Introduction

Algin is a class of water-soluble acidic polysaccharide in the cell walls of Sargassum, Kelp, and Fucus vesiculosus1. Its key constituents include polyglucuronic acid and polymannuronic acid. Due to its gel properties, algin is widely used as a thickener, emulsifier, and stabilizer in textile, food, pharmaceutical, and biological industries2. As a natural insoluble complex polysaccharide, algin has a high viscosity, high molecular weight, high degree of polymerization, and dietary fiber properties1,2,3, which hinder its absorption and utilization by organisms, thereby limiting its potential as a physiologically active ingredient. To address this limitation, algin is often subjected to degradation or derivatization methods4,5. Algin oligosaccharides, resulting from such processes, exhibit diverse biological functions, including anticoagulant6, antioxidant7,8,9, antitumor10,11, free radical scavenging, and bifidobacterial growth effects12. In comparison to algin, algin oligosaccharides offer broader application prospects and greater development value.

Presently, the preparation of algin oligosaccharides involves chemical, physical, and biodegradation methods. Biodegradation, encompassing alginate-degrading bacteria and alginate lyases, emerges as a promising venue. Alginate-degrading bacteria, typically sourced from seawater, sediments, seaweeds, marine plants, and mollusks 13,14, exhibit alginate-cleaving enzyme activity. Alginate-degrading bacteria grown on plates with sodium alginate as the only carbon source can show white circled hydrolysis circles or white annular hydrolysis circles after being infiltrated with calcium chloride for some time. Therefore, calcium chloride infiltration can be used to screen alginate-degrading bacteria for the initial screening of plates, such as the screening isolation of Formosa agariphila KMM 3901 T 15. When alginate lyase degrades algin, it produces reducing sugar. The DNS reagent can react with the reducing sugar, resulting in a reaction product that exhibits maximum absorption at 540 nm. Consequently, the amount of reducing sugar generated can be quantified using the DNS method to determine the activity of alginate lyase. Since the alginate-degrading strain always secreted the alginate lyase, the DNS method can be used to accurately determine the enzyme activity of the degrading strains16. Zhu et al.17 isolated the alginate-degrading bacterium, Flammeovirga sp. NJ-04, which showed an enzyme activity of 3343.7 U/mg. While numerous alginate-degrading bacteria have been isolated, their efficiency varies, and many fall short for industrial use. Mutagenesis and targeted cultivation of existing alginate-degrading bacteria using physical, chemical, and biological means can significantly improve the degradation efficiency of the strains. Li S et al.18 obtained two mutants P1-37 and P2-81 by PCR targeted mutagenesis, whose alginate-degrading activities were increased by 162% and 241%, respectively, compared to those of the original strains. Here we used laboratory-derived alginate-degrading strain Cobetia sp. cqz5-12 as the starting strain and conducted mutagenesis using both UV and alkylating agents. Ultraviolet (UV) mutagenesis could cause DNA molecules to form pyrimidine dimers, which hindered the normal pairing between bases, leading to microbial mutations19,20. Chemical mutagens tend to cause single base-pair (bp) changes, or single-nucleotide polymorphisms (SNPs) as they are more commonly referred to, rather than deletions and translocations21. Coberia sp. cqz5-12 is the wild strain we used to obtain the mutant strain in this paper. Cobetia sp. cqz5-12, which can degrade degrade alginate and was isolated from rotten Sargassum fusiforme and then preserved at -80℃ in our lab in the former experiment16. However, the ability of alginate degradation decreased in this wild strain according to improper storage condition. In order to recover and improve its alginate degradation capacity, we chose UV and NTG mutagenesis to mutate this wild strain, and then a mutant strain Coberia sp. cqz5-12-M1 was been selected and a preliminary analysis was conducted to determine the alginate degradation efficiency of this mutant strains. This study contributes both theoretically and materially to the advancement of the industrial production of alginate oligosaccharides.

Results

Selective breeding of the mutant strains

All strains obtained from the first round of UV mutagenesis alone were initially screened using the CaCl2 infiltration method and rescreened using the DNS method. Three mutant strains with improved degradation capacity were thus obtained (Table 1). Among these, strains Cobetia sp. cqz5-12-ZW-3.5-I-19 and Cobetia sp. cqz5-12-ZW-3.5-Q-22 both received UV radiation for 3.5 min and had the highest degradation capacity, with a D/d values of 11.00 and 10.00, respectively. Furthermore, the crude enzyme activities of their fermentation supernatants were 39.40 ± 0.03 U/mL and 40.06 ± 0.04 U/mL, respectively. However, compared with the original strains, the crude enzyme activity of the fermentation supernatants of the mutant strains Cobetia sp. cqz5-12-ZW-3.5-I-19 and Cobetia sp. cqz5-12-ZW-3.5-Q-22 were only increased by 0.79% and 2.48%, respectively.

