Science of the Total Environment 622–623 (2018) 1391–1399

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Science of the Total Environment

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Biochar alters microbial community and carbon sequestration potential

across different soil pH
Yaqi Sheng, Lizhong Zhu ⁎
Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang 310058, China
Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou, Zhejiang 310058, 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

• CO2 emission from biochar-amended

soils with two different pH levels was
studied.
• Higher biochar degradation resulted in
higher CO2 emission in acidic ferralsol.
• Biochar increased the bioavailability of
SOC and copiotrophic bacteria in
ferralsol.
• Adsorption of SOC on biochar resulted in
decreased CO2 emission in phaeozems.

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

Article history: Biochar application to soil has been proposed for soil carbon sequestration and global warming mitigation. While
Received 5 October 2017 recent studies have demonstrated that soil pH was a main factor affecting soil microbial community and stability
Received in revised form 29 November 2017 of biochar, little information is available for the microbiome across different soil pH and the subsequently CO2
Accepted 29 November 2017
emission. To investigate soil microbial response and CO2 emission of biochar across different pH levels, compar-
Available online xxxx
ative incubation studies on CO2 emission, degradation of biochar, and microbial communities in a ferralsol
Editor: J Jay Gan (pH 5.19) and a phaeozems (pH 7.81) with 4 biochar addition rates (0.5%, 1.0%, 2.0%, 5.0%) were conducted. Bio-
char induced higher CO2 emission in acidic ferralsol, largely due to the higher biochar degradation, while the
Keywords: more drastic negative priming effect (PE) of SOC resulted in decreased total CO2 emission in alkaline phaeozems.
Soil carbon sequestration The higher bacteria diversity, especially the enrichment of copiotrophic bacteria such as Bacteroidetes,
Biochar Gemmatimonadetes, and decrease of oligotrophic bacteria such as Acidobacteria, were responsible for the in-
pH creased CO2 emission and initial positive PE of SOC in ferralsol, whereas biochar did not change the relative abun-
SOC dances of most bacteria at phylum level in phaeozems. The relative abundances of other bacterial taxa (i.e.
Microorganisms
Actinobacteria, Anaerolineae) known to degrade aromatic compounds were also elevated in both soils. Soil pH
was considered to be the dominant factor to affect CO2 emission by increasing the bioavailability of organic car-
bon and abundance of copiotrophic bacteria after biochar addition in ferralsol. However, the decreased bioavail-
ability of SOC via adsorption of biochar resulted in higher abundance of oligotrophic bacteria in phaeozems,
leading to the decrease in CO2 emission.
© 2017 Published by Elsevier B.V.

⁎ Corresponding author at: Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang 310058, China.
E-mail address: [email protected] (L. Zhu).

https://doi.org/10.1016/j.scitotenv.2017.11.337
0048-9697/© 2017 Published by Elsevier B.V.
1392 Y. Sheng, L. Zhu / Science of the Total Environment 622–623 (2018) 1391–1399

