Enhancing the growth of thermophilic Bacillus licheniformis YYC4 by low-intensity fixed-frequency continuous ultrasound

Graphical abstract

Highlights

• •Low-intensity fixed-frequency continuous ultrasound could promote bacterial growth.
• •Biomass of B. licheniformis YYC4 increased by 48.95% after ultrasound treatment.
• •Sonication caused sonoporation, leading to increased bacterial membrane permeability.
• •Sonication improved metabolic activity and spore germination of B. licheniformis YYC4.

Abstract

The effect of low-intensity fixed-frequency continuous ultrasound (LIFFCU) on the growth of Bacillus licheniformis YYC4 was investigated. The changes in morphology and activity of the organism, contributing to the growth were also explored. Compared with the control, a significant increase (48.95%) in the biomass of B. licheniformis YYC4 (at the logarithmic metaphase) was observed following the LIFFCU (28 kHz, 1.5 h and 120 W (equivalent to power density of 40 W/L)) treatment. SEM images showed that ultrasonication caused sonoporation, resulting in increased membrane permeability, evidenced by increase in cellular membrane potential, electrical conductivity of the culture, extracellular protein and nucleic acid, and intracellular Ca²⁺ content. Furthermore, LIFFCU action remarkably increased the extracellular protease activity, volatile components of the culture medium, microbial metabolic activity, and spore germination of the strain. Therefore, LIFFCU could be used to efficiently promote the growth of targeted microorganisms.

1. Introduction

Bacillus licheniformis YYC4, a thermophilic protease producing strain, isolated from cigarette, has successfully been used in the solid-state fermentation of soybean meal under non-sterile conditions at a relatively high temperature of 55 °C [1]. The nutritional quality and antioxidative activity of soybean meal, as well as the structural and interfacial characteristics of its protein were significantly improved after fermentation [2]. The fermentation efficiency of B. licheniformis YYC4 on soybean meal was comparable to B. subtilis, which is well known for its ability to produce protease, and frequent use in fermenting oilseed meal to produce bioactive peptides [3], [4]. That notwithstanding, how to improve the fermentation efficiency of B. licheniformis YYC4 and its associated product quality is still a concern for many researchers (including our research team).

Ultrasound, as an emerging non-thermal physical processing technology, has received patronage in the food and microbiological industry. Although the lethal effect of ultrasonic irradiation on microorganisms have been known for almost a century, its application to promote or control their growth and metabolic activities is much more recent. The lethal or sub-lethal effect of ultrasound on microorganisms depends on its frequency and intensity. Low-frequency (20–100 kHz) and high-intensity (10–1000 W/cm²) ultrasonication can inactivate microorganisms [5], [6]. Whereas, low-frequency and low-intensity (<1 W/cm²) ultrasonic can enhance microorganism growth and metabolite production, suggesting that it could be very beneficial to the fermentation industry [7], [8], [9], [10].

The modern fermentation industry is highly competitive and innovative. Various novel processing and monitoring technologies have recently been investigated to enhance the productivity and process efficiency of food fermentation. During fermentation, complex biochemical changes occur, involving enzymes, substrate and microorganisms. Literature shows that, ultrasonication can enhance the reaction efficiency, and strengthen the reaction process due to its cavitation, mechanical, and thermal effects [11]. However, how ultrasonication stimulate activity of microorganisms, particularly at sub-lethal levels, is still not clear. Thus, in this study, we investigated the influence of low-intensity fixed-frequency continuous ultrasound (LIFFCU) on the growth of thermophilic B. licheniformis YYC4. Also, the changes in the microorganism and/or its activity as a function of the ultrasound action, and how such impacted the growth and fermentation efficiency was clarified by analyzing the morphology, membrane permeability, metabolic activity and spore germination of the organism (B. licheniformis YYC4). This research serve as a theoretical foundation and technical reference for ultrasonic industrial applications (fermentation) requiring the use of B. licheniformis YYC4.

2. Materials and methods

2.1. Microorganism, maintenance and preparation

B. licheniformis YYC4 was isolated (by our research team, Jiangsu University, China) from a Yunyan cigarette sample, manufactured by Hongyun Honghe Tobacco (Group) Co., Ltd. (Kunming, China), and preserved in the China Center for Type Culture Collection (Wuhan, China) with the identification CCTCC No. M 2,019,599 [1]. Stock culture of B. licheniformis YYC4 was kept at −20 °C in 30% glycerol with a volume ratio of 1:1. The strain was activated by inoculating 200 µL bacterial suspension into 50 mL Luria-Bertani (LB) medium (containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl, respectively) in a 250 mL flask, and incubated at 60 °C (160 rpm for 24 h) before growth curve plotting and ultrasonic treatment.

2.2. Growth curve of B. licheniformis YYC4

A 2% ratio of activated strain suspension was inoculated into sterile LB medium (50 mL) contained in 66 shake flasks (250 mL) labeled A¹ to A²², B¹ to B²² and C¹ to C²², respectively. The mixtures were incubated at 60 °C and 160 rpm. Afterward, the absorbance of culture medium was determined at 600 nm at intervals of 0.5 h (0–5 h), 1 h (5–8 h) and 2 h (8–24 h) using a spectrophotometer (UV-1100, Purkinje General Co., Ltd., Beijing, China) after dilution 10-fold using sterile medium. The growth curve of B. licheniformis YYC4 was plotted and the different growth phases of the strain were identified accordingly.

