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. 2025 Oct 21;28(8):2869–2880. doi: 10.1007/s10123-025-00737-5

Inoculation methods that enhance the survival of Bacillus strains in rice husk biochar for use as bacterial carriers

Hiroki Nakahara 1, Nobutaka Someya 1,✉, Yosuke Maeda 1, Masaharu Kubota 1
PMCID: PMC12727821  PMID: 41118014

Abstract

Storing biochar in soil is an effective method for improving the soil environment and reducing atmospheric greenhouse gases. Some strains of Bacillus species have been utilized as plant growth-promoting bacteria or biological control agents against plant diseases and pests. By colonizing biochar with beneficial bacteria, highly functional biochar that contributes to increased crop yields can be developed. In this study, we investigated the survival of type strains of eight Bacillus species (Bacillus amyloliquefaciens, B. licheniformis, B. nakamurai, B. pumilus, B. siamensis, B. subtilis, B. thuringiensis, and B. velezensis) in rice husk biochar, based on the sporulation state of vegetative cells or endospores. The bacterial density in the biochar significantly reduced when inoculated with a high concentration of vegetative cells, whereas it remained high when inoculated with a high concentration of spores. When water-washed biochar was inoculated with a high concentration of vegetative cells, survival significantly improved compared to that when unwashed biochar was inoculated. When a high concentration of vegetative cells was inoculated into the biochar water extract (biochar: water = 1:10), the bacterial densities of most species, except B. nakamurai and B. licheniformis, were significantly lower than those in alkali-sterilized water at pH 10.2 (same pH as biochar) and normal sterilized water (pH 6.7). These results suggest that the survival of Bacillus vegetative cells in biochar is inhibited by water-soluble inhibitors contained in the biochar rather than by alkaline conditions. When endospores were inoculated onto unwashed biochar, all strains maintained high viability for at least 12 weeks.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10123-025-00737-5.

Keywords: Biocontrol agents, Carbon farming, Endospores, High-functional biochar, Microbial carrier, Sporulation

Introduction

According to the Intergovernmental Panel on Climate Change, biochar is defined as a solid material generated by heating biomass to a temperature > 350 °C under conditions of controlled and limited oxidant concentrations to prevent combustion (IPCC 2019). Storing biochar on agricultural land is a promising strategy for reducing greenhouse gases such as CO₂, N₂O, and CH₄ in the atmosphere through long-term carbon sequestration and enhanced microbial processes, including increased methanotroph activity and microbial reduction of N₂O to N₂ (Joseph et al. 2021; Lehmann et al. 2021). This has attracted global attention to the impacts of biochar application on agricultural soils and crop production. Traditionally produced rice husk biochar (RHB) has been used as a soil conditioner for a long time in rice-producing countries in Asia, including Japan (Asadi et al. 2021; Ogawa and Okimori 2010). RHB usually has higher Si and K compounds, ash content, and pH (8.5–10) than other biochars, and RHB application improves the soil’s organic carbon content, cation exchange capacity, available Si and K concentrations, water content, and microbial activity (Asadi et al. 2021; Prakongkep et al. 2013).

To store a large amount of carbon in the soil and decrease CO2 in the atmosphere, it is necessary to expand and continue the application of biochar to agricultural lands. However, biochar contains few fertilizer components, and its application alone often results in limited increases in crop yield (Joseph et al. 2021). Additionally, biochar contains growth inhibitors such as alkalis, phenols, polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds, and heavy metals, which may inhibit plant growth and microbial activity in the soil depending on the application conditions (Brtnicky et al. 2021; Buss et al. 2015, 2016; José et al. 2019; Xiang et al. 2021). Biochar can serve as a carrier for microorganisms that promote plant growth and suppress diseases. Co-application of biochar with beneficial microorganisms is more likely to produce consistent growth-promoting effects than applying biochar alone (Ajeng et al. 2020; Bolan et al. 2023; Win et al. 2020).

Plant growth-promoting bacteria (PGPB) and biological control agents (BCAs) against plant diseases are among the factors that increase crop yields (El-Saadony et al. 2022). Particularly, many strains of the Bacillus and Pseudomonas genera have been reported to be effective as PGPB and BCAs (Dimkić et al. 2022; Etesami et al. 2023; Wang et al. 2024). Several strains of Bacillus species have been widely used as PGPB and BCAs against plant diseases and pests (Azizoglu 2019; Ngalimat et al. 2021; Wang et al. 2024). Bacillus species are broadly classified into two major clades: the subtilis clade, which includes B. subtilis and related species, and the cereus clade, which includes B. cereus and related species (Gupta et al. 2020). Species within the subtilis clade, such as B. subtilis, B. licheniformis, B. pumilus, and members of the operational group B. amyloliquefaciens (OGBa; including B. amyloliquefaciens, B. velezensis, B. nakamurai, and B. siamensis), have been reported in many studies as PGPB and BCAs (Ngalimat et al. 2021; Wang et al. 2024). Within the cereus clade, B. thuringiensis is widely used as a BCA for pest control (Azizoglu 2019). Bacillus strains produce a wide range of antimicrobial compounds, and many strains exhibit antimicrobial activity against plant pathogens, such as Rhizoctonia and Fusarium (Etesami et al. 2023; Fira et al. 2018).

