Study on mutagenesis of high protease producing thermophilic bacteria by UV-DES and hydrolysis of excess sludge

. Excess sludge a resource-rich organic waste, can be solubilized by thermophilic enzymes to extract proteins and anaerobic digestion of methane production to reduce sludge and use resources. At the same time, it can improve new ideas to relieve the pressure of the energy industry. To solve the problems of low enzyme-producing activity of wild strains and low hydrolysis rate of ES, this study utilized UV-DES compound mutagenesis to screen dominant mutant strains under optimized conditions, which were added to ES hydrolysis reaction system. The results showed that UV-DES mutagenesis could effectively improve the ability of S-TE and promote the hydrolysis of ES. The protease activity of the mutant strain KT16 obtained by screening was 46.7% higher than that of the original strain DC8. KT16 effectively degraded insoluble organic matter, reduced the average particle size of ES, and increased the concentrations of extracellular proteins, polypeptides, and amino acids to 1731.8, 521.3, and 510 mg·L-1, respectively. This study will provide a reference for the resource utilized.


Introduction
A great deal of sewage is produced with the rapid development of industry, which is generally treated by activated sludge method in most sewage treatment plants [1].It is estimated that by 2025, the production of excess sludge (ES) will reach 90 million tons (moisture content of 80%) annually in China [2].ES contains pathogens, heavy metal compounds, and persistent organic pollutants [3], which are prone to cause secondary pollution.Traditional disposal methods of ES, such as landfilling, incineration, and soil application, have either high processing costs or low resource utilization rate [4].ES contains abundant organic matter, such as proteins, accounting for approximately 50% of the dry weight of bacterial cells [5].The proteins isolated from ES can be used as animal feeds [6], wood adhesive, concrete foaming agent, and corrosion inhibitor, etc [7].To realize efficient extraction of protein in ES, physical, chemical, biological, and combined techniques have been proposed.The physical methods include ultrasonic, microwave [8], membrane filtration, etc.Therefore, efficient utilization of the organic components in ES under the premise of the harmless disposal has become a hot issue in the field of environmental research.
This study aims to increase protease production and ES hydrolysis efficiency from DC8 by UV-DES compound mutagenesis treatment.In addition, The bacterial agent can be used in anaerobic digestion pretreatment, promote sludge hydrolysis and improve acid production by anaerobic fermentation.The extracellular enzymes secreted by mutagenesis can quickly and effectively depolymerize and crack the sludge, thus improving the degradation rate of sludge, strengthening the production of anaerobic digestion of methane, and finally realizing the "zero emission" of sludge and the reuse of resources, and providing a new way to alleviate the energy problems.

Strain, medium, and culture conditions
Geobacillus thermodenitrificans DC8 was screened from the compost substrate (cow dung and straw) and used as the wild-type strain for mutagenesis.Before mutagenesis, the DC8 strain was activated and cultured in 100mL LB liquid medium with a shaking speed of 180 r•min -1 at 60 o C for 24 h.

Mutagenesis and screening by ultraviolet and DES combined method
The cell of DC8 was collected by centrifugation at 8000 r•min -1 for 10 min, washed twice with sterilized 0.9% NaCl solution.The final concentration was 1 × 10 8 CFU•mL -1 in 0.9% NaCl.
For UV mutagenesis, the mutagenic dosage only depended on the duration of UV irradiation.Before irradiation, the UV lamp was warmed up for 20 min.Then 10 mL of DC8 spore suspension were poured into a sterilized plate, and irradiated with 20W UV light at a distance of 25 cm for 0, 30, 60, 90, 120, 150, 180, and 210 s.Following irradiation, the sample was kept in the dark for 2 h at 4 o C.
Then the strain was treated with DES mutagenesis. 1 mL spore suspension (1 × 108 mL -1 ) was transferred into a 10 mL sterilized tube and mixed with 1 mL DES ethanol solution.In order to determine the best mutagenic reaction conditions, it adjust different mutagenic time (5,10,15,20,25,30,35, and 40 min), different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5%), and different temperature (40, 45, 50, 55, 60, 65 o C).The mixture was stirred at 200 r• min-1 for reaction.After each treatment, 2 mL 25% (w/v) sodium thiosulfate was added to the reaction mixture to stop the mutagenic process.cell suspension was properly diluted and spread onto a solid medium for 12 h at 60 o C prior to determination of the lethality.After that, the cell suspension was properly diluted and cultured on solid medium at 60 o C for 12 h, and calculated the lethality according to formula (1).

