Promotion of lipopeptide antibiotic production by Bacillus sp. IA in the presence of rice husk biochar

Abstract

The purpose of this study is to isolate the beneficial microorganisms whose growth is promoted in the presence of charcoal materials. We successfully isolated strain IA, whose growth is promoted on an agar plate with charcoal materials, and identified it as a novel strain of the Bacillus sp. The growth of strain IA in the liquid medium was promoted by the addition of both activated charcoal (AC) and rice husk biochar (RHB). Moreover, the sporulation of strain IA in the RHB medium and the antifungal activity of the culture supernatant of the RHB medium were much higher than those with AC. HPLC and MS analyses revealed that strain IA produced an antifungal lipopeptide iturin A, and the yield of iturin A in the RHB medium was 8 times higher than that in the medium without RHB. This is the first paper to describe the positive effect of RHB on microbial metabolisms.

Introduction

Biochar, produced by low-temperature pyrolysis of a biomass (rice hulls, wood, crop residues, switchgrass, etc.) under limited oxygen conditions, has attracted attention due to its abilities of environmental improvement and potential in agricultural applications.¹⁾ The addition of biochar into the soil positively affects plant growth and leads to the enhancement of agricultural productivity.^2–6) Increasing crop productivity by biochar application has been explained as an amendment of the soil structure, an improvement in nutrient adsorption, and a change in the microbial community.^5–8) The effects of biochar on soil have been reported in terms of biological properties as well as physical and chemical properties.

Ishii and Kadoya demonstrated that the application of biochar to soil increases the infection rates of mycorrhizal fungi in the host plant roots.^4,5) Graber et al. reported that the number of root-associated yeasts and Trichoderma spp. increased in the plant growth medium with citrus wood biochar as compared with the one without the biochar.⁹⁾ Another study reported that the application of biochar to the soil increased the ammonia oxidation rate via ammonia-oxidizing microorganisms at early time points in the incubation.⁸⁾ These studies reported that biochar changes a microbial community and/or promotes microbial growth in in vivo experiments. However, the direct effect of biochar on beneficial microorganisms, such as the potential of biochar to promote metabolite production via microbes, has not been clarified.

On the other hand, activated charcoal (AC), produced from various hard woods by a high-temperature steam-activation process,¹⁰⁾ has been reported to show microbial growth enhancement. Many reports have suggested that AC acts as a habitat for microbes and thus promotes their growth.^11–13) The direct effects of AC on microbial metabolism have also been reported. Ikegami et al. reported that addition of AC to a yeast culture medium causes the acceleration of both yeast growth and ethanol fermentation.^14,15) The addition of granular AC facilitated direct interspecies electron transfer between bacteria and methanogens and enhanced the conversion of waste to methane in methanogenic fermentation.¹⁶⁾ Based on these reports, AC affects microbial growth both directly and indirectly. In contrast with increased knowledge about AC, the biological activities of other carbides of biomaterial origin remain to be elucidated. Therefore, to understand the positive effects of biochar on agricultural productivity, it is necessary to reveal the direct relationships between biochar and microbes such as those demonstrated in AC.

Among biochar products, rice husk biochar (RHB) has been traditionally used as a soil ameliorant in Japanese agriculture.^17–19) Rice hulls are generated in huge quantities as an unavoidable agricultural waste material, not only in Japan, but also in numerous other countries. Most rice hulls have been disposed of by burning, and thus, searching for an efficient use for rice hulls is important for creating sustainable agriculture. Since biochar changes a microbial community in the soil as described above, RHB may directly affect microbial growth. To investigate the details of the effect of RHB on microbes and to evaluate RHB as an agricultural material, charcoal-sensitive microbes are required. In this study, we isolated and characterized the microorganisms highly sensitive to charcoal materials. To screen charcoal-sensitive microbes, we used AC as the most studied charcoal material. In addition, the effects of AC and RHB on the growth and properties of the isolated strains were compared to evaluate whether RHB, one of the least expensive charcoal materials, is a more applicable material than AC.

