Environmental Pollution 227 (2017) 98e115

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Effects and mechanisms of biochar-microbe interactions in soil

improvement and pollution remediation: A review*
Xiaomin Zhu a, b, Baoliang Chen a, b, *, Lizhong Zhu a, b, Baoshan Xing c
a
Department of Environmental Science, Zhejiang University, Hangzhou 310058, China
b
Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou 310058, China
c
Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, United States

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

Article history: Biochars have attracted tremendous attention due to their effects on soil improvement; they enhance
Received 14 November 2016 carbon storage, soil fertility and quality, and contaminant (organic and heavy metal) immobilization and
Received in revised form transformation. These effects could be achieved by modifying soil microbial habitats and (or) directly
1 April 2017
influencing microbial metabolisms, which together induce changes in microbial activity and microbial
Accepted 13 April 2017
community structures. This review links microbial responses, including microbial activity, community
structures and soil enzyme activities, with changes in soil properties caused by biochars. In particular, we
summarized possible mechanisms that are involved in the effects that biochar-microbe interactions have
Keywords:
Biochar amendment
on soil carbon sequestration and pollution remediation. Special attention has been paid to biochar effects
Soil improvement on the formation and protection of soil aggregates, biochar adsorption of contaminants, biochar-
Microbial community mediated transformation of soil contaminants by microorganisms, and biochar-facilitated electron
Carbon sequestration transfer between microbial cells and contaminants and soil organic matter. Certain reactive organic
Contaminant mitigation compounds and heavy metals in biochar may induce toxicity to soil microorganisms. Adsorption and
Interaction mechanisms hydrolysis of signaling molecules by biochar interrupts microbial interspecific communications, poten-
tially altering soil microbial community structures. Further research is urged to verify the proposed
mechanisms involved in biochar-microbiota interactions for soil remediation and improvement.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
For the purposes of soil remediation, biochar generally refers to
a carbon-rich solid that is produced by the pyrolysis of biomass in
oxygen-limited conditions (Beesley et al., 2011; Chen and Chen,
2009). Biochars are applied to soil due to their potential benefits
for carbon sequestration, soil fertility, and contaminant immobili-
zation (Cao et al., 2009; Chen et al., 2008a; Jeffery et al., 2015). The
physiochemical properties of biochar are responsible for changes in
soil character including changes in pH, nutrient maintenance, and
water retention, which can induce heterogeneous responses in
microbial species. This response can result in changes in microbial
community structure and can consequently alter soil element
*
This paper has been recommended for acceptance by Dr. Hageman Kimberly Jill.
* Corresponding author. Department of Environmental Science, Zhejiang Uni-
versity, Hangzhou 310058, China.
E-mail addresses: zhuxm@zju.edu.cn (X. Zhu), blchen@zju.edu.cn (B. Chen), zlz@
zju.edu.cn (L. Zhu), bx@umass.edu (B. Xing).
cycling and function (Biederman and Harpole, 2013; Lauber et al.,
2009; Rousk et al., 2009, 2010). There are some components in
biochar, including minerals, volatile organic compounds (VOCs),
and free radicals (Spokas et al., 2011), that can potentially influence
microbial activity, reshape the soil microbial community, and
change the soil enzyme activity that catalyzes various key biogeo-
chemical processes including soil organic matter turnover and
elemental cycles (e.g., N, P, and S) (Paz-Ferreiro et al., 2014). Due to
the various positive effects on the soil properties and microbes,
biochars are considered effective agents for soil remediation.
However, the variability among different types of biochar makes its
effects on soil remediation quite unpredictable, and the specific
mechanisms of biochar-microbe interactions are still unclear.
The use of contaminant-degrading microbe inoculation
(mycoremediation) together with biochar can enhance the bio-
logical degradation of pollutants (e.g., PAHs) (Chen and Ding, 2012;
Chen et al., 2012a; Garcia-Delgado et al., 2015), providing a prom-
ising method for soil contaminant remediation. Such a process is
considered a combination of the immobilization of the pollutants
by the biochar and the further degradation of these pollutants by

http://dx.doi.org/10.1016/j.envpol.2017.04.032
0269-7491/© 2017 Elsevier Ltd. All rights reserved.
 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
microbes. The adsorption of organic contaminants, toxic heavy
metals, and hazardous anions (e.g., ClO
4 ) by biochar immobilizes
them and prevents their leaching into the groundwater (Cao et al.,
2009; Chen and Yuan, 2011; Chen et al., 2012d; Fang et al., 2014b;
Jones et al., 2011; Yang et al., 2016b). Further transformation and
detoxification of environmental pollutants by microbes as cata-
lyzed by biochar have drawn increasing attention in recent studies
(Dong et al., 2014; Oh et al., 2013). Persistent free radicals (PFRs)
that are formed on biochar during thermal decomposition of the
feedstocks activate reactive oxygen species (ROS) (Fang et al.,
2015b; Kluepfel et al., 2014; Yu et al., 2015), and the electron
transfer between biochar and microbial cells plays an important
role in organic contaminant degradation and heavy metal trans-
formation (Dong et al., 2014; Fang et al., 2015a; Yang et al., 2016a;
Yu et al., 2015).
Biochar can participate in soil processes such as organic matter
decomposition as it takes part in the direct extracellular electron
transfer (DEET) between soil organic matter (or soil minerals) and
microbial cells, as well as in the direct interspecific electron transfer
between microbial cells (DIET) (Chen et al., 2014; Fang et al., 2014a).
The identification and quantification of the reactive components of
biochar particles that are responsible for the electron transfer be-
tween the biochar and soil microbes are essential to investigate
biochar-involved elemental cycling. The electron transfer between
biochar particles and soil minerals, organic matter, pollutant mol-
ecules, and microbial cells, as well as in response of the microbial
community to the reactive components of the biochar, is an
emerging research field that seeks to further clarify the effects of
biochar on soil biogeochemical processes.
Several studies in the past decade extensively discussed the
structure, physiochemical properties, and structure-function re-
lationships of biochar with respect to the apparent effects of bio-
char on soil (Ahmad et al., 2014; Ameloot et al., 2013; Chen and
Yuan, 2011; Chen et al., 2008a, 2012b; Lehmann et al., 2011;
Warnock et al., 2007); however, an understanding of biochar-
microbe interactions is a research gap that would link biochar
properties with many soil processes, e.g., carbon storage and
contaminant degradation. With regard to the newly found electron
transfer and free radical activation functions of biochar, the
mechanisms of biochar effects can be more clearly identified (Fang
et al., 2015b; Kappler et al., 2014). Therefore, this review aims to
seek answers for the following questions: (1) What are the most
essential physiochemical properties of biochar that influence the
microbial activity and community, and how do these properties
influence the microbes? (2) Mechanistically, how does the inter-
action of the soil microbes with different types of biochar affect soil
carbon sequestration and contaminant dissipation? Answers to
these questions are essential to identifying the cause of the het-
erogeneous effects of biochar in existing research and to develop
the required understanding to accurately predict biochar effects,
both of which are key challenges in this research field.
2. Driving mechanisms of biochar-microbe interaction in soil
Biochar affects the soil microbial activity and biomass, changes
the soil bacteria to fungi ratio and soil enzyme activity, and re-
shapes the microbial community structure (Ahmad et al., 2016;
George et al., 2012; Mackie et al., 2015; Nielsen et al., 2014;
Rutigliano et al., 2014). Note that biochar application may signifi-
cantly alter the microbial community structure even when it does
not change the overall microbial activity and biomass. To clearly
interpret the microbial responses to biochar application in soils,
gene copy numbers can serve as a more sensitive parameter than
microbial biomass (Chen et al., 2013). Various techniques are used
to test microbial activity and community structure, including
99
ergosterol extraction, quantitative real-time polymerase chain re-
action (q-PCR), fluorescence in situ hybridization (FISH), phos-
pholipid fatty acid quantitation (PLFA), molecular fingerprinting of
16S rRNA gene fragments including denaturing gradient gel elec-
trophoresis (DGGE) and terminal restriction fragment length
polymorphism (TRFLP), and high-throughput sequencing (also
known as next-generation sequencing, or NGS) of soil microbial
genes (Chen et al., 2013; Hale et al., 2014; Kolton et al., 2011; Mackie
et al., 2015; Rousk et al., 2009). Changes in the relative abundances
of Acidobacteria, Actinobacteria, Gemmatimonadetes, and Verru-
comicrobia are frequently detected using high-throughput
sequencing, under treatment with biochar (Mackie et al., 2015;
Nielsen et al., 2014). With higher resolution to the species level,
the metagenomics sequencing of microbial genes is able to realize
function annotation reflected by the soil microbial community
structure changes (Ja €ckel et al., 2004). Such a process is essential to
explain the effects of biochar on soil remediation (Chen et al., 2013;
Hale et al., 2014; Kolton et al., 2011; Mackie et al., 2015; Rousk et al.,
2009). Since the mechanisms underlying biochar's effects on mi-
crobes and related soil functions and processes are still not quite
clear, this review focuses on the synthesis of several possible
mechanisms based on the published research.
The influences of biochar on microbial activity are diverse and
seven possible mechanisms are demonstrated in the central circle
of Fig. 1 (from which points 1 to 3 can be classified into direct in-
fluences, and points 4 to 7 indirect influences): (1) biochar provides
shelter for soil microbes with pore structures and surfaces
(Quilliam et al., 2013a); (2) biochar supplies nutrients to soil mi-
crobes for their growth with those nutrients and ions adsorbed on
biochar particles (Joseph et al., 2013); (3) biochar triggers potential
toxicity with VOCs and environmentally persistent free radicals
(Fang et al., 2014a); (4) biochar modifies microbial habitats by
improving soil properties that are essential for microbial growth
(including aeration conditions, water content, and pH) (Quilliam
et al., 2013a); (5) biochar induces changes in enzyme activities
that affect soil elemental cycles related to microbes (Lehmann et al.,
2011; Yang et al., 2016b); (6) biochar interrupts microbial intra- and
inter-specific communication between microbial cells via a com-
bination of sorption and the hydrolysis of signaling molecules (Gao
et al., 2016; Masiello et al., 2013); it should be noted that biochar
may contain some molecules that can work as signals for microbial
communication; and (7) biochar enhances the sorption and
degradation of soil contaminants and reduces their bioavailability
and toxicity to microbes (Beesley et al., 2010; Qin et al., 2013;
Stefaniuk and Oleszczuk, 2016). The proposed mechanisms
involved in biochar-microbe interactions need further experi-
mental verification, and a research emphasis should be placed on
the linkage between biochar-microbe interaction mechanisms and
their environmental effects.
2.1. Biochar provides shelter for microbes
One hypothesis of the benefits of biochar for microorganisms is
that biochars can be shelters for microbes due to their pore struc-
tures. Biochars provide more habitable pore volume per unit vol-
ume than soil does (Quilliam et al., 2013a). Microbial living cells can
attach on biochar surfaces; in such cases, biochars with large spe-
cific surface areas can provide habitats for microbes as well (Abit
et al., 2012). However, the colonization of bacterial cells and
fungal hyphae has spatial heterogeneity between the external and
internal pores of biochar (Quilliam et al., 2013a). Different microbial
colonization patterns on the surfaces and in the pores of biochar
can be explained by three phenomena: 1) there is less nutrient
accessibility in biochar pores than in natural soil pores, 2) the
biochar pores can be blocked with soil organic matter (e.g., humic
100 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115