Table 1 Results of UV mutagenesis.
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The two strains obtained from the first round of UV mutagenesis, Cobetia sp. cqz5-12-ZW-3.5-I-19 and Cobetia sp. cqz5-12-ZW-3.5-Q-22, were subjected to further NTG mutagenesis to obtain mutant strains with significantly increased degradation capacity. Two mutant strains with significantly improved degradation capacity were obtained (Table 2). The NTG mutagenesis concentration used to create both the Cobetia sp.cqz5-12-ZWH-3.5-I-0.001–1 and Cobetia sp.cqz5-12-ZWH-3.5-I-0.001–5 mutant strains was 0.001 g/L. The crude enzyme activities of their fermentation supernatants were 53.98 ± 0.69 U/mL and 50.40 ± 0.55 U/mL, which were 38.09% and 28.93% higher, respectively, compared to those of the original strains. Furthermore, their D/d values were 12.00 and 11.00, respectively. After pooling the results of initial screening and rescreening, Cobetia sp. cqz5-12-ZWH-3.5-I-0.001–1 was finally selected as the target mutant strain and renamed as Cobetia sp. cqz5-12-M1.

Table 2 Results of NTG mutagenesis.
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Growth characteristics of the mutant strain Cobetia sp. cqz5-12-M1

The growth characteristics of the mutant strains were investigated using a univariate experiment (Fig. 1). The carbon sources, nitrogen sources, NaCl concentrations, temperatures, and initial pH levels were selected according to some literatures22,23. As shown in Fig. 1a, the mutant strain Cobetia sp. cqz5-12-M1 grew poorly on media having maltose, sucrose, starch, or lactose as the sole carbon source (OD600 at 27 h was only about 0.5), but grew profusely on media having yeast powder, fructose, glucose, or sodium alginate as the sole carbon source (OD600 value at 27 h was 1.2–1.6), achieving optimal growth in the presence of sodium alginate or glucose (OD600 > 1.5 at 27 h). Since the mutant strain most efficiently degraded alginate in the presence of sodium alginate, sodium alginate was finally selected as the optimal culture carbon source for this strain. As shown in Fig. 1b, the mutant strain Cobetia sp. cqz5-12-M1 exhibited lower growth rates when cultured on medium with aluminum nitrate as the sole nitrogen source, reaching OD600 values of approximately 0.2 between 12 h-36 h. In contrast, it exhibited higher growth rates when cultured on medium with yeast powder, tryptone, yeast powder + tryptone, ammonium chloride, ammonium nitrate or ammonium sulfate as the sole nitrogen source (OD600 value > 1.6 at 27 h), and the optimal growth level was exhibited in the presence of yeast powder (OD600 value up to 2.0 at 27 h). As shown in Fig. 1c, while the mutant strain Cobetia sp. cqz5-12-M1 was unable to grow normally on an NaCl-free medium, its growth rate increased with increasing concentrations of NaCl up to 30 g/L and was optimum when cultured on a medium containing 30 g/L NaCl (OD600 value of approximately 2.0 at 24 h). As shown in Fig. 1d, the mutant strain Cobetia sp.cqz5-12-M1 could grow normally between 28–40 °C. The fastest growth rate was achieved at an incubation temperature of 34 °C, with an OD600 value of 2.0 at 18 h. As shown in Fig. 1e, the mutant Cobetia sp. cqz5-12-M1 was able to grow in the pH range of 5.5–9.0, with no significant differences in growth rates across different pH values.

Fig. 1
figure 1

Growth condition optimization for the mutant strain Cobetia sp. cqz5-12-M1. A univariate experiment was executed to study and optimize the growth conditions of the mutant strain Cobetia sp. cqz5-12-M1 and a subsequent univariate experiment was conducted using the results of the former univariate experiment. Samples were taken every 3 h during incubation to determine OD600 for growth curve plotting. Three replicates were performed for each treatment, and the final data obtained were expressed as mean ± standard deviation (SD), using the medium of the uninoculated mutant strain Cobetia sp. cqz5-12-M1 as a blank control. (a) Carbon source optimization; (b) nitrogen source optimization; (c) NaCl concentration optimization; (d) temperature optimization; (e) pH optimization.

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In summary, the optimal growth of the mutant strain Cobetia sp. cqz5-12-M1 was achieved in a medium containing 5 g/L sodium alginate, 5 g/L yeast powder, 30 g/L NaCl, 2 g/L K2HPO4, 2 g/L KH2PO4, 1 g/L MgSO4•7H2O, and 0.01 g/L FeSO4•7H2O (pH 6.5, incubation temperature 34 °C). At this point, the strain quickly entered the exponential phase. At 18 h, the OD600 value of the strain could reach 2.0. Thereafter, the strain entered the stabilization phase with a slow increase in OD600 value. At 36 h, the OD600 value of the strain could reach 2.3.