Several studies have reported the use of biochar as an inoculant carrier (Ajeng et al. 2020, 2023; Bolan et al. 2023; Deng et al. 2023; Han et al. 2023; Malik et al. 2022; Xiang et al. 2022). Biochar coated with beneficial bacterial strains has the potential to become a highly functional biochar that can increase crop yield. However, the survival of bacterial strains on biochar varies depending on the bacterial species and the type of raw materials of biochars, which can inhibit bacterial survival (Yang et al. 2019). Various biochar properties, including surface area, surface charge, porosity, functional groups, and pH, can influence the adsorption and retention of microbes, including bacteria (Bolan et al. 2023). In addition, conditions that enhance the survival of Bacillus strains in biochar have been explored. For example, biochar modified with starch, amino acids, citric acid, sulfuric acid, or quaternary ammonium salts improves bacterial immobilization (Ajeng et al. 2023; Deng et al. 2023; Han et al. 2023). However, the effects of different sporulation states, such as vegetative cells versus endospores, on the viability of Bacillus in biochar remain largely unstudied.

Bacillus strains are known to undergo sporulation from vegetative cells to endospores (Tan and Ramamurthi 2014). These endospores exhibit high environmental resistance, significantly enhancing their viability and enabling them to survive high temperatures (over 100 °C) and dry conditions for several years. We initially hypothesized that the viability of Bacillus strains in biochar is higher when the inoculum contains more endospores than vegetative cells. Yang et al. (2019) reported that water-soluble compounds leached from biochar can either promote or inhibit bacterial growth, depending on the bacterial species. Based on this, we further hypothesized that vegetative cells are more susceptible to inhibition by water-soluble compounds in biochar than endospores and that their survival can be improved by using water-washed biochar. By testing these hypotheses, we aim to propose optimal biochar inoculation strategies that ensure high bacterial survival, even when using Bacillus strains with varying proportions of vegetative cells and endospores.

In this study, we used eight type strains of Bacillus species (Bacillus amyloliquefaciens, B. licheniformis, B. nakamurai, B. pumilus, B. siamensis, B. subtilis, B. thuringiensis, and B. velezensis) which are used as PGPB and BCAs and investigated the survival and viability of Bacillus strains in RHB in different endospore states of vegetative cells or endospores. We also investigated the factors that influence the survival of Bacillus strains in RHB water extract, the inoculation methods that enhance the survival of Bacillus strains, and their long-term persistence in RHB during storage.

Materials and methods

Biochar

The RHB used in this study was provided by Yanmar Energy Systems Co. Ltd. (Hyogo, Japan). To prepare the washed biochar, RHB was washed using reverse osmosis (RO) water at a tenfold (w/v) concentration with shaking at 130 rpm for 30 min. The washing water was drained, and the biochar was dried at 60 °C.

The components of the unwashed and washed biochars were analyzed by external companies. Water content, pH, electrical conductivity (EC), ash, N, P2O5, K2O, CaO, MgO, Na, Al, Mn, Fe, As, Cd, Hg, Ni, Cr, Pb, organic C, total C, and the carbon-to-nitrogen ratio in the biochar were analyzed by the Environmental Research Center Co., Ltd. (Ibaraki, Japan) using methods specified by the Food and Agricultural Materials Inspection Center, Japan. Water-soluble phenol in biochar was measured by Resonac Techno Service Co. (Ibaraki, Japan) using samples obtained by extracting biochar (unwashed and washed) with tenfold the amount of RO water, according to the JIS K 0102 28.1.3 method. Eighteen PAHs, designated as regulated AfPSGS2014:01PAK (BAuA 2014), were analyzed by the Mie Prefecture Environmental Conservation Agency (Mie, Japan) using a measurement method in accordance with ZEK 01.2–08 (gas chromatography mass spectrometry).