Fig. 1. Proposed mechanisms of biochar-microbe interactions and the environmental effects of biochar. The central circular area illustrates the interaction between biochar and
microbes, while the enclosing four boxes represent the effects of their interaction on carbon sequestration, soil processes (elemental cycling), contaminant degradation, and plant
growth. Interactions between the biochar and the microbes and its effects include: (1) biochar can act as a microbial shelter with its pore structure; (2) through sorption of nutrient
cations via functional groups, biochar can improve soil CEC and maintain nutrients for microbial growth; (3) free radicals and VOCs on biochar can be toxic to some soil microbes,
inhibit soilborne pathogens, and favor plant growth; (4) biochar can improve soil properties (e.g., pH, water content, and aeration conditions), and change the growth pattern of soil
microbes; (5) biochar can adsorb enzyme molecules, influence soil enzyme activities and elemental cycles; (6) biochar can adsorb and enhance the hydrolysis of signaling mol-
ecules, and consequently interrupt microbial communication and alter microbial community structure; (7) biochar can enhance the sorption (via biochar surface functional groups)
and degradation of soil contaminants (facilitated through electron transfer between biochar, microbes, and contaminants), which can reduce the toxicity of contaminants to soil
microbes. The interactions between biochar and soil microbes can alter the microbial community and their metabolic pathways (which can be revealed by metagenomics analysis of
microbial DNA sequencing), resulting in changed soil processes. There are interactions among different environmental effects as well.

Fig. 2. The interaction between microbial cells and biochar particles as observed with scanning electron microscopes (SEM). (A) Pine biochar inoculated with a co-culture of
Geobacter metallireducens (rods) and Methanosarcina barkeri (spheres) for a short time period (20 days, scale bar ¼ 1 mm) (Chen et al., 2014) and (B) Field-aged biochars buried in
agricultural soil for a long time period (3 years, scale bar ¼ 5 mm) (Quilliam et al., 2013a). (A) and (B) both show the attachment of microbial cells on biochar surfaces and pores. In
(C), the mineral phases have entered the pores of wheat-straw biochar (Joseph et al., 2013). The SEM images show that the mineral phases have undergone considerable reactions
that produce complex morphologies (Joseph et al., 2013).

acids), and 3) toxic substances, such as PAHs, may be present in
biochar (especially in fresh biochar) (Kasozi et al., 2010; Quilliam
et al., 2013a, 2013b). Microbial colonization on biochar surfaces
and pores is also dependent on the biochar aging process, which
can be considered a temporal heterogeneity (Quilliam et al., 2013a).
Bacterial cells from co-cultures of Geobacter metallireducens and
Methanosarcina barkeri are able to attach themselves on biochar
surfaces during a very short time period (20 days, as shown in
Fig. 2A). The colonization of soil microbes (both fungal hyphae and
single bacterial cells) on surfaces and in the pores of biochar can be
improved by adjusting the aging periods of biochar (3 years, as
shown in Fig. 2B) (Quilliam et al., 2013a).
 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115 101

2.2. Biochar supplies nutrients to soil microbes
Biochar contains a range of nutrients (e.g., K, Mg, Na, N, and P)
(Chathurika et al., 2016; Rodriguez-Vila et al., 2016) and enriches
soil nutrients via sorption that is due to its large surface area, high
pore volume, and negative surface charge (Chen et al., 2012c).
Cation exchange capacity (CEC) is a critical indicator of soil's ability
to retain cationic nutrients and supply nutrients to support mi-
crobial activity. The improved soil CEC that results from biochar
application reflects a higher nutrient retention capability and a
lower nutrient loss through leaching, which is beneficial for soil
microbial activity (Lehmann, 2007b), especially for microbes living
in soils with low organic matter content (de Andrade et al., 2015;
Laird et al., 2010; Mukherjee et al., 2011; Silber et al., 2010). The
nutrient conditions of biochar (indicated by ash content) are largely
determined by its feedstock and pyrolysis temperatures. Biochars
produced from herbaceous material (crop residues) and manure
generally have higher ash content than wood biochar and are
consequently able to supply more nutrients (Fig. 3A) (Akhter et al.,
2015). A clear rising curve between ash content with the pyrolysis
temperatures has been found for crop residue biochar and manure
biochar (Xu and Chen, 2013). The nutrients from biochar can be
released with different rates into soils (Mukherjee and
Zimmerman, 2013). In this case, biochar can act as a slow-release
fertilizer, bringing long-term benefits to soil fertility and microbi-
al growth. Notably, another reason that biochar supplies nutrients
for soil microbes might be that it regulates soil microbial functions
that are essential for nutrient cycling. For example, biochar can
enhance the abundance of rhizobacteria that are able to transform
organic S and P into bio-available forms, which further promotes
the growth of Lolium perenne (Fox et al., 2014) and are reasonably
suspected to promote the growth of other microbes that can only
utilize inorganic S and P.
Biochar stores and supplies nutrients to soil microbes via the
sorption of nutrient cations and inorganic anions with its surface
functional groups, especially oxygen-containing groups such as the
carboxylate group (Fig. 4) (Chen et al., 2015; El-Naggar et al., 2015;
Jien and Wang, 2013; Liang et al., 2006; Mukherjee et al., 2011;
Yuan et al., 2016). Some studies showed that biochar maximized
CEC at low and moderate pyrolysis temperatures, while to the
contrary, some other studies showed that biochar CEC increased
with the pyrolysis temperature (Lehmann, 2007a; Mukherjee et al.,
2011; Yuan et al., 2011). The feedstock types and pyrolysis program
parameters, including temperatures, heating rate, and holding
time, primarily determine the biochar functional groups and
consequently the capability of biochar to improve the soil CEC