Degradation characteristics of the mutant strain Cobetia sp. cqz5-12-M1

The degradation properties of the mutant strains were investigated via univariate experiments to optimize fermentation conditions (Fig. 2). As shown in Fig. 2a, the mutant strain Cobetia sp. cqz5-12-M1 exhibited varying alginate lyase activities across different fermentation times. The total enzyme activity depicted an overall trend of increasing-stabilizing-decreasing trend with increasing fermentation time. Over the initial 66 h, the mutant strain exhibited a steady escalation in total enzyme activity, peaking at 202 ± 1.98 U/mL. Subsequently, between 66–138 h, the total enzyme activity stabilized, hovering approximately 188–202 U/mL. Beyond 138 h, the total enzyme activity gradually declined, correlating with extended fermentation time. Based on the cost of time, enzyme activity of fermentation supernatant, enzyme activity of bacterial precipitation, and the change of total enzyme activity, 72 h was finally selected as the optimal fermentation time for the mutant strain. The absence of NaCl hindered the mutant strain Cobetia sp.cqz5-12-M1 from growing normally (Fig. 1c) but did not diminish the alginate cleavage enzyme activity (Fig. 2b). Instead, the total enzyme activity exhibited a pattern of initial increase followed by a decrease with rising NaCl concentration. Notably, the mutant strain showcased its highest total enzyme activity (141.43 ± 2.78 U/mL) at a NaCl concentration of 30 g/L. The choice of nitrogen source affected the total enzyme activity of the mutant strain (Fig. 2c). Among the six nitrogen sources tested, the total enzyme activity produced by the mutant strains in fermentation culture with organic nitrogen sources (yeast powder, tryptone, and lambda powder) was (slightly) higher than that of the mutant strains in fermentation culture with inorganic nitrogen sources (urea, ammonium sulfate, and ammonium nitrate). In particular, the highest total enzyme activity (166.06 ± 3.89 U/mL) was produced when yeast powder was the sole nitrogen source (Fig. 2c). The type of carbon source also affected the total enzyme activity of the mutant strain (Fig. 2d). The mutant strain Cobetia sp. cqz5-12-M1 had the highest total enzyme activity (163.21 ± 3.53 U/mL) when fermented and cultured with sodium alginate as the sole carbon source. However, no significant difference in the total enzyme activity of the mutant strains was observed when fermentation cultures were incubated with glucose, sucrose, dextran, or sodium carboxymethylcellulose as the sole carbon source (143.59–145.64 U/mL). Notably, fermentation with starch or fructose resulted in the lowest enzyme activities (around 117.61–123.77 U/mL) (Fig. 2d). At different fermentation temperatures, the total enzyme activities of the mutant strains showed a tendency to increase and then decrease with fermentation temperature, but the overall differences were not significant (Fig. 2e). The total enzyme activities recorded across temperatures from 25 to 40 °C exceeded 135 U/mL, with the highest activity at 162.67 ± 2.22 U/mL observed at a fermentation temperature of 31 °C. Moreover, the initial pH of the medium influenced not only the growth but also the total enzyme activity of the mutant strains (Fig. 2f). Elevating the initial pH corresponded with an increase in the mutant strain’s total enzyme activity. However, when the initial pH exceeded 6.5, the total enzyme activity declined with increasing pH. The mutant strain achieved its maximum total enzyme activity (159.29 ± 3.16 U/mL) at an initial medium pH of 6.5. Based on the above results, a response surface methodology (RSM) was employed to further investigate the optimal conditions for enzyme production. Through RSM, it was determined that the highest total enzyme activity reached 168.53 ± 1.24 U/mL when the mutant strain was incubated in a culture medium containing 5 g/L sodium alginate, 5 g/L yeast powder, and supplemented with 30 g/L NaCl at a temperature of 31℃.

Fig. 2
figure 2

Fermentation condition optimization for the mutant strain Cobetia sp. cqz5-12-M1. A univariate experiment was executed to study the degradation characteristics of the mutant strain Cobetia sp. cqz5-12-M1 and optimize its fermentation conditions. A subsequent univariate experiment was conducted using the results of the former univariate experiment. At the end of the fermentation period, the samples were centrifuged at 13,680 × g for 5 min. Fermentation supernatant and bacterial precipitate were collected separately. Subsequently, fermentation supernatant and bacterial precipitate enzyme activities were determined using the DNS method. Finally, the total enzyme activity was calculated. Three replicates were performed for each treatment, and the final data obtained were expressed as mean ± standard deviation (SD). (a) Fermentation time optimization; (b) NaCl concentration optimization; (c) Nitrogen source optimization; (d) carbon source optimization; (e) temperature optimization; (f) pH optimization.

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In conclusion, under optimized conditions (pH 6.5, fermentation temperature of 31 ℃), the mutant strain Cobetia sp. cqz5-12-M1 exhibited its highest degradation capacity with a total enzyme activity of 168.53 ± 1.24 U/mL after a fermentation time of 72 h. The optimal medium composition consisted of sodium alginate (5 g/L), yeast powder (5 g/L), NaCl (30 g/L), K2HPO4 (2 g/L), KH2PO4 (2 g/L), MgSO4•7H2O (1 g/L) and FeSO4•7H2O (0.01 g/L). Among these, the fermentation supernatant contributed to an enzyme activity of 120.98 ± 1.40 U/mL while the bacterial precipitate contributed to an enzyme activity of 47.55 ± 0.22 U/mL. After optimization, the crude enzyme activity in the fermentation supernatant increased by approximately 1.24-fold compared to pre-optimization levels, reaching a value of 53.98 ± 0.69 U/mL.

Effect of chemical reagents on the strain growth

The change of the chemical environment will affect the growth of the strain, and also affect the specific degradation ability of the strain to a certain substrate. In order to more clearly understand the mutant's tolerance to different environmental factors and its alginate degradation ability under different environments, filtering paper method (or Oxford cup method) and CaCl2 method were used to study the growth of the strain under different environments (Fig. 3) and alginate degradation (Table 3), respectively. The results can provide reference for the subsequent practical application of the mutant.