1. Introduction Therefore, this study aims to investigate the microbial community

change and carbon sequestration to biochar addition in soils of different
Biochar application to soil has been proposed as a promising method pH. The effects of biochar on physical, chemical, microbial parameters
for carbon sequestration, along with the co-benefits of mitigating global and CO2 emission of two different pH soils were examined to reveal:
warming, improving soil fertility, increasing crop yields and enhancing (1) the effect of the addition rate on the total CO2 emission, biochar deg-
the sorption for pollutants (Kan et al., 2016; Lehmann and Joseph, radation and priming effect (PE) of SOC under different soil pH; (2) tax-
2009). The carbon sequestration potential of biochar rests on its high re- onomic changes of bacterial to biochar addition under two soil pH level;
calcitrance against microbial decay (Lehmann et al., 2011; Spokas, (3) the effect of physiochemical properties and dominated factor on mi-
2013). Observed effects of biochar on CO2 emission and the microbial crobial communities change and carbon sequestration potential after
processes are often conflicting due to the variable properties of biochar biochar amendment. The results of this study are expected to better es-
and soil (Singh and Cowie, 2014; Zimmerman et al., 2011). Soil pH plays timate the carbon sequestration potential of biochar for large-scale
a crucial part on the release of inorganic carbonates, organo-mineral asso- application.
ciation between biochar and soil, as well as the microbial activities, thus
to affect the stability of soil organic carbon (SOC) and biochar 2. Materials and methods
(Maestrini et al., 2015; Sheng et al., 2016). It was implied biochar amend-
ment significantly decreased soil CO2 fluxes in neutral soils but increased 2.1. Soils and biochar
soil CO2 fluxes in acidic soils (Sheng et al., 2016). Biochar altered the mi-
crobial communities directly or indirectly through affecting the physico- The ferralsol soil was collected from Hangzhou, Zhejiang province
chemical processes in the soil, largely depended on the pH level in soil. (30.2 °N, 120.1 °E), the phaeozems soil was collected from Harbin, Hei-
However, the mechanisms of biochar on microbial processes under differ- longjiang province (45.7 °N, 126.6 °E). Soil samples were hand-picked
ent soil pH are still unclear. Observing the effect and mechanism of bio- to remove obvious plant debris and roots, air-dried, sieved through a
char on the microbial communities and the subsequent CO2 emission 2 mm sieve, and analyzed for basic properties including pH, soil total
under different soil pH would be crucial in predicting the carbon seques- carbon, NO3− and NH+ 4 . Rice straw was selected as the biochar feed-
tration potential and large-scale application in agriculture. stock for its abundance in China. After grounded, passed through a
Biochar application would largely affect the physiochemical proper- 0.154 mm sieve, and oven-dried overnight at 70 °C, the powder was
ties of soil and therefore microbial communities and processes. The al- compacted in a quartz boat and pyrolyzed for 2 h at 500 °C, in the
kaline nature of the biochar increased the soil pH (Feng and Zhu, tube furnace which was filled with N2 and programmed at a heating
2017; Murray et al., 2015), which would lead to more abundance of rate of 5 °C min−1. The produced biochar was then sieved to
gram-negative bacteria, as well as lower proportion of gram-positive b0.154 mm for subsequent analysis.
bacteria and fungi at higher pH condition (Pietri and Brookes, 2009; After incubation, the soil samples were freeze-dried. Soil pH was
Rousk et al., 2009). In addition, the dissolution of nutrients such as nitro- then determined in a soil/water slurry at 1:10 (w/v) ratio. NH+ 4 and
gen, phosphorus and several small molecule compounds (lactic acid, NO− 3 were detected by ion chromatography (Thermo DIONEX ICS-
succinic acid, formic acid) in biochar be affected by soil pH, thus to affect 1500, Waltham, America, and Thermo DIONEX Acquion, Waltham,
the growth of microorganism (Lin et al., 2012a; Smith et al., 2013). America). Soil total carbon (TC) was detected by TC-SSM (Shimadzu
What's more, the adsorption of native SOC and microbial enzymes, as TC-SSM analyzer, Kyoto, Japan). Water extracted organic carbon
well as the organo-mineral interactions between biochar and clay min- (WEOC) was extracted from the mixture of biochar and soil with dis-
erals via direct electrostatic interactions, H bonding, cation bridging, tilled water (1:1 w/v) for 30 min. The mixture was then centrifuged
and ligand exchange reactions would be accelerated under lower pH with a rotation speed of 3000 rpm for 15 min and the supernatant
level (Chen et al., 2013; von Lutzow et al., 2006). The adsorption and was collected and filtrated through a 0.22 μm membrane. WEOC con-
organo-mineral interaction would provide physical protection against tent was measured with a TOC-VCPH (Shimadzu TOC-VCPH analyzer,
microbial degradation and predatory soil fauna (Keith et al., 2011; Kyoto, Japan). Chemical properties of the origin soils and biochar, bio-
Lehmann et al., 2011; Nguyen et al., 2009; Zimmerman et al., 2011). char-amended soils and non-amended treatments are shown in Table
However, although physiochemical interactions played important role 1 and Table S1.
in the change of microbiome, the response of specific microbial commu- The molecular level of SOC was detected by solvent extraction. The
nities under different pH level was still unclear. methods were detailed before (Mitchell et al., 2016). 2 g soil was soni-
A range of microbial community change would occur after biochar cated for 30 min and centrifuged with a rotation speed of 3000 rpm
application. Previous studies indicated that biochar significantly for 10 min in a 40 mL vial along with 30 mL dichloromethane, then
changed most bacterial groups at genus level (Zheng et al., 2016). In- the supernatant was decanted. The procedure was repeated with
crease in the relative abundance of Actinobacteria, Bacteriodetes, 30 mL dichloromethane/methanol (1:1 v/v), followed by 30 mL metha-
Firmicutes, Planctomycetes, Gemmatimonadetes (Chen et al., 2013; Hu nol. The three extracts were combined and filtered through a 0.22 μm
et al., 2014; Khodadad et al., 2011; Kolton et al., 2011) and decrease in membrane and dried under N2. The extracts were converted to
Acidobacteria, Chloroflexi by biochar (Xu et al., 2014) were noted. The la- trimethylsilyl derivatives by reaction with 100 μL of N,O-
bile fraction in biochar and the following physical weathering of recalci- bis(trimethylsilyl)-trifluoroacetamide (Sigma) and 10 μL of pyridine
trant portions suggested selection in favor of copiotrophic organisms (Sigma) at 70 °C for 1.5 h. After derivatization, the samples were diluted
(Jenkins et al., 2017; Sun et al., 2015). In addition, taxa (i.e. Aminobacter, to 510 μL by hexane (Fisher Scientific) and detected by GC (Agilent
Bradyrhizobium Nitrosospira, Streptomyces) enriched in the biochar- 7890B, Agilent, America) linked to a quadrupole MS (Agilent 5977A,
amended soil might be attributed to the effects such as aromatic struc- Agilent, America).
ture and improving soil nitrogen conditions (Ding et al., 2013; Kolton et
al., 2017). The variation of soil pH was indicated to contribute 56.5% of 2.2. Incubation experiment
the total variation of microbial change (Xu et al., 2014). Significant pos-
itive correlation between the diversity of taxa and pH has also been de- In this experiment, 5 g soil with 0, 25, 50, 100 and 250 mg biochar
tected as a consequence of biochar treatment (Jenkins et al., 2017). were thoroughly mixed in 40 mL sample vials at the addition rate of
However, the microbial changes to biochar amendment are sometimes 0%, 0.5%, 1.0%, 2.0% and 5.0% (the ferralsol amend with 0%, 0.5%, 1.0%,
contradictory, the mechanism of microbial community changes and 2.0% and 5.0% biochar were annotated as F, FB 0.5, FB 1.0, FB 2.0 and
subsequent CO2 emission as consequences of soil pH change under bio- FB 5.0, the phaeozems amend with 0%, 0.5%, 1.0%, 2.0% and 5.0% biochar
char application were still poorly identified. were annotated as P, PB 0.5, PB 1.0, PB 2.0 and PB 5.0). Each treatment
 Y. Sheng, L. Zhu / Science of the Total Environment 622–623 (2018) 1391–1399 1393