2.3. Colony counting of B. licheniformis YYC4

The pour plate method was applied, using LB solid medium (consisting of 1.8% agar). The plate was incubated at 60 °C after serial dilution of the strain suspension using sterile water. Then, the colonies formed on the plate were counted.

2.4. Optimization of ultrasound conditions

LIFFCU equipment (Shangjia Biological Technology Co., Ltd., Wuxi, China) was used in this study as a sonication source to enhance the growth of B. licheniformis YYC4. The scheme diagram of the ultrasound device is shown in Fig. 1.

Fig. 1.

Schematic diagram of ultrasound equipment (1: Operation interface; 2: Ultrasonic generator; 3: Sample treatment vessel; 4: Thermostatic water bath; 5: Ultrasonic transducer).

The strain, cultivated to the latent prophase and anaphase as well as the logarithmic prophase, metaphase and anaphase in LB medium, respectively, was transferred into a 250 mL glass flask containing 50 mL of LB medium with a volume ratio of 10%. Then, suspension was immediately sonicated (28 kHz, 100 W, 1 h) with a fixed-frequency and continuous ultrasound mode in 3 L water. Sonicated strain solution was thereafter incubated at 60 °C, and 160 rpm for 24 h, to optimize the growth phase of B. licheniformis YYC4 to receive irradiation based on the bacterial colonies on LB plate. The effect of ultrasonic power (80, 120, 149, 160 and 180 W), frequency (22, 28, 40 and 68 kHz), irradiation mode (fixed-frequency (FF) or sweeping-frequency (SF)), and time (0.5, 1, 1.5, 2, 2.5 and 3 h) on the growth of strain was evaluated by plate colony counting method, after incubation for 24 h. Untreated strain suspension was used as the control.

2.5. Analysis of B. licheniformis YYC4 morphology

Scanning electron microscopy (SEM) was used to observe the morphological changes (with a magnification of 10,000x) in the strain subjected to ultrasound treatment, based on the method described by Bajpai et al. [12], with slight modification. The suspension of B. licheniformis YYC4 treated with the optimal ultrasonic conditions for 0, 1.5 and 3.0 h was centrifugated at 3200 xg for 15 min and washed twice with 0.1 M phosphate buffer solution (PBS, pH 7.4). The bacterial cells were fixed in 2.5% glutaraldehyde overnight at 4 °C. Then, the samples were dehydrated through graded ethanol solutions (25%, 50%, 75%, 95% and 100%, each 30 min), critical point dried, and gold coated by sputtering, followed by microscopic examination using a scanning electron microscope (S-3500 N, EDAX Inc., USA).

2.6. Effect of ultrasonication on membrane permeability of B. licheniformis YYC4

2.6.1. Measurement of cellular membrane potential

Effect of ultrasound action on the membrane potential (MP) of the strain was measured according to the rhodamine fluorescence method as described by Zhang et al. [13], with slight modification. The strain suspensions (5 mL) treated with sonication under different duration were centrifuged at 3200 xg for 15 min, washed twice with sterile saline, and then resuspended in 5 mL sterile saline. Subsequently, an aliquot of rhodamine stock solution (10 µL, 1 mg/mL in PBS) was added, after standing in dark for 30 min, and the resultant was washed and resuspended in 5 mL sterile saline. The fluorescence intensity of the suspension was measured using a fluorescence spectrophotometer (Model Cary 172 Eclipse, Varian Inc., Palo Alto, USA) at excitation and emission wavelength of 480 and 530 nm, respectively.

2.6.2. Determination of electric conductivity of culture medium and extracellular protein and nucleic acid

Electric conductivity, and extracellular protein and nucleic acid of B. licheniformis YYC4 culture (following ultrasound treatment at different time) were examined to evaluate the alterations in cellular membrane permeability after incubation for 24 h at 60 °C and 160 rpm. After centrifugation at 3200 xg for 15 min, the electric conductivity of the supernatant was measured using an electrical conductivity meter (DDS-11A, Yifen Scientific Instrument Co., Ltd., Shanghai, China). The extracellular protein and nucleic acid were estimated based on the absorbance at 280 nm and 260 nm (corresponding to the maximum absorption wavelength of protein and nucleic acid, respectively) [14]. The increase in extracellular protein and nucleic acid was calculated as follows:

$$\mathit{Increment}ofextracellularproteinornucleicacid\left(\%\right)=\left(\frac{A_{\mathit{ultrasound}}-A_{\mathit{without}ultrasound}}{A_{\mathit{without}ultrasound}}\right)\times 100$$

2.6.3. Determination of intracellular Ca²⁺ content

The suspension (4 mL) of untreated and ultrasonicated B. licheniformis YYC4 was centrifuged (4 °C, 6000 xg, 10 min), washed twice by PBS (0.01 M, pH 7.4), and then resuspended into 0.5 mL PBS (0.01 M, pH 7.4). After mixing with 3.5 μL 1 mM Fluo-4/AM (F312, Dojindo Laboratories, Japan) and incubating for 1 h at 37 °C in the dark, fluorescence intensity of the sample was measured at excitation and emission wavelength of 488 and 520 nm, respectively. Meanwhile, the sample was detected using inverted fluorescence microscope [15].

2.7. Effect of ultrasound treatment on microbial metabolism

Extracellular protease activity and volatile ingredient composition of B. licheniformis YYC4 culture treated with ultrasound or not were examined after incubation for 24 h at 60 °C and 160 rpm to explore the effect of ultrasound treatment on microbial metabolism. The bacterial metabolic activity was immediately determined after sonication.

