Eco-friendly biotransformation of penicillin G by free and immobilized marine halophilic Bacillus pseudomycoides AH1

Background Several antibiotics are partially metabolized by patients after administration and end up in municipal sewage systems. The fate of biodegradation in aquatic environments and the role of biodegradation in the devel‑ opment of bacterial resistance are poorly understood. Thus, as a crucial step in an environmental risk assessment, the biodegradability of many therapeutically significant antibiotics was investigated. Results A marine halophilic bacteria that degrades penicillin G (PEN‑G) was isolated and identified based on mor‑ phology, physio‑biochemical characteristics, and 16S rDNA sequences as Bacillus pseudomycoides AH1 (accession no. MF037698). The effects of various concentrations of PEN‑G and carbon and nitrogen sources on the biotransforma‑ tion ability at 30°C and pH 7.0 were evaluated. Cells grown in medium supplemented with glucose as an additional carbon source and yeast extract as a nitrogen source exhibited maximal PEN‑G biotransformation efficiency and rate (71.678% ±1.28 and 2.99 mg/h, respectively). The culture conditions for B. pseudomycoides AH1 cells were optimized using a Plackett–Burman design (PBD). Six key determinants (p < 0.05) significantly affected the process outcome, as deduced by regression analysis of the PBD data, and modified MSM broth achieved PEN‑G biotransformation effi‑ ciency (100%) under aerobic shaking conditions at 35°C, irrespective of HPLC analysis. Additionally, the present inves‑ tigation could strongly support the application of immobilization approaches for the removal of PEN‑G‑contaminated environmental sites.


Introduction
In recent years, many different kinds of antibiotics have been developed and used in various fields, including for the treatment of diverse microbial diseases and for stimulating the growth of poultry, livestock, and fish (Van et al. 2020).These antibiotic residues have been detected in numerous environmental samples, notably wastewater from cities (McArdell et al. 2003), sediments and sludge (Lindberg et al. 2005), and ground and surface water (Batt and Aga 2005).Antibiotic residues may disrupt the physiological processes of microbial populations or may lead important environmental groups of microorganisms to disappear from the environment (Kulik et al. 2023).
Penicillins are β-lactam antibiotics that have a broad spectrum of activity against both gram-positive and gram-negative organisms.Penicillin G (PEN-G) is especially produced on a large scale, with between 30 and 90% of it being dumped into waste streams (Wu et al. 2019).The danger of exposure to antibiotics has become a significant issue that has been repeatedly addressed in the clinical treatment of numerous illnesses.Consequently, methods for the efficient decomposition of PEN-G residues in the environment are required to lower the danger of exposure (Wang et al. 2015).
Several methods have been employed to remove β-lactam antibiotics, including photocatalysis, UV and solar-based processes, and chemical oxidation, which includes ozonation (Michael-Kordatou et al. 2018).Unfortunately, there are several drawbacks to these methods, including their high cost, the development of hazardous secondary byproducts, ecological risks, and nonsustainability (Panigrahy et al. 2020).Hence, microbial bioremediation has been considered an effective method that offers a more reliable, economical, and sustainable choice for cleaning different contaminated locations (Kamaz et al. 2019;Nas et al. 2020).Microbes that can mineralize, biodegrade, or biotransform antibiotics can also be identified (Reis et al. 2020).Additionally, bioremediation has been applied to a variety of methods, including the use of single-strain microorganisms (Shao et al. 2018), microbial consortia and genetically engineered microorganisms (Liu et al. 2017).
Physicochemical and nutritional factors could have an impact on the efficacy of bioremediation for eliminating contaminants.Therefore, to identify the optimum biodegradation conditions for pollutant removal, it is crucial to ascertain their effects and optimize the most significant conditions.Multivariable optimization investigations of different biotechnological, environmental, and chemical processes have effectively employed statistical approaches such as the use of Plackett-Burman design (PBD) (Plackett and Burman 1946;El-Borai et al. 2016;Farag et al. 2021).They are also considered to be reliable and effective methods for rapid screening of significant factors from a multivariable system with a few experiments (Favier et al. 2021).
A number of bacterial strains can biodegrade several antibiotics (Wang et al. 2015;Anan et al. 2018;Wang et al. 2020).Bacillus species are aerobic, endosporeforming, gram-positive bacteria with a low level of pathogenicity.Numerous researchers have examined the ability of Bacillus species to break down various antibiotic types (Liyanage and Manage 2015;Fiedler et al. 2019;Zhang and Wang 2021).
Cell immobilization has several advantages that could be crucial for the biodegradation of xenobiotic compounds (Chen et al. 2002;Farag et al. 2022), including increased tolerance to a range of toxic and resistant compounds in an appropriate matrix, ease of separation and reuse, high rates of volumetric reaction and high local cell concentrations (Bergero and Lucchesi 2015).
As a result, the aim of the current work was to develop a cost-effective and efficient method for the biotransformation of PEN-G through the use of a local marine bacterial isolate that can utilize PEN-G as its only source of energy and carbon.Additionally, the best parameters for maximum PEN-G biotransformation were investigated.Furthermore, to achieve efficient PEN-G biotransformation, several matrices were employed during the immobilization procedure.