Fig. 3
figure 3

Effect of disinfectants and antibiotics on strain growth. The effect of disinfectants and antibiotics on the growth of the strains was determined by using the Oxford cup method and filtering paper method. Disinfectants included 75% ethanol, 3% hydrogen peroxide, iodophor, 5% carbolic acid, and 2% phenol. Antibiotics included polymyxin, tetracycline, kanamycin, nystatin, oxytetracycline, amoxicillin, cephalosporin, chloramphenicol, vancomycin, chlortetracycline, isoniazid, terbinafine, neomycin, griseofulvin, bacitracin, gatifloxacin, salinomycin, streptomycin, and ampicillin (all of the above antibiotics were at a concentration of 10 mg/mL). Sterile water was used as a control. Three replicates were performed for each treatment, and the final data obtained were expressed as mean ± standard deviation (SD). * denotes 0.01 < P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001, and **** denotes P < 0.0001. (a) Effect of disinfectants on bacterial growth as determined by the filtering paper method; (b) effect of antibiotics on strain growth as determined by the filtering paper method; (c) effect of disinfectants on strain growth as determined by the Oxford cup method; (d) effect of antibiotics on strain growth as determined by the Oxford cup method.

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Table 3 Effect of chemical reagents on the alginate degradation capacity of the strain.
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The effects of common disinfectants and antibiotics on the growth of the original and mutant strains were assessed using the filtering paper method and Oxford cup methods to evaluate the change of resistance of mutant to common disinfectants and antibiotics (Fig. 3). Various disinfectants—75% ethanol, 3% hydrogen peroxide, iodophor, 5% carbolic acid, and 2% phenol—were analyzed for their influence on strain growth through both methods (Fig. 3a and c). As presented in Fig. 3a and c, 3% hydrogen peroxide and 5% carbolic acid significantly inhibited the growth of the original strain Cobetia sp. cqz5-12. The diameters of inhibition zones induced by 3% hydrogen peroxide and 5% carbolic acid were 7.3 ± 1.2 mm and 1.3 ± 0.6 mm, respectively, in the filtering paper method, whereas these values were 12.9 ± 1.6 mm and 26.9 ± 2.1 mm in the Oxford cup method, respectively. In addition, 3% hydrogen peroxide, iodophor, and 5% carbolic acid significantly inhibited the growth of the mutant strain Cobetia sp. cqz5-12-M1. In the filtering paper method, the diameters of the inhibition zones induced by 3% hydrogen peroxide, iodophor, and 5% carbolic acid were 10 ± 1 mm, 8 ± 1 mm, and 1.7 ± 0.6 mm, respectively, whereas these values were 19.5 ± 2.1 mm, 6.2 ± 1 mm, and 34.9 ± 0.6 mm in the Oxford cup method, respectively.

The effects of 10 mg/mL antibiotics polymyxin, tetracycline, kanamycin, nystatin, oxytetracycline, amoxicillin, cephalosporin, chloramphenicol, vancomycin, aureomycin, isoniazid, terbinafine, neomycin, griseofulvin, bacitracin, gatifloxacin, salinomycin, streptomycin, and ampicillin on the growth of the strains were assessed using the filtering paper method and the Oxford cup method (Fig. 3b and d), and the founding was made as follows: 1) 10 mg/mL tetracycline, kanamycin, nystatin, vancomycin, aureomycin, isoniazid, chloramphenicol, terbinafine, neomycin, griseofulvin, bacitracin, salinomycin, and streptomycin had no inhibitory effect on the growth of the original and mutant strains for both methods. 2) 10 mg/mL cephalosporin and gatifloxacin significantly inhibited the growth of the original strain Cobetia sp. cqz5-12. In the filtering paper method, the original strain Cobetia sp. cqz5-12 produced inhibition zones with diameters of 4.7 ± 0.6 mm and 7 ± 1 mm when cultured in media containing cephalosporin or gatifloxacin, respectively, whereas these values were 5.2 ± 1 mm and 1.5 ± 0.6 mm, respectively, in the Oxford cup method. 3) 10 mg/mL gatifloxacin significantly inhibited the growth of the mutant strain Cobetia sp. cqz5-12-M1 growth. In the filtering paper method, the mutant strain Cobetia sp. cqz5-12-M1 produced an inhibition zone with a diameter of 8.3 ± 1.2 mm when cultured in a medium containing gatifloxacin, whereas this value was 17.9 ± 0.6 mm in the Oxford cup method.

Analysis of both methods revealed undoubted alterations in the mutant strain's tolerance to specific disinfectants and antibiotics. Notably, the mutant strain decreased the tolerance to iodophor while enhancing its resistance to cephalosporins, allowing poor growth in the presence of iodophor and normal growth in the presence of cephalosporins.

Effect of chemical reagents on the alginate degradation capacity of the strain

Based on the effect of chemical reagents on the two strains’ growth (Fig. 3), the disinfectant 75% ethanol as well as 10 mg/mL antibiotics tetracycline, neomycin, griseofulvin, isoniazid, terbinafine, ampicillin, streptomycin, kanamycin, vancomycin, nystatin, chlorotetracycline, salinomycin, amoxicillin, bacitracin, chloramphenicol, oxytetracycline, polymyxin B, and cephalosporin were selected as the test reagents to determine the capacity of the mutant to degrade the alginate in different chemical environments (Table 3). Preliminary analysis of the data in Table 3 showed that all the chemicals tested inhibited the degradation of alginate by the mutant strain Cobetia sp. cqz5-12-M1 and the original strain Cobetia sp. cqz5-12 very significantly.

