World Journal of Microbiology and Biotechnology (2023) 39:87

https://doi.org/10.1007/s11274-023-03533-3

RESEARCH

Study on the mechanism of biochar affecting the effectiveness

of phosphate solubilizing bacteria
Jingkun Lu1,2 · Sainan Liu1,2 · Wenfu Chen1,2 · Jun Meng1,2

Received: 7 September 2022 / Accepted: 23 January 2023 / Published online: 2 February 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023

Abstract
Low phosphorus utilization and phosphorus fertilizer pollution are serious issues primarily affecting soil health. To inves-
tigate the effects of biochar on the growth, phosphorus solubilization, and metabolites of phosphorus-solubilizing bacteria
(PSB), rice husk biochar (RH) and rice straw biochar (RS) were incubated with Bacillus megatherium (BM1) and Bacillus
mucilaginosus (BM2), respectively. The highest phosphorus solubilization was observed in BM2 following the addition of
RS. The dissolved amount of phosphorus was 244.99 mg/L, which was 43.86% higher than that of the control group. Hence,
biochar can improve the phosphorus solubilization capacity of PSB by affecting the organic acid and polysaccharide contents,
and phosphatase activity secreted by the PSB, as the porous structure and surface characteristics of biochar ensured the
adsorption of PSB. This study can help improve the functional activity of PSB and provide basis for improving the utilization
of soil phosphorus, which in turn, aid in the development of biochar-based microbial fertilizers.

Keywords  Phosphate-solubilizing bacteria · Phosphorus solubilization · Organic acid · Phosphatase activity ·

Polysaccharide

Introduction contents in soils is crucial to improve the efficiency of plant

phosphorus utilization.
In plant metabolism, phosphorus (P) is a key nutrient Phosphate-solubilizing bacteria (PSB) plays an important
involved in photosynthesis, energy transfer, signal transduc- role in the soil and plant rhizosphere and are considered
tion, macromolecular biosynthesis, and respiration. It is one to be environmentally friendly bioinoculants in agricultural
of the main nutrients essential for biological growth and production (Hussain et al. 2019). PSBs can convert insoluble
development (Khan et al. 2010; Cordell et al. 2011; Ali et al. phosphate in soil into an available form, facilitating plant
2020). In soils, its mineral forms consist of primary minerals uptake and utilization through mineralization, solubiliza-
such as apatite, hydroxyapatite, and oxyapatite, primarily tion, and immobilization (Liu et al. 2014; Pereira and Cas-
owing to its insolubility (Sharma et al. 2013). Phosphorus tro 2014; de Amaral Leite et al. 2020). PSBs secrete low-
minerals can be converted into organic and inorganic forms molecular-weight organic acids that bind calcium, iron, and
depending on external factors in the environment, which can aluminum ions and release phosphate. Secretion of enzymes
made it available to plants and microorganisms. The lat- such as phosphatase and phytase, including ion chelators
ter is mainly present as insoluble mineral complexes, which promotes phosphorus solubilization (Rawat et al. 2021). PSB
cannot be taken up by plants (Rodríguez et al. 1999; Zhang inoculation is believed to be an effective way to achieve high
et al. 2016). Therefore, increasing the soluble phosphorus phosphate nutrition efficiency and high crop yield.
Biochar application is another method to improve soil
fertility, as biochar contains high amounts of carbonaceous
* Jun Meng material as a result of high-temperature pyrolysis of organic
[email protected] feedstock under anaerobic conditions (Lehmann et al. 2011).
It also improves soil quality and phosphorus availability by
1
Agricultural College, Shenyang Agricultural University, increasing the cation exchange capacity and pH (Bekiaris
Shenyang, China
et al. 2016). Numerous studies have indicated changes in
2
Key Laboratory of Biochar and Soil Improvement, Ministry soil microbial properties such as higher microbial biomass,
of Agriculture and Rural Affairs, Shenyang 110866, China