2.7.1. Determination of extracellular protease activity

The activity of neutral, alkali and acid protease was determined after an appropriate dilution of the culture with 0.1 M phosphate buffer (pH 7.5), borate buffer (pH 10.5) and sodium lactate buffer (pH 3.0), respectively, according to the Chinese National Standard GB/T 23527–2009, based on the hydrolysis rate of casein, with a modification of incubation temperature to 60 °C instead of 40 °C [1]. One unit of enzyme activity is defined as the amount of enzyme required to liberate 1 mg tyrosine in 1 min under assay conditions.

2.7.2. Analysis of volatile compounds of B. licheniformis YYC4 culture medium

The volatile compounds of B. licheniformis YYC4 culture medium was determined, using the method outlined by Pyo et al. [16], with some changes. A 100 μL filtrated sample (with 0.22 nm water filtration membrane) was mixed with 0.4 mL methanol, followed by centrifugation at 4 °C, 6000 xg for 10 min. The supernatant (0.4 mL) was vacuum dried in a 2 mL injection bottle. Subsequently, 80 μL methoxyamine salt pyridine solution (15 mg/mL) was added. After mixing, the resultant mixture was left to react at 37 °C for 2 h. Afterward, 100 μL derivative reagent of bis(trimethylsilyl)trifluoroacetamide (BSTFA, containing 1% trimethylchlorosilane (TMCS)) was added and reacted at 70 °C for 1 h. After cooling to room temperature, 10 μL of 2-octanol (as internal standard) was added and mixed. The volatile ingredients of untreated and ultrasonicated for 1.5 h B. licheniformis YYC4 culture medium were analyzed, using gas chromatography-mass spectrometry (GC–MS) system (Thermo Fisher Scientific, Austin, USA), LB medium (without inoculation of the strain) was used as control.

Separation of volatile compounds was performed on a HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The oven temperature was initially maintained at 70 °C for 5 min, ramped to 280 °C at 5 °C/min, holding for 5 min. Helium was used as the carrier gas with a flow rate of 1 mL/min. The injector, ion source and fourth level rod temperatures were respectively set at 250, 230 and 250 °C with a splitless mode. The mass spectral data were obtained at 70 eV in electron ionization with a mass scan range of 30–500 m/z. The obtained volatile compounds were identified by comparing the mass spectra and the retention times of reference substances, as well as the data from National Institute of Standards and Technology (NIST 08) library. Compounds were expressed as relative peak intensity (i.e., volatile compounds peak intensity/internal standard peak intensity × 100).

2.7.3. Determination of metabolic activity of B. licheniformis YYC4

The metabolic activity of B. licheniformis YYC4 was analyzed following the protocol of Sun et al. [17]. The strain in culture medium was harvested by centrifugation at 4 °C, and 3200 xg for 15 min. The concentration of B. licheniformis YYC4 was adjusted to 10⁸ cfu/mL using normal saline (0.9%). Then, bacterial suspension (5 mL) was mixed with 10 µL of 250 mg/mL iodonitrotetrazolium chloride (INT) and allowed to react at 37 °C for 30 min. The maximum absorbance of formazan at 630 nm was recorded to estimate the metabolic activity (i.e., the TCA cycle) of the strain.

2.8. Effect of ultrasound treatment on spore germination of B. licheniformis YYC4

B. licheniformis YYC4 culture was heated in boiling water for 10 min. The culture medium was diluted and coated on the LB agar plate at room temperature, and then incubated at 60 °C for 24 to form colonies.

2.9. Statistical analysis

All experiments were conducted in triplicate. Data were expressed as mean ± standard deviations (SD), and one-way analysis of variance (ANOVA) with Duncan test was performed (at p < 0.05) using SPSS 20.0 (IBM Corporation, NY, USA). All graphs were drawn using Origin Pro 2019b (Origin Lab Corporation, MA, USA).

3. Results and discussion

3.1. Growth curve of B. licheniformis YYC4

Growth curve of B. licheniformis YYC4 is presented in Fig. 2A, as baseline data for the identification of the changes in growth. It was observed that, the latent phase was within 2 h, and the logarithmic and stationary phase began from 2 h and 6 h, respectively. The short latent and logarithmic phase indicated that B. licheniformis YYC4 could easily adapt to a fresh medium, and then grow exponentially.

Fig. 2.

The growth curve of B. licheniformis YYC4 (A) and optimization of ultrasound conditions (bacterial growth phase to receive irradiation (B), ultrasonic powder (C), ultrasonic frequency and mode (D), and treatment time (E)). Error bars indicate the standard deviations of triplicate samples. Different letters indicate significant difference at p < 0.05.

