Article

pubs.acs.org/est

Detecting Free Radicals in Biochars and Determining Their Ability to

Inhibit the Germination and Growth of Corn, Wheat and Rice
Seedlings
Shaohua Liao,† Bo Pan,*,† Hao Li,† Di Zhang,† and Baoshan Xing*,‡
†
Faculty of Environmental Science & Engineering, Kunming University of Science & Technology, Kunming 650500, China
‡
Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States
*
S Supporting Information
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ABSTRACT: Biochar can benefit human society as a carbon-

negative material and soil amendment. However, negative
biochar impacts on plant germination and growth have been
observed, and they have not been fully explained. Therefore,
protocols to avoid these risks cannot be proposed. We
hypothesized that the free radicals generated during charring
may inhibit plant germination and growth. Significant electron
paramagnetic resonance (EPR) signals were observed in the
biochars derived from several types of common biomass (corn
stalk, rice, and wheat straws) and the major biopolymer
components of biomass (cellulose and lignin), but not in the
original materials, suggesting the ubiquitous presence of free
radicals in biochars. EPR signal intensity increased with
increasing pyrolysis temperature, and it was dominantly contributed by oxygen centered in the mixture of oxygen- and carbon-
centered free radicals as the temperature increased. The free radicals in biochars induced strong ·OH radicals in the aqueous
phase. Significant germination inhibition, root and shoot growth retardation and plasma membrane damage were observed for
biochars with abundant free radicals. Germination inhibition and plasma membrane damage were not obvious for biochars
containing low free radicals, but they were apparent at comparable concentrations of conventional contaminants, such as heavy
metals and polyaromatic hydrocarbons. The potential risk and harm of relatively persistent free radicals in biochars must be
addressed to apply them safely.

■ INTRODUCTION
Biochars have attracted a great deal of attention because they
provide several benefits, such as improving soil fertility,
sequestering carbon, and retaining various contaminants.1
Both lab and field studies of biochars have supported their
significant positive impacts on plant germination, growth2 and
yield,3 and thus biochars are recommended for use as soil
amendments or for seed germination. However, Spokas et al.
noted that 50% of their reviewed papers reported decreased
plant yields or no significant difference after biochar
application.4 Several previous studies reported the presence of
potential contaminants in biochars, such as metalloid elements
(such as As), toxic metals (including Cd, Cr, Cu, Ni, Pb, and
Zn), tars, polyaromatic hydrocarbons (PAHs), furans, and
dioxins.5,6 However, these conventional pollutants remained at
relatively low concentrations in biochars,2 with likely low
bioavailability because of adsorption by the biochar. Therefore,
even with the generally successful application of biochars,
extended work is still warranted to understand the mechanisms
of observed negative impacts from biochars on plant growth to
enjoy the full beneficial potential of these materials.7
Previous studies have suggested that free radicals are
generally produced during pyrolysis or by heat treatment of
organic chemicals. Dellinger and co-workers systematically
examined the generation of free radicals during the pyrolysis of
organic contaminants in the presence of transition metals. The
half-lives of these free radicals lasted as long as 5 days8 and
posed significant cytotoxicity.9 In systems without transition
metals, the pyrolysis of organic materials could also generate
free radicals. The typical example is the pyrolysis of coal
samples,10 but the stability and persistence of these free radicals
were not examined.
It is therefore very likely that free radicals will be generated
during biochar production (charring). However, no study was
conducted before to assess the presence of free radicals and
their persistence and properties in biochars, and thus critical
information is missing for the evaluation of potential risks from
biochars in association with free radicals. We thus hypothesize
Received: September 23, 2013
Revised: June 29, 2014
Accepted: July 2, 2014
Published: July 2, 2014

© 2014 American Chemical Society 8581 dx.doi.org/10.1021/es404250a | Environ. Sci. Technol. 2014, 48, 8581−8587
Environmental Science & Technology Article