According to the data in Table 3, the alginate degradation capacity of the mutant strain Cobetia sp. cqz5-12-M1 did not change significantly relative to the original strain Cobetia sp. cqz5-12 when the disinfectant 75% ethanol was present. Without the presence of 75% ethanol, the mutant strain degraded alginate about 1.14 times that of the original strain. However, when 75% ethanol was present, the degradation capacities of both the mutant and original strains were reduced to 25.2% and 24.2% of those in the absence of 75% ethanol, respectively. However, the alginate degradation capacity of the mutant strain relative to the original strain did not change significantly at this time and was approximately 1.19 times that of the original strain.

Table 3 provides a comprehensive insight into the alginate degradation ability of the mutant strain in the presence of certain antibiotics was indeed significantly different from that of the original strain. In the absence of antibiotics, the mutant strain Cobetia sp. cqz5-12-M1 exhibited approximately 1.14-fold enhancement in alginate degradation compared to the original strain Cobetia sp. cqz5-12 strain. However, the presence of antibiotics elicited various effects on the alginate degradation capacity of the mutant strain relative to the original strain. It either decreased (cases i, ii), increased (case iii), or did not change significantly (case iv). These cases are described below in detail.

i) Under the influence of neomycin, ampicillin, and chloramphenicol (10 mg/mL), both strains experienced significant inhibition. The alginate degradation capacity of the mutant strain dropped to 13%, 13.9%, and 13.9% of its capacity in the absence of antibiotics, whereas the original strain retained 18%, 17.3%, and 18.1% of its initial capacity, respectively. Moreover, the mutant strain's degradation capacity was reduced to approximately 0.90, 0.92, and 0.88 times that of the original strain.

ii) The alginate degradation capacity of both strains was highly significantly inhibited when 10 mg/mL isoniazid, streptomycin, vancomycin, amoxicillin, bacitracin, oxytetracycline, and cephalosporin were present. At this point, the alginate degradation capacity of the mutant strain was 15.3%, 18.3%, 12.3%, 13.7%, 12.7%, 14.9%, and 10.7% of its capacity in the absence of antibiotics, respectively. In addition, the alginate degradation capacity of the original strain was 18.3%, 20.8%, 14.7%, 15.2%, 14.9%, 16.8%, and 12.8%, respectively, of its capacity in the absence of antibiotic. Although the alginate degradation capacity of the mutant strain relative to that of the original strain also decreased at this time, the difference in alginate degradation capacity between the two strains was not significant. The alginate degradation capacity of the mutant strains was approximately 0.96, 1.01, 0.97, 1.03, 0.97, 1.01, and 0.96 times that of the original strain, respectively.

iii) The alginate degradation capacity of both the mutant strain, Cobetia sp. cqz5-12-M1, and the original strain, Cobetia sp. cqz5-12, was significantly inhibited in the presence of 10 mg/mL of tetracycline, terbinafine, nystatin, chlortetracycline, or salinomycin. At this point, the alginate degradation capacity of the mutant strains was 19.6%, 15.2%, 17.5%, 15.7%, and 13.1%, respectively, of its capacity in the absence of antibiotic presence. In addition, the alginate degradation capacity of the original strain was 15%, 13.9%, 14.4%, 13.8%, and 12.1% of its capacity in the absence of antibiotics, respectively. Although the alginate degradation capacity of both strains was highly significantly inhibited, the capacity of the mutant strain to degrade alginate increased to 1.49, 1.25, 1.39, 1.30, and 1.23 times that of the original strain, respectively.

iv) The alginate degradation capacity of both strains was highly significantly inhibited in the presence of 10 mg/mL kanamycin, griseofulvin and polymyxin B. The alginate degradation capacity of the mutant strains was 16.6%, 20.4%, and 12.8% of its capacity in the absence of antibiotics, respectively. The alginate degradation capacity of the original strain was 17.6%, 21.1%, and 13.5% of its capacity in the absence of antibiotics, respectively. Although the alginate degradation capacity of both strains was inhibited very significantly, the alginate degradation capacity of the mutant strain relative to that of the original strain did not change significantly at this time. At this time, the alginate degradation capacity was approximately 1.08, 1.11, and 1.08 times that of the original strain, respectively.

Discussion

Alginate-degrading bacteria offer promising potential for addressing the energy crisis caused by fossil fuel consumption by degrading brown algae to produce bioethanol24,25. In addition, these bacteria can be employed to process algal waste for algin oligosaccharide extraction26,27. However, inherent limitations in the strains, such as low vigor, hinder their practical application in bioethanol and algin oligosaccharide production. To surmount these limitations, a strategic approach combining UV and chemical NTG mutagenesis alongside univariate optimization was implemented. This aimed to improve the alginate degradation capabilities of the Cobetia sp. cqz5-12 strain, held in our laboratory, to chart a viable pathway for its industrial utilization. Mutagenesis breeding, a prevalent technique in contemporary laboratories, expedites the screening of favorable strains. For instance, Hu et al.28 employed a combination of ethyl methanesulfonate and UV mutagenesis to derive a mutant strain, Vibrio sp. 510–64, which exhibited alginatea 3.87-fold increase in alginate-degrading enzyme activity compared to the original strain. In contrast, our experiments with combined UV and NTG mutagenesis resulted in a mere 38.09% enhancement in enzyme activity for the mutant strain, showcasing limited efficacy in improving enzyme activity. Subsequent univariate optimization efforts yielded only a modest 1.93-fold increase in enzyme activity of the fermentation supernatant from the mutant strain in comparison to the original strain. Despite this improvement, the enzyme activity remained inadequate for industrial-scale application, necessitating further investigations to enhance its vigor.