3.2. Optimization of ultrasonic conditions

3.2.1. The growth phase of B. licheniformis YYC4 to receive sonication

Sonication (28 kHz, 100 W, 1 h) was applied following incubation of B. licheniformis YYC4 (for 1, 2, 3, 4 and 6 h), corresponding to the latent prophase and anaphase as well as the logarithmic prophase, metaphase and anaphase, respectively, according to the growth curve. As shown in Fig. 2B, ultrasonic treatment significantly increased the biomass of B. licheniformis YYC4 in these five phases, especially in logarithmic metaphase (16.02%, p < 0.05). It is well known that microorganisms at the logarithmic phase, are usually very active and sensitive to the external stimuli, leading to rapid growth and proliferation. Huang et al. [7] found that the highest biomass (increased by 119.23%) in seed culture was obtained when L. paracasei at the logarithmic prophase was treated with ultrasound (28 kHz, pulse on– and off-time 100 and 10 s, 100 W/L for 1 h). Huang et al. [15] also reported that, the biomass of ultrasound treated Candida tropicalis increased by 142.5% at the mid logarithmic phase, when compared with the untreated yeast. Therefore, B. licheniformis YYC4 at logarithmic metaphase was (in our case) selected, ultrasonicated, and used for the experiments.

3.2.2. Optimization of ultrasound parameters

Power is an important parameter to indicate the intensity of ultrasound. Fig. 2C depicted the influence of ultrasound power (ranged from 80 to 180 W at 28 kHz for 1 h) on biomass of B. licheniformis YYC4 in logarithmic metaphase. The results showed a gradual increase in the biomass with increasing ultrasonic power, and reached the maximum value of 36.89% at 120 W (equivalent to power density of 40 W/L), then decreased with further increase in power. A reduction in biomass was obtained at 180 W. This result is consistent with the finding of Wang et al. [18], who indicated that the power density of 40 W/L was suitable for treating Bacillus amyloliquefaciens cells. Also, Huang et al. [7] showed that power density<160 W/L had positive effects on the growth of the probiotics in the process of seed culture, illustrating that microorganisms have different sensitivities to ultrasound treatment. It was reported that low-intensity ultrasound could effectively promote the growth of microorganism, while excessive sonication results in the reverse [19]. Therefore, 120 W was regarded as the optimum ultrasonic power to treat B. licheniformis YYC4.

Physical and chemical phenomena associated with ultrasound frequency include agitation, vibration, pressure, shock waves, shear forces, microjets, compression and rarefaction, acoustic streaming, cavitation and formation of free radicals [20]. Effect of ultrasonic frequency and mode of FF (22, 28, 40, and 68 kHz) and SF (22 ± 2, 28 ± 2, 40 ± 2, and 68 ± 2 kHz) on biomass of B. licheniformis YYC4 at logarithmic metaphase is shown in Fig. 2D at fixed power of 120 W and for 1 h. The result indicated that all the frequencies (including FF and SF mode) caused an increase in the biomass. The highest increase in biomass (37.16%) was, however, achieved at FF 28 kHz (compared with the other treatments). Additionally, FF sonication caused an obvious increase in the growth of B. licheniformis YYC4 compared with SF sonication. This may be linked to the fact that SF sonication enabled the sample material resonate to limit the bacterial growth. The observation was in agreement with Zhang et al. [21], who found that the highest strain concentration and the fastest growth rate of Staphylococcus cerevisiae were acquired at FF 28 kHz, compared with SF 28 ± 2 kHz and other ultrasonic treatments. As a result, FF 28 kHz was used for the following experiments.

As shown in Fig. 2E, positive effect on biomass was achieved after ultrasonic treatment (<2 h). Sonicated sample (1.5 h) had the highest biomass, which was improved by 48.95%, compared with the other groups, implying that, a suitable ultrasonication time could enhance the contact of the strain with nutrients, thereby promoting growth of the strain [19]. The growth of B. licheniformis YYC4 was inhibited by 2.5 and 3.0 h of sonication, indicating that prolonged sonication caused negative effects on the cell growth, which might be attributed to the increasing cavitation impact or excessive permeability of the cell membrane [22]. Joyce et al. [23] reported that prolonged sonication could cause irreversible damage or even death to cells.

The growth of microorganisms depends on the composition and pH level of the media, incubation temperature and the airflow rate by shaking or dissolved oxygen [22]. The increase in growth rate of B. licheniformis YYC4 could possibly be linked to the improved transfer of small molecules in an either stagnant or relatively slow moving fluid medium, as well as the exchange of materials between intracellular and extracellular following sonication, leading to the enhancement of material and energy metabolism of the strain in logarithmic metaphase, contributing to a rapid propagation than non-sonicated sample [24], [25]. Also, Rajasekhar et al. [26] indicated that the mechanistic effect induced by low-frequency ultrasound could break the agglomerated cells and thus promote the growth of microorganisms.

3.3. Effect of ultrasound treatment on morphology of B. licheniformis YYC4

The morphological changes in B. licheniformis YYC4 treated with ultrasound (0, 1.5 and 3 h) were examined (Fig. 3). SEM images exhibited visible changes in their morphology. Untreated strain displayed a smooth and regular morphology, with a uniform rod shape. A 1.5 h sonication caused a wrinkled surface and physical damage, resulting in the formation of pores on the surface of B. licheniformis YYC4 (highlighted by arrow) which has been denoted as “sonoporation”. Sonoporation might be reversible and could enhance the transport rate of oxygen and nutrients to the cells, as well as waste products away from the cells [27]. According to Liao et al. [28], transitory, and temporary pores on cell membranes allow non-permeable extracellular substances such as proteins and macromolecules to enter cells and release of intracellular enzymes out from cells. Therefore, these pores provide important biological effects on the growth and metabolic processes of ultrasound treated cells [29], [7]. However, further increase in ultrasound time to 3 h induced more wrinkles on its surface, and obvious deformation of the strain. The cell membrane played essential role in maintaining normal physiological functions of microorganisms, including transportation of important materials and generation of ATP [30]. Once the membrane is damaged irreversibly, the growth of microorganisms can be inhibited, and even cause death.