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community composition, and diversity upon addition of bio- biochar has been found to directly increase the microbial
char to the soil. Further, biochar was also found to improve population by providing a good habitat within its large inter-
soil biological properties by enhancing the functional nal surface area and porous structure, and improves P avail-
activity of soil microorganisms and positively altering the ability in soil through PSB (Ghodszad et al. 2021).
community structure (Li et al. 2018). Its effect as a carrier
of microorganisms on the activity of the bacteria has also
been extensively studied in the past. For instance, Husna Materials and method
et al. (Husna et al. 2019) empty bundles of rice and coconut
husks, corn cob, and oil palm as carriers of phosphorus solu- Biochar production and inoculum preparation
bilizing microorganisms (PSM). Here, not only did it ensure
the survival of PSM, but also it increased the activity of the Biochar was obtained from rice husks and rice straws col-
PSM that was loaded on the coconut husk biochar. Mean- lected from the Rice Research Institute of the Agricultural
while, Hale et al. (2015) used pinewood biochar as the active College, Shenyang Agricultural University. The rice husks
carrier of Enterobacter cloacae UW5 (PGPR) to strengthen and rice straws were air-dried and ground to pass a 2-mm
the microbial abundances when it was its added to the soil sieve, then placed in a ceramic crucible, heated anaerobi-
(Hale et al. 2015). Biochar also contains different types of cally at 400 °C for 1 h in a muffle furnace, and cooled to
volatile organic compounds (VOCs) depending on the feed- room temperature. The pore properties of the resulting
stock type and pyrolysis conditions, in which it may inhibit biochar products, including the surface area, pore volume,
or enhance the activity of functional soil microorganisms. and pore diameter, were determined using a surface area
The VOCs have certain solubility in aqueous solutions, they and porosity analyzer (Micromeritics ASAP 2460, USA).
are likely to form part of dissolved organic carbon upon bio- The electrical conductivity (EC) were measured using an
char application (Spokas et al. 2011). Consequently, it may S7-Standard kit conductivity meter. pH was measured using
be used as a carbon source by PSM. Biochar may not only be a pH meter (HI2221, Hanna). The available P content was
an independent source of phosphorus to soil PSM (Parvage determined by referring to the Routine Analysis Method
et al. 2013), but it may also serve as a promoter of phospho- for Soil Agricultural Chemistry edited and released by the
rus release (Qian et al. 2019). For instance, the synergistic Professional Committee of Agricultural Chemistry of the
application of biochar with phosphorus-solubilizing micro- Chinese Society for Soil Sciences (1989). The total C and N
bial agents has been reported to have significantly increased contents were determined using an elemental analyzer (vario
the effectiveness of soil phosphorus (Zwetsloot et al. 2016). MACRO cube, Elementar, Germany). The basic properties
The VOCs contained in biochar also supported the survival of the rice husk biochar (RH) and rice straw biochar (RS)
of Bacillus mucilaginosus despite its short lifetime, hence, are shown in Table 1.
this indicates its potential as an inoculum carrier during Phosphate-solubilizing bacteria using Bacillus megath-
biochar application (Sun et al. 2015). Previous studies have erium, CGMCC1.234 (BM1) and Bacillus mucilaginosus,
suggested that organic molecules in biochar may promote CGMCC1.2326 (BM2) were obtained from the China Gen-
the growth of phosphorus-solubilizing microbes, resulting in eral Microbiological Culture Collection Center (CGMCC).
an increased bacterial activity and solubilization of insoluble BM1 and BM2 were placed in beef extract-peptone agar
phosphate (Efthymiou et al. 2018). It has also been found to medium and silicate bacterial medium, respectively. There-
significantly enhance the formation of PSB biofilms, which after, they were incubated at 30  °C until a logarithmic
contributes to better adaptation of PSB to unfavorable envi- growth in population was achieved, in which they were
ronments, including the improvement of their phosphorus
mobilization capacity (Tao et al. 2019). It is expected that
Table 1  Properties of RH and RS
the re-application of biochar to the soil following microbial
loading may aid in fully regulating the effects of biochar on RH RS
soil microorganisms.
BET Surface Area (­ m2/g) 4.01 3.82
Generally, biochar has been found to have positive effects
Total pore volume(cm3/ g) 0.0055 0.0059
on PSB. However, its effects on the function and metabo-
Average pore diameter ( nm) 5.5012 6.1950
lism of PSB remains largely unknown. As it can promote
EC (ms/m) 1.52 1.40
microbial metabolism, owing to its functional groups, sur-
pH 7.68 8.72
face charge, and porosity (Chen et al. 2019), its effects on
Available P (mg/kg) 288.20 295.40
the activity and phosphorus solubilization of PSB warrants
C% 56.04 36.72
attention. In this study, indoor incubation experiments were
N% 0.74 1.11
used to evaluate the effects of biochar on the growth, phos-
phorus solubilization capacity, and metabolites of PSB, as RH rice husk biochar, RS rice straw biochar

