Mutation-Screening in l-(+)-Lactic Acid Producing Strains by Ion Implantation

Abstract

In this paper, in order to obtain some industrial strains with high yield of l-(+)-lactic acid, the wild type strain Lactobacillus casei CICC6028 was mutated by nitrogen ions implantation. By study, it was found that the high positive mutation rate was obtained when the output power was 10 keV and the dose of N⁺ implantation was 50 × 2.6 × 10¹³ ions/cm². In addition, the initial screening methods were also studied, and it was found that the transparent halos method was unavailable, for some high yield strains of l-(+)-lactic acid were missed. Then a mutant strain which was named as N-2 was isolated, its optimum fermentation temperature was 40°C and the l-(+)-lactic acid yield was 136 g/l compared to the original strain whose optimum fermentation temperature was 34°C and l-(+)-lactic acid production was 98 g/l. Finally, High Performance Liquid Chromatography method was used to analyze the purity of l-(+)-lactic acid that was produced by the mutant N-2, and the result showed the main production of N-2 was l-(+)-lactic acid.

Introduction

Low-energy ion beam implantation, as a new mutagenesis technique [1], has attracted much attention in breeding of microorganisms and crops for its advantages, such as higher mutation rate, wider spectrum and lower injury rate of mutation than the traditional mutation methods [2]. Many high-yield strains have been obtained by ion beam implantation in industrial microorganisms [3, 4]. The biological effects may result from energy absorption, mass deposition, and charge exchange of energetic ions [5, 6]. Now, N⁺ implantation has been widely used to bombard microorganisms for obtaining industrial strain [7].

l-(+)-lactic acid (l-LA) has been used as a natural preservative in many food products since a long time ago. Currently, l-LA is widely applied in food, pharmaceuticals, textile, leather, and feed industries [8, 9]. One of the most promising application was its use for biodegradable and biocompatible polymers synthesis such as poly lactic acid (PLA) [10], as a biodegradable and thermoplastic biomaterial, PLA is commonly used as high-quality biodegradable plastics for packing and other applications, which can potentially replace polyethylene, polypropylene and polystyrene. However, this potential is dependent on whether lactic acid can be produced at a low cost [11, 12]. The superior strain with high yield of l-LA is required, for approximately 90% l-LA is produced by biotechnology [13–15] all over the world.

The purpose of this work was to screen a potential industrial strain with high yield of l-LA and high fermentation temperature by N⁺ ion implantation.

Materials and Methods

Microorganism

Lactobacillus casei CICC6028 was maintained on agar slant containing (g/l): beef extract 5, yeast extract 5, peptone 10, glucose 10, lactose 5, sodium chloride 5, agar 20; and pH 6.8.

Media

Selective medium (g/l): glucose 50, peptone 15, beef extract 15, yeast extract 7.5, sodium acetate anhydrous 5, MgSO₄·7H₂O 0.2, MnSO₄·7H₂O 0.125, CaCO₃ 10 (sterilized alone), agar 20, pH 6.8, and 1 ml/l tween-80.

Seeding medium (g/l): glucose 50, peptone 15, beef extract 15, yeast extract 7.5, sodium acetate anhydrous 5, MgSO₄·7H₂O 0.2, MnSO₄·7H₂O 0.125, CaCO₃ 10 (sterilized alone), pH 6.8.

Fermentation medium (g/l): glucose 140, peptone 15, beef extract 15, yeast extract 7.5, sodium acetate anhydrous 5, MgSO₄·7H₂O 0.2, MnSO₄·7H₂O 0.125, CaCO₃ 10 (sterilized alone), pH 6.8, and 1 ml/l tween-80.

The media used in this study were all sterilized at 115°C for 30 min.

Mutagenesis Method

Implantation sources were produced by ion beam bioengineering instrument (Patent No. CN 93103361.6, Zeng-liang Yu et al., 2000.P.R.C.). The instrument was devised by ASIPP (Chinese Academy of Sciences, Institute of plasma physics).