Fig. 3.

Effect of ultrasound treatment on morphology of B. licheniformis YYC4 (A: control; B: 1.5 h and C: 3.0 h, respectively), with a magnification of 10,000x (the red arrow highlights the pores on its surface). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. Effect of ultrasound treatment on membrane permeability of B. licheniformis YYC4

3.4.1. Cellular membrane potential

Cell membrane potential (MP) is generated by differences in the concentrations of ions on opposite sides of the cell membrane, which is indispensable for energy transduction and nutrient uptake and regarded as an important indicator for physiological activity of microbial cells [28]. As shown in Fig. 4A, ultrasonication induced a gradual increase in the MP with increasing of treatment time from 0 to 1.5 h, then decreased with further prolongation of exposure time from 1.5 to 3 h, suggesting the change in electric potential between the interior and the exterior of a biological cell. MP as an element of the proton motive force, it is involved in the generation of ATP [31]. Therefore, the increase of MP with ultrasonication (from 0 to 1.5 h) indicated an enhancement in ATP producing ability of the strain, as well as the improvement of its metabolism. Any treatment that depolarizes the cell membrane are deemed to reduce MP. Rodriguez et al. [32] reported that surfactant with antimicrobial activity could reduce MP and alter cell permeability, causing metabolic inhibition, growth arrest or cell lysis [33]. In this study, the fluorescence intensity of Rhodamine123 was directly correlated with the bacterial MP. The gradual loss of fluorescence with sonication time (from 1.5 to 3.0 h) indicates cell membrane depolarization, leading to irregular cell metabolic activity and even bacteria death.

Fig. 4.

Effect of ultrasound treatment on cell membrane potential and electrical conductivity of the culture (A), extracellular protein and nucleic acid content (B), intracellular Ca²⁺ content (C), inverted fluorescence microscope diagram of intracellular Ca²⁺ concentration (D₁: control; D₂: 0.5 h; D₃: 1.0 h; D₄: 1.5 h; D₅: 2.0 h; D₆: 2.5 h and D₇: 3.0 h, respectively) of B. licheniformis YYC4. Error bars indicate the standard deviations of triplicate samples. Different letters indicate significant difference at p < 0.05.

3.4.2. Electric conductivity of the culture medium and extracellular protein and nucleic acid

Relative electric conductivity was examined to determine the changes in bacterial membrane permeability. The results (Fig. 4A) revealed that the electric conductivity gradually increased with treatment time from 0.5 to 2.0 h, then decreased with further prolongation of ultrasonic time. This finding indicated that the appropriate ultrasonication time could improve cell membrane permeability, resulting in more intracellular electrolytes being released into the culture medium.

Changes in extracellular protein and nucleic acid of untreated and ultrasonicated B. licheniformis YYC4 were explored as parameters of membrane permeability, and the outcomes are displayed in Fig. 4B. Results indicated that ultrasound observably increased the extracellular protein and nucleic acid content, and the maximum values were achieved at 2 h, which increased by 14.81% and 9.30%, respectively, over the untreated sample. It is well known that ultrasound is a periodic longitudinal wave providing forces of diverse directions and intensities at any positions of a microbial cell at a certain moment during a vibration cycle [15]. Tangential forces generated from these forces cause tangential movement of the cell membrane, making the local cell membrane thinner or the local pores larger, resulting in increased membrane permeability and thus faster material exchange between two sides of the cell membrane [34].

3.4.3. Intracellular Ca²⁺ content

Ca²⁺ plays an important role in the regulation of cell cycle as a second messenger [35], [36]. It is reported that ultrasonic treatment has significant effect on Ca²⁺-ATP enzyme, H-ATP enzyme and several ion channels, which are very important for cell growth and reproduction [10]. Effect of ultrasonication on the intracellular Ca²⁺ content of B. licheniformis YYC4 was determined by using Fluo-4-AM and inverted fluorescence microscope. As shown in Fig. 4C, within 1.5 h of ultrasonic exposure time, the intracellular Ca²⁺ concentration steadily increased, then decreased with further prolongation in ultrasonic time. Wang et al. [37] found that low intensity ultrasound (24 kHz, 2 W, 29 °C) greatly enhanced the total Ca²⁺ content within the cells which in turn increased the yeast biomass. They realized that low-intensity sonication could alter the surface potential of the cell membrane of S. cerevisiae, activating Ca²⁺ channels and causing Ca²⁺ out of cell to flow in. The increase of cellular Ca²⁺ might trigger the cell reaction for promoting the growth and proliferation of S. cerevisiae and thus shortening logarithmic phase efficiently. Previous reports indicated that when the cell membrane got stimulated, the change in MP would make the channel relatively free to the penetration of Ca²⁺, reducing its electrochemical gradient. Shi et al. [38] noted that low-intensity ultrasonic treatment of yeast cells produced three times the amount of intracellular Ca²⁺ and efficiently shortened the logarithmic growth phase compared to non-sonicated cultures. The increase in cellular free Ca²⁺ density was ascribed to the penetration of Ca²⁺ out of cells or the release of Ca²⁺ stores [39]. However, excessive ultrasound treatment might cause unrepairable damages of the cell membrane, leading to the leakage of intracellular Ca²⁺ into culture medium.