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subsequently subjected under co-cultured conditions of 2% pressed into tablets. The spectral scanning range, resolution,
bacterial seed solution and 1% biochar (v/v) in 100 mL of and scanning rate was 4000 ­cm−1–400  ­cm−1, 4  ­cm−1, and
Pikovskaya medium. The soluble phosphorus and bacterial 50 ­cm−1, respectively.
metabolite contents were then measured.
The Pikovskaya medium supplemented with bacteria Phosphorus solubilization and pH
and biochar was used as the control, in which the medium
formula was as follows: the beef extract-peptone medium The bacterial inoculum (2%) and 1% (V/V) biochar were
(per L) was composed of 5 g peptone, 3 g beef extract, and mixed in 100 mL of the Pikovskaya medium. The mixture
5 g NaCl at pH 7.0–7.2; while the silicate bacterial cul- of biochar and PSB was triplicated and cultured at 30 °C
ture medium (per L) was composed of 5 g sucrose, 0.5 g and 160 rpm for 72 h. The Sterile water-inoculated medium
­MgSO4·7H2O, 0.1 g ­CaCO3, 1.0 g ­(NH4)2SO4, 0.005 g ­FeCl3 was used as the blank treatment and PSB was used as the
and 2 g K
­ 2HPO4 at pH 7.0–7.2. The Pikovskaya medium (per control. During fermentation, the solubilized P concentra-
L) was composed of 10.0 g glucose, 0.5 g(NH4)2SO4, 0.3 g tion was measured using the Mo-blue method (Alam et al.
NaCl, 0.3 g KCl, 0.03 g F
­ eSO4·7H2O, 0.3 g M ­ gSO4·7H2O, 2002), while the pH of the medium was measured using a
0.03 g ­MnSO4·4H2O, 0.5 g yeast extract and 5.0 g ­Ca3(PO4)2 pH meter (HI2221, Hanna, Italy).
at pH 7.0–7.5.
Organic acid analysis
Preparation of the bacterial incubation
The cultures were centrifuged and filtered through 0.22 μm
Bacteria were inoculated with 2% strains BM1 and BM2, millipore filters. The organic acid content of the filtrate was
whereas biochar was inoculated with RH and RS. Both measured using high-performance liquid chromatography
were inoculated into 50 mL of beef extract-peptone agar (HPLC, 1525-2489, Waters, USA). The separation condi-
medium and silicate bacterial medium, respectively. They tions were as follows: the high-performance liquid chroma-
were shaken in an orbital incubator in dark conditions at tography column was Hypersilc, 8 (250 mm × 4.6 mmi.d.,
160 rpm and 30 °C. The mixed supernatants were serially 5 μm); the mobile phase was 0.02N sulfuric acid, and the
diluted at different time intervals (1, 3, 6, 12, 24, and 48 h). UV detection wavelength was 210 nm; the injection vol-
Cell counting was performed using the standard plate count- ume was 10 μL, flow rate was 0.5 mL ­min−1, and column
ing method. The biochar was sterilized before addition. The temperature was room temperature. The standard curves for
bacterial growth without the biochar was used as a control. organic acids were based on oxalic, malic, acetic, citric, and
succinic acids, with retention times of 4.0, 4.9, 5.6, 6.6 and
7.3 min respectively.
Scanning electron microscopy (SEM)
Extraction of EPS and protein
The microstructure of the biochar was observed using scan-
ning electron microscopy (SEM, Regulus 8100, Hitachi The fermentation broth was dialyzed in membrane tubes to
Limited, Japan). Each treatment was performed at 30 °C and remove small molecules and inorganic ions. The collected
160 rpm for 72 h. After centrifugation and removal of the supernatant was precipitated by adding two volumes of 95%
supernatant, the mixture was freeze-dried and gold-sprayed ethanol and was left overnight at 4 °C (Hu et al. 2021). Fol-
to observe the morphology of the biochar before and after lowing centrifugation at 8000r for 10 min, the precipitate
loading, and the surface morphology of the samples was was collected, dried, and crushed to obtain crude EPS. The
observed by SEM. The scanning electron microscopy (SEM) total sugar content of the different samples was analyzed
acceleration voltage was set to 1 kV. using the phenol–sulfuric acid method (Dubois et al. 1956).
The total protein content was determined using the Bradford
Fourier transform infrared spectrometer (FTIR) protein quantification kit, according to the manufacturer's
instructions.
Spectrograms of the samples were obtained using Fourier
Transform Infrared Spectroscopy (FTIR, TENSOR27, Alkaline phosphatase (ALP) and Acid phosphatase
Bruker Optics, Germany). Each treatment was incubated at (ACP) activity
30 °C and 160 rpm for five days. The fermentation mixture
was centrifuged at 8000r for 10 min. The precipitate was The fermentation broth was centrifuged to obtain the
washed twice with sterile water, in which the remaining supernatant. Phosphatase activity was measured using
portion was dried and mixed with potassium bromide in a the Leagene® Alkaline Phosphatase Assay Kit (Sigma-
mortar and pestle prior to grinding into a fine powder and Aldrich) and Leagene® Acid Phosphatase Assay Kit

