Khaled M. Ghanem

Application of the response surface methodology for optimizing the activity of an aprE-driven gene expression system in Bacillus subtilis

Abstract The major targets for improvement of recombinant expression systems in microbial cells are gene dosage, transcriptional control machinery and, to some extent, translation. Here we show that optimization of fermentation conditions by applying statistically designed, multifactorial experiments offers an additional method for potential enhancement of gene expression systems. A chromosomally encoded fusion between the Bacillus subtilis aprE regulatory region and the E. coli lacZ gene carried by the B. subtilis host cells was used. The 2 × SG sporulation medium was used as a basal medium. Among the 11 fermentation factors we examined, the most significant variables influencing β-galactosidase expression were statistically elucidated for optimization and included peptone, MgSO4 · 7H2O, and KCl. The optimum concentrations of these variables were predicted by using a second-order polynomial model fitted to the results obtained by applying the Box-Behnken design, a response surface method. Calculated optimum concentrations were predicted to confer a maximum yield of 2,423.5 β-galactosidase specific activity units. A verification experiment performed under optimal conditions yielded 96% of the predicted specific activity value with an increase by a factor of almost 5 compared with the results obtained under basal conditions.

Microbial extraction of beet pulp pectin

Abstract Beet pulp (BP), a by product of the sugar industry in Egypt, contains about 51% holocellulose, 24% pectin, and 13% lignin of its dry weight. Different yeasts were tested for their ability to produce protopectin-solubilizing enzymes. Kluyveromyces marxianus was the most active culture from the standpoint of the pectin released (about 14% on the dry BP basis, which represents about 59% of the whole pectin in BP). Milling is the best mechanical BP pretreatment for pectin bioextraction, surpassing chemical treatments. A BP : water ratio of 1 : 16 (5 g BP : 80 ml H2O) was found to be favourable for pectin extraction. One-day-old seed culture of K. marxianus at inoculum size of 10% and incubated for 2 days under shaking, at 30°C at pH 6.0 were optimal conditions for bioextraction of BP pectin (about 63.3% of the whole pectin in BP was extracted).

Statistical optimization of cultural conditions for decolorization of methylene blue by mono and mixed bacterial culture techniques

Acinetobacter baumannii, Corynebacterium sp., Cytophaga columnaris, Escherichia coli, Pseudomonas fluorescence and Pseudomonas luteola were locally isolated bacteria from sewage Disposal Lake at Jeddah, Saudi Arabia and they can decolorize methylene blue. E. coli was the most potent MB decolorizing and to a lesser extend P. luteola. Five different media were tested to elucidate medium formulation in favor of MB decolorization by E. coli and P. luteola. Ingredients of the basal medium favored the complete decolorization of 50 μg MB/ml after 84 h of fermentation. Time course decolorization of MB by E. coli indicated that 75 h of fermentation was satisfactory to decolorize 50 μg MB/ml. It was also able to decolorize different levels of MB up to 150 μg MB/ml after 95 h of fermentation. Bacterial consortium of E. coli and P. luteola was highly efficient to decolorize MB than monoculture, where the decolorization period reduced by more than 37% and increased decolorization rate (μgMB/h) up to 58%. Statistical designs of two phase multifactorial optimization (Plackett-Burman and Box-Behnken) were carried out to optimize cultural conditions to increase the efficiency of mixed culture to decolorize 150 μg MB/ml. Under the optimized conditions the decolorization period was reduced by about 31.7% and with increased decolorization rate by 46.4%. Methylene blue can be efficiently decolorized by facultative aerobic bacteria (E. coli and P. luteola). The decolorization process was markedly influenced by the composition of the fermentation medium and concentration of MB. Mixed culture of E. coli and P. luteola was highly efficient to decolorize MB than monoculture technique. The cultural conditions were considerably optimized using statistical experimental designs of PlackettBurman and Box-Behnken.

Methane production from beet pulp

Abstract The applicability of untreated or alkali treated beet pulp (BP), a waste product of the sugar industry in Egypt, for methane production was examined. The daily methane yields varied in a stochastic manner. The values fluctuated throughout the experiment after an initial increase which occurred in the first 10–15 d. The average composition of the gas produced from the alkali treated BP was 43–71% methane and 57–29% CO 2 . On the other hand, using untreated BP, a lower methane content (31–57%) and higher CO 2 (from 69-43%) were obtained. The volatile fatty acids profile was also variable and was less stochastic as was the methane yield.

Biodegradation of Kerosene by Aspergillus flavus Using Statistical Experimental Designs

ABSTRACT The ability of different local fungal isolates to degrade kerosene in liquid medium was studied. The results showed that the percent of kerosene degradation varied among the different tested fungi and that 60–96% of kerosene was degraded after 7 days in the presence of 0.2% (v/v) of Tween 80. The absence of the surfactant led to about 28.34% decrease of biodegradation. The degradation of 2% (v/v) of kerosene by the most efficient fungus (Aspergillus flavus) was significantly influenced by the incubation period and the composition of culture medium. Statistical experimental designs were used to optimize the process of kerosene degradation by the fungus. Under optimized medium compositions and culture conditions, A. flavus degraded kerosene (100%) after 111.3 h of incubation. Optimal conditions obtained in this work provided a solid foundation for further use of A. flavus in treatment of kerosene-polluted soil. The optimized conditions were applied to bioremediate 2.5% (v/w) kerosene-polluted soil by A. flavus, and the fungus efficiently degraded kerosene after 35 days of incubation.