Materials and Methods

1. Charcoal materials used in this study

Two charcoal materials (activated charcoal and rice husk biochar) were used in this study. AC was purchased from Kanto Chemical (Tokyo, Japan). RHB was produced from rice hulls by a traditional charcoal-making process²⁰⁾ in Akita Prefecture, Japan (Fig. 1). The process was performed as follows. A burning chamber combined with a chimney was used to produce RHB. A rice-straw fire was set inside the chamber, and then the chamber was covered with a large amount of rice hulls. The rice hulls were pyrolyzed by radiant heat, and when the pyrolysis was almost complete, the fire was extinguished by water. RHB was crushed into a fine powder using a pestle and mortar for use in the following experiments.

Fig. 1. The RHB used in this study. Scale bar is 1 cm.

Rice husk ash provided from BioSilica laboratory (Mie, Japan) was used as the residue left after the complete burning of rice hulls.

2. Isolation of microorganisms with growth enhancement in the presence of charcoal materials

Microorganisms were obtained from environmental samples and isolated using 1/10 tryptic soy agar (1/10 TSA, Bacto peptone, 2 g; glucose, 0.25 g; K₂HPO₄, 0.25 g; NaCl, 0.5 g; and agar, 1.5% (w/v), in 1 L of distilled water). Isolated strains were cultured in a Luria–Bertani (LB) medium, and each of the 10 µL of culture media was inoculated at the center of 1/10 TSA supplemented with and without 5 g/L of AC and incubated at 30°C. The growth areas on the plates were compared, and the microorganisms with a larger growth area on the AC plate were screened. The screened microbes were further tested on a plate containing 5 g/L of RHB in a similar way as described above.

Isolated strains with growth enhancement were identified based on an analysis of the 16S rRNA region after PCR amplification with primers 9F and 1510R. Sequences were automatically analyzed on an ABI PRISM 3130 xl Genetic Analyzer System (Applied Biosystems, Foster City, CA, USA). The 16S rRNA gene sequences determined were compared with those retrieved from the DNA database of APORON DB-BA 12.0 (Techno Suruga Laboratory, Shizuoka, Japan) and DDBJ/ENA(EMBL)/GenBank using a BLAST homology search. A phylogenetic tree was constructed to ascertain the phylogenetic position of the isolated strain and was generated using the neighbor-joining method.²¹⁾ Gene sequencing and phylogenetic analysis were carried out at Techno Suruga Laboratory (Shizuoka, Japan).

3. Antifungal activity test against a plant pathogenic fungus

A plant pathogenic fungus, Rhizoctonia solani K1,^22,23) was used as the test organism in the antifungal activity tests. The antifungal activity of isolated strains was examined on plates as follows. A single colony of the isolated strain was inoculated at a place 2 cm from the edge of the petri plate, and a small plug (about 6 mm in diameter) of R. solani K1 was placed on the other side of the isolated microbes on the plate. After incubation at 24°C for appropriate periods, the growth-inhibition zone of R. solani K1 was observed.

4. Application of charcoal materials to a liquid culture

Microbes with antifungal activity were cultured using 20 mL of TSB (same composition as TSA without agar) supplemented with 5 g/L of AC or RHB in a 100 mL Erlenmeyer flask at 30°C with shaking (120 rpm). The total cell concentration of each sample was determined by counting the colony-forming units (CFUs) on TSA after serial dilution. Spore concentration was determined by the number of CFUs after heating at 80°C for 30 min. To check the extracellular antifungal activity, the culture media were centrifuged at 9,000×g for 10 min, and the supernatant was filtered through a DISMIC-25cs cellulose acetate membrane (0.20 µm; Advantec, Tokyo, Japan); filter-sterilized culture supernatants were used for antifungal activity tests.