The univariate optimization experiment and response surface experiment demonstrated a modest 1.24-fold enhancement in the enzyme activity of the fermentation supernatant from the mutant strain Cobetia sp. cqz5-12-M1 following optimization. This minimal enhancement in enzymatic activity may be attributed to insignificant disparities between the optimal and pre-optimization fermentation conditions: the components of the fermentation medium were consistent, and the concentration of each component was varied slightly pre- and post-optimization. Fermentation conditions had a considerable effect on strain enzymatic activity, as evidenced by many previous studies as well as this study. Therefore, we could completely change the carbon source and nitrogen source and other factors we used in this study to significantly increase the enzymatic activity.

Mutations can significantly impact a strain’s sensitivity to external factors. However, the strain’s sensitivity to the same reagents can exhibit divergent results depending on the assay method used. For instance, employing the filtering paper method, the original strain Cobetia sp. cqz5-12 displayed inhibition zone diameters of 3 ± 1 mm, 7.3 ± 1.2 mm, 2.3 ± 0.6 mm, 1.3 ± 0.6 mm, and 0 mm when exposed to 75% ethanol, 3% hydrogen peroxide, iodophor, 5% carbolic acid, and 2% phenol, respectively. In contrast, the Oxford cup method resulted in values of 0 mm, 12.9 ± 1.5 mm, 0 mm, 26.9 ± 2.1 mm, and 16.2 ± 2.6 mm for the same reagents. Comparably, the filtering paper method revealed that the mutant strain Cobetia sp. cqz5-12-M1 in the presence of 75% ethanol, 3% hydrogen peroxide, iodophor, 5% carbolic acid, and 2% phenol produced inhibition zone diameters of 0.7 ± 1.2 mm, 10 ± 1 mm, 8 ± 1 mm, 1.7 ± 0.6 mm, and 1.7 ± 1.5 mm, whereas these values were 0.5 ± 0.6 mm, 19.5 ± 2.1 mm, 6.2 ± 1 mm, 34.9 ± 0.6 mm, and 18.9 ± 3.2 mm for the Oxford cup method. In addition, the experimental results of the filtering paper method and the Oxford cup method showed that the sensitivity of the original strains to 75% ethanol, iodophor, 5% carbolic acid, and 2% phenol showed great differences depending on the assay method. The susceptibility of the mutant strain Cobetia sp. cqz5-12-M1 to 5% carbolic acid and 2% phenol also varied greatly depending on the assay type. The same phenomenon occurred for the effect of antibiotics on the growth of the strain. In the filtering paper method, 10 mg/mL ampicillin inhibited the growth of the original strain Cobetia sp. cqz5-12, producing a 2-mm diameter inhibition zone. However, ampicillin failed to inhibit the growth of the original strain in the Oxford cup method. In the filtering paper method, 10 mg/mL cephalosporin failed to inhibit the growth of the mutant strain Cobetia sp. cqz5-12-M1. However, in the Oxford cup method, cephalosporin inhibited the growth of the mutant strain, causing the mutant strain to produce an inhibition zone with a diameter of 11.9 ± 1.5 mm. In the filtering paper method, 10 mg/mL chloramphenicol, oxytetracycline, and polymyxin failed to inhibit the growth of the mutant strain. However, in the Oxford cup method, chloramphenicol, oxytetracycline, and polymyxin were able to inhibit the growth of the mutant strain, resulting in the production of inhibitory circles with diameters of 1.2 ± 1 mm, 3.9 ± 0.6 mm, and 2.9 ± 0.6 mm, respectively. Given the difference in sensitivity between the different methods, the results of the filtering paper method and the Oxford cup method were taken into account when evaluating the sensitivity of the strains to a specific reagent.

The mutant strain Cobetia sp. cqz5-12-M1 exhibited varying sensitivity to specific disinfectants and antibiotics, indicating that the mutation investigated in this study indeed impacts the growth and alginate degradation capacity of the strain. This variation may be attributed to the genetic, transcriptomic, or proteomic change, which should be confirmed through genomic, transcriptomic, and proteomic assays. Future investigations involving whole genome sequencing as well as transcriptomic and proteomic analyses are warranted to elucidate the precise mechanism underlying this variation. Overall, the findings of this study lay a theoretical foundation for subsequent in-depth studies of resistance and alginate degradation mechanisms.

Methods

Strain and media

The original strain, Cobetia sp. cqz5-12, was isolated and purified by our laboratory and is presently archived in the Chinese General Microbial Strain Conservation and Management Center under the strain conservation number CGMCC 1.1745916.

The mutant strain, Cobetia sp. cqz5-12-M1, was originated from Cobetia sp. cqz5-12 via UV mutagenesis and chemical mutagenesis in this study.

The initial growth medium comprised 5 g/L sodium alginate, 1 g/L MgSO4•7H2O, 5 g/L (NH4)2SO4, 30 g/L NaCl, 2 g/L K2HPO4, and 0.01 g/L FeSO4•7H2O16. Additionally, 1.5%-2% agar powder was added to the solid medium and sterilized at 115 ℃ for 30 min.