Fig. 3. Biochar properties as function of pyrolysis temperature and feedstock types. (A) to (D) show the changes in ash content, specific surface area (SSA), pH, and volatile matter
content (VM), respectively, with pyrolysis temperature. Different colors and shapes indicate different feedstock types, and linear (or nonlinear) regressions between biochar
properties with pyrolysis temperatures are analyzed; the regression and its 95% confidence and 95% prediction bands are illustrated (feedstock types are not distinguished, analyzed
with the software SigmaPlot). Positive relationships between pH and ash content with pyrolysis temperature are found, while negative relationships between VM content with
pyrolysis temperature is found. An increased ash content with temperature is more obvious for manure biochar and crop residue biochar than for wood biochar (A), while an
increased SSA with pyrolysis temperature is only found for wood biochar (B). “Other biochars” include distilled grain biochar and sugar cane bagasse biochar. Data are from Table S1.
102 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115

Fig. 4. Schematic diagram showing the roles of biochar functional groups (AFG ¼ acidic functional groups, SOM ¼ soil organic matter): (1) The AFG are responsible for the liming
effect of biochar which modifies the soil microbial habitat; (2) the electrostatic attraction between the carboxyl groups of biochar with the nutrient cations effectively retains
nutrients to ensure a nutrient supply to soil microbes and (3) to immobilize heavy metals, thus reducing heavy metal toxicity to microbial cells; (4) electrostatic attraction, as well as
polar and non-polar organic attraction, of humic acid molecules can result in the adsorption of soil organic matter that is beneficial for carbon sequestration (further discussed in
later chapter); (5) hydrogen bonding between eOH groups on biochar with oxygenated anions can adsorb inorganic anions to supply nutrients or reduce anion contaminant
toxicity; (6) electron transfer to form free radicals on the biochar surface can facilitate organic contaminant degradation and heavy metal transformation (to be detailed discussed in
later chapter) and can reduce contaminant toxicity to microbes.

(Table S1) (Chen et al., 2008a; Jeong et al., 2016; Mukherjee and
Zimmerman, 2013; Yuan et al., 2011). The biochar CEC was pH
dependent, increasing from low to neutral environmental pH
values (Lehmann, 2007a), which indicated possible interactive ef-
fects between the changes in the pH and the CEC in soils with
biochar. Additionally, interaction between biochars and soil min-
erals can be responsible for the long-term maintenance of the
minerals during the aging of biochar (Novak et al., 2009). For
example, a complex reaction between biochar (wheat straw) and
soil clay as well as extra added chemical fertilizers (including
CO(NH2)2, KCl and monoammonium phosphate) can result in some
unusual morphologies (not identified) on biochar surfaces, as
indicated in Fig. 2C (Joseph et al., 2013). From the above results, the
various aspects of biochar preparation including feedstock types,
pyrolysis temperatures, the aging period, and the relationships
between various biochar properties (e.g., pH and CEC) should be
fully considered during the field application of biochar to improve
the nutrient supply to soil microbes.
The aromaticity of biochar is responsible for its recalcitrant
nature against microbial decomposition (reflected by the atomic H/
C and O/C ratios), and some fractions of biochar can also function as
a carbon source for soil microbes (the quality of biochar as a carbon
source is indicated by its C/N ratio, Table S1, Fig. S1) (Demisie et al.,
2014). Biochars are typically low in available carbon for microbial
utilization because they contain higher C/N ratios than their feed-
stocks and are thus difficult to degrade by microorganisms because
of the lack in N supply. As a consequence, biochar amendment
increases the recalcitrant SOC pool, enhancing soil carbon seques-
tration (Yanardag et al., 2015). Bacteria and fungi have their own
preferences for different carbon sources and have different toler-
ances to changes in environmental factors, such as pH and water
condition (Rousk et al., 2009; Zhang et al., 2015). Compared with
bacteria, fungi are able to colonize soil macroaggregations
(>200 mm) that contain a higher SOC, ratio of C/N, and total N
(Zhang et al., 2015). This ability may be due to the advantages of the
living strategy of fungi; specifically, the translocation of nutrients
and water within a continuous hyphal network enables fungi to
colonize poor carbon sources with a high C/N, such as biochar
(Ascough et al., 2010). Therefore, biochar application that promotes
the formation of macroaggregates should favor fungi growth rather
than bacteria under the same environmental condition. Both
atomic H/C and O/C ratios commonly decrease with increased py-
rolysis temperature, indicating a more intensive aromatic and
nonpolar structure of the biochar (Fig. S1) (Hale et al., 2015; Xiao
et al., 2016). Biochar C/N ratios are mainly dependent on feed-
stock types; for example, the C/N of wood biochar is normally
higher than that of manure biochar (Cantrell et al., 2012; Chen et al.,
2008a). In addition, the increased pyrolysis temperature can cause
elevated C/N in some feedstocks due to the condensed aromatic
carbon structure (Table S1, Fig. S1B) (Cantrell et al., 2012; Spokas
et al., 2011). Therefore, the pyrolysis temperature and feedstock
type are useful when considering the stability of biochar C against
microbial decomposition in practical application.
 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
2.3. Potential toxicity of biochar to microbial cells
Certain compounds in biochar are known as microbial in-
hibitors, including benzene (the dominant product of pyrolysis
during glowing combustion of charcoal), methoxyphenols and
phenols (the products of pyrolysis of hemicelluloses and lignin),
carboxylic acids, ketones, furans (that are generally presented as
the sorbed VOCs on biochar), and PAHs (Ghidotti et al., 2017; Lyu
et al., 2016; Spokas et al., 2011). Such compounds can be identi-
fied from water or organic solvent extractions of biochar. Organic
solvent extracts from biochar contain organic compounds from
many classes, including n-alkanoic acids, hydroxyl and acetoxy
acids, benzoic acids, diols, triols, and phenols, while water extracts
from biochar contain dicarboxylic acids, aromatic organic acids and
polyol along with hydroxy acids, n-alkanoic acids, and benzoic
acids (Table 1) (Graber et al., 2010). Biochar VOCs vary with feed-
stock type, elemental composition, pyrolysis temperature, and
heating procedures and conditions (Spokas et al., 2011). Higher
concentrations and toxicities of PAHs and polychlorinated dioxins
and furans (PCDD/DF) are found in biochar generated at moderate
temperatures (300 and 400  C) than at high temperatures
(>400  C) (Lyu et al., 2016). The diversity of composition for the
VOCs species sorbed on biochar can be the main contributing factor
to the various responses of soil microbial activity to biochar.
Although volatile organic compounds (VOCs) in fresh biochar can
support the survival of some microbes (such as Bacillus mucilagi-
nosus) as a carbon source (Sun et al., 2015), they can induce po-
tential toxicity to microbes (not species-specific) when present in
high concentrations (especially for some low molecular weight
oxygenated VOCs, including acids, alcohols, and carbonyls) (Ennis
et al., 2012; Spokas et al., 2011). As microbial inhibitors, VOCs can
also induce direct toxicity to microbial soil pathogens, thus
benefitting plant growth (Graber et al., 2010). However, the inhi-
bition may not be very specific to pathogens, and the alteration of
the microbial community needs further investigation to evaluate
the environmental benefits and risks of biochar application.
Persistent free radicals (PFRs) that are generated and stabilized
during the pyrolysis of biochar, including semiquinones, phenoxyls,
cyclopentadienyls, and phenols, can also induce toxicity to mi-
crobes (Truong et al., 2010). The formation of free radicals on bio-
char includes replacing surface functional groups on metal oxides
with aromatics while metal oxides are being reduced (Fig. 5I)
(Balakrishna et al., 2009; Doong et al., 2014; Fang et al., 2014a) and
breaking chemical bonds in macromolecules (Fig. 5II) (Dellinger
et al., 2007; Liao et al., 2014). The PFRs are abundant (approxi-
mately 1017 spins g
1 pine needle biochar) and depends on the
pyrolysis temperature and time (Fang et al., 2015a). The adverse
impacts initiated by biochar PFRs were previously misidentified
because of the lack of proper methods to separate those effects
caused by free radicals from those caused by co-existing molecular
pollutants; they have yet to be taken into account in current
research (Liao et al., 2014; Truong et al., 2010).
Free radicals can induce oxidative stress in living microbial cells,
reduce cellular glutathione (GSH), glutathione peroxidase (GPx),
and superoxide dismutase (SOD) levels, and decrease cell mem-
brane integrity with the generation of reactive oxygen species
(ROS) such as hydroxyl radicals (OH), the superoxide radical anion
(O
2 ), and hydrogen peroxide (H2O2) (Balakrishna et al., 2009;
Dellinger et al., 2007; Liao et al., 2014). Free radicals interfere
with cytochrome P450s (e.g., CYP1A2, a member of the ubiquitous
superfamily of enzymes that catalyze the mixed-function oxygen-
ation of both endogenous and foreign compounds) and competi-
tively inhibit the metabolism of exogenous organic substrates
(Reed et al., 2015). Semiquinone radicals (QH) are one type of free
radicals that are found in combustion-generated ultrafine
103
particulate matter; these radicals can reduce oxygen to form su-
peroxide, which then forms H2O2 that can initiate Fenton reactions
in the presence of transition metal ions such as Fe2þ and Cu2þ
(which are ubiquitous in biological systems). These Fenton re-
actions generate OH that causes DNA strand breaks, which lead to
DNA damage (Dellinger et al., 2001). On the other hand, free radi-
cals from biochar can play an important role in the degradation of
organic pollutants with the ROS induced by the biochar, especially
the strongest species: the hydroxyl radical (OH) and the sulfate
radical anion (SO
4 ) (Fang et al., 2015a, 2015b), which will be dis-
cussed later. Both the potential toxicity of biochar free radicals on
soil microorganisms and the positive effects of biochar free radicals
on pollutant degradation are two sides of the coin to be considered
in the evaluation of the environmental effects of biochar.
2.4. Biochar modifies microbial habitats
Biochar can modify microbial habitats by improving the soil's
physical properties. Biochar porosity can decrease soil bulk density,
improve soil aeration condition (Abel et al., 2013), and control the
transport of soil microbes in biochar amended soil (Abit et al.,
2012). Biochar can increase the available water content that in-
fluences nutrient accessibility to microbial cells (Abel et al., 2013).
In addition, the biochar can increase water content at the perma-
nent wilting point, which indicates the capability of biochar, via its
high porosity, to store water unavailable to plants; keeping water in
this manner is beneficial, especially in sandy and degraded soils
(Abel et al., 2013). Moreover, the improved water retention capacity
means that there is a greater capability of the soil to hold water
against dry-wet cycles in the natural environment, which can favor
the maintenance of a stable microbial activity (Liang et al., 2014).
The pyrolysis parameters (mainly temperature, heating rate and
time) and feedstock compositions (e.g., lignin and lipid concen-
trations) of the preparation of biochar control its porosity, carbon
stability, and the surface adsorption of nutrients (Abit et al., 2012;
Cantrell et al., 2012; Chen et al., 2008a). Although an increasing
trend of specific surface area with pyrolysis temperatures is found
for wood biochar, this relationship is not found for biochars
generated from other feedstock types such as manure and crop
residue, indicating the extraordinarily important role of feedstock
type on the formation of large SSAs (Table S1, Fig. 3B) (Chen et al.,
2008a; Wang et al., 2015). The role of biochar in improving soil
properties and modifying microbial habitats can be dependent on
the feedstock types and pyrolysis procedures used in making
biochar.
Biochar can be an effective liming agent to neutralize soil pH
(Yuan et al., 2011). An increase of the soil pH by 0.2e0.3 units after
biochar application can be the main factor affecting the soil mi-
crobial community, in contrast to other chemical variables in soil
such as the total C content, total N content, electrical conductivity
(EC), and NO
þ
3 and NH4 concentrations (Nielsen et al., 2014). An
increase in the soil pH and a decrease in the toxicity of exchange-
able Al in acidic soils with biochar amendment (Qian et al., 2013)
increase the bacteria abundance in the pH range from 4 to 7; the
abundances of bacteria are positively correlated with soil pH (Leal
et al., 2015; Rousk et al., 2010). Fungi and bacteria have different
sensitivities to soil pH (Rousk et al., 2009), and bacteria are
generally more tolerant to a narrower range of pH and are more
sensitive to soil pH changes than fungi are (Rousk et al., 2010). As a
result, there are likely to be different responses from bacteria and
fungi to biochar-induced changes in soil pH, and the microbial
community of fungi and bacteria may react in different ways to
biochar-induced changes in soil pH, which can result in an alter-
ation of the overall microbial community structure. A pyrose-
quencing analysis of the soil bacterial community showed
104 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115