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(Sigma-Aldrich). Alkaline phosphatase (ALP) was plotting and correlation analysis were performed using
detected using the benzene disodium phosphate colori- Origin 2021.
metric method. Benzene disodium phosphate produces
free phenols and phosphoric acid via alkaline phos-
phatase. Under alkaline condition, phenol combines with
aminoantipyrine and oxidizes to form red quinone struc- Results
ture. Absorbance was measured at 510 nm. The standard
curve includes 0.05 mg/ml phenol 0, 0.1, 0.2, 0.3, 0.4, and Effect of biochar on bacterial growth
0.5 mL, and was supplemented with 0.55 mL d­ dH2O to
correspond to the alkaline phosphatase activity of 0, 10, To analyze the effect of biochar on the growth of the two
20, 30, 40, and 50 U/L. bacterial strains, the quantity of bacteria in the supernatant
As acid phosphatase (ACP) hydrolyzes benzene diso- of the medium was determined at different time intervals
dium phosphate to produce phenol and inorganic phos- (Fig. 1). All treatments showed an increasing trend in bac-
phorus, it was detected using benzene disodium phosphate terial growth as incubation times increased. RH and RS
colorimetry. Following alkali treatment, phenol reacts with had positive effects on BM1 growth (Fig. 1A). The biochar
4-APP, in which the resulting product is oxidized to form treated with BM1 exhibited a more significant increase in
a red quinone compound. Absorbance was measured at the bacterial population than the BM1 treatment alone. In
510 nm. The standard curve includes 10 mM phenol 0, 10, particular, the biochar treatment significantly increased
20, 30, 40, 50 μL, which were added to 1 mL of distilled the number of bacterial colonies in BM1 with increasing
water to correspond to acid phosphatase activities of 0, 10, incubation time (P < 0.05). Following the addition of bio-
20, 30, 40, 50 U/L. char, BM1 preferentially reached the maximum logarithmic
growth stage compared with the control treatment. RH and
RS negatively affected BM2 (Fig. 1B), in which the biochar
Statistical analysis treated with BM2 exhibited a more apparent reduction in
bacterial count than the BM2 treatment alone.
Each experiment triplicated. The mean and standard devia- The biochar treatment also significantly reduced the num-
tion were calculated using descriptive statistics. Univariate ber of bacterial colonies in the BM2 cells (P < 0.05). How-
analysis of variance (ANOVA) with SPSS 16.0 was used ever, the differences in bacterial growth between the RH and
to with and without assess differences biochar treatment. RS treatments found to be non-significant.
This study considered a 5% statistical significance. Data

Fig. 1  Effect of biochar addition on the growth curves of BM1 (A) and BM2 (B). RH rice husk biochar, RS rice straw biochar. All data represent
the mean ± standard error

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Fig. 2  SEM images of bacteria adsorbed by biochar in the presence on the surface of RH (A) and RS (B); C, D BM2 is attached on the
of PSB (BM1 Bacillus megatherium, BM2 Bacillus mucilaginosus, surface of RH (C) and RS (D)
RH rice husk biochar, RS rice straw biochar.). A, B BM1 is attached