5. Purification and identification of antifungal compounds

To characterize the antifungal compounds, the culture supernatant obtained by centrifugation at 12,000×g for 10 min was acidified to pH 2 with 12 N HCl and kept 4 hr. The acid precipitates were pelleted by centrifugation at 18,000×g for 30 min, and the pellet was extracted with methanol. The methanol extract was dried in vacuo. Inactive materials were removed by sequential extraction with ethyl acetate and acetone.²⁴⁾

The residue dissolved in methanol was analyzed by thin-layer chromatography (TLC) on a silica gel 60 F254 plate (Merck KGaA, Darmstadt, Germany). Plates were developed in chloroform–methanol–water 65 : 25 : 4 (v/v/v)^25,26) in triplicate. After drying, the presence of compounds on one of the developed plates was visualized under UV₂₅₄ and a ninhydrin reaction. Another plate was used in the characterization of their R_f value of antifungal compounds by TLC bioautography assay. For the TLC bioautography assay, a developed TLC plate containing 0.2 mg of the crude extract was put on a PDA plate with a small plug of R. solani K1 and incubated at 24°C. To obtain antifungal compounds, the area showing antifungal activity was cut off from the other developed TLC plate and extracted with ethanol. The ethanol extract containing antifungal compounds was dried in vacuo and resuspended in methanol.

Analyses of antifungal compounds were performed using high-performance liquid chromatography (HPLC) and electrospray ionization (ESI) time-of-flight (TOF) mass spectrometry (MS). The HPLC system consisted of a Jasco LC-2000 (Jasco, Tokyo, Japan) with a Chromolith Performance RP-18e column (4.6 mm×100 mm, Merck KGaA, Darmstadt, Germany). The isocratic eluent was a 35 : 65 (v/v) mixture of acetonitrile and water. The system was operated at a flow rate of 2 mL/min and monitored at 205 nm. ESI-TOF MS analyses were performed on a Triple TOF 5600+ system (AB Sciex, Framingham, MA, USA) equipped with ESI in positive ion mode. Partially purified samples were applied to TOF MS using a syringe pump, and ions were monitored in the range of 500 to 1200 m/z.

6. Quantification of the active constituent (iturin A)

Quantification of the active constituent (iturin A) was carried out using HPLC. A portion of the cultured medium was transferred into a tube and mixed with an equal volume of 35% (v/v) acetonitrile. The mixture was vortexed at room temperature for 10 min and then centrifuged at 9,000×g for 10 min. The supernatant was filtrated through a DISMIC-13jp PTFE membrane (0.20 µm; Advantec, Tokyo, Japan), and 20 µL of the filtrate was applied to the HPLC analyses described above. The isocratic eluent was a 35 : 65 (v/v) mixture of acetonitrile and 0.1% formic acid (aq). The yield of iturin A was determined by a calibration curve made with an authentic sample of iturin A purchased from Sigma-Aldrich (St. Louis, MO, USA).

Results and Discussion

1. Isolation and characterization of the microbe that showed enhanced growth in the presence of charcoal materials

Microorganisms isolated from environmental samples were screened under the criterion that their growth is promoted in the presence of AC. As a result of screening, the microbe that showed the largest growth area on the AC plate was selected and named “strain IA”. Isolated strain IA was further tested on 1/10 TSA supplemented with 5 g/L of AC or RHB (Fig. 2a), and the growth areas of a microbe on the plate were measured quantitatively for 5 days (Fig. 2b). AC strongly promoted the growth of strain IA, and the growth area reached the maximum on day 2. RHB also enhanced the growth of strain IA on the plate, and the growth area on the RHB plate was nearly 3 times wider than that without RHB on day 5. This extension of the growth area on the agar plate may be due to an enhanced swarming motility.^27–29) In general, it is reported that bacteria exhibit swarming motility on media solidified with 0.4–1.2% agar, and an agar concentration above 1% inhibits the swarming of many bacterial species.^28,29) In addition, Gao et al. and Venieraki et al. reported that swarming motility is important for colonization of the plant root.^30,31) Considering these facts, our finding that the swarming motility of strain IA was strongly enhanced on a 1.5% agar plate supplemented with AC or RHB suggested that the addition of these charcoal materials into the soil might promote the root colonization of strain IA in the rhizosphere.