Bacteria and preparation of the bacterial suspension

Eight bacterial type strains of the genus Bacillus were used: B. amyloliquefaciens NBRC 15535 T, B. licheniformis NBRC 12200 T, B. nakamurai CCUG 68786 T, B. pumilus NBRC 12092 T, B. siamensis LMG 27549 T, B. subtilis NBRC 13719 T, B. thuringiensis NBRC 101235 T, and B. velezensis LMG 22478 T. The NBRC strains were provided by the National Institute of Technology and Evaluation Biological Resource Center, NITE (NBRC), Japan. LMG strains were provided by the Belgian Coordinated Collections of Microorganisms (BCCM)/Laboratory of Microbiology, Ghent University (LMG) Bacteria Collection, Belgium. The CCUG strain was provided by the Culture Collection of University of Gothenburg (CCUG), Sweden. The Bacillus type strains were cultured overnight in Luria – Bertani (LB) liquid medium (Sigma-Aldrich) with shaking at 160 rpm and then stored in an LB liquid medium supplemented with 15% glycerol at − 80 °C.

The stock culture of Bacillus type strains was streaked onto a nutrient agar (NA) plate (Nutrient broth [BD Difco] supplemented with 1.5% agar) and cultured at 28 °C. To obtain bacterial suspensions with different endospore densities, the plate was incubated for 1, 2, or 7 days, and then one loopful of a colony was suspended in 2 mL of sterile water. The bacterial suspension was separated into 1 mL samples, which were placed in 1.5-mL Eppendorf tubes. A non-heat-treated (NHT) bacterial suspension containing both vegetative and endospore cells was used. The other bacterial suspension was heat-treated (HT) at 80 °C for 1 h on the heat block (Dry Thermo Unit DTU-1B, TAITEC Co., Ltd., Saitama, Japan) to create a bacterial suspension containing only endospore cells. The bacterial concentration in the suspension was determined by dilution plating on NA plates and expressed as colony-forming units (CFU). The plate was incubated at 28 °C for 1–2 days, and the colonies on the plate were counted. The sporulation rate was calculated using the following formula: sporulation rate (%) = (A/B) × 100, where A is the bacterial density of HT (endospores) and B is the bacterial density of NHT (total cells, including vegetative cells and endospores).

Antifungal activity of Bacillus strains against plant-pathogenic fungi

Soil-borne pathogens in spinach foot rot (Rhizoctonia solani MAFF 244979) and spinach Fusarium wilt (Fusarium cugenangense MAFF 150605), provided by the Genetic Resources Center, National Agriculture and Food Research Organization, Japan, were used as plant-pathogenic fungi. Fungi were cultured on potato dextrose agar (PDA) plates (BD Difco).

A single colony of the Bacillus type strain was transferred to three spots on a PDA plate at equal distances from the center. Mycelial disks (8 mm) of R. solani or F. cugenangense were cut from the edge of the growth on a PDA plate and placed in the center of the test plate. As a control, a mycelial disk was placed on a fresh PDA plate without the Bacillus type strain. The plates were incubated at 25 °C for 9 days, and the presence or absence of an inhibition zone around the colonies of the Bacillus strains was determined.

Survival of Bacillus strains in biochar

Biochar well plates were prepared by placing 0.05 g of RHB in 48-well plates (VIOLAMO VTC-P48, AS ONE Co., Osaka, Japan). Bacterial suspensions were obtained by pre-cultivating bacteria on NA plates for 1, 2, and 7 days, and bacterial suspensions with or without heat treatment were used as the inoculum. Each bacterial inoculum (180 µL) was inoculated into the biochar in the well and then incubated at 28 °C for 7 days. To determine the bacterial concentration in the biochar after incubation, 820 µL of sterile water was added to the well, and the suspension was diluted and streaked on NA plates. The plate was incubated at 28 °C for 1–2 days, and the number of colonies on the plate was counted.

Effects of biochar washing on biochar composition and bacterial survival

A bacterial suspension was prepared from colonies cultured on an NA plate for 1 day, and 180 µL of the NHT bacterial suspension was inoculated into the well containing 0.05 g of unwashed or washed RHB. The plate was cultured at 28 °C for 7 days, and the bacterial concentration in the biochar was determined.

Survival of Bacillus strains in biochar water extract

To prepare biochar water extracts, 10 g of RHB was mixed with 200 mL or 100 mL of sterile RO water (biochar: water [w/v] = 0.5:10 [Ex1] or 1:10 [Ex2]) and shaken at 130 rpm for 30 min. Then, the extract was filtered using filter paper (No. 2, ADVANTEC Toyo, Tokyo, Japan) and a 0.22-µm filter (Millex-GP PES Membrane SLGPR33RS, Merck KGaA, Darmstadt, Germany). The pH of the biochar water extract was measured using a pH meter (Seven Multi; Mettler-Toledo International Inc., Columbus, Ohio, USA). To prepare sterile water adjusted to pH 10.2 (W2), the same pH as Ex2, the pH was adjusted with 1 N NaOH and then sterilized by filtration via a 0.22-µm filter. Normal sterile water (W1, pH 6.7) was used as the control.