SEM analysis the O–H stretching vibration peaks of alcohols and phenols
associated with intermolecular hydrogen bonds developed
Following the addition of PSB, the surface of the biochar from 3423–3425 ­cm−1 (Singh et al. 2020). The vibrations
showed a distinct bacterial morphology (Fig. 2), in which observed from 2821–2825 ­cm−1 range can be attributed
the bacteria appeared as bio-aggregates in the biochar. RH to the symmetric and asymmetric extensions of aliphatic
and RS provided growth sites for both bacteria, and BM1 hydrocarbons or cycloalkanes such as -CH3 and -CH2. At
and BM2 adhered to the surface and pores of the biochar 1595–1645  ­cm−1, the biochar showed a vibrational peak
without affecting the surface structure of the bacteria. The of carboxyl C = O (Chia et al. 2012), which suggests that
corresponding surface areas of RH and RS were found to be biochar was composed of hydroxyl, aromatic, and oxygen-
4.01 ­m2/g and 3.82 ­m2/g, respectively (Table.1). The total containing groups. Normally, the peaks below 1300 ­cm−1
pore volume and average pore diameter of RS were higher are indicative of the presence of phosphorus and phos-
than those of RH, indicating that the pore size structure of phorus-containing compounds, however, the peak of tri-
RS was more advantageous compared to that of RH. calcium phosphate was observed to fluctuate between
770–778  ­cm−1, in which a stretching vibration of P-O-P
FTIR analysis was found at 549 ­cm−1 (Bekiaris et al. 2016). Notably, the
observed changes in the functional groups of the biochar
The surface functional groups of the bacteria-loaded following bacteria loading were found to be statistically
biochar were characterized using infrared spectroscopy non-significant.
(Fig. 3). All biochar treatments showed similar patterns
in the infrared spectrum, indicating that both biochar con- Changes in pH and P release
tained certain types of functional groups with nearly simi-
lar quantity. The results also showed that the C-H vibra- Prior to sterilization, the pH values of the CK, RH, and RS
tional absorption peaks of the aromatic rings of RH and were 7.04, 7.72, and 8.76, respectively. However, it was
RS appeared from 770–778 ­cm−1 (Sun et al. 2011), while reduced in the cells treated with PSB (Fig. 4A) following

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Fig. 3  FTIR spectra of RH (A) and RS (B) after adsorption of PSB (RH rice husk biochar, RS rice straw biochar, BM1 Bacillus megatherium,
BM2 Bacillus mucilaginosus 

Fig. 4  The concentrations (mg/L) of soluble P and the final pH values of different treatments in Pikovskaya medium. CK without biochar, RH
rice husk biochar, RS rice straw biochar. Different letters indicate significant differences at the P < 0.05 level

72 h of incubation. BM1, BM1 + RH, and BM1 + RS showed to that of the control (P < 0.05). Moreover, a significant
pH values of 4.79, 5.26 and 5.88, respectively, were decrease (P < 0.05) in pH was observed in the RH and RS
decreased by 2.25, 2.46, and 3.50, respectively. The pH val- treated media compared that in the single BM2 treatment, in
ues for BM2, BM2 + RH, and BM2 + RS were 5.46, 5.20, which it was particularly more rapidly decreasing following
and 5.01, respectively, which similarly decreased by 1.58, the addition of biochar compared to that before sterilization.
2.52 and 3.75 compared with the pH values prior steriliza- Figure 4B shows the amount of P solubilized in the
tion. The final pH of the RS treatment medium was signifi- medium following the mixed culturing of biochar and
cantly higher than that of the BM1 treatment alone, however, PSB. The soluble P in the medium of all treatments
it was found to have no statistical significance compared ranged from 102.04–244.99 mg/L, in which the soluble

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P content of the 102.04 mg/L and 137.53 mg/L media or inhibit organic acid production by PSB. The total
for the BM1 + RH and BM1 + RS treatments was found to amount of the five organic acids (oxalic, malic, acetic,
be significantly lower (P < 0.05) than that of the control. citric, and succinic acids) produced by bacteria ranged
The lowest P solubility was observed for BM1 after the from 22.04–662.06  mg/L, which then increased follow-
addition of rice husk biochar. Meanwhile, the soluble P ing the addition of biochar. Particularly, biochar increased
content of the medium of BM2 + RH and BM2 + RS treat- the acetic acid content of BM2 (P < 0.05), and BM2 + RH
ments were 178.05 mg/L and 244.99 mg/L, respectively, in and BM2 + RS obtained the highest acetic acid contents
which the increasing P solubilization was observed in the with 354.55 mg/L and 637.7 mg/L, respectively. However,
BM2 + RS treatment (P < 0.05), compared to that of the biochar also decreased the succinic acid content of PSB
control and BM2 + RH treatments. The soluble P content (P < 0.05). The calculated PSB production of the rice straw
in the medium was significantly higher with the addition of biochar treatment was also higher than that of the rice husk
rice straw biochar than with the addition of rice husk bio- biochar.
char (P < 0.05), and P solubility was observed to be high-
est in BM2 following the addition of rice straw biochar. Changes in EPS and protein content