The seed liquid medium contained 6 g/L sodium alginate, 5 g/L tryptone, 2.5 g/L yeast powder, 25 g/L NaCl, 0.25 g/L K2HPO4, 0.5 g/L MgSO4•7H2O, and 0.05 g/L FeSO4•7H2O (pH: 7.5; sterilized at 115 ℃ for 30 min)16.

The fermentation liquid medium consisted of 5 g/L sodium alginate, 5 g/L tryptone, 25 g/L NaCl, 0.25 g/L K2HPO4, 0.5 g/L MgSO4•7H2O, and 0.05 g/L FeSO4•7H2O (pH: 7.5; sterilized at 115 ℃ for 30 min)16.

Sodium alginate and agar powder were procured from Sigma and OXOID, respectively. Other biochemical reagents and antibiotics were obtained from Sangong Bioengineering (Shanghai) Co., Ltd.

Selection of Cobetia sp. cqz5-12 mutant strains

The original strain Cobetia sp. cqz5-12 was activated and inoculated into the seed medium, and incubated at 30 ℃ at 150 r/min for 16–18 h to obtain the seed liquid. The seed liquid was centrifuged at 1520 × g to obtain the bacterial precipitate. Subsequently, the bacterial precipitate was transferred to the fermentation liquid medium and incubated at 30 ℃ at 150 r/min for 36 h to obtain the fermentation broth. Further, the fermentation broth was subjected to centrifugation (1520 × g) to obtain the bacterial precipitate. The bacterial precipitate was washed twice in 0.85% sterile saline. The pellet was then resuspended in 0.85% sterile saline to obtain a bacterial suspension of 107 CFU/mL, and 5 mL aliquots of the above suspension were placed in a φ90 mm sterile Petri dish and irradiated under a 20 W UV lamp for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 min. The UV-irradiated strains were subsequently subjected to gradient dilution. The appropriate gradient dilutions of 100 µL were applied to the initial medium plates, quickly wrapped in tinfoil, and placed in a constant temperature incubator at 30 ℃ for 24–48 h. After single colonies had appeared, the mutant strains with the most enhanced degradation capacity were selected via an initial screening using the CaCl2 infiltration method16 and followed by screening using the DNS method. The mutant strains were further subjected to mutagenesis using 1-methyl-3-nitro-1-nitrosoguanidine (NTG). Specifically, 10 mg of NTG was dissolved in 1 mL of acetone to obtain a 10 mg/mL nitrosoguanidine master mix. The master mix was added to 5 mL of the mutant strain suspension to give final nitrosoguanidine concentrations of 0.0001, 0.001, 0.01, and 0.1 g/L, respectively. The suspensions were then incubated at 30 ℃ for 60 min and centrifuged at 2380 × g for 5 min to collect the bacterial precipitates. The precipitates were washed, diluted to the appropriate gradient, spread on plates containing initial growth medium, and incubated at 30 ℃ for 24–48 h. After single colonies appeared, the mutant strains with the most enhanced degradation capacity were obtained by initial screening using the CaCl2 infiltration method followed by rescreening using the DNS method. The mutant strain thus obtained was named Cobetia sp. cqz5-12-M1 and stored at 4 ℃.

Optimization of growth and degradation conditions of the mutant strain Cobetia sp. cqz5-12-M1

Growth condition optimization of mutant strains

Several sequential univariate experiments were performed to optimize the growth conditions of the mutant strains. Cobetia sp. cqz5-12-M1 seed liquid obtained from culture was collected by centrifugation, washed, and inoculated into the initial growth liquid medium (50 mL of liquid medium in 150 mL shake flask) containing different carbon sources (or different nitrogen sources, NaCl concentrations, temperatures, and initial pH values) at 5% inoculum, and then cultured for 36 h at 34 °C at 150 r/min. Glucose, alginate, malt sugar, yeast powder, lactose, fructose, starch, and saccharose were used as carbon sources in the carbon source optimization experiment. Yeast powder, tryptone, yeast powder plus tryptone, ammonium sulfate, aluminum nitrate, ammonium nitrate, and ammonium chloride were used as nitrogen sources in the nitrogen source optimization experiment. The concentration of NaCl used in the NaCl concentration optimization experiment were 0, 10, 15, 20, 25, 30, 35, 40, 45, 50 g/L. 28, 30, 32, 34, 36, and 40 ℃ were selected as the incubation temperature in the temperature optimization experiments, and then 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 were chosen as the initial pH values. Samples were taken every 3 h during incubation to determine OD600 that used for the growth curve plotting. Each treatment was performed in three replicates, and the medium without the mutant strain Cobetia sp. cqz5-12-M1 was used as a blank control.

