2.3.1 Plate screening

Colony suspensions irradiated to different doses were diluted to 10⁻⁵ gradient. Then, 0.1 mL diluted colony suspensions was daubed on the carboxymethyl cellulose (CMC) agar plate (0.2% NaNO₃, 0.1% K₂HPO₄, 0.05% MgSO₄, 0.05% KCl, 0.2% CMC sodium salt, 0.02% peptone, and 1.7% agar), and was stained by Gram’s iodine after a 3-d incubation at 30°C^[16].

After irradiation, the survival and mutation rates were calculated using ^[8] Survival rate = (T/U) × 100%, where T and U are number of colonies after and before heavy-ion beam irradiation.

Positive mutation rate = (M1/T) × 100%, where M1 is the total number of colonies of the positive mutant strain. A transparent circle-to-colony (HC) ratio greater than 1.30 on CMC agar screening plates indicated a positive mutation.

Negative mutation rate = (M2/T) × 100%, where M2 is the total colony forming units of the mutant strain. An HC value less than 1.10 on CMC-Na Agar screening plates indicates a negative mutation.

2.3.2 MTP fermentation screening

The strains with high HC value were selected for MTP fermentation and shaking flask fermentation^[17]. A highly efficient method was established for a preliminary screening for hyper-cellulase-producing mutants. The screening medium was inoculated with the mutated spore suspensions and cultured in MTP at 30°C and 200 r/min. The standard filter paper assay is not suitable for high-throughput determination of enzyme activity. By referring to the literatures, we developed a microplate-based method for assaying large sample volumes to screen the mutants. The reaction volume was reduced by 25 times from the 0.5 mL used in the International Union of Pure and Applied Chemistry (IUPAC) method, and the absorbance was recorded with a 96 microplate reader using a test wavelength of 520 nm^[18].

2.3.3 Enzyme production by shaking flask fermentation

A.niger H11 strains were pre-cultured for 12 h until adequate biomass was obtained. This biomass was used for subsequent enzyme production. The previous step was conducted in 250-mL Erlenmeyer flasks with 50 mL medium. The composition of the pre-culture medium (per flask) was 2 mL/L Tween-80, 7.5 g/L CMC-Na, 1.4 g/L (NH₄)₂SO₄, 2.0 g/L KH₂PO₄, 0.3 g/L CaCl₂, 0.0016 g/L MgSO₄·H₂O, 0.005 g/L FeSO₄·7H₂O, 0.0016 g/L MnSO₄·H₂O, 0.0014 g/L ZnSO₄·7H₂O, 0.002 g/L CoCl₂; the medium was autoclaved at 121°C for 30 min and then aseptically inoculated with A. niger H11 maintained on substrates such as PDA. After inoculation, the pre-culture was incubated in a shaker at 30°C and 200 r/min for 12 h. Pre-cultured A. niger H11 mycelium (2.5 mL) was inoculated aseptically into a flask, which was then incubated in a shaker (200 r/min) at 30°C. Enzyme production was performed in 250-mL Erlenmeyer flasks with 50 mL of medium which was the same to the pre-culture medium. The prepared medium was autoclaved at 121°C for 30 min prior to use.

2.3.4 Cellulase activity assays

Cellulase activities were determined as previously reported with minor modification ^[19,20]. FPA was determined using the dinitrosalicylic acid (DNS) method described by Ghose^[21] by reducing 25 times of the reaction volume. A 3 mm × 4 mm filter paper disc diluted in 40 μL 0.05 M citric buffer (pH 4.8) was digested by 20 μL of culture supernatant. The samples were incubated at 50°C for 60 min. The enzymatic reaction was terminated by adding 140 μL of DNS, followed by incubation at 95°C for 5 min. The samples were cooled in an ice bath. And then 40 μL of reaction fluid was transferred to 160 μL distilled water in a 96 microwell plate, and the absorbance was measured at 520 nm. One unit of FPA is defined as the amount of enzyme that produces 1 μg of glucose in 1 min. BGL activity was determined using the method described by Wu^[22] by reducing 25 times of the reaction volume. The reaction mixture containing 30 μL of D-salicin solution and 30 μL of enzyme solution was incubated at 50°C for 30 min. The reaction was terminated by adding 140 μL of DNS, followed by incubating at 95°C for 5 min. The samples were cooled in an ice bath. Then 40 μL of reaction fluid was transferred to 160 μL distilled water in a 96 microwell plate, and the absorbance was measured at 520 nm. One unit of BGL activity is defined as the amount of enzyme that produces 1 μg of glucose in 1 min.