Chemicals
All of the analytical-grade compounds were purchased from Sigma Chemical Co.(St.Louis, Missouri, USA).

Sampling and culture media
Ten sediment samples were collected in sterile screwcapped bottles from multiple locations, such as the Suez Government, Burllus Lake (Kafr-Elsheikh), and two samples from pharmaceutical effluents (Alexandria), Egypt.The samples were then transported to the laboratory in an ice box and kept at 4°C until further analysis.

Enrichment and acclimatization of PEN-G-degrading bacteria
Five grams of each sample was mixed in 50 ml of sterile seawater before the proper aseptic glass beads were placed in 250-ml Erlenmeyer flasks.To disperse the samples, the flasks were shaken for 30 min at 30°C in a rotary incubator shaker.Next, one ml of each sample was added separately to flasks containing 99.0 ml of sterile MSM (pH 7.0) supplemented with 50 mg/l of PEN-G.Then, the flasks were incubated for 5 days at 30°C and 160 rpm in an orbital incubator.The bacterial cultures were then subjected to gradually increasing PEN-G concentrations to acclimatize the bacterial strains to the antibiotic.After incubation, a 250-ml Erlenmeyer flask containing 99.0 ml of sterilized MSM supplemented with 100 mg/l PEN-G was loaded with one ml of fermentation liquid, which was then incubated at 30°C and 160 rpm.Similarly, for another hebdomadal acclimation, one ml of suspension from each medium cultivated for five days was added to flasks containing 99.0 ml of MSM and 150 mg/l of PEN-G.Next, 99.0 ml of fresh MSM and 200 mg/l of PEN-G were combined with 1.0 ml of each of the previous cultured media.The flasks were cultivated for 5 days at 30°C and 160 rpm.Thus, isolates with higher tolerance and remediation abilities toward the antibiotic were obtained.Next, solid MSM plates containing 100 mg/l were streaked with the enrichment suspension, and the plates were incubated for 72 h at 30°C.The isolates were also tested for their pathogenicity by subculturing on blood agar plates.For further identification, bacterial colonies of various morphological types were chosen, purified, subcultured, and kept at -20°C (Liyanage and Manage 2015).

Bacterial inoculum preparation
According to Mo et al. (2019), the standardized bacterial inoculum was prepared by transferring a full loop of bacterial isolate from a 24 h old slant into 10 ml of sterilized MSM and incubating at 30°C for 72 h on a rotary shaker (160 rpm).

Bacterial biomass determination
To assess bacterial growth, the turbidity at 600 nm was determined using a spectrophotometer to record the optical density of the bacterial isolate (Farag et al. 2021).

Quantitative evaluation of PEN-G-degrading bacteria
The analysis was carried out according to the methodology outlined by Feng (2009).After a total of 72 h of incubation, the residual concentration of PEN-G in the MSM medium, which contained 100 mg/ml of the purified strains, was assessed using UV spectrophotometry.A total of 25 ml of PEN-G solution (100 µg/ml) was added to a 50 ml volumetric bottle, and the absorbance was measured from 310 to 340 nm to determine the maximum detection wavelength.The results indicated that the maximum detection wavelength was approximately 325 nm, which is in the range detected by other studies (Feng 2009, Yang et al. 2019).
To establish the standard curve, an accurately weighed quantity of sodium benzyl penicillin was dissolved and diluted to 100 ml to obtain standard solutions with the following concentrations of antibiotics : 20, 40, 20, 40, 60, 80, and 100 μg/ml.The antibiotic absorbance was assessed at the maximum absorption wavelength (325 nm).Distilled water was used as the blank control.The absorbance values and the PEN-G concentrations had a strong linear relationship (Yang et al. 2019).
To test the biodegradability of the isolates, one ml (1.6 × 10 6 CFU) of each bacterial suspension was inoculated in a 250-ml Erlenmeyer flask containing 50 ml of fresh fermentation medium (MSM) and 100 mg/l PEN-G and then cultivated at 30°C for 72 h on an orbital shaker (160 rpm).Following incubation, a cooling centrifuge was used to centrifuge the culture broth at 6000 rpm and 4°C.Uninoculated medium was used as the control treatment.
The biotransformation efficiency of the bacterial isolates toward the antibiotic was calculated as described by Al-Gheethi et al. (2017) as follows:

Determination of residues of PEN-G biotransformation by HPLC
PEN-G and its bioremediation intermediates were detected by high-performance liquid chromatography (Agilent HC-C18 (2)) and an Ultimate XB C18 column (4.6 × 150 mm, 5 μm).All the analytical grade reagents used in the HPLC analyses were of this grade.The samples were tested at 30°C with a flow rate of 1.0 ml/min and using a methanol/phosphate mobile phase (1 mM KH 2 PO 4 , pH, flow rate: 1.0 ml/min; the injected volume of the sample was 20 µl; the wavelength of the detector was 225 nm) (Wang et al. 2020).

