Mutation of Bacillus velezensis Using Corona Discharge

Abstract

Bacillus velezensis is a kind of beneficial bacteria that is widely used in agriculture industry. Bacillus velezensis was irradiated with corona discharge generated by a needle-array high-voltage electrode. The results showed an improvement of activity of Bacillus velezensis by the corona discharge treatment was confirmed at an optimum input energy. Mutation of the Bacillus velezensis by the corona discharge treatment was also confirmed through an rRNA sequence alignment analysis. The enzyme activity of the mutated bacteria was greatly improved, which was a positive effect that can meet the production demand.

1. Introduction

Bacillus velezensis is a Gram-positive bacterium with rod shape that can produce spores. It is a kind of aerobic or facultative anaerobic bacteria, which can be widely used in air, water, human, animal intestines, vegetables and food [1,2]. In recent years, Bacillus velezensis plays an increasingly important role in the application of microorganisms [3]. Bacillus velezensis can produce spores even in adverse environments. Spores can be converted to powder without death of bacteria, which has obvious advantages over other biocontrol bacteria [4]. Therefore, some kinds of Bacillus velezensis are gradually developed and utilized in the production of enzymes, antibiotics, pesticides, surfactants, biological agents, flavor enhancers and nutrition and health products [5].

The agricultural application of high voltage is a new technology that can be used as seed germination promotion [6,7], plant growth acceleration [8,9], inactivation of bacteria in soil–liquid hydroponic media [10,11,12], and promotion of mushroom body formation [13,14]. There are many other applications of high voltage technology in agriculture industry [15]. Corona discharge is the most common form of gas discharge in high-voltage electric field. It is the partial self-sustaining discharge of gas medium in uneven electric field. The process of corona discharge is usually accompanied by the generation of ultraviolet and ozone.

Corona discharge can kill microorganisms using simpler, longer-lifetime and lower-cost devices than those for glow and dielectric barrier discharges. As the mechanism of sterilization effect with nonthermal plasma, it has been suggested that some active species, such as ozone, superoxide anion (O₂⁻), hydroxyl radical and ultraviolet rays, contribute the effect in a tube for medical purposes. The influence on cell membranes of nucleic acids is also an important part of the sterilization mechanism [16]. In the process of DNA replication, ultraviolet irradiation will cause errors in base complementary pairing, that is, base pair mismatch, which will lead to gene mutation. Most mutant Bacillus velezensis were developed by UV irradiation [17]. Corona discharge may be a feasible technique for mutagenesis of Bacillus velezensis. In this study, it was found that corona discharge can realize the mutation of Bacillus velezensis. Compared with other mutation methods, corona discharge mutation can achieve a higher mutation rate. At the same time, it has a high probability to obtain strains with fast growth rate, and its enzyme activity can be greatly improved.

2. Materials and Methods

2.1. Bacillus velezensis Strains and Media

The Bacillus velezensis strains were provided by Shandong Bluegreen Biotechnology Co., Ltd (China). The mutant strain (1–7) was obtained from untreated Bacillus (615) strain. The mutant strain (1–7) was produced by the disruption of the gene of (615) strain. All chemicals, such as iodine, potassium iodine and starch, were bought from Sinopharm Chemical Reagent Co., Ltd. The preparation of bacterial solution was carried out according to determination of feed Bacillus in feed [18].

2.2. Corona Discharge Treatment

The circuit connection diagram of the experimental system is shown in Figure 1. The test sample was placed in a treatment chamber with adjustable height. The high-voltage system was used to generate and adjust the voltage of the high-voltage outputted and display the magnitude of the amplitude, which consisted of controller and high-voltage generator (ZVI240/120, Suzhou Haiwo). The system consisted of a rectification/filtering, inverter bridge, Cockcroft–Walton circuit, etc. The output voltage varied in range from 5 kV to 240 kV at maximum output current of 5 mA.