Changes in organic acid content Figure 5 shows the results of the PSB culture using the Piko-
vskaya medium to determine the effect of biochar on extra-
Table 2 shows the results of the determination of organic cellular polymer production in PSB, including exopolysac-
acids using HPLC. It was found that biochar can promote charides and proteins. The contents of exopolysaccharides

Table 2  Nature and concentration of organic acids in different treatments (mg/L)

Oxalic acid Malic acid Acetic acid Citric acid Succinic acid Total organic acid
c a b
BM1 13.86 ± 0.40 – – 17.36 ± 2.11 22.79 ± 1.59 54.01 ± 3.69c
BM1 + RH 9.79 ± 1.63d 43.3 ± 5.15 – 2.33 ± 1.75c 4.48 ± 0.62c 59.91 ± 3.37c
BM1 + RS 15.52 ± 0.28b – – 6.53 ± 1.97b – 22.04 ± 2.04c
BM2 4.94 ± 0.62e – – 1.15 ± 0.21c 41.05 ± 7.26a 47.14 ± 6.86c
BM2 + RH 9.16 ± 0.43d – 354.55 ± 42.95b 1.25 ± 0.07c – 364.96 ± 43.31b
BM2 + RS 18.99 ± 0.65a – 637.70 ± 52.77a – 5.36 ± 0.37c 662.06 ± 52.16a

Different letters indicate significant differences at the P < 0.05 level

Fig. 5  Effects of biochar on EPS (A) and protein (B) secretion of PSB. CK without biochar, RH rice husk biochar, RS rice straw biochar. Differ-
ent letters indicate significant differences at the P < 0.05 level

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and proteins in PSB cultures increased to an extent following different biochar treatments showed a significant positive
the addition of different biochar, in which the exopolysac- correlation with ALP, ACP, EPS, and protein. However,
charides and protein contents were significantly higher in there was no significant correlation between WSP and pH.
the BM2 treatment than in the BM1 treatment. Here, the Meanwhile, correlation between pH, soluble phosphorus,
exopolysaccharide and protein contents of the CK group was and metabolites of BM2 (Fig. 7B), showed that the pH in
1.07 mg/mL, and 1.94 μg/mL, respectively. Meanwhile, the solution under different biochar treatments was negatively
exopolysaccharide content of the RH and RS groups was correlated with WSP, ACP, ALP, and EPS (P < 0.05). The
1.65 mg/mL and 1.92 mg/mL, and the protein content was soluble phosphorus content also showed a positive correla-
1.56 μg/mL and 4.16 μg/mL. The extracellular polysaccha- tion with ALP, EPS, and protein levels (P < 0.05). However,
ride and protein contents in the RS treatment were also sig- no fixed correlation between pH, WSP content and bacterial
nificantly higher than those in the RH treatment. metabolites was found in bacterial fermentation broth, which
may be attributed to the specific bacterial species.

Phosphatase activity
Discussion
The activities of acid phosphatase (ACP) and alkaline
phosphatase (ALP) of bacteria under different treatments Effects of biochar on the growth of PSB
are shown in Fig. 6. The ACP activity of BM1 was higher
under the biochar treatment than that of the control. Fol- In this study, the addition of biochar significantly increased
lowing the RS treatment, the highest ACP activity of BM1 the number of bacteria in BM1, whereas it decreased in
was 4.14 U/L, and was more effective in increasing the ACP BM2. The bacterial incubation process may have been
activity of BM1. The ALP activities of BM1 and BM2 were influenced by the biochar adsorption, as its pore structure
higher than that of the control group following the addition can serve as a habitat for PSB (Qian et al. 2013; Chandra
of biochar. The ALP of BM1 and BM2 were 19.70 U/L and et al. 2019; Ghodszad et al. 2021). The number of bacteria
26.87 U/L, respectively. in the medium following the addition of rice husk biochar
was higher than of the rice straw biochar, which may be
Correlation analysis of the effect of biochar addition attributed to the higher specific surface area of rice husk
on bacterial metabolites biochar. The average diameter and total pore volume of were
higher in RS than in RH, indicating a greater proportion of
The correlation analysis between pH, soluble phosphorus narrower pores in RH (Table.1). PSB was more likely to be
and metabolites of BM1 (Fig. 7A) showed that pH under adsorb on the surface of RH than in RS. Conversely, PSB