Table 1
Soil physical and chemical properties of the biochar-amended treatments and control after 90 days incubation.

pH NH+
4 -N NO−
3 -N TC WEOCc
(mg/kg) (mg/kg) (mg/g) (mg/L)

F 5.996 ± 0.015aa 0.639 ± 0.003b 19.65 ± 0.43b 11.39 ± 0.31a 28.82 ± 0.39b
FB 0.5 5.933 ± 0.018a 0.511 ± 0.007b 4.034 ± 0.38a 13.74 ± 0.14a 26.63 ± 0.26ab
FB 1.0 6.188 ± 0.016ab 0.286 ± 0.004ab 4.289 ± 0.23a 16.31 ± 0.26ab 25.45 ± 0.29ab
FB 2.0 6.207 ± 0.025ab 0.028 ± 0.001a 6.797 ± 0.17a 20.92 ± 0.75ab 24.11 ± 0.28ab
FB 5.0 6.758 ± 0.014b /b 7.817 ± 0.76a 34.76 ± 0.79b 21.67 ± 0.58a
P 8.237 ± 0.001c 0.055 ± 0.002a 6.247 ± 0.53a 37.68 ± 0.66bc 33.42 ± 0.07b
PB 0.5 8.274 ± 0.002c 0.044 ± 0.001a 5.253 ± 0.06a 39.02 ± 0.64bc 32.09 ± 0.04b
PB 1.0 8.325 ± 0.002c 0.040 ± 0.001a 4.762 ± 0.87a 40.67 ± 0.43bc 29.49 ± 0.46b
PB 2.0 8.334 ± 0.003c 0.034 ± 0.001a 4.725 ± 0.12a 45.19 ± 0.83c 27.41 ± 0.16ab
PB 5.0 8.407 ± 0.004c 0.021 ± 0.001a 4.362 ± 0.44a 56.46 ± 1.32c 26.80 ± 0.02ab
a
Values in the same column followed by different letters were statistically different at p b 0.05, n = 3.
b
Blow limit of detection.
c
Water extracted organic carbon.

was set with 6 replicates. Deionized water of 80% soil water holding ca- normalized, pooled in equimolar and paired-end sequenced (2
pacity was added to the vials, the vials were sealed and incubated in the × 300 bp) on an Illumina MiSeq platform (Guhe Information Technolo-
dark at 25 °C. Three empty vials were set to measure the concentration gy Co. Ltd., Hangzhou, China). Sequence reads processing was per-
of CO2 of surrounding atmosphere. The measurement of CO2 was de- formed using QIIME (version 1.9.0), including additional quality
tailed before (Sheng et al., 2016). The CO2 emission was measured trimming and demultiplexing. Operational taxonomic unit (OTU) was
with a gas-tight glass syringe (Agilent) through injecting 100 μL head- picked by Vsearch v1.11.1, followed by dereplication, cluster, detection
space CO2 from the incubation vials into the gas chromatograph (Fuli of chimeras. The barcoded 16S rRNA gene sequences were annotated
9790, Hangzhou, China). Limits of detection (LOD) of CO2 measurement into libraries by QIIME, resulting in operational taxonomic units
was 2 ppm, the relative standard deviation (RSD) were lower than 5%. (OTUs), bacterial diversity, and phenetic classification. Taxonomy was
The incubation experiments was carried over a period of 90 days, and assigned to an OTU based on its representative sequence by Greengene
CO2 evolution was measured periodically at 1, 2, 4, 7, 14, 21, 30, 40, 13.8. Calculations of alpha-diversity (including Shannon, Simpson,
50,60, 75, 90 days of incubation. Chao1) were performed by QIIME.
Phylogenetic investigation of PICRUSt was used to predict the Kyoto
2.3. Degradation of biochar and priming effects (PE) of soil organic carbon Encyclopedia of Genes and Genomes (KEGG). PICRUSt predicted
(SOC) metagenomes from 16S data by using evolutionary modeling and com-
paring with a reference genome database. Generally, OTUs of 16S rRNA
δ13C analysis by Precon-IRMS (Delta V Thermo Finnigan, Waltham, sequences were normalized by PICRUSt, and then the metagenomes
America) was used to calculate the amount of biochar mineralized predicted by PICRUSt algorithm were collapsed into clusters of
CO2 (CB) from biochar-amended soil, methods has been detailed before orthologous groups of proteins (COGs) and KEGG. Predictive COGs and
(Sheng et al., 2016). CB was calculated as: KEGGs were screened out and visualized by Statistical Analysis of
  Metagenomic Profiles (STAMP) software package v 2.1.3. The KEGG
C T δT 13 CO2 −δS 13 CO2 profiles across all samples were pairwise compared between control
CB ¼   ð1Þ and biochar-amended treatments.
δB 13 CO2 −δS 13 CO2
2.5. Statistical analyses
CT is the total C mineralized from the biochar-amended soil, δ13T CO2
is the δ13T C value of the CO2-C evolved from the biochar-amended The regression and correlation analyses were performed on SPSS
soils, δ13 13
S CO2 is the δT C value of CO2-C evolved from the control soil, 20.0 (IBM, Armonk, USA). The differences in physiochemical properties
and δ13S CO 2 is the initial δ13
T C value of the biochar. The amount of the and relative abundances of dominant communities were tested by the
soil-C mineralized (CS,B) from the biochar-amended soil and the prim- analysis of variance (ANOVA). Kyoto Encyclopedia of Genes and Ge-
ing effects (PE) induced by the biochar on mineralization of the native nomes (KEGG) pathways were drawn by using STAMP (version 2.1.3).
organic carbon in the soils were calculated as: Pearson correlation analysis was used to assess the relationships
among physiochemical properties of soils, CO2 emission and relative
C S;B ¼ C T −C B ð2Þ abundances of dominant communities. The relationship between mi-
crobial community structure and the selected environmental variables
PE ¼ C S;B −C S ð3Þ was analyzed by the redundancy analysis (RDA) to determine the inde-
pendent contributions of these selected variables to the variation in
CS is the amount of CO2-C evolved from the control soil, CT is the total community composition.
C mineralized from the biochar-amended soil, CB is the amount of C
mineralized from biochar. The positive priming effect means accelerat- 3. Results
ing of SOC mineralization by biochar. In contrast, negative priming effect
means the suppression of SOC mineralization by biochar. 3.1. Soil chemical properties