Identification of the most potent bacterial isolate
Morphological, microscopic, biochemical, and 16S rDNA gene sequence studies were used to characterize and identify the pure target isolate that was isolated from the PEN-G-enriched cultures.

DNA isolation and strain identification
The identification method mainly consisted of DNA extraction, PCR amplification, and sequencing according to Sambrook and Russell (2001).The partial 16S rDNA gene was amplified using the universal primers forward (5-AGA GTT TGA TCA TGG CTC AG-3) and reverse (5-TAC GGT TAC CTT GTT ACG AC-3).PCR amplification was performed using IU of Taq DNA polymerase.A Perkin ABI PRISM 377 DNA sequencer (Perkin Elmer, Foster City, CA, U.S.A.) was used to detect 16S rDNA sequencing fragments.The acquired nucleotide sequences were compared to those found in the NCBI database using a BLAST search.The Biotransformation rate (mg/h) = (degraded PEN−G (mg))/(fermentation period (h)) sequence was determined using the BioEdit sequence alignment editor tool (version).The neighbor-joining method was used to conduct phylogenetic analysis.

Effect of initial PEN-G concentration, carbon, and nitrogen source
To determine the effect of the initial concentrations of PEN-G, several concentrations of the antibiotic ranging from 50 to 500 mg/l were combined with sterilized MSM and incubated at 30°C for 72 h in an orbital shaker (160 rpm).Following incubation, microbial growth was assessed; the residual PEN-G concentration in the cellfree culture was then assayed, and the PEN-G biotransformation efficiency and rate were calculated.The effects of 0.1% additional carbon sources (glucose, fructose, sucrose, maltose, lactose and starch) and 0.1% nitrogen sources (yeast extract, casein, tryptone, peptone, NH 4 NO 3 , KNO 3 , (NH 4 ) 2 SO 4 , NH 4 Cl and NaNO 3 ) on PEN-G biotransformation were also tested in MSM supplemented with 300 mg/l PEN-G.All experiments were conducted at least in triplicate, and the mean values were calculated.

Plackett-Burman experimental design
An effective approach for identifying factors that have a major impact on the biotransformation of PEN-G by the most potent bacterial strain is the Plackett-Burman experimental design (Plackett and Burman 1946).Eleven variables were tested in 13 trials for the current investigation.High (+) and low (-) levels were tested for each variable (Table 1).Every experiment was carried out in duplicate, and the response was determined by averaging the PEN-G biotransformation.
The main effect for each variable was calculated by the following equation: In the given context, ∑xi represents the variable main effect, Σpi+ and Σpi-are the PEN-G biotransformation responses in trials with high and low concentrations of the independent variable (Xi), respectively, and N represents the number of trials divided by two.The factors that were significant at the 95% level (P<0.05) were chosen because they were considered to have significant effects on the biotransformation of PEN-G and thus were used for further optimization.Using regression analysis, the biotransformation coefficients were calculated.The effects of different factors on biotransformation were examined using p values and F tests.

Cell immobilization procedures By Entrapment
According to Farag et al. (2015), entrapment was carried out in 3% sodium alginate or K-carrageenan solution.A volume of approximately 3 ml (OD 600, 1.0) of bacterial suspension was introduced into sterile alginate or k-carrageenan solution.The gel-mixing cells were drawn out using a sterile syringe and put into a cross-linking solution (2% KCl for k-carrageenan and 2% CaCl 2 solution for alginate) to create circular beads with a diameter of 3 to 4 mm.After two hours of hardening, the beads were rinsed with sterile distilled water.After that, the beads were added to previously sterilized MSM medium containing PEN-G, and the mixture was shaken (160 rpm) at 35°C.

By adsorption
For cell adsorption, several solid support matrices (luffa pulp, polyurethane foam, and charcoal cubes) were utilized after sterilization at 121°C for 30 min.Before being immobilized, 2 ml of bacterial cells (OD 600 , 1.0) was added to sterile MSM containing PEN-G.The mixture was then incubated at 35°C in the dark in an orbital incubator.Subsequently, each support matrix was introduced, and the mixture was incubated at the optimal temperature on a rotating shaker (160 rpm) (El-Borai et al. 2022;Farag et al. 2022).