Orthogonal test
In this test, L9 (3 3 ) orthogonal experiment form, orthogonal experimental factors and levels were carried out.The grouping situations were shown in Table 1.

Protein determination
The total protein of the sludge was determined by in four steps: digestion, distillation, absorption and titration.By adding concentrated sulfuric acid and catalyst to the sludge, fully shaking and heating digestion and decomposition, the organic nitrogen in the sludge is converted into ammonium sulfate, alkalized and distilled to free ammonia, then absorbed with boric acid, and then titrated with hydrochloric acid standard titration solution, the consumption of hydrochloric acid is recorded, and the protein content is calculated.The tic proteins in the supernatant were determined using a modified BCA method.In 25 μL of the supernatant sample, 25 μL solution D, 37℃ for 30 min, then 1 mL working solution, 37 ℃ for 30min, finally left at room temperature for 10min, and the absorbance at 562nm was measured.The standard curve was drawn with the standard solution of bovine serum protein.Solsoluble protein units were mgCOD / L, and 1g soluble protein was converted to 1.5g COD / g protein.

Amino acid content determination
Amino acids are determined by automatic amino acid analyzer.The principle is to use cation exchange chromatography separation-ninhydrin column derivative method to analyze the type and content of amino acids.

Measurement of polypeptide content
The content of peptides in the supernatant was determined using the biuret reagent assay.The sample was first diluted to an appropriate concentration, after which 1 mL of supernatant was added to the tube, followed by 4 mL of double urea reagent.After sufficient shaking, the colorimetric measurement was performed at room temperature (20~25℃) for 30min and at 540nm.Tube without protein solution was used as blank control solution.The average value of the three groups was determined, with the protein content as the abscissa and the light absorption value as the ordinate.
The standard curve made is shown in Figure 2-5, with the standard equation of y=0.0549x-0.0032,R 2 =0.9999.

Measurement of genetic stability of the mutant strains
The mutant strains were inoculated by streaking on agar plate every other day for a total of 10 generations.Flask fermentation was carried out in each five generation, and the enzyme activity of protease was measured to check its stability.

Residual sludge by hydrolysis of the mutant strain was compared with the original strain
Hydrolysis was performed under this reaction conditions T=60℃, the solid content of the sludge was 4%, T=8h, the inoculum of the hyperthermophilic crude enzyme was 20%,

Analytical methods
Protease activity was determined by Foline-Phenol method [9]with casein as standard.

Results and Discussion
During UV irradiation, the irradiation time was the unique variable factor, whereas the other parameters like radio frequency power and distance were fixed.For chemical mutagenesis, besides time, the affecting factors also include the concentration of mutagen and reaction temperature.Fig. 1(b) shows the relationship of DES treatment time and lethality.When the DES mutation time was 30 min, the lethality of the strain reached 75%.Therefore, the optimal mutation time for DES was determined to be 30 min.Fig. 1(c) shows the relationship of DES concentration and lethality.High lethal rate will lead to chromosomal abnormalities, which is not conducive to screening positive strains.At DES concentration greater than 2%, almost all strains were inactivated by the high concentration of DES.Thus 2% was the best mutagenic concentration for DES.It can be seen the relationship between DES mutagenesis temperature and lethality from Fig. 1(d).The results showed that 55-60 ℃ was the optimal temperature range for the growth of the strain.At time, the strain was sensitive to DES and the lethality increased rapidly.So the optimal mutation temperature was 60 ℃.

Orthogonal experiment results
Based on the single factor test, L9 (3 3 ) orthogonal test table was adopted.A represents temperature, B represents time, C represents concentration, and lethality was used as the evaluation index of the orthogonal test.The results of the orthogonal test are shown in Table 2.
According to the range R, the sequence of the effects of mutation temperature, time and DES concentration on the lethality is C > B > A. The results showed that DES concentration had the greatest effect on lethality, while the factor that had the least effect on lethality was the mutagenic temperature.The optimal combination of DES mutagenic conditions was A1B1C1, i.e., a mutagenic temperature of 55 ℃, a mutagenic time of 25 min, and a DES concentration of 1.5%.Under the optimized compound mutation condition, three parallel validation tests were carried out, and the lethality reached 78.8 ± 1.5%, which was consistent with the results of orthogonal analysis.