2.4. High-throughput sequencing analysis Generally, biochar addition increased soil pH in both soils except
ferralsol amended with 0.5% biochar, with the greatest increase of
Metagenomic DNA was extracted from 500 mg freeze-dried soil 0.825 and 0.16 at the rate of 5.0% for ferralsol and phaeozems, respec-
Samples using the PowerMax (stool/soil) DNA isolation kit (MoBio Lab- tively (Table 1). The increase of pH in ferralsol was more drastic than
oratories). The V3-V4 hypervariable region of the 16S rRNA gene was that in phaeozems because of the higher buffer ability of phaeozems
amplified from genomic DNA by primers. Purified amplicons were whose initial content of total carbon was higher (Murray et al., 2015).
1394 Y. Sheng, L. Zhu / Science of the Total Environment 622–623 (2018) 1391–1399

−
The concentration of NH+ 4 and NO3 was highest in untreated ferralsol, More biochar C was mineralized in ferralsol (Fig. 3), the highest min-
NH+ 4 content decreased significantly (p b 0.05) as the biochar addition eralization rate of biochar appeared on the first day. Biochar degrada-
rate increased. NO− 3 content drastically decreased from 19.65 mg/kg in tion followed an order of FB 5.0 N FB 2.0 N FB 1.0 N FB 0.5 over the
untreated ferralsol to 4.03 mg/kg in FB 0.5, then arose as addition rate whole 90 days of incubation in ferralsol. However, degradation rate of
increased. In contrast, only a slightly decrease in the concentration of biochar showed an opposite trend on the first day in phaeozems, the
−
NH+ 4 and NO3 were observed after biochar application in phaeozems. degradation rate of biochar began to increase at higher addition rate
The total carbon (TC) content and water extractable organic carbon over time. Following sharp declines after the transient high degradation
(WEOC) was higher in phaeozems (Table 1). Total carbon (TC) content rate of biochar on the first day, second peak of biochar degradation was
was 37.68 mg/g in phaeozems, which was almost 2.31 folders higher observed on the 4-7th day of the incubation except the addition rate of
than ferralsol. Biochar significantly increased TC content from 11.39 to 5.0%. After the 30th day of the incubation, the degradation rate of bio-
34.76 mg/g and 37.68 to 56.46 mg/g for ferralsol and phaeozems at char decreased to a relative stable phase for both soils.
the rate of 5.0%. While WEOC decreased as the biochar addition rate in- Biochar addition initially accelerated the SOC mineralization in acid-
creased in both soils, biochar significantly (p b 0.05) decreased WEOC by ic ferralsol, this positive priming effect (PE) was facilitated at higher bio-
7.6%, 11.7%, 16.3% and 24.8% in ferralsol at the rate of 0.5%, 1.0%, 2.0% and char addition rate (Fig. 4a). The highest cumulative PE was 4.33, 7.41,
5.0%, respectively. For phaeozems, biochar decreased WEOC by 4.0%, 16.71 and 26.66 μg for 0.5%, 1.0% 2.0% and 5.0% biochar- amended
11.7%, 17.9% and 19.8% at the rate of 0.5%, 1.0%, 2.0% and 5.0%. ferralsol, respectively. The positive PE were transient and shifted to neg-
Biochar amendment altered the molecular-level composition and ative PE as time went by except ferralsol amended with 5.0% addition
distribution of native soil organic matter (Fig. 1). The proportion of rate of biochar. Negative cumulative PE were observed on the 4th, 7th
most aliphatic hydrocarbon and carbohydrates by solvent extraction and 20th day of the incubation for ferralsol amended with 0.5%, 1.0%
was higher in phaeozems (Tables S2, S3), resulted in higher bioavail- and 2.0% biochar. Compared with ferralsol, the treatments of
ability. After biochar amendment, the proportion of most aliphatic hy- phaeozems exhibited negative PE through the whole incubation. Soil
drocarbon and carbohydrates such as palmitic acid, lignoceric acid, amended with higher biochar addition rate (5.0%) led to a 2–3 fold
stearic acid, trehalose and glycerol monostearate significantly increased higher negative PE than the other treatments.
(p b 0.05) in ferralsol at the end of the incubation (Table S2). However,
as the addition rate increased, palmitic acid, stearic acid and trehalose 3.3. Shift of microbial community composition under biochar application
significantly decreased in phaeozems (Table S3). For example, the pro-
portion of palmitic acid decreased from 7.093% to 5.565% and 5.494% at Biochar amendment showed higher OTU richness and bacterial di-
the addition rate of 1.0% and 5.0%, respectively. More aromatic com- versity in acidic ferralsol, but showed slightly decrease in phaeozems.
pounds such as 2, 6-Bis (tert-butyl) phenol were shown in ferralsol The untreated phaeozems exhibited 2.38 folders higher OTUs than un-
(Table S4), aromatic compounds (i.e. benzenepropanoic acid, phthalic treated acidic ferralsol (Table 2). Changes in both bacterial community
acid and quinoline) significantly increased as the biochar addition rate structure and composition were found under different biochar addition
arose. rate by high-throughput sequencing. The three replicates of each treat-
ment were generally assembled together and distinct from each other
(Fig. S1). Furthermore, comparison of the sites effect prior to biochar
3.2. CO2 emission, degradation of biochar and priming effect (PE) of SOC treatment showed significant differences in community structure, em-
phasizing the importance of original soil properties on microorganism.
The cumulative amount of C mineralized from the biochar was sig- The predominant phyla in soils were Proteobacteria,
nificantly higher in ferralsol than that in phaeozems. Biochar signifi- Gemmatimonadetes, Firmicutes, Chloroflexi, Bacteroidetes, Actinobacteria,
cantly increased the total CO2 emission in the acidic ferralsol at all Acidobacteria, Cyanobacteria, Nitrospirae and TM7. These taxa accounted
addition rates (p b 0.05), the total C mineralized was significantly higher for N 89% of the bacterial sequences in all treatments (Table S5). As
in the 5.0% biochar treatment than other treatments (Fig. 2). At the end shown in Fig. 5, the relative abundance of Firmicutes decreased under
of the incubation, CO2 emission in ferralsol increased from 80.36 mg to biochar treatments in ferralsol, Acidobacteria decreased except the in
83.16, 84.92, 91.43 and 108.54 mg at the rate of 0.5%, 1.0%, 2.0% and the FB0.5, while Chloroflexi, Gemmatimonadetes, Bacteroidetes and
5.0%, respectively. In contrast, the total CO2 emission was suppressed Actinobacteria increased. Acidobacteria was the most sensitive phylum
in the alkaline phaeozems after biochar application, as the addition to biochar in ferralsol, with the relative abundances decreasing from
rate increase, the suppression of CO2 emission increased. At the end of 10.43% to 9.50%, 7.52% and 6.90% for FB 1.0, FB 2.0 and FB 5.0, respective-
the incubation, biochar decreased CO2 emission by 3.85%, 5.30%, 6.34% ly. Firmicutes and TM7 were also significantly decreased by biochar
and 14.9% at the rate of 0.5%, 1.0%, 2.0% and 5.0%. amendment, from 27.24% and 10.42% to 17.76% and 2.13% for 5.0%