Scanning electron microscopy (SEM)
The biocatalysts were rinsed with sterilized distilled water and vacuum dried to remove moisture.After being sliced and coated with gold, the samples were investigated using a scanning electron microscope (JEOL JEM-2100 F; JEOL Ltd., Japan).

Biotransformation of different initial PEN-G concentrations by immobilized cells
To determine the optimal initial concentration of PEN-G (0.2 to 3.5 g/l), the immobilized cells were introduced to the optimum medium supplemented with various concentrations of PEN-G and incubated in an orbital shaker incubator (160 rpm) at 35°C.Following incubation, a predefined volume was aseptically collected at regular intervals, and the concentration of residual PEN-G was measured.Every treatment was carried out in triplicate.

Reuse and storage of immobilized cells
Cells immobilized within the most suitable material support were incubated at 35°C.Following incubation, the culture medium was removed from the flask for determination of residual PEN-G, while the immobilized cells were kept inside for another cycle.The previously described procedure was carried out many times; fresh sterile MSM supplemented with PEN-G was added, and the cells were incubated.At the end of each run, the residual concentration of PEN-G was calculated.

Isolation of high-efficiency PEN-G-degrading bacteria
After acclimatization and purification, a total of four isolates that showed the ability to grow on PEN-G as the sole carbon source were chosen for the next investigation.BL-1, NSC-1, PW-1, and NSC-2 were isolated from Burullus drains, the New Suez canal, pharmaceutical wastes, and the New Suez canal, respectively.The plants were cultured on sterile MSM supplemented with 100 mg/l PEN-G as the only carbon and energy source for 72 h at 30°C and 160 rpm to compare their remediation capabilities.Following incubation, microbial growth (absorbance at 600 nm) and the residual PEN-G concentration were assessed.The data (Fig. 1) revealed that all bacterial isolates recorded optical density for growth in the range 0.8-1.Also they were capable of utilizing more than 60% PEN-G.Furthermore, among all the isolates that were investigated, the bacterial strain BL-1 showed the greatest ability to remediate PEN-G, with biotransformation efficiencies and rates of 98.0% and 1.361 mg/h, respectively.Consequently, the BL-1 strain was selected as the target isolate, despite its slightly least growth (0.8) among the tested isolates, as it had the highest biotransformation capability.Moreover, the isolate showed no hemolysis when subcultured on blood agar, thus confirming no pathogenicity, while the others showed slight hemolysis.The purity of the isolate was confirmed by microscopic examination of the morphological uniformity of cells from a single colony subcultured on solid media plates.

Strain identification
Based on morphological, microscopic, and biochemical evaluations, the most potent bacterial isolate (BL-1) that degrades PEN-G was identified (Additional File; Table 1).
In addition, this identification has been verified by a molecular method based on the study of the 16S rDNA gene sequence.The sequence's nucleotide sequence analysis was carried out at the NCBI server's Blast N site.The sequence of isolate BL-1 was 652 bp (Fig. 2), 100% similar to that of members of the genus Bacillus, and it was identified as Bacillus pseudomycoides AH1 and deposited in GenBank with accession number MW052694.

Determination of PEN-G biotransformation by HPLC
PEN-G biotransformation was analyzed by HPLC 90 min after injection with a standard (noninoculated) sample of 100 mg/l PEN-G and another sample of culture medium containing the same concentration of PEN-G and previously inoculated with Bacillus pseudomycoides AH1 (Fig. 3).This method could be used to determine the concentration of PEN-G in aqueous media.Fig. 3A shows the chromatogram of a standard PEN-G solution (in medium without inoculation), whereas Fig. 3B shows the PEN-G residues (in medium inoculated with Bacillus pseudomycoides AH1).Based on a comparison of the two chromatograms, PEN-G had a 4.15 min retention time (Fig. 3A), which decreased, and the appearance of two additional peaks with varying retention times (2.221 and 3.284 min, respectively) is shown in Fig. 2B.These analytical results imply that the Bacillus pseudomycoides AH1 strain degrades PEN-G and that two biotransformation products are produced in solution throughout the process.

