Preparative Biochemistry and Biotechnology

ISSN: 1082-6068 (Print) 1532-2297 (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb20

Influence of biochar application on potassium-

solubilizing Bacillus mucilaginosus as potential
biofertilizer

Sainan Liu, Wenzhu Tang, Fan Yang, Jun Meng, Wenfu Chen & Xianzhen Li

To cite this article: Sainan Liu, Wenzhu Tang, Fan Yang, Jun Meng, Wenfu Chen & Xianzhen
Li (2017) Influence of biochar application on potassium-solubilizing Bacillus mucilaginosus
as potential biofertilizer, Preparative Biochemistry and Biotechnology, 47:1, 32-37, DOI:
10.1080/10826068.2016.1155062

To link to this article: http://dx.doi.org/10.1080/10826068.2016.1155062

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Published online: 25 Feb 2016.

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PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
2017, VOL. 47, NO. 1, 32–37
http://dx.doi.org/10.1080/10826068.2016.1155062

Influence of biochar application on potassium-solubilizing Bacillus mucilaginosus as

potential biofertilizer
Sainan Liua, Wenzhu Tangb, Fan Yangb, Jun Menga, Wenfu Chena, and Xianzhen Lib
a
Biochar Engineering Research Centre, Shenyang Agricultural University, Shenyang, China; bSchool of Biological Engineering, Dalian Polytechnic
University, Dalian, China

ABSTRACT KEYWORDS
Biochar can enhance soil fertility to increase agricultural productivity, whereas its improvement in soil Bacillus mucilaginosus;
microbial activity is still unclear. In this article, the influence of biochar on the cell growth and the biochar; biofertilizer; cell
potassium-solubilizing activity of Bacillus mucilaginosus AS1153 was examined. The impact on cell growth growth; feedstock;
potassium
is related to the biochar-derived feedstocks and the particle size of biochar. Both intrinsic features and
inner component fraction can promote the cell growth of B. mucilaginosus AS1153. The potassium-
solubilizing activity was increased by 80% when B. mucilaginosus was incubated in conjunction with the
biochar derived from corn stover. The survival time of B. mucilaginosus also was prolonged by adsorption
in biochar. The experimental results suggested that the biochar containing B. mucilaginosus could be
used as a potential biofertilizer to sustain crop production.

Introduction nutrient availability.[11] However, the possible connection

Although chemical fertilization can increase crop yield in the
agricultural process, the exponentially increased fertilizer
consumption throughout the world has caused a serious
environmental problem.[1] Recently, some microbes have been
reported to release potassium in accessible form from
potassium-bearing mineral in soil.[2] They could provide an
alternative technology to make potassium available for plant.[3]
Bacillus mucilaginosus could improve the potassium
nutritional status in soil and had a beneficial effect on the
growth of Sudan grass.[4] The inoculation of B. mucilaginosus
together with maize and wheat plants resulted in the mobiliza-
tion of potassium from waste mica, which in turn acted as a
source of potassium for plant growth.[5] Co-inoculation of
waste mica with B. mucilaginosus and Azotobacter chroococ-
cum showed a promising and alternative option for utilizing
it as potassium fertilizer to crops.[6] Thus potassium-solubiliz-
ing bacteria have extensively been used as biofertilizer in
China because its use can reduce agrochemicals and support
eco-friendly crop production.[7] However, its effectiveness as
a biofertilizer was not better in the agricultural practice
because of the low microbial activity in soil.
Biochar, produced by pyrolysis of biomass in the absence
of oxygen, has created a lot of researches and commercial
interests in recent years.[8] It has been described as a possible
means of enhancing soil fertility.[9] Such improvement in soil
fertility has been explained mainly by increasing pH in acidic
soil and enhancing nutrient-holding capacity.[10] However,
those results are variable and dependent on the experimental
setup and soil property.[11] In fact, changes in microbial abun-
dance also were observed when the biochar was applied to soil
amendment,[12] which have an effect on improving crop
between the biochar and microbial activity remains poorly
elucidated.[12] So far, no experimental data directly support
that biochar has an impact on increasing soil microbial
activity. In this article, the biochar impacting on cell growth
of B. mucilaginosus as well as its potassium-releasing activity
was studied. It was first reported that the biochar could
improve the microbial activity of B. mucilaginosus, which
can be used as a potential biofertilizer to enhance agricultural
activities.
Experimental
Strain, materials, and culture conditions
Bacillus mucilaginosus AS1153 was purchased from the China
General Microbiological Culture Collection Centre and
cultured in the glucose medium at 180 rpm and 30°C.
When the biochar impacting on the cell growth was studied,
B. mucilaginosus was incubated in NB medium at 30°C and
180 rpm.
Potassium feldspar powder was purchased from Dabieshan
Mining Co., Wuhan, China, which was sieved at a 0.149 mm size
and washed with distilled water until the washings were clear.
The glucose medium was consisted of (per liter) 10 g
glucose, 1.5 g (NH4)2SO4, 0.1 g yeast extract, 0.1 g KCl, 0.1 g
Na2HPO4, 0.1 g MgSO4, and 1 g CaCO3 at pH 7.0. The NB
medium was composed of (per liter) 3 g beef extract, 5 g
peptone, and 5 g NaCl at pH 7.0. The potassium-solubilizing
medium was composed of (per liter) 10 g sucrose, 1 g
Na2HPO4, 0.5 g (NH4)2SO4, 0.5 g MgSO4, 1 g CaCO3, 0.2 g
yeast extract, 0.02 g FeC13, and 10 g potassium feldspar
powder at pH 7.0–7.4.