Fig. 2. The growth of strain IA on a 1/10 TSA plate with charcoal materials. (a) The growth of strain IA was assessed on a 1/10 TSA plate with/without charcoal materials. (b) The swarm areas of IA cultured on 1/10 TSA (open square) and 1/10 TSA supplemented with AC (filled circle) and RHB (filled triangle) were measured. Error bars represent standard deviations for n=3.

Isolated strain IA formed creamy-white colonies on TSA (Fig. 3a) and was a Gram-positive rod-shaped bacterium (Fig. 3b); furthermore, an endospore formation was observed under microscopy (data not shown). Based on the sequence of the 16S rRNA gene (1,476 bp: deposited in DDBJ with accession no. LC438405), it is proposed that this strain belongs to the genus Bacillus and is most closely related to Bacillus siamensis PD-A10^T (GQ281299),³²⁾ with 99.4% similarity, as shown in Fig. 3d. Differences in 9 bases of the 16S rDNA base sequence were observed between B. siamensis PD-A10^T and strain IA, and thus, the charcoal-sensitive strain IA was identified as a novel strain of Bacillus sp.

Fig. 3. Characterization of isolated strain IA. (a) Colony appearance of IA on TSA. Scale bar is 1 mm. (b) Gram stain of IA. Scale bar is 5 µm. (c) Antifungal activity of strain IA (right) against R. solani K1 (left) on 1/10 TSA. (d) Phylogenetic relationships of strain IA, some species of the genus Bacillus, and related taxa based on 16S rRNA gene sequence analysis. The branching pattern was generated using the neighbor-joining method. Bootstrap values (expressed as percentages of 1,000 replications) are shown at the branching points. Bar indicates 0.01 substitutions per nucleotide position.

2. Effect of charcoal materials on the growth of strain IA in a liquid culture

As AC and RHB affected the growth of strain IA on the plate culture, the effects of AC or RHB supplementation on the growth of strain IA in the liquid culture were also evaluated. The total cell number, spore cell number, and pH values on day 5 are shown in Table 1. The total cell concentration in the RHB medium was significantly higher than that in TSB. The sporulation ratios of strain IA in TSB, the AC medium, and the RHB medium were 0.08%, 0.10%, and 91.1%, respectively. The number of spore cells of strain IA in the RHB medium was significantly higher than in TSB and the AC medium. The values were increased much more by RHB than by AC, and the effect on spore number was especially remarkable, with the spore number in the RHB medium almost 10,000 times higher than that in the AC medium. The pH values in the AC and RHB media increased from 7 to over 8, but the value in TSB did not change during the incubation (pH 7). Bacillus species are well known to produce and secrete various enzymes, such as protease,³³⁾ which digests peptone in a medium and releases free amino acids, followed by the release of ammonia.^34,35) For this reason, the higher pH values in the AC and RHB media than in TSB suggest that the amount of protease produced by strain IA in the AC and RHB media was higher than that in TSB.

Table 1. Effect of AC and RHB on the growth of strain IA

	Culture medium of strain IAa)			Ratio
	TSB (A)	with RHB (B)	with AC (C)	B/A	C/A
Total cell (CFU/mL)	6.5×106	5.4×107*	1.1×107	8.3	1.8
Spore cell (CFU/mL)	5.3×103	4.9×107*	1.1×104	9.3×103	2.2
pH	7.1	8.4	7.7

^a) Dunnett’s test was used to compare the differences between the experimental groups (RHB and AC media) and the control group (TSB). Difference was assessed with two-side test with an alpha level of 0.05. Asterisk indicate a significant difference (p<0.05).