Optimization of PEN-G biotransformation Effect of initial PEN-G concentration and additional carbon and nitrogen sources
The effect of different initial PEN-G concentrations on PEN-G biotransformation was assessed.The highest biotransformation rate (2.26±0.04mg/h) was observed at 300 mg/l, and the lowest (0.46±0.01 mg/h) was observed at 500 mg/l (data not shown; Additional file 2).In contrast, as the initial PEN-G concentration increased, microbial proliferation decreased.The present findings generally showed that the inclusion of carbon substrates increased the cell density and decreased the efficiency of PEN-G biotransformation (Fig. 4A).However, glucose was an exception; its addition slightly increased the cell density (0.754) and biotransformation efficiency and rate (61.450% ± 0.45 and 2.56 mg/h, respectively) compared with those of the control.
The results of the addition of an additional nitrogen source revealed that the addition of yeast extract resulted in a maximum PEN-G biotransformation efficiency and rate (71.678% ±1.28 and 2.99 mg/h, respectively) (Fig. 4B).Repressed PEN-G biotransformation efficiency was observed in a medium lacking a nitrogen source, in which the efficiency was approximately 36.40% of the maximum efficiency detected in the presence of yeast extract.Therefore, yeast extract was chosen as the nitrogen source for the experimental design and was used to assess a variety of physiochemical factors impacting the ability of B. pseudomycoides AH1 cells to biotransform PEN-G.

Optimization of PEN-G biotransformation by Plackett-Burman design
The presence of many variables needs to be taken into consideration because optimizing the fermentation conditions of the culture medium is considered a laborious and complex process.Therefore, the Plackett-Burman design was employed to estimate the most significant factors affecting PEN-G biotransformation.The PEN-G biotransformation efficiency (%) was assessed in the present study (Table 2).Trial number 11 had the highest PEN-G biotransformation efficiency (99.989%), whereas trial number 10 had the lowest PEN-G biotransformation efficiency (38.558%).This in turn demonstrated that performing the optimization step was needed.
The PEN-G biotransformation efficiency (%) was negatively impacted by high levels of glucose and incubation time within the test ranges but was positively impacted by all other factors (Fig. 5).ANOVA was used to evaluate the adequacy of the model and the impact of the medium components on the percentage of PEN-G biotransformation by B. pseudomycoides AH1 cells, as listed in Table 3. Fisher's statistical test (F test) was utilized to determine the model's statistical significance.Consequently, a component was regarded as significant if it had a statistical confidence above 98% (P = 0.02).Based on multiple linear regressions of the Plackett-Burman design data (Table 3), six investigated independent factors were found to have a significant impact (p < 0.05) on process performance.All of these variables were predicted to have the greatest impact on PEN-G biotransformation efficiency (%).
To demonstrate the significance of each investigated independent variable on the process output in ascending order, a Pareto chart (Fig. 6) was developed.
The first-order models for power generation were determined to be appropriate after ANOVA was performed.A linear model equation was then presented to determine the optimal levels of these variables for PEN-G biotransformation efficiency (%) and can be written as: where Y is the response of the PEN-G biotransformation efficiency (%), and the concentrations of the independent variables are represented as X 1 :X 11, as reported in Table 1 in the Materials and Methods section.
Within the limits of the experimental conditions, the PEN-G biotranformation efficiency (%) may be predicted via the Plackett-Burman design.In addition, there is considerable agreement among the predicted and actual response levels (Fig. 7).For the PEN-G biotransformation efficiency (%), the highest experimental value was 99.653, whereas the predicted response was 99.159.
By applying the Plackett-Burman experimental design, a validation experiment was carried out in triplicate to verify the performance of B. pseudomycoides AH1 cells.The PEN-G biotransformation efficiency was 99.653 ± 1.5%, showing a 3.12-fold increase.There was a 0.59% difference between the predicted and actual results.

PEN-G biotransformation by immobilized B. pseudomycoides AH1 cells
To evaluate the effect of the immobilization approach on PEN-G biotransformation by B. pseudomycoides AH1, cells were batchwise cultured at 35°C in 250 ml flasks containing 50 ml of optimized MSM supplemented with 450 mg/l PEN-G.Samples were taken every 8 h for up to 96 h.According to the results (Fig. 8), PEN-G was degraded to differing degrees by the cells immobilized on various supports or in the gel matrix.The average biotransformation rates of cells immobilized in alginate beads, polyurethane foam, and luffa pulp were greater than those of free cells in comparison to those of other materials (12.5, 11.50, and 11.45 mg/h, respectively).However, free cells exhibited greater PEN-G biotransformation than immobilized cells in K-carrageenan and on charcoal cubes and nut particles.Based on these findings, the immobilization method employing Ca-alginate-entrapped cells was chosen for achieving the greatest level of PEN-G biotransformation.
Cells of B. pseudomycoides AH1 immobilized in alginate beads and polyurethane foam and cultured on optimized media supplemented with PEN-G (450 mg/l) were collected and investigated by SEM (Fig. 9).SEM revealed unevenly distributed, tightly packed cells.

Effect of different initial PEN-G concentrations on PEN-G biotransformation using immobilized B. pseudomycoides AH1 cells
Different PEN-G concentrations (200-3500 mg/l) were applied to the immobilized cells to determine their tolerance limits (Fig. 10).The findings indicated that immobilized B. pseudomycoides AH1 cells could degrade PEN-G in 36 h at a biotransformation rate of 41.67 mg/h when the initial concentration was 1500 mg/l.However, when the PEN-G concentration was increased to 3500 mg/l, the biotransformation efficiency of PEN-G decreased and eventually decreased to its lowest value (17.20%).

