b). This result suggests that G+ and G− bacteria were
HD of As-spiked soils (73 and 73). The B/F ratio decreased in most REs- dominant in the soil microbial population rather than actinomycetes and
spiked soils compared with the control soils. fungi.
The plot of PCA score variation according to PLFA biomarkers in REs The plot of PCA score variation on total PLFA under contamination
treatments showed a significant distinction in different treatments. The with different REs after 180-day incubation showed one key factor in the
shift in the microbial community can be clearly seen in Fig. 6 and Table 4 identified overall dataset (96.23% of total variance) (Fig. 7). The
for bacteria, actinomycetes, and fungi. REs amendment apparently greatest proportion of the variance is explained by the significant PLFA
affected microbial community structure. The positive PC1 values at 30 d changes under RE-spiked soils compared to the control soils. The
7
Fig. 5. Microbial phospholipid fatty acid content (n = 42) under RE stress. Data are shown as means with standard deviation. Different letters in individual
treatments indicate significant differences (p < 0.05) among the control and different treatments. Control represents background soils; HD and LD represent high and
low doses of RE amendment, respectively. No significant differences (p > 0.05) were found among different REs treatments at 30 and 180 d.
Table 4
1
Specific phospholipid fatty acid (PLFA) content (nmol g− soil) as Gram-positive bacteria (G+), Gram-negative bacteria (G− ), actinomycetes, and fungi (mean ±
standard deviation, n = 3).
RE Treatment G+ G− Actinomycetes Fungi G+ /G− B/F
30 d
Control 42.96 ± 6.01a 0.56 ± 0.06a 54.32 ± 7.06a 3.28 ± 0.49a 76.72 ± 9.97b 13.27 ± 1.72a
Cd LD 40.22 ± 4.02ab 0.46 ± 0.06b 47.30 ± 4.73ab 2.49 ± 0.32b 87.44 ± 12.24ab 16.34 ± 1.63a
HD 41.91 ± 5.45ab 0.44 ± 0.06bc 46.88 ± 6.56ab 2.48 ± 0.32b 95.26 ± 12.38ab 17.08 ± 2.56a
Pb LD 42.42 ± 5.09a 0.44 ± 0.04bc 46.11 ± 5.53ab 2.42 ± 0.29bc 96.42 ± 13.50ab 17.71 ± 2.48a
HD 41.31 ± 6.20ab 0.42 ± 0.04bc 44.50 ± 6.23b 2.35 ± 0.26bc 98.36 ± 9.84ab 17.76 ± 2.31ab
Cr LD 40.91 ± 5.73ab 0.43 ± 0.05bc 42.85 ± 4.71b 2.27 ± 0.25bc 95.15 ± 14.27ab 18.21 ± 2.00ab
HD 42.80 ± 4.71a 0.42 ± 0.05bc 41.96 ± 4.20b 2.17 ± 0.30bc 101.90 ± 12.23a 19.92 ± 1.99ab
Ni LD 38.86 ± 5.83ab 0.41 ± 0.06bc 40.26 ± 4.43b 2.13 ± 0.21bc 94.79 ± 12.32ab 18.44 ± 2.21ab