An antifungal activity test using strain IA was carried out because Bacillus spp. are well known to produce a variety of antifungal lipopeptides, such as surfactin, iturin, and fengycin.^36–40) The isolated strain IA showed strong antifungal activity against the plant pathogenic fungus R. solani K1 (Fig. 3c). This result suggests that strain IA would protect plants from phytopathogenic fungi in soil.

The antifungal activity of the culture supernatant of strain IA was analyzed. The culture supernatants listed in Table 1 were used for the antifungal activity test against the plant-pathogenic fungus R. solani K1. As a result, a clear inhibitory zone was observed only around the stainless cup containing the culture supernatant from the RHB medium (Fig. 4), suggesting that RHB induced the production of antifungal compounds.

Fig. 4. Antifungal activity of the strain IA culture supernatant against R. solani K1. A plug of R. solani K1 (left) and a sterile stainless cup with a culture supernatant (right) were placed on the PDA and cultured at 24°C. Each stainless cup contained 200 µL of sterilized water (a), a supernatant of the TSB culture medium (b), a supernatant of the TSB culture medium supplemented with AC (c), or a supernatant of the TSB culture medium supplemented with RHB (d).

The addition of two charcoal materials (AC and RHB) to agar or the liquid culture of strain IA exhibited different effects; AC significantly promoted swarming motility only on the agar culture, while RHB promoted both swarming motility on the agar culture and growth and sporulation in the liquid culture. Moreover, the culture supernatant of the RHB medium strongly inhibited the growth of R. solani K1, but that of the AC medium did not. In the screening for charcoal-sensitive microorganisms using AC, we successfully isolated strain IA, whose growth is promoted in the presence of RHB as well as AC. According to the results, both AC and RHB have the ability to promote swarming motility on an agar culture, while only RHB had the ability to promote growth, sporulation, and antibiotic production in the liquid culture. Several reasons can be considered as to why the two charcoal materials exhibited different effects on strain IA growth and antibiotic production. One possible reason is the different resources of the two charcoals. Activated charcoals are generally made from palm shells and hard woods, while RHB is produced from rice hulls. RHB has been reported to contain diverse trace minerals derived from rice plants, and these trace minerals may contribute to the biological activities of RHB. It has been reported that rice husk ash (RHA) contains various elements, such as Si, Al, Fe, Mn, Mg, Ca, Na, K, Ti, and P.^41,42) Among these, the ferrous ion and manganese ion were reported to promote lipopeptide production by Bacillus spp.^43,44) In fact, the promotion effects of growth and sporulation in TSB supplemented with 5 g/L of RHA were equal to or higher than those in the RHB medium. The total and spore cells of strain IA in the RHA medium were 1.6 and 1.2 times higher than that in the RHB medium, respectively. Therefore, minerals in RHB may contribute to the promotion of growth, sporulation, and antibiotic production by strain IA.

In order to investigate whether these promotion effects of RHB depend on the quality of rice hulls and/or production processes, strain IA was cultured using RHB produced by different manufacturers in Wakayama and Mie Prefectures in Japan. Both RHBs promoted the growth, sporulation, and the antibiotic production of strain IA, as did the RHB made in Akita Prefecture, suggesting that these beneficial effects are common among RHBs. Further studies are needed in order to elucidate the mechanism(s) of the promotion of growth and antibiotic production by RHB.

3. Identification of the antifungal substance produced by strain IA

Figure 5 shows the TLC analyses of RHB-supplemented TSB culture supernatants. The result of the TLC test indicated the presence of several bands via UV visualization (Fig. 5a). As strain IA belongs to the Bacillus species, we tried to detect antifungal lipopeptides. To identify the antifungal lipopeptides, developed TLC plates were visualized via ninhydrin reaction. Three bands with R_f values of 0.19, 0.37, and 0.75 were observed via ninhydrin reaction (Fig. 5b). According to the bioautography assay against R. solani K1, only the substance with an R_f value of 0.37 showed antifungal activity on the developed TLC silica plate (Fig. 5c).