Effect of reuse and storage of immobilized cells
B. pseudomycoides AH1-immobilized cultures were reused for 10 cycles.The gel beads containing the cells could entirely degrade PEN-G by the fifth cycle, with a biotransformation rate equal to 41.64 mg/h.However, the biotransformation efficiency and rate titer decreased in subsequent cycles (Fig. 11).The results showed that for five batches in succession, the amount of PEN-G substantially decreased in the immobilized cells.After five cycles of repetition, cell leakage occurred, and the efficiency of PEN-G biotransformation decreased.The stability and activity of microorganisms are also significantly affected by prolonged storage.The PEN-G biotransformation efficacy of immobilized B. pseudomycoides AH1 after storage at 30°C for ten weeks was tested (Table 4).The data indicated that with increasing storage time, the removal of PEN-G decreased gradually from 99.87% to 73.24%, which indicated that the immobilized biocatalyst had good storage stability.

Discussion
Penicillin is considered an inexpensive β-lactam antibiotic that exhibits a broad spectrum of antimicrobial action.Ecosystems and human health may be negatively impacted by penicillin residues, which have the potential to be hazardous, leading to the development of penicillin-resistant strains.Currently, the removal of penicillin from water is an important issue.Therefore, it is crucial to isolate a potential local microbial strain that withstand, grow, and degrade PEN-G even at extremely high initial concentrations.
The present study has several advantages compared to other anaerobic/aerobic biodegradation and abiotic techniques.In addition, the use of a single bacterium rather than a microbial consortia, a high rate of PEN-G biotransformation, efficient biotransformation, and aerobic process execution are highly important.There are few papers  discussing the biotransformation of antibiotics in general and PEN-G in particular in the most recent reviews of the literature.The LB-1 strain succeeded in nearly completely eliminating PEN-G, with the biotransformation efficiency reaching 99.65% after 48 h of incubation.However, the strain showed the least growth (0.8) among the tested isolates which may be related to accumulation of some metabolites from the degradation process which can act as suppressors for cell growth (Wang et al. 2015).Moreover, the use of microorganisms in biodegradation processes is safe, energy saving and eco-friendly.The present study also showed the ability of four bacterial isolates from different locations to grow well on media supplemented with penicillin G as the sole carbon source.Most microorganisms cannot use antibiotics as a sole carbon source and depend on cometabolism by adding additional carbon sources for reproduction (Wang et al. 2021).
Few bacterial strains have been found to degrade PEN-G, such as Klebsiella pneumoniae Z-1 (Wang et al. 2015), Phanerochaete chrysosporium (Mullai and Vishali 2007), Actinobacillus pleuropneumoniae (Hathroubi et al. 2015), and Paracoccus sp.KDSPL-02 (Wang et al. 2020).The most potent strain reported in this study was Bacillus pseudomycoides AH1.This strain is one of nine novel 454 species of the Bacillus cereus group, as reported by Liu et al. (2017).Numerous researchers have documented Bacillus cereus resistance to β-lactam antibiotics (Kim et al. 2015;Fiedler et al. 2019;Nguyen et al. 2022).Bacterial strains comparable to B. pseudomycoides MT32 have been isolated from soil samples in the Tenth Rhamadan district of Sharkia Governorate, Egypt, which is an industrialized area contaminated with heavy metals (El-Saadony et al. 2019).Liyanage and Manage (2015) reported that B. cereus, Enterobacter ludwigii and Enterobacter sp. were used for sulfanomide and amoxicillin degradation.Additionally, Al-Gheethi and Ismail (2014) demonstrated that four different β-lactam antibiotics (ampicillin, cefalexin, cefuroxime, and amoxicillin) can potentially be degraded by Bacillus subtilis strains.To date, a review of the literature has not revealed any information concerning the ability of Bacillus pseudomycoides to degrade penicillin G.
The bacterial strains, along with the fermentation conditions, have a significant impact on the rate of biotransformation.The PEN-G biotransformation efficiency decreased with increasing initial concentration of PEN-G, possibly due to substrate inhibition, as demonstrated by Mullai and Vishali (2007).Another potential explanation for the reduced PEN-G biotransformation  might be the diminished activity of the β-lactamase enzyme at high levels of substrate (PEN-G) (Nguyen et al. 2022).The PEN-G biotransformation efficiency slightly increased with the addition of glucose (0.1%).The cell growth of B. pseudomycoides AH1 in our study is also slightly increased when using additional glucose from that reported by using PEN-G alone as a carbon source, indicating that the strain can use also PEN-G alone as a carbon source for growth.Anan et al. (2018), used glucose as an additional carbon source for Achromobacter xylosoxidans strain Cef6 to achieve maximum sodium ceftriaxone biotransformation.Additionally, Wang et al. (2015) suggested that the decrease in the biodegradation rate of PEN-G with the addition of glucose might be due to the preference of Klebsiella pneumoniae Z1 for utilizing external organic glucose rather than antibiotics.In contrast to our results, Leng et al. (2016) reported that the addition of glucose increased the cell density and decreased rate of tetracycline biotransformation in Stenotrophomonas maltophilia DT1 cells.In cell cultures, sucrose was shown to support high rates of growth, although low PEN-G biotransformation efficiency (%) was also observed.It is commonly known that sucrose is a disaccharide that is formed when the monosaccharides glucose and fructose are combined.This combination of molecules can offer high-sufficient carbon sources to support the growth of bacteria.The current findings are in contrast with earlier research by Sudha et al. (2022), who found that the addition of sucrose as an additional carbon source increased the oxytetracycline degradation efficiency of Lysinibacillus sp.strain 3+I, up to a maximum of 14%.Tan et al. (2022) additionally found that, among the six carbon sources, potassium acetate exhibited the greatest rates of tetracycline biodegradation by the yeast strain Cutaneotrichosporon dermatis M503, whereas glucose, sucrose, and maltose gave lower rates of biodegradation.Therefore, the species determines the best carbon source, and our isolate could represent a novel B. pseudomycoides variant.The majority of bacterial cells are able to employ multiple compounds as their sole supply of nitrogen.The addition of yeast extract to the MSM increased the biotransformation efficiency.The maximum rate of PEN-G biodegradation by Paracoccus sp.KDSPL-02 was achieved using a combination of NH 4 NO 3 and yeast extract (Wang et al. 2020).Similarly, the addition of yeast extract to cultures of Klebsiella strain TR5 enhanced tetracycline degradation (Yin et al. 2020).