The structure of the treatment corona discharge electrode and sample holder is shown in Figure 2. The needle–plate electrode was used for corona discharging. The needle electrode was evenly distributed above the plate. The diameter of the needle was 1 mm, and the length of the needle was 65 mm. The samples were placed in a glass Petri dish for high-voltage corona discharge treatment. The radius of the dish was 90 mm, and the thickness was 1 mm. The distance d marked in Figure 2 was the distance between needle plate and the sample, which can be changed in different experiments.

The condition of bacterial treatment was corona discharge. The discharge current can be measured by the measuring module shown in Figure 1. The discharge distance between needle and plate can be adjusted in the range of 0–100 mm, and the discharge voltage can be adjusted in the range of 0–100 kV. The treated sample was placed between the needles and the plate.

The photo of actual sample treatment is shown in Figure 3. The picture on the left shows the photo of corona discharge of positive needles and negative plate, and the one on the right shows the photo of treating bacterial liquid by corona discharge. The corona discharges appeared in the vicinity of all needle tips. The current waveforms showed the corona discharge flowed as almost direct current. The radical species and charged particles in the corona discharge flowed to the Petri dish as drift and diffusion phenomena.

2.3. Corona Discharge Current and Lethality Rate

We designed a three-factor and three-level orthogonal experiment as shown in

Table 1. Treatment conditions and lethality of the treated bacteria.

Number	Sample	Voltage	Time	Current	Number of Viable Bacteria	Lethality Rate
1	None	5 kV	/	48 μA	/	/
2	None	10 kV	/	118 μA	/	/
3	None	12 kV	/	201 μA	/	/
4	Powder	5 kV	5 min	55 μA	(7.00 ± 2.67) × 108	−23.5%
5	Powder	10 kV	30 min	121 μA	(5.33 ± 0.89) × 108	5.99%
6	Powder	15 kV	15 min	199 μA	(4.93 ± 0.78) × 108	13.1%
Control	Powder	/	/	/	(5.67 ± 0.50) × 108	/
7	Liquid	5 kV	30 min	50 μA	(2.67 ± 0.44) × 108	38.3%
8	Liquid	10 kV	15 min	137 μA	(2.33 ± 0.16) × 108	46.2%
9	Liquid	15 kV	5 min	231 μA	(1.93 ± 0.24) × 108	55.4%
Control	Liquid	/	/	/	(4.33 ± 0.45) × 108	/
10	Medium	5 kV	15 min	46 μA	(1.50 ± 0.47) × 108	76.1%
11	Medium	10 kV	5 min	118 μA	(1.80 ± 0.60) × 108	71.3%
12	Medium	15 kV	30 min	204 μA	(0.57 ± 0.16) × 108	91.0%
Control	Medium	/	/	/	(6.27 ± 0.25) × 108	/

. In this table, a distance of 8 mm between the needle and plate was chosen. ‘Powder’ means the powder bacteria were used as samples, placed in the Petri dish and treated in the corona discharge between the needle and the plate. Additionally, ‘Liquid’ means the samples were bacterial fluid, and ‘Medium’ means the bacteria were evenly coated on NA medium as treatment samples. ‘None’ means there was nothing between the needle and the plate. ‘Time’ means the treatment time of the sample in the corona discharge. When dealing with different forms of bacteria, the corona discharge current, which was measured as shown in Figure 1, under different voltages as shown in Table 1. We also calculated the bacterial lethality rate under different treatment conditions using the following equation:

$${\eta }_L=\frac{n_c-n_t}{n_c}\times 100\%$$

(1)

where ${\eta }_L$ is the lethality rate, $n_c$ is the bacteria number of control sample and $n_t$ is the inactivated bacteria number by corona discharge treatment. The inactivation rate was obtained as follows. The bacterial solution was diluted to ${10}^{-7}$ (1st dilution) and ${10}^{-8}$ (2nd dilution) in the super clean workbench (SW-CJ-2F, AIRTECH). After 18 h of culture, the bacterial solution was taken out to count the number of bacteria. The average number of two culture dishes with the same dilution gradient were taken and the number of viable bacteria was calculated according to following equation:

$$N=\frac{\sum C}{\left(n_1+0.1n_2\right)d}$$

(2)

where N is number of bacteria in the sample; C is sum of the number of bacteria on the plate; and $n_1$ and $n_2$ are sums of the number for 1st dilution and 2nd dilution, respectively. Lastly, d is the dilution factor of the 1st dilution.