Figure 1. EPR signals detected in the biochars produced from rice straw (A, B, and C), cellulose (D, E, and F) and lignin (G, H, and I). The EPR
signal intensities (A, D, and G), that normalized the intensities (DI/N) (B, E, and H) and g factors (C, F, and I) are presented. Both the DI/N and g
factors indicated that these free radicals were stable for over one month. Data for corn stalks and wheat straws are similar to that of rice straw and are
presented in SI Figure S1. The peak widths (ΔHP−P/G) of all five materials are presented in SI Figure S2.

that free radicals will be generated during biomass charring, and
they are relatively persistent. As a result, these radicals will
inhibit plant growth. Typical biomass and model materials were
used to produce the biochars. An electron paramagnetic
resonance (EPR) spectrometer was used to understand the
generation and properties of free radicals in these biochar
samples. This line of research will provide useful information
for long-term, safe applications of biochars.
continuously flowing N2 during the whole process (1.5 L/min
during the whole heating and cooling processes until reaching
50 °C). The resulting biochars were stored in sealed plastic
bags and placed in the dark until further use. Two mg of
biochar particles were loaded into a micropipette (from
Germany, 1.0 mm in i.d., 1.5 mm in o.d., and 125 mm in
length) and sealed with vacuum grease (Germany) at one tip.
These loaded biochars were continuously monitored for free