Table 1
Organic compounds frequently detected from various biochar types as VOCs, organic solvent extracts, or water extracts of biochar.
 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115 105

significant correlation between the soil pH and the soil bacterial
community composition. The relative abundances of Acidobacteria,
Actinobacteria, and Bacteroidetes were largely driven by the range of
soil pH values (3.5e9.0) at the continental scale from North to
South America (Lauber et al., 2009). Increasing the soil pH with
biochar application altered the abundance, diversity, and compo-
sition of nitrifying bacteria in soil and changed soil nitrification as a
consequence (Zhang et al., 2017). Pyrolysis temperature domi-
nantly determined biochar pH values, which usually rise with
increased pyrolysis temperature for various feedstocks (Fig. 3C).
This trend is mainly attributed to the decreased number of acidic
functional groups (AFG) and decreased volatile matter (VM) con-
tent (Fig. 3D) (Mukherjee et al., 2011), yet there is an increased
alkalinity (due to carbonates and alkaline ash content) of biochar
106 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
Fig. 5. Schematic of the mechanisms of PFR formation and free radical generation on biochar, including: I) the interaction between organic compounds containing oxygenous
functional groups and metal oxidation, and II) the breaking of chemical bonds in macromolecules during heating and cooling. The figure is modified from the reference (Fang et al.,
2014a; Liao et al., 2014).
with increased pyrolysis temperature (Smebye et al., 2016; Yuan
et al., 2011). Therefore, high temperature biochars are normally
more effective at improving the soil pH (Cantrell et al., 2012; Cao
and Harris, 2010; Mukherjee et al., 2014; Yuan et al., 2011).
2.5. Biochar alters soil enzyme activity
Most of the elemental turnover in soil, which determines the
nutrient bioavailability and includes turnover of C, N, P, and S, is
catalyzed by enzymes. Soil enzymatic activities respond faster than
other soil variables to soil management and disturbance introduced
into soil and are therefore indicators of biological changes and soil
quality (Bandick and Dick, 1999). Enzyme activities respond to
biochar application in various ways, mainly depending on the types
of enzymes and biochar, the biochar application rate, and soil
properties (Table S2). The decreased microbial abundance and ac-
tivity of soil enzymes in relation to organic matter decomposition
can enhance C sequestration (Luo and Gu, 2016). Possible mecha-
nisms involved in the influence of biochar on enzyme activity
include but may not be limited to: (1) biochar adsorbs extracellular
enzyme molecules and/or substrates on the surface or blocks the
reaction site of enzymes (Bailey et al., 2011) in a way regulates their
apparent affinity with substrates (Paz-Ferreiro et al., 2015); (2)
biochar influences enzyme activity with changes in soil physi-
ochemical properties (especially pH) (Zimmerman and Ahn, 2010);
and (3) biochar releases some small molecules that are speculated
to act as allosteric regulators or inhibitors of specific enzymes (such
as a possible up-regulation of beNeacetylglucosaminidase activity
with ethylene) (Bailey et al., 2011).
The sorption (binding) of enzymes on biochar and soil organic
matter can change the kinetic properties of enzyme activity
(Lammirato et al., 2011; Nannipieri et al., 2012) and therefore is the
most important mechanism regulating the soil enzyme activity
(Zimmerman and Ahn, 2010). The adsorption efficiency of the
enzyme and substrates depends on the biochar structure: adsorp-
tion of enzyme molecules on biochar surfaces is considered to be
driven by non-coulombic forces between the uncharged regions of
the protein and the uncharged regions of the biochar surface, and
the adsorption of small molecular polar substrates (e.g., disaccha-
ride) on charred fractions (especially activated carbon) is stabilized
via hydrogen bonding to polar surface groups (e.g., COOH, SO4H,
PO4H) on the sorbents (Lammirato et al., 2011). Changes in surface
functional groups in aged biochar alter the adsorption capacity of
enzyme and substrates, thus affecting enzyme activity (Gibson
et al., 2016). For example, the oxidation of aromatic carbon and
the introduction of aliphatic C-H groups on biochar during the
abiotic aging process improved laccase and peroxidase activities
and enhanced fungal respiration (Gibson et al., 2016). Biochar can
reduce the activation energy (Ea, related to the temperature
sensitivity of an enzyme) of an enzyme-catalyzed reaction and
down-regulate the enzymatic sensitivity to temperature changes
(in terms of Q10), resulting in enhanced activities of b-glucosidase
and arysulfatase (Paz-Ferreiro et al., 2015). A reduced Ea under
biochar application indicates higher substrate affinity (in term of
apparent Km) and less temperature sensitivity of the enzyme,
possibly due to sorption of substrates and enzymes on biochar (Paz-
Ferreiro et al., 2012). On the other hand, soil enzymes respond
quickly to soil management (e.g., organic matter amendment)
 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
(Bandick and Dick, 1999), and thus changes in soil properties with
biochar application can reasonably influence soil enzyme activities.
For the third mechanism, inhibitors released from biochar can
interfere with enzyme-catalyzed reactions as well: for example,
during pyrolysis, plant biochars can release a number of benzofu-
rans, polycyclic aromatic hydrocarbons, and heterocyclic com-
pounds, which are inhibitory compounds to soil enzymes
(Lammirato et al., 2011).
2.6. Biochar affects intra- and interspecific communication of
organisms
Biochar modifies microbial cell-to-cell communication via the
sorption of signaling molecules (e.g., an N-acyl-homoserine
lactone, AHL) (Masiello et al., 2013) and, perhaps more importantly,
by enhancing the hydrolysis of AHL (Gao et al., 2016). The inter-
cellular signaling molecule N-3-oxo-dodecanoyl-L-homoserine
lactone is an AHL that is used by many gram-negative soil bacteria
(e.g., nitrogen-fixing plant symbionts and pathogens that cause soft
rot in plants) to regulate gene expression and intraspecific
communication (Masiello et al., 2013). When sorption is the main
mechanism, this effect is dependent on pyrolysis temperature,
which influences the biochar adsorption capacity (Masiello et al.,
2013). High-temperature biochar (700  C), with higher specific
surface area (SSA), adsorbed the signaling molecule (AHL) and
interrupted the inter-cell transfer of the signal to a larger extent
than moderate temperature biochar (300  C), with a much lower
SSA, did (Masiello et al., 2013). By adjusting the soil pH, biochar is
able to inhibit or promote the exchange of signaling compounds
that regulate specific soil microbial activity (Warnock et al., 2007).
Additionally, the increase in soil pH induced by biochar can drive
the hydrolysis of the signaling AHLs, thereby reducing the number
of bioavailable AHLs and consequently inactivating the bacterial
cell-to-cell communication (Gao et al., 2016). While some signaling
molecules for bacterial communication (such as AHL) are pH-
sensitive, some fungal signals (such as farnesol, a fungal auto-
inducer) are less sensitive to pH, possibly leading to shifts in the
fungi to bacteria ratio (and a change in the soil microbial commu-
nity structure) with biochar application (Gao et al., 2016).
Biochar can modify microbe-to-plant communication in the
rhizosphere (Harel et al., 2012), affect the competition between
beneficial soil microbes and soilborne pathogens (Akhter et al.,
2015), and trigger systemic plant defenses to soilborne pathogens
(Akhter et al., 2015; Elad et al., 2011). Application of biochar suc-
cessfully induced resistance against two foliar fungal pathogens
(Botrytis cinerea and Leveillula taurica) for both pepper and tomato
plants (Elad et al., 2010). This action is regulated by either the
enhanced interaction between plant roots and the plant-growth-
promoting rhizobacteria (PGPR) or fungi (PGPF) (Harel et al.,
2012) or by the direct toxic effects of certain biochar components
on microbial pathogens (Graber et al., 2010). Both wood biochar
and green waste biochar, when amended to the potting medium,
stimulated a range of general defense pathways of strawberry
plants and as a result suppressed anthracnose disease caused by the
fungal pathogens Botrytis cinerea, Colletotrichum acutatum, and
Podosphaera aphanis (Harel et al., 2012). It was found that higher
application rates (3%) of biochar induced higher defense ability
against the pathogen-caused plant disease than low application
rates (1%) of biochar did (Harel et al., 2012). Moreover, there are also
