The majority of antibiotics now used in clinical practice are cephalosporins (Das et al. 2019). A. chrysogenum is the primary strain utilized in the industrial manufacture of cephalosporin C (CPC). The advantages of CPC include its strong antibacterial activity and wide antibacterial range. The yield and titer of cephalosporins obtained from A. chrysogenum, which is generated by Penicillium chrysogenum as well, are significantly lower than those of penicillin (Liu et al. 2022). The production and price of CPC are crucial for the cephalosporins-antibiotic sector because it is the main source of 7-ACA. The Ministry of Science and Technology of China has named CPC fermentation as one of the main scientific and technological achievements during the past 30 years due to the ongoing demand for strain enhancement for A. chrysogenum, a producer of CPC. Due to the drawbacks of conventional methods for A. chrysogenum strain improvement and the broad use of molecular biology, genetic engineering has emerged as an effective strategy for modifying the antibiotic-producing strain and developing high-yield mutant strains (Hu and Zhu 2016). The current study attempted to optimize culture medium components and environmental circumstances in order to increase CPC production by the examined isolate. Different L-methionine and soybean oil were evaluated for the main production culture. The parameters chosen for optimization via RSM were the degree of acidity (pH), the size of the bacterial inoculum, along with the incubation period. The software used for this step was Design Expert v. 7.0 as previously reported (Ibrahim et al. 2019; El-Housseiny et al. 2021). Following that, CPC production was increased through mutagenesis utilizing gamma (ɣ) radiation. The efficacy of CPC as an antibacterial agent was bioassessed against the microorganism Staphylococcus aureus; strain number ATCC 25923 at the end of each run.

Not only the composition of the culture medium but also the surrounding environment frequently impact the biosynthesis of certain active metabolites by microorganisms (El-Housseiny et al. 2021; Ibrahim et al. 2019). L-methionine and soybean oil are significant components in CPC production media, that act a big role in increasing CPC production. As it supplies sulphur for the synthesis of cephalosporin, L-methionine is a preferred medium ingredient for cephalosporin biosynthesis (Gohar et al. 2013). Besides, L-methionine is the major stimulant to the arthrospore formation that is correlated with CPC production, and is important in induction of four enzymes in CPC biosynthesis (Lotfy 2007; Martín and Demain 2002). Concerning soybean oil, it contains different amino acids from which the CPC biosynthesis process begins (Martin and Demain 1980). Soybean oil supplementation increases CPC production but has little effect on morphology, despite the fact that in batch or fed-batch cultures it occasionally seems like morphology and productivity are connected (Sándor et al. 2001). Consequently, for the main production medium, different L-methionine and soybean oil concentrations were tested as described previously in Additional file 1: Table S1. The optimum media was that which contained 3 g/L L-methionine and 4.7% v/v soybean oil.

The ambient fermentation conditions required to be optimized once the culture media for the manufacture of CPC were optimized. This was achieved using RSM, a helpful mathematical and statistical method for choosing experimental design, showing the relatedness of the components and the ideal integration of parameters, as well as for predicting responses (Ahsan et al. 2017). The Design Expert® v. 7.0 was used for experiment design, mathematical representation, statistical univariate analysis, production of response surfaces, contour, and diagnostic plots.

The 3D response surfaces and respective contour plots provide comprehensive information on the interrelationship between two of the variables, hence allow the prediction of the settings conducive to the highest production (Ghribi et al. 2012). With the help of these plots and a numerical optimization algorithm, the inoculum size of 1% v/v, pH of 4, and incubation period of 4 days were discovered to be the ideal conditions for maximum CPC production. When CPC was produced using the proposed levels, the inhibition zone measured 24.667 mm, which was extremely near to the value estimated by the model (25.6617 mm), demonstrating the validity of the model. This value was calculated using a standard curve using standard cephradine, which has an antibacterial activity of 39.89 µg/mL.

The results were methodically examined by ANOVA, and it proved the adequacy of the model. It also evinced the significant influence the factors possess when producing CPC (Nisha et al. 2023). The P value decides whether the results are significant or not (Lang et al. 1998). The Fisher's F-test demonstrates this with a very low probability value [(P value < 0.000)] (Inamdar et al. 2013). The F value was 48.32, revealing the model’s significance. This F value had 0.01% probability of occurring due to chance. The multivariate models turned out to be significant thus can be used to precisely represent CPC production, according to the ANOVA results for the models. Furthermore, adjusted coefficient of determination (Adj R2) results demonstrated a high level of agreement with Pred R2. The difference should be less than 0.2 (Nisha et al. 2023). In our model, R2 value of 94.89% (0.9489) showed that the model was highly significant. The CV represents the precision of the treatment comparison. As the CV value increases, the experiment's reliability falls (Ghribi et al. 2012). The low value of the study revealed the great accuracy of the experimental results. Adequate (Adeq.) precision is evaluates the signal-to-noise ratio. An adequate precision ratio of 21.475 was obtained in this model, indicating an appropriate signal. This model is useful for navigating the design space.

The effects of each variable on their own and in combination have an impact on the production of the antibiotic. Tools that make it easier to fully understand the design space influenced by these factors include 3D surface plots and 2D contour plots. As a result, graphical summaries for case statistics were made, to validate the derived models. The Box-Cox plot not only guides the decision regarding which power law transformation to use, but it also suggests whether or not the response should be measured using a different scale. Because the current lambda was within the 95% confidence interval, no transformation was recommended. The Predicted versus Actual plot is applied to compare experimental response values with model predictions to evaluate the results quantitatively. The Residuals versus Run plot displays the residuals in relation to the experimental run order (El-Housseiny et al. 2021). According to the findings of this investigation, these produced graphs demonstrate the models' validity.