When the free ligand Fluo-4 combines Ca²⁺ in B. licheniformis YYC4, the produced fluorescence can be observed by an inverted fluorescence microscope to show its intensity and intracellular distribution. As shown in Fig. 4D1-D7, ultrasound treatment (from 0 to 1.5 h) caused a steady enhancement in fluorescence intensity and quantity, then a reduction was obtained with the further increasing of sonication (from 1.5 to 3.0 h), which was consistent with the results in Fig. 4C.

3.5. Effect of ultrasound treatment on metabolism of B. licheniformis YYC4

3.5.1. Extracellular enzyme activities

Fig. 5A showed that the extracellular enzyme activities of neutral, alkaline and acid proteases of the B. licheniformis YYC4 culture steadily increased with ultrasonic time, reaching the maximum values at 1.5 h, which were substantially enhanced by 16.89%, 63.64%, and 32.77%, respectively. While further prolongation of sonication induced a gradual reduction in their activity (by 21.85%, 31.82% and 22.37%, respectively) at 3 h of exposure. This phenomenon was attributed to the improved cellular membrane permeabilization and the increment of biomass of the strain induced by appropriate intensity ultrasonic stimulation resulted in more proteases secreted into the culture than that without ultrasonic treatment [30]. Furthermore, literature reported that suitable ultrasound treatment could increase the activity of enzymes by altering their structure and thus increasing the affinity among enzymes and substrates [7], [40]. Shi et al. [38] found that low-intensity ultrasound enhanced the fermentation and proteinase activity of S. cerevisiae. However, prolonged ultrasound treatment could decrease the biomass of the strain in the culture medium and inactivate the protease [41]. Ma et al. [42] examined the effect of the energy-gathered ultrasound on the activity of Alcalase. They found that the activity of Alcalase was increased by 5.8% after cavitation (80 W and 4 min), along with a slight increase of tryptophan on its surface, as well as an increase of α-helix (5.2%) and a reduction of random coil (13.6%) in the secondary structure of Alcalase. These alterations might make Alcalase more regularity and flexibility, which were helpful for the improvement of Alcalase activity. Moreover, ultrasound can change the characteristics of substrates and thereby accelerating/enhancing the reaction between enzyme and substrate [22].

Fig. 5.

Effect of ultrasound treatment on extracellular protease activity (A) and metabolic activity (B) of B. licheniformis YYC4. Error bars indicate the standard deviations of triplicate samples. Different letters indicate significant difference at p < 0.05.

3.5.2. Volatile compounds of the culture medium

The effect of ultrasonication on volatile compounds of B. licheniformis YYC4 culture was investigated by GC–MS. As shown in Table 1, LB medium contained 89.324 mg/mL volatile ingredients, including alcohols, organic acids, amines, amino acids, alkanes, esters, and organic heterocycles, with their contents of 5.487, 6.957, 6.766, 64.085, 0.226, 2.518 and 3.285 mg/mL, respectively. However, after inoculating B. licheniformis YYC4 and incubating for 24 h (with or without ultrasound treatment), both alkanes and esters in the culture were not detected. The content of alcohols, organic acids, amines, amino acids, and organic heterocycles in the culture inoculated B. licheniformis YYC4 and incubated for 24 h (without ultrasonication) was 2.661, 5.497, 5.163, 44.809 and 2.684 mg/mL, respectively, with a total content of 60.814 mg/mL, showing a decrease of volatile ingredients compared with LB medium. Further, the content of the above-mentioned ingredients was 4.377, 10.802, 8.139, 71.925 and 3.921 mg/mL in ultrasound treated B. licheniformis YYC4 culture after 24 h of incubation, respectively, with a total amount of 99.164 mg/mL, exhibiting a significant increase in their content by 64.49%, 96.51%, 57.64%, 60.51%, and 46.09%, compared with the untreated sample. The significant changes in the volatile substances after inoculation of B. licheniformis YYC4 and cultivation for 24 might be that volatile nutrients in the medium could be consumed as a result of the microbial growth and reproduction. This also was ascribed to the produced new ingredients (e.g., L-threitol, benzoic acid, phenylacetic acid, 2-ketoisocaproic acid, N,N-dimethylformamide, thymine, guanine and so on) through microbial metabolism. Additionally, the metabolic ability of B. licheniformis YYC4 might be enhanced by ultrasonic action and thus improved bacterial ability to convert nutrients from the medium into alcohols, acids, amines, amino acids, and organic heterocycles. DPA, a substance released during bacterial spore germination, was detected in the B. licheniformis YYC4 culture. Ultrasound treated sample had a higher DPA content than that without ultrasound, indicating that ultrasound promoted spore germination of B. licheniformis YYC4. Similarly, Wang et al. [18] found that the endospores germination rate of sonicated B. amyloliquefaciens was 67.33%, significantly higher than the control (with a 15.67% of germination rate).

Table 1.

Effect of ultrasound treatment on the volatile compounds of B. licheniformis YYC4 culture (mg/mL).