The traditional approaches for medium optimization, known as OFATs, require a great deal of time and are not appropriate for assessing a large number of independent parameters.Consequently, a crucial statistical method known as the Plackett-Burman experimental design is usually employed to determine the key variables in a multivariable system and to provide the basis for additional optimization (Anan et al. 2018).The results of the experimental design revealed that a higher glucose concentration has a negative impact on the biotransformation efficiency, which may be a result of reduced glucose adsorption by the strain or detrimental osmotic pressure (Dike et al. 2014).The effect of temperature and pH on the biotransformation process was also revealed by the design in which both parameters positively affected the biotransformation process.The optimum temperature and pH were 35°C and 8, respectively, similar to the results reported by some authors who also reported that the stability of penicillin is usually lower in alkaline media than in neutral and acidic media (Wang et al. 2021).The results obtained from the optimization methods revealed that free cultures of Bacillus pseudomycoides AH1 had a good ability to degrade PEN-G in a medium containing an initial concentration of 450 mg/l, which was superior to the results of other investigations (Wang et al. 2015;Yang et al. 2019).The determination coefficient was used to evaluate the model's goodness of fit (R2).In this instance, the calculated R2 value was 0.9998, whereas the adjusted R2 value was 0.9974.The two values are close to one another, confirming good model fitting.As stated by Guilford and Fruchter (1973), a regression model with an R2 value near 1.0 is considered to have a remarkably strong correlation.The model in the study is appropriate, as demonstrated by the strong correlation established between the predicted and experimental results.These results are in good agreement with other authors' reports of the use of statistical optimization techniques and mathematical modeling for enhancing the biodegradation of antibiotics and phenolic compounds (Anan et al. 2018;Chris Felshia et al. 2018;Yang et al. 2020;Farag et al. 2021;Favier et al. 2021).
A few drawbacks of biodegradation/biotransformation technology include microbial cell death, substrate inhibition, and a lack of ability to withstand extreme conditions (Lou et al. 2019).Numerous authors have presented data on the viability and efficacy of antibiotic biological degradation in immobilized microbial cells (Wang et al. 2020;Al-Dhabi and Arasu 2022;Xia et al. 2023).According to Wang et al. (2020), immobilized Paracoccus sp.KDSPL-02 cells in alginate beads had greater effectiveness than free cells in the breakdown of PEN-G.Furthermore, Liu et al. (2023) enhanced the capability of Alcaligenes sp.R3 cells to remove tetracycline by immobilizing tetracycline in both polydopamine and carboxymethylcellulose.Consequently, immobilized cells are utilized more frequently and have greater biotransformation efficiency than free cells (Zur et al. 2016).Among all the tested supports, immobilized cultures of Bacillus pseudomycoides AH1 in alginate beads had the highest PEN-G biotransformation efficiency, which was even greater than that obtained from free cultures.This might be because of the structure of the alginate beads, which provide an internal living environment for microorganisms to shield them from outside contaminants and improve the probability of surviving in an antibiotic-contaminated environment (Zhao et al. 2019).Furthermore, alginate is an anionic polysaccharide that has outstanding chemical and physical stability, stable properties, and a low toxicity profile.It additionally enhances the possibility of reuse and decreases the risk of cell leakage or contamination in the fermentation medium (Ramakrishnan and Prakasham 2011;Rivas-Sanchez et al. 2022).The investigated bacterium can completely degrade PEN-G by immobilized cultures in alginate beads when the initial antibiotic concentration is raised to 1500 mg/l, which is a higher concentration than that reported by Wang et al. (2020), who reported that entrapped Paracoccus sp.KDSPL-02 cells in alginate beads entirely degraded PEN-G at an initial concentration of 1200 mg/l, and the cells maintained their degradation efficiency up to 2000 mg/ml.In accordance with our findings, Phanerochaete chrysosporium has the ability to tolerate PEN-G concentrations of up to 2000 mg/l; however, its degradation capability is limited to 61.4% (Mullai and Vishali 2007).A key aspect that influences the efficacy of biotransformation over time in industrial treatment is the reuse of immobilized cultures (Lin and Cheng 2020).Immobilized cultures of Bacillus pseudomycoides AH1 in alginate beads were reused for several cycles since reusing immobilized microbial cells could save money and could be considered together with storage stability as a key parameter of their utilization in practice (Wang et al. 2015).Bacillus pseudomycoides AH1-immobilized cultures maintained their biotransformation efficiency until the fifth cycle, after which the efficiency decreased.Cell inactivation, cell leakage, the buildup of hazardous substances inside the carrier, and mass transfer restrictions might all be the reasons for this decline (Partovinia and Rasekh 2018).Immobilized cultures of Bacillus pseudomycoides were also stable under storage conditions and could conserve 73.23% of its biotransformation efficiency after 10 weeks.Similarly, immobilized Bacillus subtilis cultures are extremely stable during storage and can be efficiently and continually utilized for three cycles to remove chlortetracycline (Zhang and Wang 2021).Zhang and Wang (2021) showed, in line with our findings, that immobilized Bacillus subtilis exhibited excellent storage stability and that as the storage time increased, the percentage of chlortetracycline removed decreased from 78.83% to 38.52%.These findings therefore emphasize the significance of the use of immobilized B. pseudomycoides AH1 to improve PEN-G biotransformation.