Suspensions of Bacillus type strains were prepared from colonies cultured on NA plates for 1 or 7 days. A bacterial suspension without heat treatment (180 µL) was inoculated into 2 mL of each buffer (W1, W2, Ex1, and Ex2) and cultured at 28 °C for 7 days. The bacterial concentration in the buffer was then determined.

Long-term survival of Bacillus strains in biochar

To prepare an endospore suspension, a bacterial suspension was prepared from the colonies grown on the NA plate over 7 days. Exactly 180 µL of the HT bacterial suspension was inoculated into the well containing 0.05 g of unwashed RHB. The plate was cultured at 25 °C for 2, 5, and 12 weeks, and the bacterial concentration in the biochar was determined. The survival rate was calculated using the following formula: survival rate (%) = (A/B) × 100, where A is the bacterial density at 2, 5, or 12 weeks and B is the bacterial density under NHT conditions on day 0.

Statistical analysis

Statistical tests for bacterial densities were performed using the t-test or one-way analysis of variance (ANOVA), Dunnett’s test, and Tukey–Kramer test (P < 0.05). The survival rate and sporulation rates were arcsine-transformed before performing a t-test or one-way ANOVA, Tukey–Kramer test (P < 0.05). Two-way ANOVA was performed to detect the effect modification (P < 0.001) of Bacillus species and period after inoculation in bacterial densities in the biochar. All tests were performed using EZR in R commander version 1.68 (Kanda 2013).

Results

Antifungal activity of eight Bacillus species against Rhizoctonia solani and Fusarium cugenangense

In this study, we investigated the antifungal activity of eight Bacillus species used for plant growth promotion and disease control against the plant pathogens R. solani and F. cugenangense (Supplementary Fig. S1 and Table S1). Clear inhibition zones were formed for B. nakamurai CCUG 68786 T and B. siamensis LMG 27549 T against R. solani. B. velezensis LMG 22478 T formed an inhibition zone in the confrontation culture; however, hyphal elongation was observed in the inhibition zone as culturing progressed. Clear inhibition zones were observed against F. cugenangense in B. velezensis LMG 22478 T, B. nakamurai CCUG 68786 T, B. siamensis LMG 27549 T, and B. subtilis NBRC 13719 T.

Survival of Bacillus species in biochar

The sporulation rate of the bacterial strains varied depending on the Bacillus species and the duration of culturing on NA plates (Table 1). The sporulation rate of the 1-day preculture was low for all strains. For most strains, the sporulation rate increased with the passage of preculture days. The bacterial suspension obtained from the bacteria cultured on NA plates for 1 day had a low endospore density, and the survival of the bacteria in the biochar was also low (Fig. 1a and b). The bacterial suspension obtained from the bacteria cultured on NA plates for 7 days increased the endospore density, and the survival of the bacteria in the biochar improved (Fig. 1e and f). However, the sporulation rates and bacterial survival in biochar of B. siamensis LMG 27549 T, B. pumilus NBRC 12092 T, B. licheniformis NBRC 12200 T, and B. thuringiensis NBRC 101235 T were lower than those of other strains, but the sporulation rates and survival in biochar increased when inoculated bacterial suspensions were obtained from 7-day preculture (Table 1 and Fig. 1).

Table 1.

Sporulation rate (%)a of eight type strains of Bacillus species in bacterial suspension obtained from preculturing on nutrient agar plate for different incubation periods

Bacillus type strain 1 day 2 days 7 days
B. velezensis LMG 22478 T 17.0 ± 5.7 ab 96.6 ± 4.4 a 96.1 ± 1.3 a
B. amyloliquefaciens NBRC 15535 T 17.9 ± 6.9 ab 45.7 ± 16.2 abc 103.1 ± 3.5 a
B. nakamurai CCUG 68786 T 34.3 ± 4.1 a 91.2 ± 3.7 ab 103.0 ± 1.9 a
B. siamensis LMG 27549 T 21.8 ± 6.5 ab 24.0 ± 20.8 bc 63.3 ± 7.6 b
B. subtilis NBRC 13719 T 6.2 ± 5.7 b 75.1 ± 9.8 abc 96.0 ± 0.8 a
B. pumilus NBRC 12092 T 3.3 ± 2.7 b 6.2 ± 5.4 c 49.6 ± 2.2 b
B. licheniformis NBRC 12200 T 0.0 ± 0.0 b 17.5 ± 8.7 c 55.7 ± 4.6 b
B. thuringiensis NBRC 101235 T 0.2 ± 0.2 b 56.2 ± 1.8 abc 58.7 ± 1.4 b
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aSporulation rate (%) = (A/B) × 100, where A is the bacterial density of heat treatment (only endospores) and B is the bacterial density of nonheat treatment (vegetative cells and endospores)

bValue indicates mean ± standard error (n = 4–6). Different letters indicate significant differences (P < 0.05) according to the Tukey–Kramer test; values were arcsine-transformed before performing statistical tests

Fig. 1.