14
(a) F
14 (b) P
12 F-1 12 P-1
F-5 P-5
10 10

8 8
Percent

Percent

6 6

4 4

2 2

0 0

0 100 200 300 400 500 600 700 800 900 0 100 200 300 400 500 600 700 800 900
Molecular weight Molecular weight

Fig. 1. Distribution of small molecular compounds (molecular weight b 1000) extracted by solvent in ferralsol (a) and phaeozems (b) amended with biochar across different addition rate
at the end of the incubation.
 Y. Sheng, L. Zhu / Science of the Total Environment 622–623 (2018) 1391–1399 1395

Cumulative CO2 emission (mg C kg-1 soil)

Cu mu lative CO2 e mission (mg C k g-1 soil)

120
(a) F 120
(b) P
FB0.5 PB0.5
FB1.0 PB1.0
100 FB2.0 100 PB2.0
FB5.0 PB5.0
80 80

60 60

40 40

20 20

0 0
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

Incubation time (days) Incubation time (days)

Fig. 2. Total CO2 release (mg C kg−1 soil) in (a) ferralsol, (b) phaeozems across 0.5%, 1.0%, 2.0% and 5.0% biochar addition rate over 90 days of incubation. The ferralsol amend with 0%, 0.5%,
1.0%, 2.0% and 5.0% biochar were annotated as F, FB 0.5, FB 1.0, FB 2.0 and FB 5.0, the phaeozems amend with 0%, 0.5%, 1.0%, 2.0% and 5.0% biochar were annotated as P, PB 0.5, PB 1.0, PB 2.0
and PB 5.0.