various influences of different biochar types (wood and green waste
biochar) on the competition between arbuscular mycorrhizal fungi
(AMF) and the soilborne pathogens (e.g., Fusarium oxysporum f. sp.
lycopersici) in the mycorrhization on tomato roots (Akhter et al.,
2015). The variability in the amount and types of biochar VOCs
107
that can act as microbial inhibitors (Spokas et al., 2011) may be the
reason for the uncertainty in biochar-triggered pathogen resis-
tance. To better reveal the mechanisms, the identification and
quantification of reactive compounds from biochar, which are able
to interfere with microbial communication, is an important
research field.
2.7. Biochar reduces the toxicity of contaminants to soil microbes
Biochar as a soil ameliorant can decrease the toxicity of soil
contaminants to soil microbes (Koltowski et al., 2017). Willow
biochar (pyrolyzed at 700  C) can reduce the microbe mortality
while increasing the reproduction of Folsomia candida in soils
contaminated with heavy metals and organic pollutants (PAHs) and
can decrease the leachate toxicity to the bacterium Vibrio fischeri
(Koltowski et al., 2017). The immobilization of soil contaminants
(including heavy metals such as Al, Cd, Co, Cr, Mn, and Ni, and
organic pollutants such as PAHs) on biochar and the consequent
reduction of their bioavailability can be the main reason for the
alleviated toxicity of soil contaminants to microbes and the
elevated microbial biomass (Qian et al., 2013, 2015; Seneviratne
et al., 2017; Zielinska and Oleszczuk, 2016). Rice straw biochar
application (5% application rate) can result in an increase of the
organic-bound fraction of heavy metals (Cd, Cu, Pb, and Zn) by up to
68% (Lu et al., 2017). The reduced heavy metal stress on N-fixing
bacteria (Bradyrhizobium japonicum) could further benefit the N
supply for plant growth (Seneviratne et al., 2017). In addition to the
effect of reducing the toxicity of contaminants to soil microbes, thus
enhancing microbial activity, interactions between the biochar and
soil microbes have more profound effects on the environmental
fate of soil contaminants, including their immobilization and
degradation, which are to be discussed in a later chapter of this
review.
3. Soil carbon storage under biochar-microbe interaction
Biochar can enhance soil carbon sequestration by various
mechanisms. In addition to the stable carbon structure and its own
recalcitrant nature (Guo and Chen, 2014; Xiao et al., 2014), biochar
can affect carbon cycle dynamics that involve microbial participa-
tion, leading to either positive or negative priming effects on native
organic matter, which represent enhanced or inhibited degradation
of native soil organic carbon (SOC), respectively, when extra carbon
sources are added (Bamminger et al., 2014; Cely et al., 2014;
Kuzyakov et al., 2000; Zimmerman et al., 2011). The interactions
between biochar, soil microbes, and native SOC account for the
stability of biochar C and native SOC, in addition to having an
overall effect on carbon sequestration (Fig. 6). The balance between
the decomposition and sequestration of biochar C results in an
overall contribution of biochar to the recalcitrant C pool. In addi-
tion, the effects of biochar on the microbial decomposition and
physical protection of native SOC determine the fate of native SOC.
The stability of biochar C, effects of biochar on the stability of native
SOC, and the possible mechanisms accounting for C sequestration
are the focuses of this chapter.
3.1. Carbon stability of biochar
As biochars are recalcitrant for microbial degradation, they are
considered stable carbon pools and have carbon sequestration po-
tential. However, biochars produced from different conditions may
have different stability, which can be reflected by the amount of
aromatic structure in the biochar, as indicated by the H/C and O/C
atomic ratios, and the amount of volatile matter which can be
108 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
Fig. 6. Schematics showing the interactions between biochar, soil microbes and native
SOC, in particular their effects on soil carbon stability and the decomposition of biochar
C and native SOC. The volatile matter (VM) on biochar can be used by microbes as a C
source and contributes to microbial respiration. Some soil microbes can utilize aro-
matic C structures as well. Native SOC provides soil microbes with a major C source,
and the resulting microbial decomposition of native SOC contributes to microbial
respiration. On the other hand, biochar and soil microbes can protect native SOC from
decomposition by forming soil aggregates with certain functional groups on the bio-
char and the microbial secretion of glomalin, respectively. The resulting soil aggregates
of organic matter, together with the aromatic C structure of biochar as a recalcitrant C
pool in soil, can contribute to the C sequestration.
utilized by soil microbes as a carbon source (Fig. 6). The reaction of
soil microbes to biochar and hence the stability of biochar are
driven by the feedstock types and pyrolysis temperatures of the
biochars (Ameloot et al., 2015; Steinbeiss et al., 2009). Maize stover
biochar had higher C, N, and P content as well as higher carbon
stability than rice and wheat straw biochars did; as indicated by its
aromatic C¼C structure, it also had less carbon mineralization
during one year (Purakayastha et al., 2015). Oilseeds (castor bean
seeds) contain high amounts of aliphatic components, and the
biochar generated from them has some quickly decomposing
compounds that result in greater mineralization than found in
wood biochar (Rittl et al., 2015). On the other hand, pyrolysis
temperatures can result in variable carbon structures of biochar,
generally leading to enhanced carbon stability with increased py-
rolysis temperature (Xiao et al., 2014).
Biochar stability affects the microbial decomposition of biochar,
which is defined as biological aging (Czimczik and Masiello, 2007;
Zimmerman, 2010). It is suggested that labile and semi-labile
fractions make up the non-stable biochar fractions, and the char-
acteristics of stable and non-stable fractions of biochars determine
their resistance to chemical and biological degradation (Masek
et al., 2013). Microbes involved in the transformation of aromatic
compounds may be the first colonizers and decomposers of fresh
pyrogenic carbon, including biochar (Zimmermann et al., 2012); as
such, these microbes influence the carbon stability. For example,
biochar application has caused increases of the relative abundance
of chitin and cellulose degraders (Chitinophaga and Cellvibrio,
respectively) and aromatic compound degraders (Hydrogenophaga
and Dechloromonas) on the genus level, likely being induced by the
enrichment of aromatic compounds such as phenol, methylphenol,
and dihydroxybenzenes in biochar (Kolton et al., 2011). Enrichment
of the chitin-, cellulose-, and aromatic compound-decomposing
microbial genera indicated their important role in the biochar
biological aging process.
3.2. Effects on native organic carbon stability
There is abundant evidence that biochar application can affect
the stability of native soil organic carbon (SOC) in terms of a
“priming effect” (Ameloot et al., 2014; Jien et al., 2015; Zimmerman
et al., 2011). This effect can be caused by microbial community
changes that are induced by biochar, which in turn increase or
decrease the decomposition of native SOC (thus determining the
stability of the native SOC). The effect may also be caused by the
physical protection of native SOC on biochar (Fig. 6) (Ameloot et al.,
2013). An alteration of the soil microbial community to a higher
fungi-to-bacteria ratio can suppress the degradation of native SOC
and cause a large negative priming effect up to 68% (Bamminger
et al., 2014). The priming effects of biochars on native SOC are
dependent on the feedstock characteristics and pyrolysis condi-
tions, which determine the difficulty of utilizing biochar organic
carbon fractions as a carbon source by soil microbes.
The dominant properties of biochar that can influence the
priming effects on native SOC include the carbon content, carbon
aromaticity, volatile matter content, the amount of fixed and easily
oxidized carbon components, and various surface properties (Cely
et al., 2014). Due to the differences in carbon stability, woodchip
biochar had a negative priming effect, while biochars generated
from wheat husk and sewage sludge caused a positive priming
effect in short-term (45 days) incubations (Cely et al., 2014). In
contrast to the role of the aromatic carbon structures of biochar in
the priming effect, the volatile matter content of biochar (i.e., labile
carbon fraction) correlated positively with the initial biological