Lactobacillus casei CICC6028 was cultured for 36 h on agar slant, then the cells of two loops were inoculated into 50 ml seeding medium containing in a 250 ml flask and cultured on a rotary shaker (100 ± 5 r/min) at 34°C for 36 h. The cells were centrifuged and rewashed twice using physiological saline and then diluted as the concentration was about 10⁵ cells per milliliter. The cells suspension of 0.1 ml were spread on the sterilized Petri dish (90 mm) to ensure that they can form a single-cell layer, then they were desiccated by filtrated air on a clean bench. Finally the above plates were treated by nitrogen ion beam under dry and vacuum environment. At the same time, in order to evaluate the survive rate of vacuum-dependent and dose-dependent, three samples were prepared. Sample one was not exposed to vacuum nor ion beam; sample two and three were set in the target chamber to make sure they were all exposed to vacuum environment. Only sample two exposed to ion beam but sample three was not treated with the ion beam. The death induced by vacuum and ion beam implantation will be eliminated by comparing the three samples.

Calculation of the Survival Rates

Survival rates which were mentioned for many times in this paper were caused by vacuum (S1), different energies (S2) and different doses of N⁺ implantation (S3) respectively. They were calculated as follows:

A is the average number of the colonies in the selective plates formed by the cells suspension without ion beam treatment, B C D indicate the average number of the colonies formed by the cells suspension which were treated by vacuum, different energies and different doses of N⁺ implantation, respectively. n1, n2, n3, n4 indicate the dilution times of the cells suspension.

Mutation Rate Determination

The mutant criterion was defined as follows: the strains whose production of l-LA were 5% higher/lower than that of the control strain were concerned as positive/negative mutation, while others were non-mutation.

The mutation rate was calculated as M/N.

M: Number of the strains whose production of lactic acid were 5% higher/lower than that of the control; N: total number of colonies on the plates.

The final data of mutation rate was derived from the average of three independent experiments.

Strain Selection Methods

Initial selection: The suspension of cells treated by ion beam was spread on the selective medium plates at 34°C for 48 h. The colonies formed transparent halos were selected and inoculated into the slants and cultured in 34°C for 36 h and then maintained at 4°C.

Flask experiments: The strains obtained from the initial selection were cultured in the seed medium for 36 h and then inoculated in the fermentation medium. The strains with high yield of l-LA were selected by measuring the yields of l-LA after 96 h incubation.

Analytical Methods

The concentration of calcium lactate was tested by EDTA titration method [16]. The optical purity of l-LA was measured by High Performance Liquid Chromatography (HPLC) using an uBondpak C18 column (Waters, USA). The mobile phase composition was CH₂OH: H₂O: H₃PO₄ (10:90:0.3, V:V:V) and the flow rate was 0.8 ml/min. The detection was made in the UV range at 210 nm at the temperature of 25°C. The standard l-LA was purchased from SINGMA (St. Louis, USA). The residual sugar was quantified by the 3,5-dinitrosalicylic acid method [17].

Results and Discussion

Parameter Determination of N⁺ Implantation

It was well known that, the nitrogen element is the main composition of DNA. As one source of mutagenesis, N⁺ had higher mutation frequency and wider mutation spectra than other ions such as Ar⁺, H⁺, He⁺[18], so it was the most common ion used in ion beam implantation [19]. In this paper, N⁺ was chose as the ion source for ion implantation. It was reported that the mutation rate was related to the survive rate during strain breeding and high mutation rate would obtained when the survive rate was about 20% [20]. In ion beam implantation, the cell death was caused by three factors which were various doses of ion beam implantation, different energy of N⁺ and exposure time under vacuum environment. However, only the various doses of ions implanted in the cells can cause gene mutation [17, 21].