addition rate, respectively (Table S5). On the other hand, no significant CO2 emission incorporated degradation of biochar and mineralization
differences were observed for biochar amended phaeozems except for of SOC, the effect of biochar on the total emission depended on the
Proteobacteria, which showed a slightly decrease in comparison to the sum of biochar mineralization and the PE of SOC induced by biochar.
control except PB 1.0. In addition, Actinobacteria and Chloroflexi also sig- Biochar degradation and the initial positive PE of SOC contributed to
nificantly increased for PB 5.0. the increased CO2 emission in acidic ferralsol. Over time, although the
The differences of diversity and richness of soil bacterial communi- positive PE shifted to negative except FB5.0, biochar degradation was
ties could primarily be explained by significant increase of pH in acidic larger and played a more important role. In contrast, the negative PE
ferralsol, whereas in phaeozems, TC is dominated factor for soil bacterial of SOC induced by biochar was higher than its own degradation in alka-
communities after biochar amendment. The RDA result indicated that line phaeozems.
biochar could strongly shift the soil microbial community by changing The cumulative amount of C mineralized from the biochar was sig-
soil chemical properties. The first two axes of redundancy analysis nificantly higher in soil with lower pH. The CO2 release from the carbon-
(RDA) together explain 47.6% and 34.9% of the total variation in the bac- ates of biochar was largely depended on soil pH. The transient high
terial communities (at the family level) of ferralsol and phaeozems, re- degradation rate of biochar followed by a sharp decline in acidic soil in-
−
spectively. In total, pH, TC, NH+4 , NO3 and WEOC could explain 53.8% dicated a faster abiotic release of carbonates. Besides, since acidic
and 46.1% of the soil microbial community composition variation for ferralsol exhibited lower proportion of aliphatic hydrocarbon and
ferralsol and phaeozems, respectively (Table S8). The importance of higher aromatic compounds, the bioavailability of SOC would be
variables on microorganism were followed as pH N TC N WEOC N NH+ 4 lower. Previous researches also showed the presence of significant
N NO− 3 in ferralsol, whereas in phaeozems TC and pH could explain char like aryl and O-aryl C of SOC in acidic soil (Farrell et al., 2013). As
10.4% and 7.40% of the variance in soil bacterial community composition a result, the labile fraction in biochar induced higher proportion of ali-
(Fig. 6). phatic hydrocarbon and carbohydrates in ferralsol, thus to increase
the bioavailability of sample and change the preferential substrate utili-
4. Discussion zation by soil microbiome, leading to higher microbial diversity and bio-
char degradation in ferralsol. Besides, the microbiome degrading char-
4.1. Higher CO2 emission, degradation of biochar and priming effect (PE) of like substance in soil with low pH would be more adaptive in the min-
SOC in acidic ferralsol eralization of recalcitrant fraction in biochar, also contributed to the
long-term biochar degradation. In contrast, the adsorption of WEOC
As the addition rate increased, biochar induced higher CO2 emission on biochar decreased the concentration of easily degradable substances
in acidic ferralsol, largely due to the higher biochar degradation, while such as stearic acid and trehalose in phaeozems, resulted in lower bio-
the more drastic negative priming effect (PE) of SOC resulted in de- availability of SOC, thus to decreased the microbial activity and degrada-
creased total CO2 emission in alkaline phaeozems. Generally, the total tion of biochar.

3.5 3.5
Respiration rate (mg CO2-C kg soil d )

Resp ir ation r ate(m g CO2-C kg soil d )

-1

-1

(a) (b)
3.0 FB0.5 3.0 PB0.5
-1

-1

FB1.0 PB1.0
2.5 FB2.0 2.5 PB2.0
FB5.0 PB5.0
2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
-10 0 10 20 30 40 50 60 70 80 90 -10 0 10 20 30 40 50 60 70 80 90
Incubation time (days) Incubation time (days)

Fig. 3. Respiration rate of biochar (mg CO2-C kg−1 soil) in (a) ferralsol, (b) phaeozems across 0.5%, 1.0%, 2.0% and 5.0% biochar addition rate over 90 days of incubation.
1396 Y. Sheng, L. Zhu / Science of the Total Environment 622–623 (2018) 1391–1399

120
(a) FB0.5 PB0.5
(b)
FB1.0 PB1.0
80
FB2.0 PB2.0
FB5.0 PB5.0
Primed soil-C ( g)

40

-40

-80

-120

-160
-10 0 10 20 30 40 50 60 70 80 90

Incubation time (days)

Fig. 4. Primed soil organic carbon of ferralsol and phaeozems across 0.5%, 1.0%, 2.0% and 5.0% biochar addition rate over 90 days of incubation (a). Positive primed soil organic carbon (SOC)
indicated higher carbon mineralization of SOC in biochar-amended soil; negative primed SOC indicated decreased SOC mineralization. Silica-alumina mineral inserted to the pore space of
biochar through organo-mineral interactions in ferralsol at 5.0% biochar addition rate at the end of the incubation by scanning electron microscope (b).

Biochar addition initially accelerated the SOC mineralization in acid- 2012b) and stabilize SOC compared to relatively fresh biochar (Luo et
ic ferralsol, but induced drastic negative PE in alkaline phaeozems al., 2011). The occlusion of clay was indicated by in Fig. 4b. The silica-
through the whole incubation. Liming effect and co-metabolism was alumina mineral gradually inserted into the pore space of biochar at
supposed to be responsible for the initial positive PE. Since biochar sig- the 90th day of incubation, which would occur via adsorption (Singh
nificantly increased the pH of ferralsol, the growth of microbiome et al., 2012) and ligand exchange reactions (Joseph et al., 2010). The
would be greatly facilitated, thus to accelerate the mineralization of organo-mineral interaction was more important in the long-term soil
SOC. Whereas in phaeozems, the pH only met a slight change. The easily C sequestration by biochar.
mineralized labile fraction in biochar resulted in increased microbial In conclusion, the degradation of biochar was responsible for the en-
biomass and the simultaneous enzyme production (Kuzyakov et al., hanced CO2 emission in ferralsol, while the adsorption of native SOC at-
2009; Maestrini et al., 2015). The decreased cumulative positive priming tributed to the emission reduction of CO2 in phaeozems. pH was the
effect and correspondent decreased biochar degradation at the 7th day of main parameter in determining the degradation of biochar and PE of
incubation also indicated the co-metabolism. Although co-metabolism SOC through carbonates dissolution, affecting organo-mineral interac-
also existed in phaeozems, the decreased WEOC through adsorption tions, as well as changing the composition of SOC and microbiome.
and lower aliphatic hydrocarbon in biochar-amended phaeozems result- Since the key role of microbial community in regulating dynamics of
ed in the reduction in the accessibility of C by the microbes and enzymes CO2 emissions, studies on microbial community change under two dif-
(Lehmann et al., 2005; Lehmann et al., 2011; Zimmerman et al., 2011). ferent pH levels and its relation with CO2 emission should be conducted.
The toxicity effects of some compounds such as phthalic acid and PAHs
in biochar (Qian and Chen, 2014; Spokas, 2013) also led to the negative 4.2. pH induced microbial community composition change
PE by reducing soil microbial activity in phaeozems.
Due to the depletion of labile fraction in biochar and the stabilization The increase in bacterial diversity after biochar amendment could be
of SOC by organo-mineral interactions, the PE shifted from positive to prior attributed to the alkalinity of biochar (liming effect) in ferralsol. As
negative in ferralsol over time. This organo-mineral interaction was
also affected by soil pH. As previous studies indicated, complexation of TM7 Chloroflexi Nitrospirae Firmicutes
carboxyl/hydroxyl functional groups of organic matter and iron oxide Gemmatimonadetes Bacteroidetes Proteobacteria
surfaces via ligand exchange increases with decreasing pH (Gu et al., Cyanobacteria Actinobacteria Acidobacteria
1.0
1994; von Lutzow et al., 2006). This was proved by the earlier shift to
negative PE of SOC at lower addition rate. Furthermore, the aging of bio-
char developed negative charge through oxidation reactions would in- 0.8
crease sorption of SOC to biochar particles (Cao et al., 2009; Lin et al.,
Relative abundance