Fig. 6  Effects of biochar on acid phosphatase (A) and alkaline phosphatase (B) activities of PSB. CK Without biochar, RH rice husk biochar, RS
rice straw biochar. Different letters indicate significant differences at the P < 0.05 level

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Fig. 7  Correlation analysis of pH, soluble phosphorus and metabo- **P ≤ 0.01, *P ≤ 0.05. WSP Soluble phosphorus content, ALP alka-
lites in BM1 (A) and BM2 (B) after biochar addition. Asterisks line phosphatase, ACP acid phosphatase, EPS extracellular polysac-
indicate the significance of the relevant differences. ***P ≤ 0.001, charide

may be more readily adsorbed on the pores of the RS than However, no correlations were found between the acid pro-
in RH. duction of PSB and pH. Simultaneous with the changes in
The role of functional groups on the surface of the bio- the organic acid content, changes in the pH in BM1 and
char may also be another factor as the presence of carboxyl, BM2 were relatively consistent, owing to the alkaline nature
amine, and amide groups on the surface of biochar facili- of the biochar itself, that neutralizes the acid in the medium,
tates the adhesion and proliferation of bacterial cells (Lee and provides a buffering effect on the solution during the
et al. 1994). The role of functional groups on the surface of incubation process (Anderson et al. 2011; Yaashikaa et al.
the biochar may also be another factor as the presence of 2020). Another plausible reason is the development of other
carboxyl, amine, and amide groups on the surface of bio- types of organic acids such as gluconic acid and tartaric acid
char facilitates the adhesion and proliferation of bacterial in the culture medium (Rawat et al. 2021). As the types and
cells (Lian et al. 2008). Therefore, compared with BM1, concentrations of organic acids produced by different PSB
the flocculated material produced by BM2 is more likely species differ significantly, the acidity coefficient and abso-
to be adsorb onto the biochar and influence the amount of lute content of organic acids may also be important to be
bacterial growth in the fermentation broth. However, these considered, in which Arnett (1964) suggested that the lower
changes were statistically non-significant, in which similar the acidity coefficient and pH, the easier it is for the acid
functional groups may have present in the biochar and PSB. to ionize hydrogen ions (Arnett et al. 1964). In this study,
with an acidity coefficient of 4.76 pKa, acetic acid ionized
fewer hydrogen ions than oxalic acid 1.27 pKa. Although
Effects of biochar on phosphorus the organic acid content was high, the pH decrease may not
solubilization ability and metabolites of PSB be significant.
A significant negative correlation between pH and P
Increasing the P solubilization rate of PSB combined with solubilization in BM2 was found. Similarly, previous stud-
biochar may be an effective way to increase its P solubi- ies have also found a negative correlation between pH and
lization rate. In this study, biochar had significant effects soluble phosphorus, suggesting that acidification may con-
on the soil pH and acid production. It reduced the pH of tribute to P solubilization (Park et al. 2011). However, a
the PSB medium and increased BM2 organic acid produc- negative correlation between pH and P solubilization may
tion. Previous studies have indicated that the decrease in not be appropriate for all PSBs, as no significant correla-
pH may be attributed to an increase in organic acids, which tions were found in BM1. Hence, the solubilization of P
may be related to P solubilization (Wei et al. 2017). The may not be an independent process, and would depend on
addition of biochar increased the acid production of PSB, the dissolution of calcium phosphate (Ca-P) and release of
particularly the amount of acetic acid produced by BM2. phosphorus, which are triggered by a combination of pH