Strain screening
After mutating the original strains according to the optimized compound mutant conditions, a total of 139 mutant strains were selected.The size of the hydrolysis circle on the skim milk solid medium was then observed and compared with the original strain.Compared with the original strain, 21 mutant strains with larger hydrolysis circles were selected for shake screening.Twenty-one mutant strains were grown in liquid mediumin shake flasks at 60℃ and 180 for 24 hours at rmp / min, and the supernatant was centrifuged to measure protease activity.Figure 2 shows that the 19 strains with protease activity were higher than the original strain, and the protease activity of KT 1, KT 16 and KT 20 was 45.8%, 45.1% and 38.3% higher than the original strains, respectively.

Fig. 2 Degradation rates of mutagenic strains and the original strains
To verify that compound mutagenesis is better than single mutagenesis, [11] mutagenized B. subtilis by UV (4 W, 254 nm) irradiation to improve protease activity.Univariate experiments showed that the optimal lethality was achieved with the irradiation time of 4min.The results showed that UV-1-11 increased its protease activity by 35.5%, at a lesser rate compared to the mutant strain obtained by compound mutagenesis in this study.This may be due to the poor efficacy of the single mutation approach.After a certain strain is subjected to the same mutation method for a long time, its growth cycle will be prolonged and its metabolism will slow down, which is not conducive to industrial production.Compound mutations can partly reduce the blindness of single mutation methods and improve the positive mutation rate.Later, used a combined mutagenesis of UV and He-Ne laser (10mW,632.8nm).The protease activity of the mutant strain LA-UV-1-11 was increased by 41.39%, which was similar to the experimental results.Therefore, compound mutations work better than single mutations and are an effective way to improve bacterial strains.

Genetic stability of mutant strains
KT1, KT16, and KT20 screened after compound mutation were inoculated into agar medium for subculture for passaging culture for 10 consecutive generations.Then the protease activity of the supernatant was determined as shown in Fig.It can be seen from Fig. 3 that the protease activity of mutant KT16 could still reach 416.8 U• mL -1 after 10 consecutive generations.Compared with the first generation, the protease activity slightly increased by 3.3 U• mL -1 , with small fluctuations.However, the enzyme activity of mutant KT1 and KT20 decreased after 10 passages.In addition, the growth rate of these two strains gradually slowed down during the cultivation process.The results indicated that the genetic characteristics of KT16 were more stable and more suitable for the next step of the experiment.

Changes in protein, amino acids and polypeptide content in the remaining sludge
Proteins, peptides and amino acids are the main components of extracellular organic matter, and their concentration and distribution reflect the hydrolysis and solubilization of extracellular organic material by Figure 4 shows the changes in extracellular protein, peptides and amino acid concentrations during hydrolysis.With the increase of time, the sludge reaction system with DC8 and KT 16, which significantly promoted the dissolution of organic matter.In the residual sludge reaction system supplemented with DC8, the amounts of protein, peptides, 5,533.6 and 241.3mg/L, respectively, increased after 8 hours of hydrolysis, and all increased gradually over time.For the residual sludge treated with KT 16, the three indexes increased by 1933.8,521.3 and 510 mg/L, respectively, while the content of protein and peptides decreased within 6 -8 hours.This may be due to the increased protease activity and accelerated reaction of thermophilic bacteria after mutation, resulting in a shorter reaction process that predecomposes the protein into polypeptides and further decomposes the peptides into amino acids.Protein concentrations were determined by the Lowry method based on reactions with peptide bonds.However, proteases produced by thermophilic bacteria can disrupt the peptide bonds and reduce their concentration.In other words, if the increase in peptide bonds is less than the protease disruption, the measured protein concentration will decrease.Furthermore, the protease activity in the excess sludge becomes increasingly low during hydrolysis.

Conclusion
The mutant strain KT16, screened by UV-DES compound mutagenesis, showed a 46.7% increase in protease activity over the original strain DC8.The mutagenesis improved the ability of S-TE, possibly because different SNP/InDel mutations altered the sequence of amino acids, which affected the synthesis and expression of relevant proteins.The content of protein, peptides, amino acids, and SCOD in ES hydrolyzed with DC8 and KT16 increased compared to the original sludge, but the hydrolysis effect of KT16 was better than that of DC8.Mutagenesis promotes efficient hydrolysis of ES, which is an important way to realize the resourceful utilization of sludge.

Fig. 1
Fig. 1 Lethal rate diagram of strains under different conditions of UV and DES.

Table 1
Factors and levels of bacteria orthogonal test

Table 2
The orthogonal results of DES mutation conditions.