Table 2 0.6
Alpha diversity per sample of bacterial community at the end of the incubation.

Samples OTUs Shannon Simpson Chao1

a
0.4
F 1401±126a 7.623 ± 0.018a 0.9856 ± 0.0001b 1344 ± 34a
FB 0.5 1424 ± 28ab 7.508 ± 0.424a 0.9821 ± 0.0108ab 1479 ± 21ab
FB 1.0 1484 ± 128a 7.657 ± 0.540a 0.9812 ± 0.0147ab 1449 ± 110ab
FB 2.0 1652 ± 126b 7.774 ± 1.253a 0.9828 ± 0.1008a 1544 ± 102b 0.2
FB 5.0 1605 ± 481ab 7.412 ± 0.390a 0.9822 ± 0.0049ab 1493 ± 476ab
P 4732 ± 561 cd 9.932 ± 0.743b 0.9967 ± 0.0003b 4409 ± 307 cd
PB 0.5 4305 ± 118c 9.686 ± 0.271b 0.9942 ± 0.0004b 4498 ± 166 cd 0.0
PB 1.0 4885 ± 263d 10.02 ± 0.032b 0.9970 ± 0.0002b 4616 ± 233d F FB0.5FB1.0 FB2.0FB5.0 P PB0.5 PB1.0PB2.0PB5.0
PB 2.0 4363 ± 111 cd 9.868 ± 0.284b 0.9963 ± 0.0018b 4165 ± 53c
PB 5.0 4770 ± 365 cd 9.972 ± 0.013b 0.9972 ± 0.0002b 4323 ± 256 cd
Fig. 5. Relative abundance of 10 dominant microbial taxa at phylum level of ferralsol and
a
Values in the same column followed by different letters were statistically different at p phaeozems revealed by 16S rRNA high-throughput sequencing across 0.5%, 1.0%, 2.0% and
b 0.05, n = 3. 5.0% biochar addition rate over 90 days of incubation.
 Y. Sheng, L. Zhu / Science of the Total Environment 622–623 (2018) 1391–1399 1397

1.0
1.0
(a) NH4+ (b) WEOC
FB0.5-1 NH4+ P-1

WEOC FB0.5-2 NO3-

FB0.5-3 PB0.5-1
P-3 P-2
F-1 PB0.5-2

RDA2 (12.8%)

RDA2 (7.2%)
F-3
F-2 PB0.5-3
FB1.0-2 PB1.0-2
FB1.0-1 NO3-

FB1.0-3 PB1.0-1

PB2.0-1
FB5.0-1
PB5.0-1
FB2.0-3 FB5.0-2 PB2.0-2
PB2.0-3
FB2.0-1 PB5.0-2
pH
PB1.0-3 PB5.0-3
FB2.0-2 TC FB5.0-3
pH TC

-1.0
-1.0

-1.0 1.0 -1.0 1.0

RDA1 (34.8%) RDA1 (27.7%)
−
Fig. 6. Redundancy analysis (RDA) of bacterial community of and environmental variables (pH, WEOC, NH+ 4 , NO3 ) for individual samples from ferralsol (a), phaeozems (b) and biochar-
amended soils across different addition rate (0.5%, 1.0%, 2.0% and 5.0%). WEOC, water extracted organic carbon.