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Page 10 of 12 World Journal of Microbiology and Biotechnology (2023) 39:87

reduction and carboxylic acid synthesis (Deubel et al. 2000; of PSB. This study demonstrated the effectiveness of bio-
Khan et al. 2009). Further, the relationship between pH and char as an immobilizer for phosphate-solubilizing bacte-
P solubilization may not be significant, as it may vary from ria, in which it enhanced the P solubilization rate of PSB.
one bacterial species to another (Chen et al. 2019). Hence, this will help aid future research on biochar-based
Other metabolites of BM1 and BM2 were significantly microbial fertilizers, including the development of further
and differentially affected by biochar. For instance, biochar improvements to positively impact soil health.
significantly increased the polysaccharide content of PSB,
owing to the active substances and functional groups on the
surface of biochar, which produces specific reactions with
microbial extracellular polymers and promote the growth of Conclusions
bacterial biofilms (Bueno et al. 2018). Biochar also enhances
the formation of PSB biofilm, aids in the better adaptation Biochar was shown to have significant effects on PSB,
of PSB to adverse environments, and enhances phospho- in which it particularly increased the growth of BM1.
rus mobilization capacity (Tao et al. 2019). Although no However, the adsorption effects was found to be differ-
organic P source was used in this study, phosphatase activity ent among PSBs depending on the physical properties of
was detected in the culture media. This may be attributed to RH and RS, as it has been found to also have significant
the kinetics of the formation of both enzymes. Measurable effects on growth of bacteria. The addition of biochar sig-
amounts of alkaline phosphatase may be formed when inor- nificantly improved the phosphorus solubility of BM2.
ganic phosphorus becomes limiting in the medium, espe- Although there was no significant correlation between the
cially when the enzyme forms large amounts of alkaline growth numbers of different bacteria and phosphorus solu-
phosphatase at a maximum rate (Torriani 1960). When the bilization capacity, RH and RS had significant effects on
sole source of phosphorus in soils are insoluble inorganic the organic acid, phosphatase, and polysaccharide contents
phosphates, it can convert organic phosphorus to inorganic secreted by PSB, which are important factors for improv-
phosphorus from the decomposing microbial population, ing the phosphorus solubilization capacity of the PSB. RS
in which it can participate in the microbial metabolic pro- showed the most significant performance in phosphorus
cess once the inorganic phosphorus fraction is taken up by solubility, phosphatase activity, and polysaccharide con-
microorganisms. tent in BM2. With this, this study further understanding
It is inferred that the BM2 significantly increases P sol- on the application of biochar and PSB in soil and crops,
ubilization following biochar addition, as it triggers the which may aid in the development and improvement of
release of BM2 organic acids, particularly acetic acid. The biochar-based microbial fertilizers in the future.
decrease in pH of the medium was beneficial for increasing
Acknowledgements  The authors would like to thank Mr. Jun Meng
the P solubilization capacity of BM2. The mechanism by and Mr. Wenfu Chen for their help on the experimental design. The
which biochar increases the solubility of P in PSB consists authors also appreciate the valuable suggestions from the reviewers.
of several phenomena: through the addition of neutraliz-
ing agents, which increases the acid production capacity Author contributions  JL, JM and WC designed the study and wrote
the manuscript. JL and SL collected and analyzed the data. All authors
of some acid-producing microorganisms. The buffering contributed to the article and approved the submitted version.
capacity of the medium involved in the complex process
of solubilization enhancement of insoluble phosphate, in Funding  This study was financially supported by the Key Projects of
which microenvironments in the buffered state can inhibit the Ministry of Agriculture and Rural Affairs (NK2022180602), the
Science and Technology Plan Project of Shenyang, China (22-317-
or stimulate the release of organic acids from microorgan- 2-08), the Earmarked Fund for Modern Agroindustry Technology
isms (Takeda et al. 2006). Biochar also has an important Research System, China (No. CARS-01-51).
buffering role in the P solubilization process of PSB that
promotes the acid production capacity of PSB. The pres- Data availability  All data generated or analysed during this study are
included in this published article.
ence of functional groups also influences the solubility
of P, as biochar contains hydroxyl and carboxyl groups, Declarations 
which play a role in P solubilization (Yuan et al. 2017;
Chen et al. 2019). In addition, the increase in the carbon Competing interests  The authors declare that they have no conflict
source in the medium following the addition of biochar of interest.
can also be one of the factors, as PSB also uses the carbon
source in the medium to promote metabolic processes,
leading to an increase in P solubilization.
This study aimed to investigate the mechanisms by
which biochar affects the phosphorus solubilization effect

13
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