CONTACT Xianzhen Li xianzhen@mail.com School of Biological Engineering, Dalian Polytechnic University, Ganjingqu, 1 Qinggogyuan, Dalian 116034, PR China.
© 2017 Taylor & Francis

Biochar preparation and washing method
Biochar was produced from corn stover, corncob, rice husk,
and bamboo, respectively, with the pyrolysis method as
described elsewhere[13] and lightly crushed and sieved to
obtain a uniform of 0.25–2.0 mm particle size. The biochar
derived from corn stover was used in all tests in this article
unless otherwise stated.
In washing process of biochar, 24 g of sample was mixed
with 250 mL of the distilled water, 1 mol/L HCl, 1 mol/L
NaOH, or acetone and stirred at room temperature for 3 hr.
After repeated washing for four times, the biochars were
rinsed with the distilled water until the conductivity of rinsing
liquor was lower than 50 µS/dm. The washed biochars were
dried at 105°C overnight for testing. The washed liquor with
distilled water and acetone was, respectively, collected together
for use, in which the acetone-washing liquor was evaporated
and dissolved in the equal volume of the distilled water.
Effect of biochar on cell growth of B. mucilaginosus
AS1153
Overnight culture of B. mucilaginosus AS1153 in the glucose
medium was inoculated at 2% of inoculum in the NB medium
containing 0.6% biochar derived from corn stover, corncob, rice
husk, and bamboo, respectively, and incubated at 30°C and
180 rpm for 12 hr. The survival cell amount in the NB medium
with and without addition of biochar was determined by the col-
orimetric Methylthiazolyldiphenyl-tetrazolium (MTT) method.
Cell adsorption in biochar
After overnight incubation in 10 mL of the glucose medium at
30°C and 180 rpm, the culture fluid of B. mucilaginosus
AS1153 was mixed with 0.06 g of sterilized biochar derived
from corn stover, corncob, bamboo, and rice husk, respect-
ively, and incubated at 30°C and 180 rpm for 1 hr. The cell
numbers in the culture fluid were determined by the colony
count method.[14] The adsorbed cell amount in the biochar
was calculated by the difference in the culture fluid between
with and without biochar addition.
Time course of growing B. mucilaginosus in NB
medium with and without biochar addition
The overnight culture of B. mucilaginosus AS1153 in the glucose
medium was inoculated at 2% of inoculum in 10 mL of NB
medium containing 0.06 g of biochar and incubated at 30°C
and 180 rpm. The culture fluid was sampled at an interval time
for cell content determination by the colorimetric MTT method.
The control was performed by incubation of B. mucilaginosus
AS1153 in the NB medium without biochar supplementation.
Cell growth of B. mucilaginosus in medium with
different particle sizes of biochar
After cultured in the glucose medium overnight, B. mucilagi-
nosus AS153 was inoculated at 2% of inoculum in the NB
medium supplemented with different sizes of biochars and
PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 33
incubated at 30°C and 180 rpm for 12 hr. The living cell
amount was determined by the colorimetric MTT method.
The particle size of biochar derived from corn stover used in
this experiment includes smaller than 0.178, 0.178–0.42,
0.42–0.84, and 0.84–2 mm, which was designed as C1, C2,
C3, and C4, respectively.
Effect of washed biochar on cell growth of
B. mucilaginosus AS1153
The overnight culture of B. mucilaginosus AS1153 was inocu-
lated at 2% of inoculum in the NB medium containing 0.6%
of the washed biochar. After incubation at 30°C and
180 rpm for 12 hr, the survival cell content was analyzed by
the colorimetric MTT method. The unwashed biochar was
used as a reference for testing cell growth and the control
was performed in the NB medium without addition of biochar.
Effect of washed liquor from biochar on cell growth of
B. mucilaginosus AS1153
After incubation in the glucose medium at 30°C and 180 rpm
overnight, B. mucilaginosus AS1153 was inoculated at 2% of
inoculum in the NB medium, which was prepared with the
distilled water and the washed liquor from biochar, respect-
ively. After strain AS1153 was incubated at 30°C and
180 rpm for 12 hr, the survival cell content was analyzed by
the colorimetric MTT method.
Survival stability of B. mucilaginosus in the presence
of biochar
The 2 mL of overnight culture of B. mucilaginosus AS1153 in
the glucose medium was added in the tube containing 0.4 g
of biochar and incubated at 30°C. The survival cell content
was measured by the colorimetric MTT method every 1 hr. The
overnight culture of B. mucilaginosus AS1153 without biochar
added was also incubated at 30°C and sampled for determin-
ing survival cell content every 1 hr.
Effect of biochar on potassium-solubilizing activity of
B. mucilaginosus AS1153
The overnight culture of B. mucilaginosus AS1153 in the
glucose medium was inoculated at 2% of inoculum in the
potassium-solubilizing medium containing 0.6% biochar.
After incubating at 30°C and 180 rpm for 5 days, the culture
fluid was centrifuged at 6,000 rpm and 4°C for 20 min. The
soluble potassium in the culture supernatant was determined.
The control was performed by incubating B. mucilaginosus
AS1153 in the potassium-solubilizing medium without biochar
supplementation. The reference was performed by incubating
the potassium-solubilizing medium containing 0.6% biochar
without inoculation of B. mucilaginosus AS1153.
Chemical analysis
The survival cells were determined by the colorimetric
MTT method as described before.[15] Briefly, MTT
34 S. LIU ET AL.