The main research content of this paper is the lethality and variation under corona discharge, so there is no experiment on the sterilization boundary conditions of corona discharge.

We performed a range analysis of the mean lethal rate, as shown in

Table 2. Range analysis of lethality rate.

Sample		Voltage		Time
K (Powder)	17.26	K (5 kv)	11.17	K (5 min)	10.73
K (Liquid)	6.93	K (10 kv)	9.46	K (15 min)	8.76
K (Medium)	3.87	K (15 kv)	7.43	K (30 min)	8.57
K’ (Powder)	5.75	K’ (5 kv)	3.72	K’ (5 min)	3.57
K’ (Liquid)	2.31	K’ (10 kv)	3.15	K’ (15 min)	2.92
K’ (Medium)	1.29	K’ (15 kv)	2.47	K’ (30 min)	2.85
R (Sample)	4.46	R (Voltage)	1.24	R (Time)	0.72

. The K value is the sum of data of a certain factor and a certain level. The K’ value is the average of the corresponding K values. The R value is the range of this factor.

According to Table 2, R (Sample) was the largest, meaning the greatest influence on the bacterial lethal rate was the treatment sample, followed by the treatment voltage, and the least influence was the treatment time. According to Table 1, it can be known that the lethal rate increased with the increase of voltage and treatment time.

The bacterial activity in the powder state was very low and basically in a dormant state. Therefore, it was barely affected by corona discharge, and the lethal rate was very low. Activity of bacteria coated on NA medium was higher than that of bacterial fluid, so the lethal rate was the highest under the treatment of corona discharge.

2.4. Enzyme Activity Analysis

The enzyme activity was obtained as follows. The 2 mL solution to be tested was taken into a centrifuge tube, centrifuged for 10 min at the speed of 10,000 r/min and diluted the supernatant 10 times with normal saline. After the solution reacted at (60$\pm$0.2) °C for 5 min, the absorbance was measured by an ultraviolet spectrophotometer λ with a wavelength of 660 nm. The absorbance A was determined rapidly, and the enzyme concentration C was calculated as shown in Equation (3), carried out according to national standards [19].

$$C=\left\{\begin{array}{c}\left(0.304-A\times 0.386\right)\times N, 0.14\le A<0.3 \\ \left(0.2359-A\times 0.1589\right)\times N, 0.3<A<0.8 \end{array}\right.$$

(3)

where N is the dilution ratio and A is the absorbance (U/g). Then the enzyme activity is expressed as follows:

$$U=C\times N$$

(4)

3. Results

In the discharge-treated bacteria, the morphology of many strains was very different from the control strain. It is speculated these strains mutated under the action of discharge. In a 10⁻⁶ solution, 29 mutant strains were obtained. Compared with the case where 14 mutants were obtained in a 10⁻⁴ solution when UV treatment was used [17], the variation rate was greatly improved. The amylase activities of these strains were tested, and the results are shown in Figure 4. It can be seen from the figure that enzyme activity of eight strains (No. 5~12) of these bacteria greatly improved, indicating beneficial variation occurred in this treatment. Corona discharge may affect the stability of enzyme—molecular conformation and the dissociation state of enzyme–molecular polar groups so as to improve enzyme activity of the strain.

The greater the activity of colony, the higher the mortality under corona treatment under the same conditions. Through the observation of strains, it can be seen that under the same activity conditions, the greater the mortality, the higher the probability of variation. Compared with the mutation methods described in another paper [17], the mutation probability of corona mutation method is greater and the difference of production speed is more obvious. As shown in Figure 5a, two kinds of bacteria were compared after 1 day of culture. The single colony (No. 1-7), marked by a red circle, was selected for the next experiment. On the right, the bacteria were treated with NA medium under 15 kV voltage, and on the left, were the untreated control strains. It can be seen with the naked eye that the treated bacteria (No. 1-7) have a larger area in the culture dish than the control (615). However, from Figure 5b, the reproduction speed of the treated strain was much higher than that of the control strain.