3.2.1 The micro-cultivation technique

The micro-cultivation technique is based on MTP. The relationship between the FPA of cellulase produced by MTP fermentation and shaking flask fermentation for A.niger H11 is shown in Fig. 3(a), which proves that the MTP fermentation can replace the shaking flask fermentation for large scale strain selection. The optimization of key factors of the MTP fermentation, i.e. the loading volume and the fermentation time, are shown in Fig.3(b). The FPA of cellulase is the highest at loading volume of 1.5 mL and the fermentation time of 60 h.

Fig. 3Full-screen View

The establishment of micro-cultivation technique. (a)The correlation determination of FPA between MTP fermentation and shaking flask fermentation; (b) The influence of loading volume and fermentation time on FPA.

3.2.2 The high-throughput assay technique

The high-throughput assay technique is based on the microplate reader. The relative standard deviation (RSD) examined for the feasibility study of detecting the reducing sugar content using 96-well MTP is 4.19%. An RSD of < 5% demonstrates that the 96-well MTP can be used for detecting the reducing sugar content, and further calculating FPA of cellulase (Table.2). The correlation determination of reducing sugar content between microplate reader and ultraviolet spectrophotometer (Fig.4a) proves that the microplate reader can replace the ultraviolet spectrophotometer for detecting the reducing sugar content. The detection results under different wavelengths (Fig.4b) and additive amounts of DNS (Fig.4c) show that, judged from the correlation coefficients, the detective wavelength of 520 nm and DNS additive amount of 140 μL are the best for detecting the reducing sugar content. Fig. 5 shows the glucose standard curve plotted under the two optimum conditions, with a liner equation of y=3.6307x−0.5544 and the correlation coefficient of R²=0.9983.

Fig.4Full-screen View

The establishment of high-throughput assay technique. The correlation determination of reducing sugar content between microplate reader and ultraviolet spectrophotometer (a), and the optimization of detection wavelength (b) and DNS additive amount (c), using 96-well MTP.

Fig. 5Full-screen View

The glucose standard curve

The two techniques supplemented each other to form a complete screening system to lay a foundation for large scale non-rational strain selection.

3.2.3 Screening results after the progressive irradiation

The mutants screened out from the plate were filtered by the MTP fermentation. The primary screening results after MTP fermentation are given in Table 3. Seventeen mutants showed marked cellulase over-production records after the MTP fermentation. An increase in the irradiation dose led to increased number of positive mutants.

3.2.4 Screening results after the second screening by shaking flask fermentation

The seventeen mutants were re-screened by shaking flask fermentation. The A.niger H11201 showed higher FPA and BGL activities than the initial strain A.niger H11 after the second screening with shaking flask fermentation.

As shown in Fig.6(a), after 96-h fermentation, the FPA of A. niger H11 and H11201 are 273.88 ± 31.40 and 379.99±37.54 U/mL, respectively; while their BGL activity are 821.19±49.60 and 1340.42±122.71 U/mL, respectively. The FPA and BGL activity of A.niger H11201 are 38.74% and 63.23% higher than those of A.niger H11, respectively. Fig.6(b) shows the cellulase production of A.niger H11201 at different hours of fermentation. Both the FPA and the BGL activity reach the maximum after being fermented for 96 h. The mycelium of A.niger H11201 accumulated at the early stage of growth, and it is good for cellulase production after growing 96 h. The fermentation conditions might be changed along with the extension of fermentation time, as a result, the FPA and BGL activity reduced at 120 h. The A.niger H11201 was passaged to nine generations, and the FPA detected in every generation did not differ significantly (Fig.6c). Therefore, the A.niger H11201 is genetically stable.

Fig. 6Full-screen View

The screening results by shaking flask fermentation. (a) The comparison of cellulase production between A.niger H11 and A.niger H11201; (b)Time courses of cellulase production of A.niger H11201; (c) The genetic stability analysis of A.niger H11201.