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
was dissolved in the phosphate-buffered saline at 5 mg/mL
and filtered to sterilize and remove a small amount of insoluble
residue present in some batches of MTT. This MTT solution
was mixed with the microbial culture at a ratio of 1:10 in micro-
plates and incubated at 30°C for 1 hr. The equal amount of
dimethyl sulfoxide was added to all wells and mixed thoroughly
to dissolve the dark blue crystals. After a few minutes at room
temperature to ensure that all crystals were dissolved, the plates
were read at 510 nm. Plates were normally read within 1 hr of
adding isopropanol.
Figure 1. Adsorption of Bacillus mucilaginosus AS1153 in biochar derived from
Soluble potassium was analyzed as following: After 10 mL various feedstocks (filled) and cell growth in the presence of those biochars
of culture supernatant was mixed with 20 mL acidic solution (clear). The control was performed in the NB medium without biochar addition
of nitric acid and perchloric acid at a ratio of 4:1, the mixture at 30°C.
was nitrated by heating. When the color of nitration liquid was
changed to canary, the nitrification was stopped and cooled. similar results were obtained despite the decrease in microbial
The nitration liquid was made up to 50 mL volume with dis- growth by about 30% in comparison with the unwashed
tilled water, and the potassium was determined with an atomic biochars (data not shown).
absorption spectrometer. One of the most commonly given reasons for this corre-
lation between the biochar application and the increased
Statistic analysis microbe abundance was the porous property of biochar that
can provide a significant microbial habitat niche.[17] Therefore,
All experiments and assays described in this article were the capability of biochars to adsorb B. mucilaginosus was also
performed in triplicate unless otherwise stated and the data evaluated when B. mucilaginosus was incubated together with
were subjected to one-way analysis of variance to detect the biochar derived from different feedstocks. As shown in
statistical significance. Figure 1, the adsorptivity of biochar to cells of B. mucilagino-
sus AS1153 was related to the source from which the biochar
was produced. The biochar sourced from corn stover showed
Results the maximum adsorptivity to cells of B. mucilaginosus AS1153
Influence of biochars on cell growth of B. when compared with the biochar derived from other biomass,
mucilaginosus and its adsorptivity which coincides with its promotion on cell growth. However,
the biochar derived from corncob also showed the similar
Although the biochar is often claimed to improve the soil adsorptivity to biochar derived from corn stover but lower
fertility, its roles in increasing microbial activity to produce stimulated activity on the cell growth of B. mucilaginosus.
biofertilizer are still unclear.[10] Thus, the promotion of Therefore, the biochar derived from corn stover was used
biochar on potassium-solubilizing B. mucilaginosus was for the following study.
studied in this article, considering that it is usually used as a
model strain in research on silicate mineral weathering.[16]
It was reported that the intrinsic properties of the biochar Cell growing process of B. mucilaginosus with biochar
are dependent on the biochar derived under pyrolysis addition
conditions in the production process, which probably result
The time course of cell growth of B. mucilaginosus in the NB
in the diverse influences on microbial activity.[8] Thus, the
medium with and without biochars is shown in Figure 2. The
biochar derived from different biomass was used to study its
application of biochar derived from corn stover in the culture
influence on cell growth of potassium-solubilizing bacteria
could strongly promote the cell growth of B. mucilaginosus
B. mucilaginosus in the NB medium. As shown in Figure 1,
B. mucilaginosus AS1153 has grown much better in the
medium with biochar than that in the medium without
biochar addition. The significant difference in the cell growth
of B. mucilaginosus AS1153 was observed in the presence of
biochar derived from different feedstocks including corn
stover, corncob, rice husk, and bamboo. All the tested biochars
could stimulate the cell growth of B. mucilaginosus and their
capability to promote the cell growth was dependent on the
feedstock for biochar production. The biochars sourced from
both corn stover and rich husk showed strong promotion on
the cell growth of B. mucilaginosus. The maximum cell growth
was obtained when the biochar derived from corn stover was
Figure 2. Time course of Bacillus mucilaginosus AS1153 growing in the NB
applied to B. mucilaginosus. To exclude the influence factors of medium supplemented with (�) or without (•) the biochar derived from corn
nonbiochar, the washed biochars were examined and the stover at 30°C.