Figure 1 showed the survive rate of the cells exposed to different energy of N⁺ ion beam implantation with dose of 15.6 × 10¹⁴ ions/cm². The survival rate decreased slowly when energy was less than 5 keV. However, it decreased sharply when the energy during 5 to 10 keV, and then the curve of survival rate trend ranged not obviously when energy surpassed 10 keV. In order to obtain higher positive mutation and higher survival rate, 10 keV was chosen as the optimal energy according to the theory stated in the Ref. [22]. Figure 2 showed the strain survive rate which decreased with the exposure time increased under vacuum environment. It illustrated that the vacuum environment had a great harm to cells.

Fig. 1.

Effect of energy on survival rate of Lactobacillus casei CICC 6028

Fig. 2.

The survival rate of L. casei CICC 6028 in vacuum

Figure 3 showed the dose-dependent survival rate in L. casei CICC6028 under the energy of 10 keV. The survival rate decreased significantly when the implantation doses were 0–10 × 2.6 × 10¹³, but it increased slightly when the implantation doses were 10–50 × 2.6 × 10¹³ ions/cm², and it decreased again when the implantation doses were 50–180 × 2.6 × 10¹³ ions/cm². The shape trend looks like a “saddle”, when the implantation doses rang from 10 × 2.6 × 10¹³–180 × 2.6 × 10¹³ ions/cm². This was just the characteristic survival curve of N⁺ implantation [21, 23]. Figure 4 showed the relationship between dose-dependent survival rate and mutation rate, when cells were exposed to the various doses of N⁺ with the energy of 10 keV. It was found that the positive and negative mutation rates were all low at the lower doses (30 × 2.6 × 10¹³ ions/cm²). The highest mutation rate was observed when dose was 50 × 2.6 × 10¹³ ions/cm². But the mutation rates were lower under higher implantation doses (70 × 2.6 × 10¹³, 100 × 2.6 × 10¹³, 120 × 2.6 × 10¹³ and 160 × 2.6 × 10¹³ ions/cm²) than that of when the dose was 50 × 2.6 × 10¹³ ions/cm². It was an interesting phenomenon that the highest mutation rate was appeared just at the ridge of the dose-dependent survival rate curve. So, in order to obtain higher mutation rate, the dose of 50 × 2.6 × 10¹³ ions/cm² was chose.

Fig. 3.

Effect of N⁺ implantation doses on survival rate of L. casei CICC 6028

Fig. 4.

Effect of N⁺ implantation doses on mutation rate of L. casei CICC 6028

Screening Mutated Strains with High Yield of l-LA

To screening the high yield of l-LA strains the transparent halos approach was studied. Figure 5 showed the transparent halos formed by the mutants on the selective medium plate. Because the selective medium contains CaCO₃, l-LA produced by colonies would neutralize the CaCO₃, and the transparent halos will be formed. In general, the diameters of transparent halos were depended on l-LA yield, the more l-LA were yielded the lager diameters would be formed [24]. However, it was found that some strains with large diameters had low yield of l-LA in this study (Table 1). The reason of this phenomenon was still unknown. It was speculated that this phenomenon might be due to the formation of the following two reasons. First of all, cells might be damaged during the exposure to ion beam. The damaged cells could not proliferate immediately after they were spread to the plates, they would take some times to repair this damage. In addition, l-LA was a primary metabolite, its’ synthesis was accompanied with the growth of the cells. It means, the high yield strains may not perform as well as they did in normal station, although they had the potential to produce large amount of l-LA. Secondly, the difference between solid medium and liquid medium on producing l-LA should be considered, and the difference might make some of the strains with high-yield l-LA be missed. Considering these disadvantages, it can be conclude that, transparent halos method was not suitable for this strain, and shake flask fermentation was chose as the initial screening method.

Fig. 5.

Transparent halos formed by the mutants on the selective medium plate

Table 1.