Fig. 1

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Survival of eight type strains of Bacillus species in rice husk biochar. A bacterial suspension was obtained by pre-culturing bacteria on a nutrient agar plate for 1 day (a, b), 2 days (c, d), or 7 days (e, f), followed by suspension in sterile water. Each inoculum was either nonheat-treated (NHT) (a, c, and e) or heat-treated (HT) at 80 °C for 1 h (b, d, and f) and inoculated onto biochar. The day of bacterial inoculation was designated day 0. The bacterial density in the biochar was determined after incubation at 28 °C for 7 days (n = 3). N.D. indicates no detection. Error bar indicates standard error. Asterisks indicate significant difference (P < 0.05) between 0 and 7 days according to t-test, and n.s. indicates no significant difference

Effects of biochar washing on biochar composition and bacterial survival

The RHB used in this study had a pH of 10, a carbon content of approximately 30%, and a low concentration of fertilizer components (Table 2). The EC of the biochar after washing was slightly lower than that before washing, and the levels of harmful substances such as heavy metals, phenols, and 18 PAHs were extremely low and did not decrease after washing (Table 2 and Supplementary Table S2).

Table 2.

Components in unwashed and washed rice husk biochar (n = 1)

Components Unwashed biochar Washed biochar
pH 10.5 10.1
EC (mS cm−1) 0.771 0.371
Ash (%) 53.5 57.2
Water content (%) 9.8 3.1
Total N (%) 0.23 0.30
Total P2O5 (%) 0.17 0.15
Total K2O (%) 0.70 0.66
Total CaO (%) 0.26 0.17
Total MgO (%) 0.06 0.07
Organic C (%) 31.3 33.9
Total C (%) 31.5 33.9
C/N ratio 136 113
Na (%)  < 0.1  < 0.1
Al (%)  < 0.1  < 0.1
Mn (%) 0.03 0.02
Fe (%)  < 0.01 0.01
As (mg kg−1) 1  < 1
Cd (mg kg−1)  < 0.5  < 0.5
Hg (mg kg−1)  < 0.1  < 0.1
Ni (mg kg−1)  < 5  < 5
Cr (mg kg−1) 16 15
Pb (mg kg−1)  < 1  < 1
Water-soluble phenols (mg L−1)a  < 0.05  < 0.05
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aWater-soluble phenols was measured in water extract of unwashed and washed biochar

Compared to the biochar before washing, bacterial survival improved when the washed biochar was inoculated with a nonheated bacterial suspension with a low sporulation rate (Fig. 2). However, the bacterial densities of B. amyloliquefaciens NBRC 15535ᵀ and B. siamensis LMG 27549ᵀ were significantly lower than those of the other strains, even in water-washed biochar (Fig. 2c).

Fig. 2.

Fig. 2

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Effects of biochar washing on bacterial survival of eight type strains of Bacillus species. A bacterial suspension was obtained by pre-culturing bacteria in a nutrient agar plate for 1 day. The bacterial density in the inoculum of nonheat-treated (NHT) and heat-treated (HT) suspensions was determined in advance. The NHT bacterial suspension was inoculated onto unwashed biochar (a) or biochar after washing with reverse osmosis water (b). The day of bacterial inoculation was designated day 0. Survival rates (c) were calculated using the following formula: survival rate (%) = (A/B) × 100, where A is the bacterial density at 7 days and B is the bacterial density under NHT conditions at day 0. The bacterial density in the biochar was determined after incubation at 28 °C for 7 days (n = 3). N.D. indicates no detection. Error bar indicates standard error. Survival rates were arcsine-transformed before performing statistical tests. Different letters indicate significant differences (P < 0.05) according to the Tukey–Kramer test. Asterisks indicate significant difference (P < 0.05) between unwashed and washed biochar according to t-test, and n.s. indicates no significant difference

Survival of Bacillus species in biochar water extracts

To investigate factors in biochar that inhibit Bacillus survival, we tested the survival of strains in different concentrations of biochar water extracts (Ex1 and Ex2) and sterile water adjusted to a pH of 10.2 (W2). In bacterial suspensions prepared from colonies after preculturing for 1 day, the total bacterial concentration in the NHT group was high, whereas the bacterial concentration in the HT group was significantly reduced; therefore, the bacterial suspension comprised almost entirely vegetative cells and a few endospores. In all strains except B. nakamurai CCUG 68786 T and B. licheniformis NBRC 12200 T, when the vegetative cell suspensions were inoculated, the bacterial densities were significantly reduced in the high-concentration biochar water extract (Ex2) compared to W1 and W2 (Fig. 3a–h). In bacterial suspensions prepared from colonies after preculturing for 7 days, the bacterial concentration after HT was higher than that after preculturing for 1 day; therefore, the endospores were present in high concentrations in the bacterial suspension. When the sporulated bacterial suspensions were inoculated, bacterial survival in Ex1 and Ex2 improved (Fig. 3i–p).