AS1153, whereas the biochar application could not change the
cell-growing profile and not shorten the growing time. The
survival cell content in the NB medium with biochar addition
was much higher than that without biochar over the culturing
time. Therefore, the capability of biochar to promote the cell
growth was also confirmed by the significant increase in the
cell content in the process of B. mucilaginosus AS1153 growing
in the NB medium when the biochar derived from corn stover
was supplemented.
Effect of particle size on cell growth of
B. mucilaginosus
A key physical feature of biochar is its large surface area, which
can provide the refuge being benefited for soil microorganism
and binding important nutritive ions.[10] The better cell
growth of B. mucilaginosus was observed in conjunction with
the ground biochars (about 1 mm in size) than that with the
crushed biochar (about 2 mm in size; data not shown). This
suggested that the promotion of biochar on cell growth was
probably dependent on the particle size of the biochar because
of the increased surface area.[10,12] The comparison in the cell
growth of B. mucilaginosus in the NB medium was performed
in the presence of the biochar derived from corn stover with
various particle sizes. As shown in Figure 3, the survival cell
mass was negatively interrelated with the particle size of the
added biochars. The large surface area and volume of pores
were thought to provide a significant habitat for microbes.[18]
Effect of washed biochar and its washed liquor on cell
growth of B. mucilaginosus
The biochar application conclusively has a positive effect on
microbial activity of B. mucilaginosus (Figures 1 and 2), but
there is a little knowledge about why biochar benefits the cell
growth. One reason was likely connected to the intrinsic
properties of biochar.[12] Thus, the comparison in the cell
growth of B. mucilaginosus was necessary to be performed in
the presence of biochar that was washed with different
solvents.
To remove the potential competent composite in the bio-
char thoroughly, the biochar derived from corn stover was
Figure 3. Effect of the particle size of biochar on the cell growth when Bacillus
mucilaginosus was cultured at 30°C in the presence of the corn stover biochars
with various particle size including smaller than 0.178 mm (C1), 0.178–0.42 mm
(C2), 0.42–0.84 mm (C3), and 0.84–2 mm (C4). The control was performed in
the NB medium without the addition of biochar.
PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 35
washed with different solvents. After that, the cell growth of
B. mucilaginosus AS1153 in the presence of the washed
biochar was determined, respectively. As shown in Figure 4a,
the cell growth of B. mucilaginosus AS1153 in the NB medium
supplemented with the washed biochar was lower than that
supplemented with the unwashed biochar but higher than
that without biochar addition. This suggested that the pro-
motion of cell growth by the washed biochar was lower than
that by the unwashed biochar, presumably because of the
potential utilization of the biochar components by the
microorganism.[19]
The early researches were usually focused on the biochar
properties.[17] Thus, the washed composites from biochar were
studied in this article to determine if it could stimulate the
cell growth. As shown in Figure 4b, the effect of the washed
liquor with distilled water and acetone on cell growth of B.
mucilaginosus AS1153 was determined, respectively. All the
washed liquors from biochars could promote the cell growth
of B. mucilaginosus AS1153. However, it is interesting that
the survival cell content in the NB medium with the unwashed
biochar was equal to the total cell amount in both the washed
biochar, and its washed liquor indicated that the promotion of
cell growth by the washed biochar together with its washed