■ EXPERIMENTAL SECTION
Materials. Three common types of biomass, namely corn
stalks and rice and wheat straws, were used in this study. This
biomass was collected from farm fields, dried, chopped and
ground to <0.3 mm. The two major components of biomass,
which are cellulose (Sigma-Aldrich) and lignin (alkali, Sigma-
Aldrich), were used as two model biopolymer materials.
DMPO (5,5-dimethyl-1-pyrroline-N-oxide, ≥ 99.0%, GLC)
and DPPH (2,2-diphenyl-1-picrylhydrazyl) were obtained from
Sigma-Aldrich and used without further purification.
Biochar Production and EPR Measurements. All five
biomass materials were pyrolyzed for 2 h at temperatures of
200, 300, 400, and 500 °C and purged with N2.11 In brief, the
feedstocks were wrapped up in tinfoil. The biomass materials
were placed in a muffle furnace with a temperature increase rate
of 15 °C/min and were pyrolyzed at 200, 300, 400, or 500 °C
for 2 h. The treated samples were kept in the furnace until they
had cooled to 50 °C. The muffle furnace was provided with
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radical signals on an EPR spectrometer (Bruker, A300−6/1, X-
band) with a single cavity, a modulation of 100 kHz and
microwave frequencies of 9.2−9.9 GHz. The typical parameters
for EPR measurement were as follows: the sweep width was
100 G, the modulation amplitude was 1.00 G, and the
resolution in the X axes was 1024 points. For solid particles, the
EPR microwave power was set specifically to 31 dB (or 0.131
mW) and the sweep time was 81.92 ms. For liquid samples, the
EPR microwave power was 9 dB (or 21 mW) and the sweep
time was 20.48 ms. The g factor values (the property of the
electron in a certain environment as explained in Supporting
Information (SI))12,13 were estimated by using the Bruker
WinEPR Acquisition and Microsoft Office Excel. EPR signals
were recorded from 1 h to 30 days after biochar production.
DPPH was used as a standard because of its similar spectral
profile to the EPR signals detected in biochars. The intensities
of the spectra were reported in DI/N, which was calculated as
the double-integrated (DI) intensity of the EPR spectrum
divided by the normalization constant N. The N value was
dx.doi.org/10.1021/es404250a | Environ. Sci. Technol. 2014, 48, 8581−8587
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Figure 2. An in situ observation of EPR signals during biochar production when using biomass (A and C) and biopolymers (B and D). Normalized
intensities (DI/N) (A and B) and g factors (C and D) are presented here and peak widths (ΔHP−P/G) are presented in SI Figure S3. The gray area
indicates the charring process and the white background is the cooling process.
calculated by taking the (conversion time · receiver gain · (Anzahl
der Datenpunkte − 1))/(sweep width) according to the software.
In Situ Pyrolysis. To examine the free radical generation
process, all three crop biomass types and two biopolymer
materials were separately pyrolyzed in situ in the EPR
spectrometer at 200 °C under N2 protection (at 1.5 L/min
during the whole heating and cooling processes until reaching
room temperature). These materials were loaded into quartz
tubes, and the EPR signals were continuously monitored during
the charring (2 h) and cooling processes (5 h).
Germination Tests. The biochar produced from rice straw
at 500 °C was selectively used in this experiment because the
biochars generated from all three crops showed very similar
EPR characteristics. Corn, wheat and rice seeds were used for
germination and plant growth experiments. Fifty wheat seeds,
20 corn seeds, or 50 rice seeds were placed in one Petri dish
(90 mm diameter) on a filter paper moistened with deionized
water. Twenty mL of water were added to the Petri dish.
Biochars were added at rates of 0.0, 0.5, 1.0, 2.0, 4.0, and 6.0 g/
Petri dish14 with three replicates. These rates were equivalent to
0, 10, 20, 40, 80, and 120 t/ha on an area basis of 10 cm soil
depth, or 0%, 0.03%, 0.06%, 0.12%, 0.24%, and 0.36% on a soil
mass basis at a soil particle density of 1.6 g/cm3. All Petri dishes
were covered and incubated in the dark at 25 °C ± 0.5 °C for 5
days, and then the germination percentage, root length and
shoot length were recorded.
·OH Capture in Aqueous Solutions. The biochar