Application of a five level central composite design to optimize operating conditions for electricity generation in a microbial fuel cell

Abstract In this work, a five level central composite design (Box–Wilson design) was employed to optimize the operating conditions for the generation of electricity in a microbial fuel cell. The following three variables were studied: temperature, initial anodic compartment pH and salt bridge component concentrations (agar and KCl). The optimal voltage yield was 17.34% greater than that observed under basal conditions and was achieved with a temperature of 32 °C, constant pH of 7.0 and salt bridge component concentrations of agar: 8.0 g/100 ml and KCL: 2.9 g/100 ml. The maximum recorded voltage at an external resistance of 30 Ω was 861.27 mV. The current density was 2.16 mA/m2, the power density was 1887.49 mW/m2, and the columbic efficiency was 24.12%.

Some cultural conditions for maximum bioextraction of beet pulp pectin

Abstract The bioextraction of the beet pulp pectin by Kluyveromyces marxianus was inhibited by ferrous sulphate penta hydrate and potassium dihydrogen phosphate, but stimulated by magnesium sulphate hepta hydrate salt. The pectin yields were also influenced by the addition of some enzymatic activators and some natural additives such as yeast extract. The characterization of both microbiologically and chemically extracted pectin samples indicated that the former had higher percentages of galacturonic acid, methoxyl groups and a higher degree of esterification and thus possesses superior qualities to chemically-extracted pectin.

Production of di-(2-ethylhexyl) phthalate by Bacillus subtilis AD35: Isolation, purification, characterization and biological activities

The emergence of extensive antibiotics resistant bacteria increased the demands for finding out new sources of antimicrobial agents. Marine niches were reported to be rich in many competent producers of significant bioactive compounds. On the course of screening program for new antimicrobials, a Bacillus strain was isolated from Alexandria sea shores, Egypt. According to the morphological, cultural and biochemical characteristics, 16S rRNA sequence analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the strain was identified as Bacillus subtilis and designated as B. subtilis AD35. One phthalate derivative namely Di-(2-ethylhexyl) phthalate (DEHP) was purified from the crude extract of B. subtilis AD35 by high performance liquid chromatography (HPLC) and the structural elucidation of this compound was confirmed on the basis of gas chromatography-mass spectroscopy (GC-MS), Fourier infrared spectroscopy (FT-IR) and UV spectrum. The results of MIC of the purified DEHP were as follow: 16 μg/ml (Salmonella typhimurium), 32 μg/ml (Methicillin resistant Staphylococcus aureus, MRSA), 0.25 μg/ml (Listeria monocytogenes), 0.5 μg/ml (Aeromonas hydrophila), 8 μg/ml (Staphylococcus aureus), 4 μg/ml (Staphylococcus epidermidis), 4 μg/ml (Escherichia coli), and 8 μg/ml (Pseudomonas aeruginosa). DEHP produced by B. subtilis AD35 up to a concentration of 2500 μg/ml exhibited no cytotoxic effect against normal Vero cells. In addition, it did not show an antiviral activity against HAV or a significant growth inhibitory effect toward human colorectal adenocarcinoma and human mammary gland adenocarcinoma cell-lines.

Statistically optimized ceftriaxone sodium biotransformation through Achromobacter xylosoxidans strain Cef6: an unusual insight for bioremediation

The present study underlines a unique promising approach toward efficient biotransformation of ceftriaxone sodium (Ceftx), a highly frequent prescribed cephalosporin antibiotic, by a newly bacterium namely Achromobacter xylosoxidans strain Cef6 isolated from Ceftx contaminated raw materials in pharmaceutical industries. A three step sequential statistical‐mathematical approach (Plackett‐Burman design [PBD], Central Composite Design [CCD], and ridge‐canonical analyses) was anticipated to optimize the biotransformation process. Ceftx concentration and medium volume: bottle volume ratio, two key determinants, significantly (p < 0.05) affected the process outcome deduced by regression analysis of PBD’ data. CCD and ridge‐canonical analyses localized the optimal levels of Ceftx concentration and medium volume: 250 ml bottle volume ratio to be 0.39 and 7.973 g Ceftx/L modified tryptic soy broth achieving Ceftx biotransformation (100%) after 39 h under aerobic static conditions at 30 °C, irrespectively deduced via HPLC analysis. Impressively, only one of five Ceftx byproducts was detected by the end of the biotransformation process. To the best of authors’ knowledge, this is the first report addressing a detailed study regarding efficient biotransformation of Ceftx by single bacterium not bacterial consortium under aerobic conditions. Present data would greatly encourage applying this approach for decontamination of some Ceftx contaminated environmental sites.

Some fermentation parameters influencing single cell protein production by Saccharomyces uvarum Y-1347

Abstract Saccharomyces uvarum Y-1347 was used for single cell protein (SCP) production in a medium containing clarified beet molasses as the sole carbon source. The growth and SCP production of the yeast were markedly affected by the composition of the culture medium, the pH, the age of the culture and the rate of shaking. Maximum yeast production and high SCP yield were achieved at the late growth phases (7-days old cultures), when the initial pH of the medium was adjusted to 7·0 and the culture was shaken at 250 rpm.