liquor is equal to that by its unwashed biochar. This suggested
that the component washed from biochar had a promotion
activity on the cell growth of B. mucilaginosus. Steiner et al.[20]
Figure 4. Effect of the washed biochar derived from corn stover with different
solvents (a) and its washed liquor (b) on the cell growth of Bacillus mucilaginosus
AS1153 in the NB medium at 30°C. The cell growth in the NB medium supple-
mented with the unwashed biochar was determined as a reference test and
the control was performed in the NB medium without the addition of biochar.
36 S. LIU ET AL.
also found that a fraction leached from biochar in some cases
could stimulate the microbial activity and increase the
microbial abundance in soil. However, the promoting activity
on microbial growth in the washed component by acetone
was significantly lower than that by distilled water (Figure 4b).
This was different from the previous study of frequently
referred to as a volatile matter.[20]
Survival stability of B. mucilaginosus adsorbed in
biochar
The increasing evidence supported that the biochar strongly
impacted the cell growth,[12] whereas it is interesting that
B. mucilaginosus cells leaked from the biochar also showed
a high cell growth in the next liquid culture even in the
absence of biochar (data not shown). The changes in survival
content of B. mucilaginosus A1153 adsorbed in the biochar
derived from corn stover were determined over the incubat-
ing time at 30°C. As shown in Figure 5, the survival cell
content in the biochar was higher than that in the liquid
culture fluid. There were survival cells occurred in biochar,
even though B. mucilaginosus was incubated at 30°C for 5 hr.
However, almost all cells of B. mucilaginosus in the liquid
culture were not survivable at that time. Thus, the biochar
could sharply prolong the survival time of B. mucilaginosus
AS1153.
Potassium-solubilizing activity of B. mucilaginosus
After incubation in the NB medium with and without biochar
derived from corm stover, the potassium-solubilizing activity
of B. mucilaginosus AS1153 was determined, respectively.
The potassium-solubilizing activity was increased by about
80% in the presence of biochar derived from corn stover in
comparison with that without biochar supplementation, in
which the potassium probably leached out from biochar was
deducted by the reference test, whereas the mobilization of
potassium was increased from 12.3 to 46.0% in the presence
of B. mucilaginosus without supplementation of biochar.[21]
These suggested that B. mucilaginosus adsorbed in the bio-
chars derived from corn stover could be used as a sustainable
biofertilizer to improve plant growth.
Figure 5. Changes in the survival cells of Bacillus mucilaginosus AS1153 in
the biochar derived from corn stover (•) and liquid culture (�) when incubation
at 30°C.
Conclusion
Biochars derived from various feedstocks showed the diversity
in promotion on the cell growth of B. mucilaginosus AS1153.
The capability of biochar to impact on the microbial activity
of B. mucilaginosus was dependent on the biochar’s particle
size. The experimental results showed a promotion on the
cell growth of B. mucilaginosus by both intrinsic features
and inner components. Biochar derived from corn stover
could increase the potassium availability for plant growth by
stimulating the microbial activity of the potassium-solubilizing
B. mucilaginosus. Biochar could prolong the survival time
under normal condition and improve the growing activity of
B. mucilaginosus, which benefits B. mucilaginosus adsorbing
in biochar as biofertilizer for agricultural process.
Funding
This work was supported by Natural Sciences Foundation of China
(31371742), Special Fund for Agroscientific (marine) Research in the
Public Interest (201303095, 201405003).
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