Fig. 3.

Fig. 3

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Survival of eight type strains of Bacillus strains in biochar water extract. A bacterial suspension was obtained by pre-culturing bacteria in a nutrient agar plate for 1 day (a–g) or 7 days (i–p), followed by suspension in sterile water. The bacterial density in the inoculum of nonheat-treated (NHT) and heat-treated (HT) suspensions was determined in advance. The NHT bacterial suspension was inoculated in sterile water (W1, pH 6.7), high-pH sterile water (W2, pH 10.2), and biochar water extracts (biochar: water [w/v] = 0.5:10 [Ex1, pH 9.8] or 1:10 [Ex2, pH 10.2]). The day of bacterial inoculation was designated day 0. The bacterial density in the biochar was determined after incubation at 28 °C for 7 days (n = 3). B. velezensis LMG 22478 T (a, i); B. amyloliquefaciens NBRC 15535 T (b, j); B. nakamurai CCUG 68786 T (c, k); B. siamensis LMG 27549 T (d, l); B. subtilis NBRC 13719 T (e, m); B. pumilus NBRC 12092 T (f, n); B. licheniformis NBRC 12200 T (g, o); and B. thuringiensis NBRC 101235 T (h, p). N.D. indicates no detection. Error bar indicates standard error. Different letters indicate significant differences (P < 0.05) according to the Tukey–Kramer test

Long-term survival stability of Bacillus strains in biochar

We investigated the long-term survival of the Bacillus strains in unwashed biochar by inoculating it with endospore-rich bacterial suspensions. The survival of bacteria in biochar varied by strain, with B. velezensis LMG 22478 T, B. amyloliquefaciens NBRC 15535 T, B. siamensis LMG 27549 T, B. subtilis NBRC 13719 T, and B. thuringiensis NBRC 101235 T exhibiting survival rates of 80–100% of the inoculated bacterial density up to 12 weeks after inoculation, whereas B. nakamurai CCUG 68786 T, B. pumilus NBRC 12092 T, and B. licheniformis NBRC 12200 T exhibited survival rates of 65–79% (Fig. 4). Effect modifications (P < 0.001) in both Bacillus species and periods in bacterial densities in the biochar were detected by two-way ANOVA.

Fig. 4.

Fig. 4

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Long-term changes in bacterial density (a) and survival rate (b) of eight type strains of Bacillus strain in biochar. A bacterial suspension was obtained by pre-culturing bacteria in a nutrient agar plate for over 7 days, followed by suspension in sterile water. The bacterial density in the inoculum of nonheat-treated (NHT) and heat-treated (HT) suspensions was determined in advance. The HT bacterial suspension was inoculated onto unwashed biochar. The day of bacterial inoculation was designated day 0. The bacterial density in the biochar was determined after incubation at 25 °C for 2, 5, and 12 weeks (n = 6). Survival rates were calculated using the following formula: survival rate (%) = (A/B) × 100, where A is the bacterial density at 2, 5, or 12 weeks and B is the bacterial density under NHT conditions at day 0. Error bar indicates standard error. Asterisks indicate significant differences (P < 0.05) from bacterial densities of NHT inoculation at day 0 in each Bacillus strain according to Dunnett’s test. Effect modifications (P < 0.001) in among Bacillus species, periods, and combination of Bacillus species and periods in bacterial densities in the biochar were detected by two-way ANOVA