Conclusion
Organic pollutants, especially antibiotics, are substances of high environmental concern globally due to the severe issues they generate.Consequently, the present study reported a new source for PEN-G removal from a marine environment.Several bacterial strains were isolated and screened for their ability to degrade PEN-G, and the most potent strain was then phylogenetically identified as Bacillus pseudomycoides AH1 (accession no.MF037698).The goal of using free and immobilized cells was to enhance the biotransformation efficiency of PEN-G.Immobilizing bacterial cells in alginate beads is an easy, affordable, and environmentally friendly way to degrade the majority of contaminants found in wastewater.To enhance efficiency and outcomes, this study could offer more ecologically friendly materials that may be combined and used in wastewater treatment systems.

Fig. 1
Fig.1PEN-G degradation by marine bacterial isolates using PEN-G as the sole carbon and energy source.Bacterial cultures were cultivated at 30°C under shaking conditions (160 rpm) for 72 h.Data are expressed as the mean ± SD

Fig. 2
Fig. 2 Phylogenetic tree showing the relationships between Bacillus pseudomycoides AH1 and closely related strains

Fig. 3
Fig. 3 HPLC results of (A) standard sample and (B) culture medium of Bacillus pseudomycoides AH1

Fig. 4
Fig. 4 Influence of additional carbon sources (A) and nitrogen sources (B) on PEN-G-biotransformation and growth.Bars indicate the standard deviation of the mean.

Fig. 8 Fig. 9
Fig. 8 PEN-G biotransformation by immobilized B. pseudomycoides AH1 cells at different incubation times.The values represent the means ± SDs of 3 replicates

Fig. 10
Fig. 10 PEN-G biotransformation efficiency and rate different PEN-G concentrations.The experiments were performed in triplicate, and the data are expressed as the mean ± SD

Table 1
Variables and their levels utilized in the optimization experiment

Table 2
Plackett-Burman experimental design for the evaluation of variables affecting PEN-G biotransformation efficiency (%)

Table 3
Statistical analysis of the effects of Plackett-Burman design on PEN-G biotransformation efficiency (%)

Table 4
Effect of long-term storage of immobilized B. pseudomycoides AH1 cells on PEN-G biotransformation.The experiments were performed in triplicate, and the data are expressed as the mean ± SD