oxygen demand (BOD), implying that positive priming effects can
be due to the microbial utilization of the labile carbon fraction
within the applied substrate, which can enhance the microbial
growth that controls the positive priming effect on native SOC
(Ronsse et al., 2013). On the other hand, biochar application can
enhance the effectiveness of microbial energy utilization by
simultaneously decreasing the metabolic quotient (qCO2) and
increasing the soil microbial biomass (or in some cases, by reducing
soil respiration with unchanged microbial biomass) (Bamminger
et al., 2014; Zheng et al., 2016), which accounts for one mecha-
nism of the negative priming effect of biochar on native SOC. The
decreased qCO2 of soil microbes can be achieved by modifying the
thermodynamic parameters of soil enzyme activities with biochar
applications (Paz-Ferreiro et al., 2015).
The oxidation of biochar surfaces during the aging process in-
troduces oxygenated functional groups (e.g., carboxylic and ester
groups) on biochar surfaces (Novak et al., 2009; Qian and Chen,
2014); therefore, it can enhance the stability of native SOC by
physically protecting soil aggregates (Fig. 6) (Heitkoetter and
Marschner, 2015). The introduction of the oxygenated functional
groups on biochar surfaces can lead to enhanced biochar and soil
CECs (Hale et al., 2011; Lin et al., 2012), and higher sorption of soil
DOM on biochar surfaces can provide acidic functional groups and
further increase the surface charge of the biochar-SOM aggregates
(Fig. 6) (Heitkoetter and Marschner, 2015). Moreover, the
oxidation-introduced carboxylic functional groups on aged biochar
surfaces serve as additional binding sites, which can intimately
attach mineral phases composed of Al, Si, and Fe and form large
aggregates (Lin et al., 2012; Qian and Chen, 2014). As a conse-
quence, biochar is considerably involved in the preservation of the
soil humus, including humic acids (HAs), fulvic acids (FAs) and
humins (HMs) in soil, which can be ascribed to the presence of
abundant oxygenated functional groups that favor soil aggregation
formation via sorption (Hua et al., 2015).
To evaluate the effects of biochar application on the soil carbon
sequestration and the deposition of native SOC, it is crucial to
distinguish between the microbial decomposition of organic
 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
carbon from biochar and that from native SOC. For this purpose, a
two-component model can be used to indicate the carbon stability
of biochar carbon and native SOC in a system receiving biochar
application (Galvez et al., 2012; Murray et al., 2015; Zimmerman
et al., 2011), as shown in equation (1):
-k1t -k2t
Cmineralized (%) ¼ f  (1
e ) þ (100
f)  (1
e ) Eq. (1)
where Cmineralized is the carbon fraction that is cumulatively
mineralized, f is the carbon fraction (%) of the active or fast turnover
carbon pool (native SOC), k1 and k2 represent the mineralization
rate constants of the active and resistant carbon pools (biochar
carbon), respectively, and t is the incubation time in days. The mean
retention time (MRT) of the respective carbon pools can be calcu-
lated as the inverse of the mineralization rate constants k1 and k2,
i.e., 1/k1 and 1/k2 (Murray et al., 2015). After a given application of
biochar, the shifts of the parameters within this model can indicate
changes in carbon stability and the effectiveness of carbon
sequestration. This model indicates that less than 3% of the applied
biochar was lost through CO2 evolution, with an MRT of
600 years at an annual mean temperature of 26  C (or, 3264 years at
10  C) (Major et al., 2010). The model also indicates that biochar can
reduce the mineralization rate constant of native SOC and increase
its MRT, especially in acidic soil (Galvez et al., 2012).
3.3. Mechanisms of biochar-enhanced soil carbon sequestration
The factors involved in the maintenance of soil carbon stability
with biochar application (Fig. 7) include: (1) the recalcitrant nature
of biochars, due to their aromatic carbon structure and the crystal
silicon structure in silica-carbon complexes formed during pyrol-
ysis (Guo and Chen, 2014; Xiao et al., 2014); (2) the interaction
between surface oxygenated functional groups such as carboxyl
with soil minerals are favorable to maintaining the carbon stability
of biochar during the aging process (Chen et al., 2015; Li et al.,
2014); (3) the adsorption of soil organic matter that is beneficial
for soil aggregate formation (George et al., 2012); (4) the availability
Fig. 7. Proposed carbon sequestration mechanisms affected by biochar, including: (A)
the stable carbon structure of biochar (aromatic carbon structure and crystal silicon
structure in silica-carbon complexes) (Guo and Chen, 2014; Xiao et al., 2014); (B) the
reaction of biochar with soil minerals, which forms a complex structure that protects
biochar from microbial degradation (Chen et al., 2015; Li et al., 2014); (C) biochar
adsorption of soil organic matter (SOM), forming aggregates that protect the degra-
dation of SOM (George et al., 2012); (D) protection of soil aggregates by fungal hyphae
and the secreted glomalin (King, 2011); (E) modification of soil enzyme activities that
control soil organic carbon decomposition (Paz-Ferreiro et al., 2015). These processes
can reduce the CO2 emission from the decomposition of biochar and SOM.
109
of soil arbuscular mycorrhizal fungi that are able to secrete glo-
malin, the protein that promotes soil microaggregation due to its
impact on hydration (King, 2011); and (5) the influences on the soil
microbial community and the modification of soil enzyme activities
that control soil organic carbon decomposition (Paz-Ferreiro et al.,
2015). Among these factors, the protection of soil aggregates and
modification of enzyme activities are related to microbial activity
and are to be mainly discussed here.
Biochar can act as a binding agent of soil organic matter during
the formation of aggregates and can thus promote the macro-
aggregation (George et al., 2012) that enhances the stability of soil
aggregates (Sun and Lu, 2014). The number of stable macroaggre-
gates (>250 mm) can be increased by approximately 25% in the case
of a sandy soil after 80 days of incubation, which is helpful for soil
pore water retention, soil carbon sequestration, and microbial
protection (Awad et al., 2013). The aggregation of large-size soil
particles is partly due to the adsorption of soil organic compounds,
such as humic acids, on biochar surfaces (Kasozi et al., 2010).
Changed patterns of soil aggregation will alter microbial commu-
nity structures in soil, since gram-positive and gram-negative
bacteria tend to possess different strategies in occupying soil par-
ticle fractions. There is a greater abundance of gram-positive bac-
teria in silt and clay fractions, which are older and have more
microbially processed soil organic matter, while there are more
gram-negative bacteria in fractions with larger (>200 mm) aggre-
gates, as these bacteria are fond of fresh plants as C sources (Zhang
et al., 2015). Similarly, fungal abundance was found to be reduced
with the decreasing size of soil aggregation due to the C substrates
being less available in soil fractions with aggregates smaller than
200 mm (Zhang et al., 2015).
The effects of biochar amendment on soil microaggregation are
not only related to the direct adsorption of soil organic matter on
biochar surfaces but also to the alteration of the microbial com-
munity (especially fungi) that produces proteins such as glomalin,
hydrophobins, and chaplins that positively influence micro-
aggregate formation and stability (King, 2011; Rillig et al., 2005).
Glomalin, produced by a phylogenetically narrow range of arbus-
cular mycorrhizal fungi within the order Glomales, has the function
to increase the hydrophobicity and stability of microaggregates
(Rillig et al., 2002). Hydrophobins, occurring widely in numerous
basidiomycetes and ascomycetes, take vital parts in mycelia for-
mation and microaggregate protection (Kershaw and Talbot, 1998).
Chaplins, released mainly by streptomycetes in phylum Actino-
bacteria, are proteins of bacterial origin that generate hydrophobic
soil surfaces (Gebbink et al., 2005; Sawyer et al., 2011). The hy-
drophobic nature of the above proteins prevents water penetration
into soil organic matter and results in microaggregate formation
(Rillig, 2005). Thus, biochar-enhanced soil carbon sequestration can
be reasonably attributed to biochar functions on soil microbiota
that maintain soil microaggregate stability through the protein
secretion.
4. Fate of soil contaminants under biochar-microbe
interactions
4.1. Immobilization of contaminants by biochars
Biochar is an effective sorbent that immobilizes organic con-