HD 39.61 ± 4.75ab 0.38 ± 0.04bc 37.10 ± 5.19bc 2.11 ± 0.21bc 104.24 ± 11.47a 18.95 ± 2.08ab
Cu LD 34.34 ± 5.15ab 0.37 ± 0.04c 36.53 ± 5.11bc 1.98 ± 0.24bc 92.82 ± 13.92ab 17.53 ± 2.63ab
HD 32.71 ± 3.60b 0.36 ± 0.04c 35.35 ± 4.24c 1.97 ± 0.30bc 90.86 ± 11.81ab 16.79 ± 1.68ab
As LD 30.44 ± 4.57b 0.35 ± 0.05c 30.61 ± 4.29cd 1.92 ± 0.23c 86.96 ± 11.31ab 16.03 ± 1.76ab
HD 29.17 ± 4.38bc 0.34 ± 0.05c 30.52 ± 4.27cd 1.67 ± 0.20c 85.79 ± 9.44ab 17.67 ± 2.65ab
Multi LD 27.57 ± 2.76bc 0.31 ± 0.04c 25.94 ± 3.37d 1.66 ± 0.22c 88.94 ± 9.78ab 16.80 ± 2.52b
HD 21.05 ± 2.95c 0.30 ± 0.03c 24.45 ± 2.80d 1.54 ± 0.17c 70.16 ± 9.12b 13.86 ± 1.52b
180 d
Control 94.38 ± 10.38a 1.64 ± 0.20a 124.43 ± 16.18a 4.32 ± 0.52a 57.55 ± 5.75b 22.23 ± 3.11a
Cd LD 50.85 ± 5.08b 0.96 ± 0.12b 79.37 ± 8.73b 2.89 ± 0.43b 52.97 ± 7.94b 17.93 ± 2.69b
HD 47.51 ± 6.18bc 0.94 ± 0.12b 78.68 ± 11.80b 2.91 ± 0.35b 50.54 ± 6.57b 16.65 ± 2.33bc
Pb LD 46.15 ± 5.08bc 0.95 ± 0.14b 77.70 ± 10.10b 2.83 ± 0.42b 48.58 ± 6.80b 16.64 ± 1.66bc
HD 42.69 ± 5.12bc 0.89 ± 0.11b 75.73 ± 8.33b 2.76 ± 0.28bc 47.96 ± 7.19b 15.79 ± 2.37bc
Cr LD 41.00 ± 4.92c 0.86 ± 0.12b 73.95 ± 9.61bc 2.72 ± 0.35bc 47.67 ± 4.77b 15.39 ± 1.54bc
HD 40.39 ± 4.04c 0.83 ± 0.09b 72.66 ± 8.72b 2.59 ± 0.36bc 48.66 ± 7.30b 15.91 ± 1.59bc
Ni LD 38.26 ± 5.36cd 0.83 ± 0.08b 67.09 ± 8.72bc 2.59 ± 0.34bc 46.09 ± 5.07b 15.09 ± 1.96bc
HD 35.01 ± 3.50cd 0.79 ± 0.11b 64.87 ± 9.73bc 2.58 ± 0.36bc 44.31 ± 5.76b 13.87 ± 1.39bc
Cu LD 34.33 ± 5.15cd 0.59 ± 0.07c 62.08 ± 6.83bc 2.48 ± 0.25bc 58.19 ± 8.15b 14.08 ± 1.97bc
HD 33.53 ± 3.35cd 0.59 ± 0.08c 56.74 ± 7.38c 2.43 ± 0.29bc 56.83 ± 6.82b 14.04 ± 1.54bc
As LD 33.43 ± 4.01cd 0.46 ± 0.05c 46.82 ± 5.15c 2.34 ± 0.26bc 72.67 ± 7.99a 14.48 ± 2.03bc
HD 32.87 ± 3.62cd 0.45 ± 0.06c 46.48 ± 6.04c 2.12 ± 0.23c 73.04 ± 8.76a 15.72 ± 2.04bc
Multi LD 32.04 ± 4.81cd 0.41 ± 0.04c 45.91 ± 4.59c 2.14 ± 0.28c 78.15 ± 10.94a 15.17 ± 1.67bc
HD 30.04 ± 4.51d 0.42 ± 0.05c 40.55 ± 5.27c 2.03 ± 0.26c 71.52 ± 8.58a 15.00 ± 1.95c
HD and LD are high and lows doses of RE amendment, respectively. B/F is the ratio of bacteria to fungi values. Superscript letters in individual treatments indicate
significant differences (p < 0.05) among the control and treatments.
variance for control soils occupied only 1.53% and positioned on posi 4. Discussion
tive PC1 and PC2. The variance of PC1 can be divided into two groups.