Volatile compounds	LB medium	Culture medium of the strain
		Without sonication	Sonication
Alcohols
Ethylene glycol	0.025 ± 0.02a	0.011 ± 0.01b	0.013 ± 0.01b
1-Heptanol	4.475 ± 0.85a	2.537 ± 0.27b	4.051 ± 0.11 a
Inositol	0.927 ± 0.15	–	–
L-Threitol	–	0.113 ± 0.08b	0.313 ± 0.02a
Thiodiglycol	0.060 ± 0.02	–	–
Organic acids
Propionic acid	0.006 ± 0.004a	0.003 ± 0.001a	0.005 ± 0.001a
Succinic acid	0.574 ± 0.17b	0.460 ± 0.05b	0.755 ± 0.03a
Malic acid	0.850 ± 0.04a	0.504 ± 0.12b	0.778 ± 0.05a
Benzoic acid	–	0.010 ± 0.006a	0.018 ± 0.003a
Phenylacetic acid	–	0.174 ± 0.11a	0.226 ± 0.07a
Piperidinic acid	2.376 ± 0.31a	1.085 ± 0.23b	2.064 ± 0.13a
Glycolic acid	0.063 ± 0.05b	0.259 ± 0.11a	0.276 ± 0.07a
Palmitic acid	0.933 ± 0.04a	0.475 ± 0.21b	1.145 ± 0.34a
Stearic acid	0.966 ± 0.33a	0.422 ± 0.17b	1.004 ± 0.03a
Ritalin acid	0.536 ± 0.06b	0.646 ± 0.21b	1.862 ± 0.27a
2-Ketoisocaproic acid	–	0.087 ± 0.01b	0.225 ± 0.03a
3-Hydroxyisovaleric acid	–	0.042 ± 0.01a	0.065 ± 0.04a
α-Hydroxyglutaric acid	–	0.446 ± 0.03b	0.834 ± 0.01a
4-Hydroxyphenylacetic acid	–	0.089 ± 0.06a	0.125 ± 0.06a
4-Hydroxybutyric acid	–	0.006 ± 0.001	–
3,4-Dihydroxybutyric acid	0.044 ± 0.02a	0.054 ± 0.01a	0.085 ± 0.03a
2,3-Dihydroxybutyric acid	–	0.013 ± 0.01a	0.019 ± 0.01a
2,4-Dihydroxybutyric acid	0.106 ± 0.10a	0.072 ± 0.01a	0.109 ± 0.04a
2,3,4-Trihydroxybutyric acid	0.134 ± 0.02a	0.122 ± 0.09a	0.216 ± 0.13a
2,3-Dihydroxy-2-methylpropanoic acid	0.181 ± 0.02a	0.158 ± 0.08a	0.234 ± 0.14a
2-Aminopropanedioic acid	0.188 ± 0.11b	0.155 ± 0.03b	0.373 ± 0.06a
2,6-Pyridinedicarboxylic acid	–	0.215 ± 0.12b	0.384 ± 0.07a
Amines
Methylamine	1.107 ± 0.12a	0.786 ± 0.14b	1.276 ± 0.32a
Ethylamine	3.922 ± 0.05b	2.712 ± 0.13c	4.312 ± 0.15a
Ethanamide	0.868 ± 0.16a	0.595 ± 0.02b	0.857 ± 0.18a
Ethanolamine	0.047 ± 0.04a	0.027 ± 0.09a	0.039 ± 0.01a
N-Ethyl acetamide	0.487 ± 0.07a	0.338 ± 0.01b	0.497 ± 0.03a
N,N-Dimethylglycine	0.068 ± 0.02a	0.064 ± 0.02a	0.081 ± 0.08a
N-Butyl phthalimide	0.189 ± 0.11a	0.136 ± 0.07a	0.231 ± 0.01a
N,N-Diethyl acetamide	0.078 ± 0.01a	0.059 ± 0.01a	0.077 ± 0.01a
4-Chloro-2,5-Dimethoxyphenethylamine	–	0.070 ± 0.02b	0.105 ± 0.01a
N,N-Dimethylformamide	–	0.376 ± 0.01b	0.664 ± 0.18a
Amino acids
Serine	3.056 ± 0.12a	0.204 ± 0.21b	0.278 ± 0.02b
Glycine	2.128 ± 0.09b	1.604 ± 0.07c	2.635 ± 0.32a
L-Valine	2.591 ± 0.19a	1.345 ± 0.51c	2.050 ± 0.05b
L-Glutamic acid	2.958 ± 0.21b	2.857 ± 0.17b	4.585 ± 0.24a
L-Alanine	4.609 ± 0.29a	3.518 ± 0.32b	5.366 ± 0.14a
L-Leucine	9.037 ± 0.36a	0.108 ± 0.01c	1.705 ± 0.09b
L-Threonine	2.800 ± 0.16b	2.087 ± 0.08c	3.130 ± 0.02a
L-Methionine	2.392 ± 0.41b	2.420 ± 0.16b	3.876 ± 0.16a
L-Tryptophan	3.638 ± 0.21b	2.590 ± 0.17c	4.100 ± 0.09a
L-Ornithine	2.128 ± 0.04c	3.402 ± 0.11b	5.411 ± 0.03a
L-Tyrosine	2.763 ± 0.08c	3.852 ± 0.34b	6.340 ± 0.14a
L-Lysine	8.609 ± 0.14c	8.760 ± 0.01b	13.196 ± 0.13a
L-Phenylalanine	4.915 ± 0.16b	4.189 ± 0.03c	6.596 ± 0.25a
L-Isoleucine	2.830 ± 0.06a	0.557 ± 0.21b	0.927 ± 0.33b
L-Aspartic acid	2.666 ± 0.17c	3.505 ± 0.21b	5.441 ± 0.01a
L-5-hydroxyproline	4.636 ± 0.07b	3.436 ± 0.24c	5.488 ± 0.07a
Homocysteine	0.053 ± 0.03a	0.029 ± 0.01a	0.038 ± 0.01a
Homoserine	2.276 ± 0.46a	0.346 ± 0.50b	0.763 ± 0.02b
Alkanes hydrocarbons
Decane	0.169 ± 0.02	–	–
2,3-Dimethyl-1,3-cyclohexadiene	0.057 ± 0.001	–	–
Esters
Glycerol monostearate	2.518 ± 0.08	–	–
Organic heterocyclic
Urea	0.098 ± 0.01b	0.510 ± 0.11a	0.628 ± 0.03a
Uridine	0.190 ± 0.08a	0.040 ± 0.01b	0.049 ± 0.03b
Uracil	0.095 ± 0.01a	0.111 ± 0.03a	0.105 ± 0.01a
Thymine	–	0.083 ± 0.04a	0.127 ± 0.11a
Guanine	–	0.334 ± 0.13b	0.592 ± 0.05a
2-Deoxyribonucleic acid	0.033 ± 0.01a	0.025 ± 0.02a	0.037 ± 0.1a
2-Octanone	2.665 ± 0.09a	1.355 ± 0.13c	2.180 ± 0.21b
3-Amino-2-piperidone	0.204 ± 0.05a	0.032 ± 0.06b	–
Cyclophenylpropylene proline diketopiperazine	–	0.194 ± 0.02a	0.203 ± 0.01a