produced from rice straw at 500 °C was again used in this
experiment. A DMPO (a freshly prepared 0.3 M solution in
0.01 M PBS solvent) spin trap was used for capturing hydroxyl
radicals (•OH) in aqueous solutions. The biochar particles
were mixed with water (20 mL) at rates of 0.0, 0.5, 1.0, 2.0, 4.0,
and 6.0 g/Petri dish, and the system was saturated with oxygen
by bubbling air in for 10 min. The mixture was sonicated for 10
min (AS20500BT Ultrasonic Cleaner, 100% power, room
temperature) to disperse the colloids, and then it was
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centrifuged for 8 min (2000 r/min). An aliquot of supernatant
(100 μL) was mixed with DMPO (100 μL). Fifty μL of the
mixture was transferred into a micropipette (1.0 mm in i.d., 1.5
mm in o.d., and 125 mm in length) and the micropipette was
sealed at one end with vacuum grease. This micropipette was
inserted into the standard resonator for high sensitivity
continuous-wave EPR measurements.
The concentration of DMPO−OH adducts was determined
by finding the intensity of the second line (at low magnetic
field) for its representative 4-line spectra and comparing it with
the intensity of standard TEMPOL (4-hydroxyl- 2,2,6,6-
tetramethylpiperidine-1-oxyl, a stable radical). The TEMPOL
concentration was verified by an Agilent UV−vis spectropho-
tometer by using an extinction coefficient of 13.2 ± 0.1 M−1
cm−1 at 436 nm.15
■ RESULTS AND DISCUSSION
Free Radicals Were Detected during Biomass Char-
ring. Significant EPR signals were observed in all the biochars
(Figures 1 and S1). The original materials did not show any
detectable EPR signals, while increasing signal intensities were
observed with increasing pyrolysis temperature. More interest-
ingly, these signals were stable with generally less than a 10%
signal decrease after one month (Figure 1), which indicated the
persistent nature of these detected free radicals. Similar stable
free radical signals were also observed for biochars produced
from pine (Pinus yunnanensis) and chitin (data not shown),
suggesting that free radical generation during charring could be
very common.
The EPR signals contributed by transition metals could be
excluded, because all detected transition metals (such as Fe, Cu,
Cr, Zn, and Pb) fell into a mg/kg range (SI Table S1), which
did not have obvious EPR signals. Free radicals are reportedly
produced during the heating process, and they can therefore
have potential health and environmental risks.16,17 Although
common free radicals have very short half-lives within a range
dx.doi.org/10.1021/es404250a | Environ. Sci. Technol. 2014, 48, 8581−8587
Environmental Science & Technology
Figure 3. EPR signals for typical DMPO−OH adducts (A) and the intensity variation of the second line with the trap time (B). Biochars were used
at rates of 0.0, 0.5, 2.0, and 6.0 g/20 mL of water to be consistent with the germination test. R represents the samples after aeration and
ultrasonication.
of 10−9−10−4 s (such as 10−9 s for •OH),18 the free radicals
generated near the solid particle surface may have strong
interactions with the particles, and steric hindrance can make
free radicals stable and persistent.19 The relatively long-lived
free radicals in the biochars are similar to the environmentally
persistent free radicals that were recently reported by Dellinger
and co-workers.8,20
Typical model compounds (biopolymers) of biomass, such
as lignin and cellulose, were also pyrolyzed to compare their
EPR signals with biomass. A very similar EPR spectral pattern
was observed in biomass biochars. Lignin generally had EPR
signals that were 5 times higher than that of cellulose, most
likely because of its aromatic structure. This result may suggest
a very important contribution from the lignin in the biomass to
EPR signals.
The g factor of EPR signals is a useful parameter for
identifying the type of free radicals.21,22 As indicated in Figure 1
and SI Figure S1, a very similar g factor change was observed
for all three biomass types. The g factors decreased with
increasing pyrolyzing temperatures in a range from 2.0053 to
2.0036. Previous studies have suggested that g factors >2.0040
are typical for oxygen-centered radicals, such as semiquinone
radical anions.21 Carbon-centered radicals, such as aromatic