The variances of Pb-, Cr-, Cd- and Ni-spiked soils were negatively posi 4.1. Effect of soil parameters on bioavailability of REs
tioned on PC1 and positively positioned on PC2. However, the variances
for multi-REs-, As- and Cu-spiked soils were positioned conversely In Arctic permafrost-affected soils, summer active layer thawing is a
except Cu-spiked soils positioned close to 0 on PC2. This also can be driver of elements liberation, depending on solubility and redox chem
supported from Table 4 that each PLFA values in multi-REs-, As- and Cu- istry of each element (Perryman et al., 2020). In western Russian Arctic
spiked soils were statistically different from the other REs-spiked soils soils, Pb, Cu and As are characterized by high content of sulfide minerals
and the control. (Antcibor et al., 2014) while Cr, Cd and Ni mostly originated from
bedrock (Ji et al., 2019). The accumulation of these REs usually showed
8
a relatively higher content in deeper layer. The origins of REs in current

topsoil mostly derived from industrial emission. The elements used in
our study, Pb, Cr, Cu, Cd, Ni, and As, are not actively absorbed by or
ganisms for cold-adapted organisms in the tundra biome. Therefore, the
bioavailability of these REs is prone to biochemical processes other than
physiological diffusion in soils (Ernst, 1996).
Arctic permafrost keeps the soil cold and prevents deep water
drainage, segregating plants from mineral soils and remain the soil
saturated to the surface (Weintraub and Schimel, 2003). Therefore, less
mobility of REs in this layer indicates lower REs bioavailability. In the
present study, most REs (Pb, Cr, Cu, Cd and Ni) bioavailable concen
trations decreased from 30 to 180 days of incubation (Fig. 2). As
exhibited significantly higher bioconcentration at the end of the incu
bation period, which may be attributed to the higher mobility of reduced
As3+ in our sandy soil without abundant negatively charged clay min
erals. The bioavailable fraction of REs decreases with increasing time
may be due to the REs transformed to relatively organic binding forms
(Jalali and Khanlari, 2008; Jayarathne et al., 2017). With increasing
incubation time, soil organic matter degradation occurred slowly in
anaerobic condition, making the slow release of REs bound to soil
organic matter. This reduction of releasing REs from soil organic matter
may consequently reduce bioavailability of REs. Additionally, we found
a significant decrease in microbial abundance was affected by spiked
REs (Table 4). The declining bioavailable concentrations also corre
sponded with the increase in microbial abundance at 180 days of in
cubation. Overall, the RE levels tested in the current study negatively
influenced soil active microorganisms and further influenced C
biogeochemical cycles. The bioavailability of As in the HD appeared to
be higher than that of other REs in soil solution (Fig. 2). This might be
due to As5+ being reduced to the more toxic As3+ form in reducing en
vironments (Awasthi et al., 2017). In acidic environments of organic
soil, soil organic ligands prevent the sorption of arsenite on ferrihydrite
more than that of arsenate (Caporale and Violante, 2016), which also
resulted in increasing As solubility in our study. Adsorption in acidic
environments is a more predominant process than the precipitation of
Fig. 6. Loading plot of principal component analysis for specific phospholipid soil solid phase in declining metal ion concentration in soil solution and
fatty acid (PLFA) component scores according to PLFA content after 30 (a) and occurs reversely in alkaline environments (Yong et al., 1993). However,
180 (b) days of incubation. Different arrowhead colors represent different Lodygin et al., (2020) found a significant migration ability of Cd2+,
biomarkers: blue for G+ bacteria, red for G− bacteria, green for actinomycetes, resulting from permafrost degradation and entry of trace metals with
and black for fungi. The raw data of specific PLFA is shown in Table S3. low affinity to humic acids in Arctic peatland while higher sorption of
Pb2+ to humic acids was observed in surface horizon of Arctic peat soils.
The bonding mechanism between REs and organic matters in Arctic
organic soils needs a further study to clarify its influence on their
bioavailability.
Moreover, the bioavailability of REs in the present study was also
significantly influenced by REs amendment, which further influenced
the soil microbial community structure and activity (Chu, 2018; Xie
et al., 2016). The spiked-REs concentrations may have toxic effects,
including inhibition of enzyme activity and DNA transcription, cell
membrane destruction, and albuminous degeneration (Gülser and
Erdoğan, 2008; Roane et al., 2009). Notably, the bioconcentrations of
multi-REs-spiked soils were higher than those of single-RE-spiked soils
were. Despite the possibility that relatively higher RE concentration in
multi-REs-spiked soils than single RE spiked soil, a previous study has
shown that soil organic matter could play a key role in reduction of RE
bioavailability (Rieuwerts et al., 1998). Therefore, the competitive
adsorption sites among REs can increase the bioavailability of co-
contaminated REs soils compared with the single-RE treatment.