According to the ANOVA results, pH and incubation length were also found to have a significant effect on CPC formation. A longer incubation normally improves the organism's development and growth-related activities up to a point, after which there may be a decline in bacterial activity due to nutritional inadequacy (Gohar et al. 2019). In the presented investigation, maximum CPC production was predicted to be obtained after 4 days incubation. The incubation period has a significant impact on A. chrysogenum's ability to produce cephalosporin because it causes the fungal mycelium to differentiate into several morphological forms, which in turn affect the biosynthesis of cephalosporin. Swollen hyphal fragments are the most appropriate morphological form for the synthesis of cephalosporin. Following that, there is a decrease in cephalosporin production as a result of the transformation of inflated hyphal fragment into conidia. Conidia are not related to the production of cephalosporin. (Gohar et al. 2019). Another crucial factor in the expansion of the microorganism and the synthesis of secondary metabolites is the size and age of the inoculum. The performance of the fermentation for the generation of secondary metabolites is significantly influenced by the physiological state of the inoculum when it is transferred to the production stage (Gohar et al. 2019).

Results, using RSM with optimized medium, suggested that the optimum pH is 4. The very brief CPC manufacturing period was caused by the higher pH value attained. Although regulating pH in the shake flask method is difficult, lowering the starting pH is a typical strategy (Cuadra et al. 2008). Given the facts, pH is a crucial factor to consider while studying CPC biosynthesis, as we have seen that the manufacturing of this antibiotic only occurs within a specific pH range (López-Calleja et al. 2012). In the current study, lowering the initial pH was a helpful method that likely enabled for lower pH values to be maintained for a longer period of time throughout culture. This raised CPC yields by 29% and production time from 1 to 2 days. Our optimum pH is similar to that obtained in other statistical study done for Egyptian soil Acremonium chrysogenum isolate (Lotfy 2007). It is important to know that the transfer of the results obtained in this study to the industrial production process is to be tested in further investigations. This will definitely be verified during scaling up of CPC production using a pH-controlled fermenter in our future studies.

In strain development, mutation was induced to boost the beneficial microbial secondary metabolite output (Parekh et al. 2000). Mutation and screening for overproducing microbial mutants resulted in not just high production but also inexpensive costs (Adrio and Demain 2006). AT to CG transversions are the most effective techniques to increase yield. Hitherto, no chemical agent is known to be capable of inducing AT to CG transversions (Baltz 2001). AT to CG transversions, on the other hand, were discovered in cells exposed to gamma radiation (Xie et al. 2004). One type of ionizing radiation, gamma rays are thought to be the most energetic radiations. Gamma radiation causes mutations by breaking single or double-strand DNA through deletion or structural abnormality (Huma et al. 2012). It is also employed depending on the biological materials being used and the desired outcome of the work in order to select the best doses. High doses are utilized in sterilization, medium doses in decontamination, and low doses in mutagenesis (Hoe et al. 2016).

For fungi, gamma irradiation is a powerful mutagenic agent (Mutwakil 2011). In our study, A. chrysogenum was exposed to gamma rays, which resulted in ten morphologically altered colonies. These colonies were tested for bioactivity against standard Staphylococcus aureus ATCC 25923. The effect of gamma irradiation on cell growth was found to be dose-related. The percentage of killing increases as the gamma radiation dose increases. The 99.99% killing was achieved at a dose of 2 KGy. The highest CPC production was found with mutant AC8. When grown in optimal conditions and medium, this mutant demonstrated a 3.46-fold increase in activity when compared to the wild-type. Similar to this, Aspergillus flavus and Aspergillus ochraceus gamma-irradiated stains at 2 KGy produced two times more mycotoxins than control strains did (Ribeiro et al. 2011). The same findings were observed with Aspergillus niger, a powerful producer of numerous crucial industrial enzymes that is genotypically enhanced by gamma radiation exposure. After an 80 Krad dosage, mutants of Aspergillus niger demonstrated increased glucose oxidase synthesis (Zia et al. 2012). After being exposed to gamma radiation, Aspergillus niger, which produces the enzymes α- and β galactosidases, produced two times as much of these enzymes (Awan et al. 2011). Additionally, a cellulase-producing mutant of Aspergillus niger increased the production of carboxymethyl cellulase and filter paper cellulase at a dose of 2 KGy (Mostafa 2014).

Furthermore, our research showed that the mutant AC8 showed genetic stability after being cultured repeatedly. These findings make gamma radiation, in comparison to other mutagens, an efficient method for causing mutation. In conclusion, maximum CPC concentration was obtained using CPC2 medium containing 3 g/L L-methionine and 4.7%v/v soybean oil. In this study, the results showed that using RSM for optimization of CPC production by Acremonium chrysogenum W42-I is a competent and useful tool. The use of RSM enabled us to achieve optimal culture conditions with a small number of experimental attempts. The optimum environmental conditions (inoculum size 1% v/v, pH 4 and an incubation period 4 days), resulted in about 4.43-fold rise in CPC production reaching 39.89 µg/mL. The final model accurately predicted data points that matched the experimental values. These results demonstrated that RSM and experimental design are effective strategies for enhancing environmental variables that increase the production of CPC by the studied isolate.

Mutagenesis caused by gamma irradiation could result in production-related morphological alterations in A. chrysogenum W42-I. Following re-plating of the mutants, the same morphological modifications were observed, and the CPC production of mutant AC8 remained constant after each culture. When grown in optimal conditions and medium, an AC8 mutant demonstrated a 3.46-fold increase in activity when compared to the wild-type. As a result, the A. chrysogenum mutant AC8 is a promising industrial strain for the synthesis of CPC.