taminants and heavy metals through various mechanisms (Ahmad
et al., 2014; Chen and Chen, 2009; Chen et al., 2008a, 2008b, 2012b,
2012c). Electrostatic attraction, polar and non-polar organic-
attraction to the carbonized phase of biochar, and partitioning to
the non-carbonized phase of biochar are forces involved in the
interactions of biochar with organic contaminants (Chen and Chen,
2009; Chen et al., 2008a; Huang and Chen, 2010). Ion exchange,
110 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
anionic metal attraction, precipitation, and cationic metal attrac-
tion are efficient mechanisms for the interaction of biochar with
inorganic contaminants (Ahmad et al., 2014, 2016; Cao et al., 2009;
Qian and Chen, 2013; Xu and Chen, 2015). In some cases, biochar
can be even more effective than activated carbon to immobilize
heavy metals, e.g., a study found six times more effective Pb
sorption (up to 680 mmol Pb kg
1) by dairy manure-derived bio-
char than activated carbon, mainly attributed to Pb precipitation
(84e87%) in form of bePb9(PO4)6 and Pb3(CO3)2 as well as surface
sorption (13e16%) via C¼C (p-electron) and eOePb bonds through
interactions with biochar (Cao et al., 2009). The sorption of certain
herbicides (such as simazine) in micropores or on the surfaces of
biochar prevents their accessibility to microbial cells, extracellular
enzymes, and plants, in addition to preventing them from leaching
into the groundwater (Jones et al., 2011). A simple modification of
biochar with iron oxide can endow such magnetic biochar with a
hybrid sorption capability of organic pollutants and phosphate,
which enables biochar a multifunctional material for agricultural
and environmental applications (Chen et al., 2011).
Recent research reveal the novel roles of biochar on alleviating
the aluminum (Al) toxicity (Qian and Chen, 2013) by modifying the
speciation of Al(OH)2þ or Al(OH)3þ, rather than by a direct elec-
trostatic attraction of Al3þ with negatively charged sites on biochar
(Qian et al., 2013). The oxidation of biochar surfaces during the
aging process can provide extra binding sites for heavy metals Cd
and Al, though the competition sorption between Cd and Al exist
(Qian and Chen, 2014; Qian et al., 2015). Novel roles of biogenic
silicon in biochars are found to alleviate Al phytotoxicity and to
prevent plant uptake of Al, indicating that Si particles can reduce
the amount of soil exchangeable Al and that Si released from bio-
chars can form Si-Al compounds in the epidermis of wheat roots
(Qian et al., 2016). The above research show the necessity to further
investigate the elemental composition of biochar and the role of
biochar on the biogeochemical cycles of soil elements, which can be
involved with soil microbes.
4.2. Influences on contaminant transformation and dissipation
Evidence has accumulated that biochars are able to act as
electron shuttles between microorganisms and pollutants to
enhance microbial degradation (Fig. 8) (Yu et al., 2015). Biochar can
bridge the electron transfer from the microbial cells to the mineral
(Fig. 8) (Kappler et al., 2014), acting as the direct electron acceptor
of acetate for microbial extracellular respiration and growth (Yu
et al., 2015) and as the electron donator to stimulate the microbi-
al reduction of the Fe(III) oxyhydroxide mineral ferrihydrite (Fig. 8)
(Kappler et al., 2014). When the reduction occurs as a result of the
electron transfer from Geobacter sulfurreducens, biochar may
further serve as an electron donator for the metabolism of other
microorganisms, in such way to catalyze biochemical processes
(Kappler et al., 2014; Yu et al., 2015). The presence of biochar has
significantly enhanced the degradation of pentachlorophenol (PCP)
by a species of bacteria (Geobacter Sulfurreducens), which is hardly
able to degrade PCP alone; this enhancement was ascribed to
biochar-facilitated electron transfer between microbial cells and
PCP molecule (Yu et al., 2015).
The electron transfer capabilities of biochar are influenced by
the pyrolysis temperature; the effects are mainly attributed to the
phenolic moieties formed under low temperatures and quinonyl
groups formed under moderate and high pyrolysis temperatures
(Fig. 8) (Kluepfel et al., 2014). The pool of redox-active moieties
(RAMs) in biochar is dominated by different components and is
quantified by its electron exchange capability (EEC), which is
mainly composed of the electron-donating capability (EDC) of the
electron donators, e.g., phenolic moieties in biochars, and the
electron-accepting capability (EAC) of the electron acceptors, e.g.,
quinonyl moieties (Kluepfel et al., 2014; Yang et al., 2016a). Biochar
could store hundreds of micromoles of electrons per gram of mass,
depending on the feedstock and hydrolysis temperatures during
the process of accepting electrons, and could further donate them
to minerals, causing their reduction (Kappler et al., 2014). It is
suggested that the redox activity of biochar was related to the
water-soluble organic components in biochar suspensions and
dissolved organic matter (DOM), which is composed of phenols
(Graber et al., 2014). In contrast to the dissolved compounds that
can be extracted with water from biochar particles, the solid phase
of biochar particles was recognized to be responsible for the elec-
tron transfer that caused the microbial ferrihydrite reduction
(Kappler et al., 2014). Direct contact between microbial cells,
pollutant molecules, and biochar particles through surface
adsorption can favor the electron transfer between them and
further accelerate the pollutant degradation, which is a process that
needs further investigation. Changes in the redox activity of biochar
are able to enhance the direct interspecific electron transfer be-
tween microbial cells (DIET) and direct extracellular electron
transfer between microbial cells with minerals, organic matter,
contaminants, and heavy metals (DEET), resulting in the degrada-
tion of organic pollutants (e.g., pentachlorophenol) and the
decomposition of organic matter (e.g., humic acids) (Fig. 8).
Together with RAMs, the electrical conductivity (EC) of biochar
contributes to its role as an electron shuttle between microbial cells
and pollutants, with a proposed mechanism that involves the for-
mation of a p-p network within the graphite-like aromatic struc-
ture of biochar, which has a high EC. The shuttle function of biochar
is responsible for the electron transfer to the organic compounds
(e.g., PCP) that are adsorbed on its surface (Chen et al., 2014; Yu
et al., 2015), as shown in Fig. 8. Biochar had various ECs, depend-
ing on feedstock type and pyrolysis temperatures, roughly ranging
from 2.11 to 4.41 mS cm
1 g
1 in pine biochar and much higher
(from 216 to 2217 mS cm
1) in manure biochar (Cantrell et al., 2012;
Chen et al., 2014).
In addition to the biochar-facilitated microbial degradation of
hazardous pollutants, biochar can reduce the adverse effects from
organic contaminants and heavy metals on soil microbes and plants
by adsorbing such hazards and further directing electron transfer
between them (Choppala et al., 2012). For metal(loid) ions, such as
Cr and As, whose toxicity depends on their valence, the regulation
of electron transfer by biochar may control their fate and risks
(Fig. 8). Biochar DOM serves both as electron donor and acceptor to
reduce toxic Cr(VI) to the less toxic and relatively immobile Cr(III)
and to oxidize As(III) to As(V). This function is most likely due to the
high reactivity of Cr with many functional groups in biochar DOM
that are able to donate electrons to reduce Cr(VI) and oxidize As(III)
(Choppala et al., 2012; Dong et al., 2014). Losses of Cr(VI) from
suspensions of biochar-applied soil and decreased toxicity, induced
by Cr, to sunflowers has been found as well (Choppala et al., 2012).
By immobilizing Cr and reducing its concentration in soil pore
water, biochar application can mitigate Cr contamination and
enhance soil microbial activity and sunflower plant growth
(Choppala et al., 2012).
The activation of persistent free radicals formed in biochar
(Fig. 5) is commonly considered to be the mechanism of biochar-
facilitated contaminant degradation (Fang et al., 2014a, 2015a,
2015b). The oxidation of biochar during the aging process re-
leases redox-active, acidic and phenolic organics, and the interac-
tion with transition metals produces PFRs, making biochar
continuously participate in redox reactions and hazardous organic
compound degradation (Fang et al., 2014a; Graber et al., 2014). Free
radicals in biochar exhibited excellent reactivity to generate OH
with H2O2 decomposition, consuming approximately 12 spins of
 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115 111