Although there is no consensus on RE mobility in different soils, some
studies have reported a lower solubility for Pb than other metals (Tye
et al., 2004; Bruemmer et al., 1986; Cerqueira et al., 2011). However,
the partition coefficient (ratio between CDGT and CTotal) of Pb did not
Fig. 7. Score plot of principal component analysis of total phospholipid fatty
acid biomarkers depicting soil microbial community distribution under show any significant differences with other REs (Table S2). This indis
contamination with different REs. Control represents background soils; HD and crimination of partition coefficient of all REs is due to the much higher
LD represent high and low doses of RE amendment. Red and blue circles mobility of metallic elements in highly acidic soils. However, the rela
represent similar clusters of principal component scores. tionship between each RE mobility and bioavailability of each RE in
9
Arctic soil organisms and vegetation was rarely reported. grouped with Zn in the periodic table, these REs-containing enzymes do
not function properly (Shaw et al., 2004). Ekmekçi et al., (2008) also
4.2. MBC profile in RE restriction reported toxic REs causing membrane damage and destruction in
cellular organelles and biomolecules. MBC formation was impeded by
From the data obtained during the incubation experiment, organic excess REs in polluted soils, inhibiting microbial population growth.
matter decomposition by microbes was inhibited by the spiked REs with However, this will not lead to decreased mineralization of organic C but
regard to the total evolved CO2− C and decreasing microbial respiration may increase the evolved CO2− C per MBC unit. The relatively higher
rate (Fig. 3). Our results were similar to those of previous studies qCO2 in spiked soils in the present study suggested that excess REs
(Romero-Freire et al., 2016; Enya et al., 2020; Cui et al., 2019). The impeded the formation or function of MBC, whereas SOC use was not
organic topsoil is considered to be the most active fraction of soil C to influenced. Increased microbial qCO2 suggested that more energy was
effect soil respiration (Vesterdal et al., 2012); therefore, the initial required by microorganisms to tolerate REs contamination and avoid
increased soil respiration may explain the available soil organic matter inhibition (Fließbach et al., 1994). Moreover, after long-term REs stress,
being consumed quickly by the microbes at the beginning so that CO2 some microorganisms may adapt and tolerate RE contamination by
was increasingly emitted. However, accessible C later in the incubation changing C utilization patterns (Prabhakaran et al., 2016; Gadd and
period remained limited for mineralization, leading to lower CO2 Griffiths, 1977). However, in the Arctic organic soils used for all treat
emissions. An incubation study of the fabric organic horizon from ments in our study, microbial qCO2 values decreased after 180 days
Alaskan tundra soils showed that the decomposition of slow and passive compared with that at 30 days, indicating that microbial C use was
soil organic C pools may be limited by fewer bacterial and fungal en affected by REs toxicity. Other microbial processes, including bio
zymes (Hale et al., 2019). Therefore, with increasing incubation time, precipitation with sulfate-reducing bacteria, may change RE mobility
microbial biomass in soils could be reduced and further limit the release (Fang et al., 2011), however, bioprecipitation are rarely reported in the
of CO2. The inhibition of respiration could be due to disrupted enzyme Arctic topsoil.
actions and cell fusion (Gülser and Erdoğan, 2008; Roane et al., 2009).