Degradation characterization and condition optimization of mutant strains

A series of univariate experiments and response surface experiment were used to assess the degradation characteristics of the mutant strain and optimize its fermentation conditions. The Cobetia sp. cqz5-12-M1 seed liquid obtained from the culture was collected by centrifugation, and the pellet was washed and inoculated into the fermentation liquid medium (50 mL of liquid medium in 150 mL shake flask) for different fermentation times, NaCl concentrations, nitrogen sources, carbon sources, temperatures, or initial pH values at 5% inoculum, and incubated at 34℃ at 150 r/min. In the fermentation time optimized experiment, samples were determined at a 12 h interval and the longest time was 156 h. The concentration of NaCl used in the NaCl concentration optimized experiment were 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 g/L. The nitrogen source used in the nitrogen source optimized experiment were yeast powder, tryptone,, lamb’s cabbage powder, carbamide, ammonium sulfate, and ammonium nitrate. Alginate, glucose, sucrose, starch, glucan, fructose, and sodium carboxymethylcellulose were chosen as carbon source in the carbon source optimized experiment. 25, 28, 31, 34, 37, and 40 ℃ were selected as the fermented temperature in the temperature optimized experiments, and then 5, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10 were chosen as the initial pH values in the pH optimized experiment. Upon completion of incubation, the samples were centrifuged at 13,680 × g for 5 min. Subsequently, fermentation supernatant and bacterial precipitate were collected. Enzyme activities of the fermentation supernatant and bacterial precipitate were determined using the DNS method16. Finally, the total enzyme activity was calculated. Each treatment was performed in triplicates. Based on the results of the aforementioned univariate experiment, a response surface experiment (RSM) was conducted. In this RSM, carbon source, nitrogen source, temperature, and NaCl concentration were chosen as the experimental factors, while the total enzyme activity was selected as the response surface value.

Effect of chemical reagents on strain growth

Effect of disinfectants and antibiotics on the growth of strains as determined by the filtering paper method

Initially, 100 µL of each of the original strain Cobetia sp. cqz5-12 and the mutant strain Cobetia sp. cqz5-12-M1 bacterial fresh cultures was spread on optimal growth medium plates. Each plate that had been coated with the bacterial solution was divided into equal portions with a marker pen, and the names of the chemical reagents were labeled within each equal portion. Chemical reagents included disinfectants and antibiotics. The disinfectants included 75% ethanol, 3% hydrogen peroxide, iodophor, 5% carbolic acid, and 2% phenol. The antibiotics included polymyxin, tetracycline, kanamycin, nystatin, oxytetracycline, amoxicillin, cephalosporins, chloramphenicol, vancomycin, aureomycin, isoniazid, terbinafine, neomycin, griseofulvin, bacitracin, gatifloxacin, salinomycin, streptomycin, and ampicillin (all of the above antibiotics were at a concentration of 10 mg/mL). Sterilized small round filtering papers were individually dipped into glass dishes containing various chemicals or sterile water (control) under sterile conditions. After infiltration, the filtering papers were removed with sterile tweezers. Excess chemical reagent liquid was drained from the inside of the glass dish to ensure that the filtering papers contained approximately the same amount of disinfectant liquid. Finally, the filter papers were attached to the corresponding areas of the plates coated with the bacterial solution and incubated at 34 ℃ for 24 h. All experiments were done in parallel triplicates.

Effect of disinfectants and antibiotics on strain growth as determined by the Oxford cup method

The method used for this procedure is identical to that described in the above-mentioned filtering paper method, with the only variation being the replacement of sterile small round filtering paper with a sterile Oxford cup. The sterile Oxford cup was placed in the center of the corresponding area of the petri dish, and 200 µL of various chemical reagents were pipetted into the Oxford cup in the corresponding area of the petri dish.

Effect of chemical reagents on the alginate degradation capacity of the strain

Freshly cultured single colonies of the original strain Cobetia sp. cqz5-12 and the mutant strain Cobetia sp. cqz5-12-M1 were transferred to plates containing optimal fermentation medium coated with 100 µL of the chemical reagents to be tested, and incubated at 31 ℃ for 48 h. At the end of incubation, 5% CaCl2 was slowly dripped into the area around each colony. After infiltration for 20–25 min, the diameter of the hyaline circle D and the diameter of the colony d were measured to calculate the D/d ratio of each single colony. The test chemicals included 75% ethanol, tetracycline, neomycin, griseofulvin, isoniazid, terbinafine, ampicillin, streptomycin, kanamycin, vancomycin, nystatin, chlorotetracycline, salinomycin, amoxicillin, bacitracin, chloramphenicol, oxytetracycline, polymyxin, and cephalosporins (all of the above antibiotics were at a concentration of 10 mg/mL). SPSS was used for significance analysis and single factor ANOVA test was used.

Determination of alginate lyase activity by DNS method

First, 1 mL of appropriately diluted protein solution mixed with 1 mL of 0.6% alginate substrate solution in a 5 mL centrifuge tube16. The mixture was water-bathed at 40 ℃ for 20 min. Subsequently, 1.5 mL of DNS reagent was added, followed by heating in a boiling water bath for 10 min and immediate cooling to room temperature16. Subsequently, the absorbance values at 540 nm (OD540) were determined with three replicates for each point. Enzyme activity unit was defined as the amount of enzyme required to catalyze the production of 1 µg of reductant per min of substrate under the experimental conditions. The enzyme activity (EU, U/mL) per mL of protein solution was calculated as follows:

$$ {\text{Enzyme}}\;{\text{activity}}\;{\text{E}} = {\text{A}} \times {\text{n}}/{\text{t}} $$

where A is the mass (in µg) corresponding to the OD540 value measured by the DNS method on the glucose standard curve, n is the dilution ratio of the enzyme solution, and t is the time of the enzyme reaction.