Discussion

Biochar contains phenols, organic acids, PAHs, carbohydrates, and various other organic compounds (Brtnicky et al. 2021; Buss et al. 2015, 2016; José et al. 2019; Xiang et al. 2021). Several studies have reported that RHB also has a high pH and contains trace amounts of biological inhibitors such as heavy metals, phenols, and PAHs (Dunnigan et al. 2017; Prakongkep et al. 2013). Higher pyrolysis temperatures produce more aromatic moieties, which are more difficult for soil microbes to metabolize (Dunnigan et al. 2017; Lehmann et al. 2021). Typically, biochar produced at relatively low-pyrolysis temperatures (< 500 °C) is rich in low-molecular-weight PAHs with 2–3 rings, whereas biochar produced at high temperatures (> 500 °C) produces high-molecular-weight PAHs with 5–6 rings (Han et al. 2022). Water-soluble organic compounds (WSOC) from biochar produced at pyrolysis temperatures < 400 °C contain more dissolved organic carbon, such as phenols, than WSOC from biochar produced at temperatures above 400 °C and inhibit the growth of cyanobacteria (Graber et al. 2014; Smith et al. 2016). Biochar has the potential to suppress phytopathogens such as bacteria, fungi, oomycetes, and nematodes (Poveda et al. 2021). Therefore, biochar may also inhibit the growth of microorganisms other than phytopathogens.

Bacillus strains undergo sporulation during culture, forming endospores that are resistant to environmental stresses, such as heat and drought (Tan and Ramamurthi 2014). Several studies have reported the survival of Bacillus strains in biochar (Ajeng et al. 2023; Deng et al. 2023; Yang et al. 2019). Yang et al. (2019) reported that the survival of Bacillus strains in biochar varied depending on the species, strain, and type of biochar. However, the effect of sporulation of Bacillus strains on the viability of bacteria in biochar remains unclear. In this study, we investigated the viability of eight Bacillus type strains on RHB based on their sporulation status. For all strains, the sporulation rate of the 1-day preculture was low, and the survival of the bacteria in the biochar was also low (Table 1 and Fig. 1). However, in the bacterial solution prepared from the 1-day pre-culture, the bacterial density was higher in NHT (containing vegetative cells and endospore at 6 log CFU g−1 or higher) than in HT (only endospore at 2 log CFU g−1 or lower), and some strains (e.g., B. nakamurai, B. subtilis, and B. thuringiensis) survived to some extent even in the inoculum containing many vegetative cells (Fig. 1a and b). For most strains, the sporulation rate increased with the passage of preculture days, and the survival of the bacteria in the biochar improved. These results suggest that sporulation of bacterial species may affect bacterial survival on biochar.

Yang et al. (2019) reported that Bacillus strains such as B. pumilus, B. licheniformis, B. mucilaginosus (syn. Paenibacillus mucilaginosus), and B. megaterium (syn. Priestia megaterium) showed decreased survival in the artificial medium containing water-soluble compounds of corncob biochar compared to the artificial medium containing washed biochar, suggesting that water-soluble compounds in biochar may be responsible for the inhibition of bacterial survival. In this study, we investigated the components of biochar before and after washing, as well as their effects on bacterial survival. Compared to the biochar before washing, bacterial survival improved when the washed biochar was inoculated with a non-heated bacterial suspension with a low sporulation rate (Fig. 2). Therefore, when using a bacterial suspension containing a high proportion of vegetative cells, bacterial survival may be improved by inoculating into water-washed biochar.

Additionally, we tested the survival of strains in different concentrations of biochar water extracts (Ex1 and Ex2) and sterile water adjusted to a pH of 10.2 (W2). In most strains, when the vegetative cell suspensions were inoculated, the bacterial densities were significantly reduced in the high-concentration biochar water extract (Ex2) compared to W1 and W2 (Fig. 3). These results suggest that the water-soluble inhibitors contained in biochar, rather than the high pH, are involved in inhibiting the survival of vegetative cells. Based on the results of these experiments, water-washed biochar may improve bacterial survival by removing water-soluble substances that inhibit the survival of vegetative cells. Smith et al. (2016) reported that water extracts of biochar contain phenols, organic acids, PAHs, carbohydrates, guaiacol, pyrocatechol, furans, and ketones, which inhibit the survival of the blue-green alga Synechococcus. In particular, it has been reported that toxic WSOC extracted from pinewood-derived lower temperature biochars (< 400 °C) include mono-, di-, and trisubstituted phenolic compounds and guaiacol derived from lignin, while toxic WSOC extracted from cellulose-derived biochar include acidic compounds (e.g. butanoic acid and pentanoic acid), pyrocatechol, and PAHs (e.g. anthracene and naphthalene). In this study, we did not identify specific inhibitors of the tested Bacillus strains through component analysis (mainly targeting PAHs) of biochar before and after water washing (Table 2 and Supplementary Table S2). Future research will focus on identifying such bacterial inhibitors, which may be estimated by analyzing the constituents of biochar water extracts.