Consequently, soil microbial changes and metabolic dysfunction in mi 4.3. Microbial community structure
croorganisms may lead to limited microbial reproduction (Xu et al.,
2019). Suppression of microbial respiration was evident in the organic MBC in our study was found to be correlated well with microbial
soils of the current study, especially for multi-REs-contaminated soils. total PLFA (Fig. 5) since PLFAs can represent total microbial abundance
This dysfunction and reduction in microbial processes may have directly (Zhang et al., 2017; Narendrula-Kotha and Nkongolo, 2017). Total PLFs
resulted from the microbial population being inhibited by REs (Xie et al., were observed to be the highest in the top of active layer and tended to
2016). In several soils, CO2 respired and nutrients release from miner decrease with depth in Ellesmere Island, Canadian Arctic (Yoshitake
alization cycles through a smaller “active fraction” with short turnover et al., 2006). A previous study found that the soil respiration rate was
times (Trumbore, 1997). While the pattern for Arctic organic soils is not highly associated with the ratio between G+ and G– bacteria, whereas
that simple, the contribution of soil material decomposition partially G+ bacteria were negatively correlated with soil respiration rate
derived from plant material such as moss, sedge, and lichen, and thence (Whitaker et al., 2014). In this way, G− bacterial groups may play a
there is a large pool of organic matter as labile C whose decomposition is more considerable role in soil microbial respiration than other microbial
constrained by several factors such as inundation and low temperature. groups. SOC was not significantly correlated with any soil microbial
Due to simplicity of ecosystem in Artic tundra, the inhibition of multi- functional group, which agrees with the results of Xu et al., (2019). In
REs-contaminated soils presented in this study will prevent the nutri our study, fungal abundance decreased in REs-contaminated soils with
ents from degradation of C in soils to threaten the whole ecosystem. relatively higher microbial qCO2, which suggests that microbial func
MBC is an indispensable fraction of organic C pools in soils, tional groups change their C use efficiency following the alteration of the
participating in soil nutrient and C cycles (Shentu et al., 2014). microbial community under REs stress.
Compared with the rapid response of microbial respiration in the present Soil microbial populations can be stressed by toxic RE levels and
study, MBC varied slowly. Soil C utilized by microbial functional groups different REs (Abdu et al., 2017). A shift in microbial PLFA functional
may exhibit similar patterns. Among all treatments, the MBC values of groups was observed in microbial biomarkers (Fig. 6). The composition
the LD and HD of multi-REs-spiked soils were the lowest (Fig. 4), which of the microbial community was also affected by spiked REs (Fig. 7). The
may have resulted from a reduction in MBC formation. RE-induced in influence was higher in multi-REs-, As-, and Cu-spiked soils compared
hibitions may be associated with the energy alteration from MBC pro with the control (Table S1). Some microbial groups can tolerate RE
duction to cellular function maintenance under REs stress in soil stress better compared with other microbial functional groups, which
microorganisms (Fließbach et al., 1994), which increased the energy results in alterations to microbial community structure (Azarbad et al.,
demand of microbial functional groups living in stressed environments. 2016). Besides, MBC was mostly involved in C cycles facilitated by fungi
In our study, the significant difference (p < 0.05) between control and and bacteria, which is important for soil organic C decomposition
REs-spiked soils were observed in MBC/SOC, especially for As- and (Petrisor et al., 1964). The RE-spiked soils in the present study nega
multi-REs spiked soils (Table 3). Besides, no relationship between SOC/ tively affected the fungal population but not G+ and G− bacteria, which
SN and MBC (r = 0.32, p > 0.05) in RE-contaminated soils was found. are more tolerant to REs than fungi. Our results show that fungi
This shows that REs toxicity restrained the connection between SOC and appeared to be more sensitive to REs than bacteria.
MBC as previously reported (Xu et al., 2019). However, the limitation of The alteration in microbial PLFA composition might be associated
our study is not able to reflect how soil MBC functioned with RE because with the C utilization profile (Ibekwe and Kennedy, 1998). A previous
bio-available REs may affect microbial activity and C utilization patterns study has reported that Pb had no toxic effects on bacterial growth and
and consequently influence the formation of metal-organic complex. diversity in boreal forest soil, and bacteria had a higher tolerance to Pb
Each RE affects microbial activities differently due to differences in than fungi (Hui et al., 2012), which agrees with our results. The dif
toxicity (Chen et al., 2014; Hattori, 1992). Toxic effects primarily ferentiation of C use can lead to shifts in bacterial and fungal abundance
included, (i) stressing essential biological functional groups of enzymes, (Deveau et al., 2018). Therefore, the decreased fungal population may
(ii) blocking active conformation of biomolecules, and (iii) displacement have been due to shifts in microbial C use. In two major principal
or substitution of essential metal ions in the biomolecules by another components of microbial PLFA composition (Fig. 6), significantly
metal ion, resulting in inhibition of soil enzymatic activities (Ochiai, different profiles were found for the fungal biomarkers 18:2ω6 and
1977). Some toxic REs (e.g., Pb2+, Cu2+, Ni2+ and Cd2+) may substitute 18:1ω9c. Xu et al., (2019) found that the biomarkers negatively affecting
Zn2+ for metalloenzymes. Despite some metals, such as Cd, being by REs contained C18:1ω9, iC17:0, C17:0, aC17:0, C16:0, and
10
10MeC16:0. However, a single factor cannot be absolutely represented and soil respiration measurement. Wenjuan Wang: Helping with data
by the PCA results due to the highly inter-related soil physiochemical sorting and statistical analysis. Chuanlan Li, Yu Huang: Helping mi
properties. A suppressive effect on microbial PLFAs is stronger at higher crobial biomass carbon analysis. Daishe Wu, Xianchuan Xie: Helping
REs concentrations, reflecting a decrease in microbial population the manuscript editing, analysis of specific phospholipid fatty acids and
(Azarbad et al., 2016). Our loading plot results indicated that REs the funding support for experiment.