Fig. 5. TLC analyses of a crude extract of the IA culture supernatant. The plate developed in chloroform–methanol–water 65 : 25 : 4 (v/v/v) was visualized under UV₂₅₄ (a) and a ninhydrin reaction (b). A TLC bioautography assay against R. solani K1 was carried out using a developed TLC plate containing 0.2 mg of crude extract (c).

The antifungal substance produced by strain IA showed a spot with an R_f value of 0.37 according to TLC analysis and bioautography assay, while Romero et al. reported that iturin A has an R_f value of 0.3 under the same solvent system.³⁹⁾ To confirm whether this antifungal substance is iturin A, an authentic sample of iturin A was also developed under TLC, and the R_f value (0.37) matched that of antifungal substances produced by strain IA.

For further analysis, these antifungal substances were partially purified by preparative TLC and analyzed using HPLC. As shown in Fig. 6b, five peaks were observed in the chromatogram of the extracted antifungal substance from strain IA; these peaks were also observed in the chromatogram of the authentic iturin A sample (Fig. 6a), suggesting that these compounds were iturin A, consisting of heptapeptides linked to a β-amino fatty acid chain with a length of 14 to 17 carbons.³⁶⁾

Fig. 6. HPLC analyses of an authentic iturin A sample (a) and the antifungal compound produced by strain IA (b). ESI-TOF MS analyses with ESI in positive ion mode of peak a1 in a chromatogram of an authentic iturin A sample (c) and peak b1 in a chromatogram of the antifungal compound produced by strain IA (d).

ESI-TOF MS analysis showed three different molecular weights of antifungal substances. The mass spectrum in chromatographic peak b1 in Fig. 6b showed [M+H]⁺ ions at m/z 1,043 (Fig. 6d); that in peaks b2 and b3 showed [M+H]⁺ ions at m/z 1,057, and that at peaks b4 and b5 showed [M+H ]⁺ ions at m/z 1,071. These results indicate that peaks b1–5 are iturin A, and they belong to iturin A homologues with an acyl chain with a different methylene group number.³⁶⁾

TLC, HPLC and MS analyses revealed that strain IA produced an antifungal lipopeptide iturin A. Moreover, the strong antifungal activity (Fig. 4d) by the culture supernatant of the RHB medium suggested that RHB has a promotional effect on iturin A production.

4. Effect of RHB dosage on iturin A production

To examine the dose effects of RHB on iturin A production, we investigated the concentration dependency of RHB for the production of iturin A in the TSB medium. The iturin A concentration increased with an increase in RHB dosage (Fig. 7). The production of iturin A reached a plateau at a concentration of 30 g/L of RHB, and the maximum yield was about 120 mg/L. This value was almost 8 times greater than that in the medium without RHB.

Fig. 7. The effect of RHB content on the yield of iturin A with strain IA. Error bars represent standard deviations for n=3.

Strain IA produced an antifungal lipopeptide iturin A, and the yield of iturin A was promoted in the presence of RHB. Some Bacillus species (Bacillus amyloliquefaciens AT-332, B. amyloliquefaciens ssp. plantarum D747, Bacillus subtilis HAI-0404, and B. subtilis Y 1336) are already commercially available as biocontrol agents in Japan.⁴⁵⁾ These agents function only as protectors against plant pathogens. However, our results suggest that a combination of RHB and strain IA is a potent biocontrol agent that functions as both an agricultural inoculant and a soil ameliorant.

Conclusion

In this study, we successfully isolated the novel strain IA with enhanced growth in the presence of charcoal materials. Moreover, RHB showed significantly more beneficial effects than AC. This is the first report to describe the effect of RHB on the growth, sporulation, and antibiotic production of strain IA. The combinatorial effects of strain IA and RHB will contribute to not only agriculture but also various biotechnology fields.

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