The increase of growth area is visible to the naked eye. The quantitative measurement of growth rate is illustrated by the number of living bacteria in the latter part of the article.

Two strains (615 and 1-7) were cultured for 18 h and counted, as shown in

Table 3. Comparison of the number of viable bacteria after 18 h of culture.

Strain	Initial value	NB1	NB2	SM3
615	1.0 × 107	5.7 × 108	2.5 × 108	1.1 × 108
1-7	1.0 × 107	7.6 × 109	1.0 × 109	4.5 × 108

. They were cultured on NB medium two times, which were recorded as NB1 and NB2, respectively. The growth rate of treatment group (1-7) was significantly higher than that of control group (615). In order to verify the growth rate of the strain in actual production, we carried out the culture experiment on soybean meal medium, which was recorded as SM3. It can be seen that the growth rate of the treatment group (1-7) was still significantly higher than that of the control group (615), indicating the mutation greatly improved the performance of the strain.

Results of 16S rRNA sequence alignment of the two strains are shown in Figure 6. Query is the sequence of strains labeled 1-7, and Sbjct is the sequence of strains labeled 615. The red rectangles in the figure show the sequence of the two strains is different.

4. Discussion

The cell membrane is a dielectric material. When the cell membrane is in the normal environmental state, it maintains a low potential difference. When the cell is in the high-voltage corona-discharge environment, the potential difference of the cell membrane has a chance to change and appear with a transmembrane potential. When the transmembrane potential exceeds the critical value, the cell membrane collapses. When high voltage is applied, the cell membrane is affected by the electric field force, and molecules or ions on the cell membrane collide, attract and repel, resulting in holes on the cell membrane. Cell electroporation destroys the balance of molecular interaction in the membrane. When the discharge intensity is large and the time is long enough, the collapse of cell membrane is unrecoverable, which has an obvious lethal effect on bacteria. In the state of low bacterial activity or dormancy, the mortality of cells under discharge is relatively low. Under the condition of high bacterial activity, the mortality rate is high, and the probability of variation is also increased accordingly.

Corona discharge occurs near the tip of the point body and radiates a large amount of ultraviolet light. At the same time, it will have a chemical reaction to produce ozone, nitrogen dioxide, nitric oxide, etc., accompanied by high-frequency pulse current and objective energy loss. In the application of corona discharge in the customization of materials, it can be seen that plasma-generated short-lived reactive oxygen and nitrogen species (RONS) are believed to be key agents for the variety of microorganisms [20].

The plasma has a great impact on the cells below them. Due to mass deposition, energy exchange and charge transfer of implanted ions and intracellular molecules, ion implantation causes serious etching of biomaterials [21]. Plasma is a surface effect on organisms, and the penetration depth of active particles produced by plasma in biomaterials is lacking quantitative interpretation. The radiation damage of high-energy ions in organisms is actually the comprehensive result of a series of low-energy events.

According to Figure 6, the 16S rRNA strand of the mutant strain was missing compared to the control strain. The 16S rRNA strand accounts for about 80% of bacterial DNA. Therefore, it is speculated that the cause of gene strand deletion may be the breakage of a bacterial DNA gene strand caused by external corona discharge. Gene deletion was caused in the process of bacterial replication and repairing of DNA. The stronger the external stimulus, the higher the probability and number of gene breaks. Bacteria cannot survive without repairing DNA. No harmful changes were found in the mutant strains. The genetic stability of mutant strains also needs further experiments to verify.

5. Conclusions

In this study, we tried to use corona discharge to mutate Bacillus velezensis to obtain strains with better performance. The results show higher mutation rate can be obtained under corona discharge. After the test of mutant bacteria, it has faster growth rate and higher amylase activity, which is very beneficial to improve the production rate of bacteria. At the same time, corona discharge is also an easy process. If it is applied in the production of large-scale chemical plants, it can better increase the profits of the plant.

The relationship between the change of gene sequence and the increase of growth rate is a good way to explain mutagenesis. Gene interpretation and gene editing require a lot of work, which will become the next research content.