Data are expressed by means ± SD of three independent replicates. Different letters indicate significant difference at the level of p < 0.05 by SPSS analysis.

3.5.3. Metabolic activity of B. licheniformis YYC4

Living cells can generate H⁺ under the dehydrogenases in the electron transfer system of TCA cycle. H⁺ can restore INT to a stable red formazan. Thus, metabolic activity of B. licheniformis YYC4 can be judged by the production rate of formazan. As shown in Fig. 5B, the absorbance at 630 nm increased gradually with ultrasonic time, and reached the maximum at 1.5 h (increased by 17.51% over the control), indicating that appropriate ultrasonication could induce microorganisms and improve their metabolic activity. Abesinghe et al. [43] noticed that ultrasound application during lag phase (1685 J/mL) and logarithmic phase (561.6 J/mL) reduced the fermentation time of buffalo’s milk by 32 min (12.5%) and 40 min (15.7%), respectively, by improving the metabolic activities of lactic acid bacteria. However, further prolongation of sonication time caused a notable reduction in the absorbance, especially at 2.5 and 3 h of ultrasound treatment (decreased by 3.63% and 8.64% over the control, respectively), suggesting reduction in the production rate of formazan, and weakening of the metabolism, and/or a significant damage in the strain caused by excessive ultrasound treatment.

3.6. Effect of ultrasound treatment on spore germination of B. licheniformis YYC4

As shown in Fig. 6, the number of B. licheniformis YYC4 spores decreased from 5.57 × 10⁶ to 4.37 × 10⁶ cfu/mL with increasing ultrasound time (0 to 1.5 h), then increased to 7.27 × 10⁶ cfu/mL with further prolongation to 3 h. The decrease might be attributed to that low-intensity ultrasound treatment could promote germination of B. licheniformis YYC4 spores, however, germinated spores had weakened resistance to the adverse conditions, leading to be killed by heat treatment in boiling water for 10 min. While the increase in number of spores with further ultrasonic irradiation might be that the more spores were produced to resist the strong ultrasound stress. There have been some studies on the facilitation effect of ultrasound on endospores germination. Most of them focused on ultrasound assisted inactivating endospores, combined with heat, pressure, electricity and radiation [44], [45], [46]. These studies indicated that it was effective to inactivate endospores using combined approaches because ultrasound played the role of promoting endospores germination while those extreme conditions deactivating the vegetative cells [47].

Fig. 6.

Effect of ultrasound treatment on spore germination of B. licheniformis YYC4. Error bars indicate the standard deviations of triplicate samples. Different letters indicate significant difference at p < 0.05.

4. Conclusion

The results in this study manifested that low-intensity ultrasonication considerably promoted the growth of B. licheniformis YYC4. Under the optimal ultrasonic parameters (28 kHz, 120 W, and 1.5 h), with a fixed-frequency continuous ultrasound treatment in 3 L water, the biomass of B. licheniformis YYC4 at logarithmic metaphase significantly increased by 48.95% after incubation for 24 h. The combined effects of ultrasonication on the strain contributed to increase in biomass. For example, low-intensity ultrasound treatment caused an improvement in cellular membrane permeability and metabolic capacity of the strain. Additionally, it also promoted germination of spores. This study verified the feasibility of applying ultrasound technology in the field involving microorganisms, such as fermentation, to improve fermentation efficiency. Also, the findings of the current work could be beneficial in setting the theoretical basis and technological support for the utilization of LIFFCU in the related industry.

CRediT authorship contribution statement

Chunhua Dai: Investigation, Data curation, Formal analysis, Writing – original draft. Zhenzhen Shu: Investigation, Data curation, Formal analysis, Writing – original draft. Xueting Xu: Investigation, Data curation, Formal analysis, Writing – original draft. Pengfei Yan: Writing – review & editing. Mokhtar Dabbour: Writing – review & editing. Benjamin Kumah Mintah: Writing – review & editing. Liurong Huang: Supervision. Ronghai He: Supervision. Haile Ma: Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.