Relationship of diameters of transparent halos and yield of l-LA

Strains	DTH (mm)	DC (mm)	DTH/DC	YLF (g/l)
1	0.585	0.150	3.90	114.40
2	0.700	0.210	3.33	44.14
3	0.700	0.190	3.68	98.19
4	0.685	0.240	2.85	121.61
5	0.905	0.205	4.41	24.32
6	0.815	0.175	4.66	128.81
7	0.950	0.230	4.13	86.48
8	0.850	0.275	3.09	25.22
9	0.845	0.200	4.23	95.48

DTH diameters of transparent haloes; DC diameters of colonies; YLF yield of l-LA by fermentation

On the basis of shake flask fermentation, the mutant N-2 was obtained. As a potential industrial strain, the stability of the mutant was crucial, so the stability production of the mutant was studied. Its yields of l-LA were constant throughout six generations at an average of 136 g/l (Table 2). This result demonstrated the mutant was stable in l-LA producing.

Table 2.

Stability of the mutant N-2

Generations	1	2	3	4	5	6
Yield (g/l)	137	132	133	139	136	139

Comparisons of the Mutant N-2 and the Original Strain

Good industrial strains should bear relatively high fermentation temperature, for if the fermentation temperature was high it will saved much on cooling water during industrial producing. Figure 6 showed the l-LA yield of mutant N-2 and the original strain at various temperatures. It indicated that the yield of l-LA of mutant N-2 was higher than original strain at the same temperature. The optimum fermentation temperature of the mutant N-2 was 40°C, it increased by 6°C compared to the original strain whose optimum fermentation temperature was 34°C. These suggested the mutant take great advantages in applying in industrial fermentation.

Fig. 6.

l-LA yield of mutant N-2 and the original strain at various temperatures

The fermentation kinetics of original strain and mutant N-2 were studied respectively under the same fermentation conditions. As showed in Fig. 7, the fermentation kinetics curve trends of the two strains were similar. During the fermentation process, with l-LA’s production, the glucose was consumed and residual sugar was decreased. The curves of concentration of l-LA accumulation and residual sugar degradation were similar between mutant N-2 and original strain L. casei CICC6028 during the first 12 h. However, after 12 h, the accumulation of l-LA increase of the mutant strain was faster than that of the original strain and the residual sugars were significantly different. The l-LA production of mutant N-2 reached to 136 g/l with residual sugar dropped to 9.5 g/l while the original was only 98 g/l l-LA with residual sugar as high as 38 g/l at 120 h.

Fig. 7.

Fermentation course of L. casei CICC6028 and mutation N-2 in 250 ml flasks

HPLC was used to analyze the production by N-2. As showed in Fig. 8, the ridge was appeared during 4.50–5.00 min (Fig. 8b) of the fermentation sample, and the ridge appearance time was also 4.50–5.00 min (Fig. 8a) of the standard l-LA. By comparing to the standard samples and the fermentation samples, it was concluded that the production was l-LA, and there was no miscellaneous peak of other organic acid (Fig. 8b), which indicated the main production of mutant N-2 was l-LA and there was no other acid almost.

Fig. 8.

HPLC chromatogram of l-LA produces by mutant N-2 a Standard samples (from left to right): malic acid; l-LA; fumaric acid b Fermentation sample

Conclusion

This work was performed to obtain a potential industrial strain of L. casei to produce high yield of l-LA. By studying the parameters of N⁺ implantation, it was found that when the output power was 10 keV and the implantation doses of N⁺ was 50 × 2.6 × 10¹³ ions/cm² the original strain had higher positive mutation rate. The initial screening methods were also studied in this work; it was found that the transparent halos method was unavailable, so the initial screening was performed by shake flask fermentation. With the optimum energy and doses of the ion beam, the mutant N-2 was screened whose optimum fermentation temperature was 40°C and the l-LA production was 136 g/l. Compared to the original strain whose optimum fermentation temperature was 34°C and the l-LA production was 98 g/l, the mutant N-2 had a great advantages as a potential industrial strain.