the bacterial diversity was the highest in neutral soils and lower in acid- to degrade recalcitrant polymers (Kirby, 2005). Previous studies also in-
ic soils (Fierer and Jackson, 2006), soil with higher biochar addition rate dicated biochar amendment was characterized by an increase in the rel-
led to higher soil pH and bacterial diversity. What's more, since a more ative abundance of Actinobacteria (Kolton et al., 2011; O'Neill et al.,
gram-negative bacteria-dominated community in neural and alkaline 2009). The relative abundances of Anaerolineae, which were assumed
soils (Feng et al., 2010) was proposed, copiotrophic phylum like to be a potential degrader of relatively recalcitrant C compounds such
Chloroflexi, Gemmatimonadetes, Bacteroidetes significantly increased as phenol (Zheng et al., 2016), met significantly increases in biochar
after biochar amendment. In addition, biochar also led to a significant treatments. At the family level, Azoarcus group which could grow with
decline in abundance of Acidobacteria and TM7, which was reported to aromatic compounds as the only source and Sphingomonas also in-
dominate in low pH soils (Hanada et al., 2014; Verastegui et al., 2014). creased in phaeozems (Rabus, 2005; White et al., 1996). Furthermore,
The metabolic quotient and enzymatic activity would also be affected those acromatic degrading bacteria would directly affect the utilization
by the pH, thus to affect the utilization of the substrates and CO2 emis- of recalcitrant C in biochar, thus to alter the PE of SOC the total CO2 emis-
sion. Previous studies indicated the utilization of glucose and N-acetyl sion in the long-term.
glucosamine by Chloroflexi was low at low pH (Miura et al., 2007). Al- In conclusion, soil pH would directly alter the bacterial diversity as
though the results of ferralsol confirmed the importance of pH, the well as indirectly changed microbiome through affecting other physico-
slightly decrease of bacteria diversity in phaeozems after biochar addi- chemical variables such as bioavailability of SOC. The alternation of SOC
tion appeared to contradict with this, implying there may be other bioavailability incorporated by the compositional heterogeneity of bio-
mechanisms. char highlighted the importance of the relationship between soil pH
Soil pH would indirectly alter microbial communities by affecting and nature of SOC on carbon sequestration. Previous study also indicat-
the molecular-level composition of native soil organic matter. Com- ed high C/N ratio led to low microbial N availability and suppressed soil
pared to alkaline soil, acidic ferralsol exhibited lower proportion of ali- C mineralization (Liu et al., 2016). The observed difference in bacterial
phatic hydrocarbon and higher aromatic compounds, resulted in diversity by soil pH would in turn affect the stability of biochar and
different microbiome. Although biochar decreased water extracted or- soil as well as the CO2 emission. As the response patterns of bacterial di-
ganic carbon (WEOC) through adsorption (Fidel et al., 2017) in both versity to physicochemical factors was synergistic effects, figuring out
soils, the proportion of aliphatic hydrocarbon and carbohydrates in- the combined effect of variables would be crucial in large-scale biochar
creased in ferralsol after biochar amendment, leading to higher bioavail- application.
ability of the substrate. Previous researches also showed higher
substrate limitation of bacteria in the acidic arenosol (Sheng et al., 4.3. Combined effect of physicochemical properties on microbial communi-
2016). As a result, the phylum Proteobacteria which exhibited ties change and carbon sequestration
copiotrophic attributes and were found abundant in soils with high C
availability (Fierer et al., 2007; Verastegui et al., 2014) increased in Soil pH was dominated factor in evaluating the richness of bacteria,
ferralsol but significantly decreased in phaeozems. In contrast, CO2 emission, as well as the priming effect. Although significant rela-
Acidobacteria which was enriched in soils with low resource availability tionship had been found for other variables, it could be ascribed to the
and associated with lower SOC mineralization rate (Fierer et al., 2007) co-variation of pH since the significant correlation (p b 0.01) among
decreased in ferralsol after biochar amendment. This was in consistence each variable (Table S3). This was in consistent with previous findings
with previous study which indicated lower substrate limitation and which found SOC and C/N ratio was significant in shaping the soil bacte-
higher gram-negative bacteria proportion in biochar-amend acidic rial community composition due to their high correlations with soil pH
soils (Sheng et al., 2016). In contrast, decrease of several aliphatic hy- (Xu et al., 2014; Zheng et al., 2016). Previous studies also suggested that
drocarbons as well as the WEOC after biochar addition contributed to microbial biogeography is controlled primarily by soil pH (Fierer and
higher diversity of oligotrophic bacteria such as Actinobacteria and Jackson, 2006; Rousk et al., 2010). Higher pH by biochar amendment re-
−
Chloroflexi in phaeozems. sulted in lower NH+ 4 content and higher adsorption of NO3 in both soils,
Besides, recalcitrant aromatic compounds such as quinoline, benzoic as well as higher activity of nitrobacteria, resulted in a relative higher
acid and phthalic acid increased in both soils after biochar addition, concentration of NO− 3 in ferralsol when the addition rate increased. Fur-
resulting higher abundance of bacteria to degrade refractory and aro- thermore, the decreased correlation coefficient between pH and PE at
matic compound (Table S5). The higher presence level of Actinobacteria the end of the incubation indicated the relative short-term effect of
in both biochar-amended ferralsol and phaeozems was due to its ability soil pH acted as a prior factor stimulating CO2 of SOC. This was in
1398 Y. Sheng, L. Zhu / Science of the Total Environment 622–623 (2018) 1391–1399

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ganic carbon in untreated and chemical treated biochars. Chemosphere 87, 151–157.
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This work was supported by the National Natural Science Founda- eral reactions of biochars in ferrosol: an investigation using microscopy. Plant Soil
tion of China (21477111 and 21621005), and the National Key Technol- 357, 369–380.
Liu, S.W., Zhang, Y.J., Zong, Y.J., Hu, Z.Q., Wu, S., Zhou, J., et al., 2016. Response of
ogy Research and Development Program of the Ministry of Science and
soil carbon dioxide fluxes, soil organic carbon and microbial biomass carbon
Technology of China (2015BAC02B00). to biochar amendment: a meta-analysis. Glob. Change Biol. Bioenergy. 8,
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Appendix A. Supplementary data Luo, Y., Durenkamp, M., De Nobili, M., Lin, Q., Brookes, P.C., 2011. Short term soil priming
effects and the mineralisation of biochar following its incorporation to soils of differ-
ent pH. Soil Biol. Biochem. 43, 2304–2314.
Supplementary data to this article can be found online at https://doi. von Lutzow, M., Kogel-Knabner, I., Ekschmitt, K., Matzner, E., Guggenberger, G.,
org/10.1016/j.scitotenv.2017.11.337. Marschner, B., et al., 2006. Stabilization of organic matter in temperate soils: mecha-
nisms and their relevance under different soil conditions - a review. Eur. J. Soil Sci. 57,
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