In this study, the sporulation rate of the bacterial strains varied depending on the Bacillus species, and there were some strains with low sporulation rates in 7 days preculture (B. siamensis LMG 27549 T, B. pumilus NBRC 12092 T, B. licheniformis NBRC 12200 T, and B. thuringiensis NBRC 101235 T), compared to other strains (Table 1). Additionally, the highly vegetative cell suspensions (precultured for 1 day) of B. amyloliquefaciens NBRC 15535 T and B. siamensis LMG 27549 T, whose viabilities were significantly inhibited even in the washed biochar (Fig. 2b), also inhibited the survival in Ex1 and Ex2 (Fig. 3b and d), suggesting that the viability of vegetative cells and their susceptibility to inhibitors differ depending on the bacterial species. It is also important to select Bacillus strains that exhibit higher survival in biochar environments. In this study, we investigated bacterial survival following inoculation with different sporulation states; however, the sporulation state of the bacteria within the biochar at the endpoint remained unknown. Future studies should examine the physiological state of the bacterial cells in biochar and determine whether vegetative cells undergo sporulation, endospores germinate, and cells proliferate.

To produce a highly functional biochar with beneficial microorganisms, the long-term survival stability of the microorganisms in the biochar is an important factor. In this study, all strains survived in unwashed biochar for at least 3 months when inoculated in a sporulated state (Fig. 4). Biochar modified with additives such as starch, amino acids, or organic acids is often used to enhance bacterial stability (Ajeng et al. 2023; Deng et al. 2023; Han et al. 2023). These modified biochars may further improve the long-term viability of Bacillus strains. Developing techniques to enhance bacterial survival, such as removing specific inhibitory compounds from biochar or replacing them with beneficial modifiers, could be a promising direction.

Korai et al. (2018) reported that there was no difference in greenhouse gas emissions between unwashed and washed biochar, regardless of feedstock. They also reported that washed biochar enhanced soil quality, carbon fractions, and the performance of ammonium and nitrates in the soil. The abundance of surface functional groups, such as hydroxyl and carbonyl groups, is useful for the adsorption of microbes (Ajeng et al. 2020; Bolan et al. 2023), whereas PAHs and WSOC in biochar and high pH conditions may limit microbial activity and increase carbon stability (Weng et al. 2022; Zhu et al. 2019). However, the effects of washed biochar on these parameters remain unclear. Win et al. (2020) reported that when a combination of biochar and B. pumilus TUAT-1 was applied to soil, TUAT-1 was readily detected in the rhizosphere with greater abundance and more effectively altered the microbial communities in the rhizosphere and endorhizosphere than after TUAT-1 treatment alone. Highly functional biochar immobilized with beneficial Bacillus strains may contribute to the stability of beneficial microorganisms in the soil. Future studies should investigate the effects of highly functional biochar on soil carbon, microbial communities, and plant growth and disease control.

Conclusions

Sporulation in the genus Bacillus has been well-studied and utilized in various industrial applications. Bacillus spp. can survive for a long time in severe environments in the form of endospores. Most Bacillus-based products, including biofungicides and bioinsecticides, are distributed in the form of powdery materials containing endospores. In this study, we demonstrated that the viability of eight strains of Bacillus species in biochar varied markedly depending on the sporulation state. The survival of vegetative cells may be reduced by water-soluble inhibitors contained in biochar. Additionally, we demonstrated that Bacillus strains can survive for long periods in biochar by inoculating the bacterial suspension in the endospore state.

To our knowledge, this is the first report to clarify the viability of both vegetative cells and endospores in biochar. Future research is needed to identify the inhibitors of bacteria present in biochar. The findings of this study provide useful information for producing highly functional biochar with a high density of beneficial bacteria. When inoculating biochar with Bacillus strains, which exhibit different proportions of endospores and vegetative cells depending on the culture method, bacterial survival in the biochar can be maintained by selecting an appropriate inoculation strategy. Specifically, when using a bacterial suspension rich in endospores, high viability can be maintained even without washing the biochar with water. In contrast, when the suspension contains a large number of vegetative cells, bacterial survival can be improved by inoculating water-washed biochar.

Supplementary Information

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Acknowledgements

The authors appreciate Yanmar Energy Systems Co. for providing biochar. The authors also appreciate Environmental Research Center Co., Resonac Techno Service Co., and Mie Prefecture Environmental Conservation Agency for analyzing the biochar components.

Authors’ contributions

All authors contributed to the conception and design of this study. Material preparation and supervision were performed by NS and MK. Investigations and data collection were performed by HN, NS, and YM. HN wrote the first draft of the manuscript, and all authors commented on the review and editing of the manuscript. All the authors have read and approved the final version of the manuscript.

Funding

This study was supported by the Green Innovation Fund Projects of the New Energy and Industrial Technology Development Organization (NEDO), Japan.

Data availability

The datasets used or analyzed in the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

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Data Availability Statement

The datasets used or analyzed in the current study are available from the corresponding author upon reasonable request.

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