Fig. 8. Schematic diagram showing mechanisms of biochar acting as an electron shuttle enhancing direct interspecific electron transfer between microbial cells (DIET) and direct
extracellular electron transfer between microbial cells with minerals, organic matter, contaminants, and heavy metals (DEET) (Chen et al., 2014; Dong et al., 2014; Kluepfel et al.,
2014; Xu et al., 2016; Yu et al., 2015). Electrons can be transferred from one microbial cell to the component with an electron-accepting capacity (EAC, e.g., quinone) in biochar,
shuttled between EAC and the component with an electron-donating capacity (EDC, e.g., phenol) and be accepted either by another microbial cell, the organic contaminant
(pentachlorophenol), the hematite minerals, or humic acids, enhancing their transformation or degradation. The graphite-like structure on biochar can realize the conductivity.
Electron transfer occurring in biochar dissolved organic matter (DOM) facilitates the transformation of metal(loid)s to nontoxic forms: Cr(VI) to Cr(III) and As(III) to As(V).

free radicals to trap one [OH] molecule (Fang et al., 2015b); sulfate changes and predators) to ensure microbial growth that contrib-
radicals (SO
4 ), formed by persulfate activation, could efficiently utes to contaminant degradation; (2) persistent free radicals could
degrade contaminants including 2-chlorobiphenyl (2-CB), poly- assist biochar as a catalyst and as an electron shuttle to enhance the
chlorinated biphenyls (PCBs), and diethyl phthalate (DEP) (Fang electron transfer between the microbial cells and pollutant mole-
et al., 2014a, 2015a, 2015b). cules, thus facilitating heavy metal transformation and organic
As a practical application, the combination of biochar and mi- pollutant degradation (Kappler et al., 2014; Yu et al., 2015).
crobial remediation strategies is proposed as an effective method
for soil organic contaminant remediation (e.g., for soil PAH pollu-
5. Perspective and conclusions
tion management) (Chen and Ding, 2012; Chen et al., 2012a;
Garcia-Delgado et al., 2015). Using biochar as carriers of PAH-
Heterogeneous effects on soil remediation are found from the
degrading bacteria (Pseudomonas putida, Pseudomonas aeruginosa,
complex combination of biochar and soils with various properties.
Acinetobacter radioresistens, and an unidentified indigenous bac-
Through the massive research with respect to biochar effects on soil
terium), the immobilized-microorganism technique (IMT) finds its
physiochemical characters and microbial activities, the most
effective application on alleviating soil PAHs contamination (Chen
influential factors point to feedstock types and pyrolysis tempera-
et al., 2012a; Galitskaya et al., 2016). For the strategy of combined
ture, which determine biochar properties including surface prop-
biochar-microbial remediation, biochars are used as a pretreatment
erties, structures, elemental composition, redox capacity,
to immobilize and concentrate organic contaminant PAHs, followed
conductivity, pH, CEC, and VOCs. Such properties play key roles in
by an inoculation of PAH-degrading microbes (e.g., white rot fungus
microbial activity and soil process. According to the specific pur-
Phanerochaete chrysosporium and Pleurotus Ostreatus) to perform
poses, different biochar types should be considered. To improve soil
the final degradation of the PAHs in soil (Chen and Ding, 2012;
pH, especially for acidic soils, the utilization of wood biochar pro-
Garcia-Delgado et al., 2015). The sequential application of biochar
duced under high temperature can be proper as it loses most of the
and white rot fungi has induced higher degradation of phenan-
P P acidic functional groups during pyrolysis. To enhance soil fertility,
threne, anthracene, fluoranthene, pyrene, 3-rings, 4-rings, and
P crop residue biochar and manure biochar with high pyrolysis
13 PAH than the single addition of this species of fungi (Chen and
temperatures have the priority, because their high ash content can
Ding, 2012; Garcia-Delgado et al., 2015). Two possible mechanisms
improve soil CEC, facilitating nutrient maintenance for microbial
that could be involved in the influence of biochar on the microbial
and plant growth. To enhance carbon sequestration, high temper-
dissipation of contaminants: (1) the attachment of microbial cells
ature biochar, especially from woods, should be considered because
to biochar particles and biochar-enhanced soil microaggregates
they generally have stable carbon structure and high C/N ratio that
that provide suitable habitats and prevent harsh environmental
make them recalcitrant to microbial decomposition. To control
changes (such as pH changes, water retention, temperature
soilborne pathogens, low temperature biochars may have the
112 X. Zhu et al. / Environmental Pollution 227 (2017) 98e115
advantages, because the relatively high small molecular VOCs on
such biochar can be microbial inhibitors. Moreover, moderate
temperature biochar with relatively higher adsorption capacity and
electron transfer capability can be considered for the purpose of soil
contamination remediation.
Understanding the key roles of biochar properties on microbial
activity in different soil types is essential to answering the most
challenging questions, such as: (1) Under which conditions can the
application of biochar reach the desired benefits, and how can the
trade-offs between various environmental effects of biochar be
balanced? (2) Why, mechanistically, is a given biochar beneficial in
some soils but not in other soils? (3) How can the biological effects
of biochar be better predicted? Focused on certain key functions of
biochar, this review synthesizes the biochar-microbe interaction
mechanisms pertinent to those questions, covering: (1) the mi-
crobial community modification by biochar, via alteration of
nutrient availability and soil characters, (2) the electron transfer
between microbial cells and contaminants facilitated by biochar,
(3) the potential microbial toxicity triggered by biochar, (4) the
carbon stability in biochar with regard to the role of microbial
degradation and the effects of biochar on the soil organic carbon
stability, and (5) the mechanisms of biochar-modified plant resis-
tance to soilborne pathogens. New developments in the meta-
genomics analysis of soil microbe genes will no doubt be pivotal to
uncovering the hidden dimensions of biochar-microbe interactions
that link biochar properties with microbial functions. Under-
standing the mechanisms of the interaction between biochar and
soil microbes is essential to revealing the mechanisms of the het-
erogeneous effects of biochar on soil remediation.
Competing financial interests
The authors declare that they have no competing interests.
Acknowledgements
This project was supported by the National Natural Science
Foundation of China (Grant 21425730, 21537005, 21621005, and
21607125), The National Key Technology R&D Program
(2015BAC02B01), and the Postdoctoral Science Foundation of China
(Grant 2015M581943). Special thanks give to the anonymous re-
viewers who contribute greatly to improve the quality of this
review.
Appendix ASupplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.envpol.2017.04.032.
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