radicals, possess a g factor that is usually less than 2.0030.22 A
combination of carbon- and oxygen-centered radicals has g
factors of approximately 2.0030−2.0040.21 Although a specific
experimental design is needed to accurately identify the
chemical structures of the free radicals detected in our biochars,
the g factor change with the increasing pyrolysis temperature
suggests that these free radicals were likely oxygen-centered
radicals (such as phenoxyl and semiquinone radicals) at low
charring temperatures and then became mixed with carbon-
centered radicals at elevated charring temperatures.
Pyrolysis/Charring In Situ. To understand the generation
of free radicals in biochars, an in situ observation of EPR signals
was conducted at 200 °C. The pyrolysis process was indicated
as gray background in Figure 2. Two stages with decreased EPR
signal intensities were noted. The first one was before 30 min.
This stage may involve free radicals generated from the
breakdown of side chains.23 These outer-surface free radicals
may quickly react and dissipate, as suggested by the initial
decrease in the EPR signal intensity. Continuous pyrolysis
caused the accumulation of a large quantity of free radicals on
the limited surface area. Interactions between these free radicals
may result in the apparent decrease of EPR signals between 60
and 120 min.
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It is important to note that a sharp increase in EPR signals
was observed during the cooling process between 120 and 150
min. The shearing of rigid structures can reportedly result in
the breakdown of chemical bonds, and thus promotes the
generation of free radicals.24 The cooling process of biochars
may shrink macromolecule structures at different directions,
break up chemical bonds and generate additional free radicals.
The g factor change provided additional information to
understand the above-mentioned process. The g factors
decreased from over 2.0050 to 2.0043 during pyrolysis,
suggesting that the initially generated oxygen-centered free
radicals were mixed with carbon-centered free radicals. During
the cooling process (120−150 min), a sudden increase in the g
factor was observed in concert with an increased EPR signal.
Thus, oxygen-centered free radicals mostly contribute to the
sharp increase of free radical concentrations. These free radicals
may have resulted from the breaking of C−O bonds25 or
oxygen incorporation into broken C−C bonds.26
Thirty min after cooling (150 min after the initial pyrolysis),
both EPR intensities and g factors were rather stable. This
finding is consistent with the data presented in Figure 1. These
free radicals originated from macromolecules instead of small
molecules (SI Figure S4). Steric hindrance in the generated free
radicals made them difficult to move in the molecular chain and
it was not easy for them to react with other free radicals and/or
molecules, which stabilized them.19 The weak EPR signal of
cellulose as indicated by DI/N values again suggested that
cellulose contributed little to free radical production in
biochars, at least at a low temperature (200 °C).
·OH in Aqueous Solutions as Induced by Biochars.
With the above detected abundant free radicals in biochars, we
hypothesize that the presence of biochars in the aqueous phase
may induce reactive oxygen species (ROS) generation, which
may cause negative effects and should be incorporated into a
biochar risk analysis. Rice straw treated at 500 °C was
selectively used as the tested sample, and the original rice
straw was used as the control. To exclude the interference of
EPR signals in biochar particles and possible reactions between
the ·OH capture reagent (DMPO) and solid particles, the
supernatants of the solid particles (containing colloids) were
collected and immediately used in ·OH detection.
Typical 4-line EPR spectra of DMPO−OH adducts in the
solution of the tested samples were captured (Figure 3).27 A
very weak DMPO−OH signal was detected in all the samples at
the initial DMPO addition. The signals increased up to 6 times
in the biochar sample within 1000 min, but they did not
increase for the control. The presence of ·OH in the control
dx.doi.org/10.1021/es404250a | Environ. Sci. Technol. 2014, 48, 8581−8587
Environmental Science & Technology Article