contamination influenced microbial community composition (Fig. 6).
Furthermore, multi-REs contamination had a higher effect on microbial Declaration of Competing Interest
profiles. Microbial biomass is positively correlated with soil C and ni
trogen contents over the successional chronosequence in Arctic soils The authors declare that they have no known competing financial
(Yoshitake et al., 2006; Yoshitake et al., 2018). Therefore, changes in interests or personal relationships that could have appeared to influence
microbial community composition may exacerbate the negative effect the work reported in this paper.
on soil C and nitrogen availability in Arctic organic soil.
Acknowledgements
4.4. Conclusions
We thank the four anonymous reviewers for taking the time to re
Determining alterations in microbial communities is important in view our manuscript and provide helpful comments that greatly
Arctic surface soils, which are sensitive to seasonal climates and soil C contributed to the improvement of this manuscript. We would like to
pools (Hale et al., 2019; Schuur et al., 2008; Schuur et al., 2015). Mi thank Miss Yu Su from the School of Visual Arts at BFA Computer Art for
crobial biomass C, N, P, and plant uptake rapidly increase in Arctic soils helping with data visualization. We are also grateful for Prof. Jeff
during the thawing season, which is the most stressful period for mi Schoenau from Department of Soil Science, University of Saskatchewan,
crobes in surface soils due to the physiological effects of rising temper Canada, for providing valuable suggestions and editing to our manu
ature and changing osmotic pressure when wet-up occurs (Schmidt script. This work was supported by grants from the Russian Foundation
et al., 2007; Schimel and Clein, 1996). Following snow thaw, trophic for Basic Research (18–44-890003, 19–416-890002 and 19–05-50107),
interactions under meltwater, i.e., rapidly increasing mesofaunal pre the Saint-Petersburg State University ("Urbanized ecosystems of the
dation via enhanced soil pore space connectivity, could also contribute Russian Arctic: dynamics; state and sustainable development", grant
to the turnover in microbial species or functional groups (Wallenstein number: 39377455), the National Natural Science Foundation of China
et al., 2007). Previous studies have reported a significant shift in the soil (52070094).
microbial community in Arctic tundra during the thawing period
(Buckeridge et al., 2013; Buckeridge and Grogan, 2008). Fungal biomass Appendix A. Supporting information
was reported to have significantly decreased during this time. In our
study, the toxicity of REs had a significantly negative effect on fungal Supplementary data associated with this article can be found in the
populations, more than on other microbial functional groups, resulting online version at doi:10.1016/j.jhazmat.2020.124430.
in a decline in the fungal abundance. Therefore, different fungus in
Arctic soils may have variable responses to REs. References
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Journal of Hazardous Materials 408 (2021) 124430

Contents lists available at ScienceDirect

Journal of Hazardous Materials

Response of carbon and microbial properties to risk elements pollution in

Control 8.56 ± 1.23a 0.32 ± 0.02a 27.03 ± 0.06a 0.49 ± 0.06a

3.3.1. Microbial PLFAs 3.3.2. Microbial group structure

values were found in the control soil as G+ bacteria, 94 nmol g− 1 soil;

a relatively higher content in deeper layer. The origins of REs in current

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