might result from the nonradical nucleophilic reaction of

deionized water with DMPO28 as described in the following
equation:

DMPO + H 2O → DMPOH − OH
→ DMPO − OH + H+
The first reaction was a nonradical nucleophilic reaction
followed by dehydrogenation. This reaction was most likely
why the signal was time-independent for the control. The above
hypothesis was also confirmed by the fact that the EPR signal
was not altered after aeration and ultrasonication (Figure 3B).
However, the ultrafine particles in the solution of biochar
samples might continuously stimulate ·OH production, which
was subsequently trapped by DMPO. DMPO−OH adducts
were then broken down after 1000 min.29,30
The above-discussed biochar-induced free radicals may be
described as follows: the electrons were transferred from
biochar-free radicals to O2, forming O2·−, which dismutates with
H+ to form H2O2. With the aid of the transition metals in
biochars (SI Table S1), H2O2 could easily be changed to ·OH,
which is then captured by DMPO through an adducting
reaction (DMPO−OH).
Germination and Evans Blue Tests. The above
discussion suggested that biochars can generally contain free
radicals and these free radicals can promote the generation of ·
OH in the aqueous phase. However, the possible risks from free
radicals in these biochars were never examined before as the
investigators did for PAHs and toxic metals.31,5 Thus, it is vital
to study the potential risks of pyrolysis-generated free radicals
in biochars. To reveal these potential risks, a germination test Figure 4. Germination (A), root length (B), and shoot length (C) of
was performed by using rice straw-derived biochar (500 °C) as corn, wheat, and rice as affected by biochars. Different letters above the
an example. Significant negative impacts on the germination, bars suggested that these values are significantly different at P < 0.05
root and shoot lengths were observed in most systems (SPSS 13.0).
containing biochar (Figure 4). The germination test was
conducted by using corn, wheat and rice as model agricultural
crops. A clear dose−response relation could be observed for the comparable to those in the supernatant before storage (IV in SI
germination, mean root length, or mean shoot length. The Figure S7A). To distinguish the toxic impacts between
stimulation of corn and wheat growth was observed as conventional pollutants and free radicals further, we conducted
suggested by their increased root and shoot lengths when an Evans Blue experiment to examine the integrity of the
small amounts of biochars (0.5 and 1.0 g/Petri dish) were used. plasma membranes in plant roots.33,34 All the roots in the three
This phenomenon was understood to come from the crops were stained with a blue color in Petri dishes containing
introduced nutrients (e.g., the macronutrients C, N, P, K, Ca, biochars produced at 500 °C (Figure 5), even for the samples
Mg, and the micronutrients Na, Fe, Zn, Mn, Cu, and B) from with growth stimulation (II in Figure 5B), but not in the system
biochars.31 Various results were previously reported in regards without biochars.33 Although plant growth (shoot and root
to the impact of biochars on plant germination and growth. length) was inhibited in the system with biochars at 200 °C in
Stimulation,14 inhibition14 and nonsignificant impacts2,32 were addition to the supernatant from 500 °C-produced biochar
all observed. It is easy to understand that the apparent net (most likely because of the presence of heavy metals, PAHs and
result is dependent on the type (biomass sources) and tars, as shown in SI Figures S6B and S6C), no plasma
condition (concentration, aging and water chemistry con- membrane damage was observed in these two systems (SI
ditions) of the biochar that is applied and the plant species. Figure S6). Therefore, the observed membrane damage most
Previous studies mostly focused on heavy metals and PAHs likely resulted from the free radicals induced by biochars.35,36
to explain the possible risks of biochars.6−8 According to our Biochars have attracted a great deal of research and public
measurements, biochars produced at 200 °C contained rather attention, because of their applications as fertilizer, the
low levels of free radicals (Figure 1), but they had the highest sequestering of greenhouse gases, and pollutant immobilizers.
PAH concentrations and relatively high concentrations of heavy Previous studies have assessed the potential risks of biochars as
metals in the supernatant (see SI Tables S1 and S2 for more soil amendments through analyses of heavy metals, poly-
explanations). No germination inhibition in corn was observed aromatic hydrocarbons, and tars. This current study clearly
for this biochar (SI Figure S7A), indicating that the heavy indicated that the free radicals with the relatively long lifetime
metals and PAHs detected in this study have limited risk to and in biochar particles could induce ROS in water, and they could
impact on germination. In addition, the supernatant of 500 °C have negative impacts on plant growth. Significant inhibitions
biochar after 30-d of storage showed no germination inhibition on plant seed germination in addition to root and shoot
although the concentrations of heavy metals and PAHs were elongation were observed. The plasma membrane damage in
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Environmental Science & Technology
Figure 5. An Evans Blue test on the roots. All the corn (A), rice (B), and wheat (C) roots were stained with blue color in the system with biochars,
suggesting the general plasma membrane damage of roots by biochars. The biochars were applied at rates of 0.0 (I), 0.5 (II), 1.0 (III), 2.0 (IV), 4.0
(V), and 6.0 (VI) g/Petri dish. The roots were washed carefully and the particles attached to the root surface (representatively indicated by white
cycles) may be the damaged tissue.
plant roots also suggested that the toxic impact originated from
free radicals. Significant free radical signals and toxic impacts
were observed for biochars after more than one month of
storage, suggesting the long-lasting risks of these free radicals
(SI Figure S10). We call for investigative attention to the risks
of biochars, which should be evaluated with great care, with
special attention given to nonconventional pollutants such as
these relatively persistent free radicals. Biochars produced by
different methods will have different characteristics including
persistent free radical properties, and thus their risks must be
evaluated individually. It is also possible that the free radicals
observed in biochars and the aqueous phase in the presence of
biochars is less harmful with proper handling. For example,
biochars produced at a lower temperature showed lower risks
under plant germination, which is a useful guide for biochar
application. In addition, these free radicals may be inactivated
by natural organic matter and/or clay particles after applying
biochars to soils. Further investigations are currently ongoing to
quench these persistent free radicals in biochars, thus ensuring
their safe application.
■
ASSOCIATED CONTENT
*
S Supporting Information
Eleven figures and two tables are presented in the Supporting
Information. This material is available free of charge via the
Internet at http://pubs.acs.org/.
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■ AUTHOR INFORMATION
Corresponding Authors
*Phone: 86-871-65102829; fax: 86-871-65170906; e-mail:
panbocai@gmail.com.
*Phone: 413-545-5212; fax: 413-545-3958; e-mail: bx@umass.
edu.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This research was supported by the National Scientific
Foundation of China (41222025, 41273138), the Program for
New Century Excellent Talents in University, the Chinese
Ministry of Education, the Recruitment Program of Highly-
Qualified Scholars in Yunnan (2010CI109), and the USDA-
AFRI Hatch program (MAS 00982). The authors thank all five
anonymous reviewers for their valuable suggestions to improve
our manuscript.
■
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