Production and structural characterization of eco-friendly bioemulsifier SC04 from Saccharomyces cerevisiae strain MYN04 with potential applications.

BACKGROUND: Bioemulsifiers are natural or microbial-based products with the ability to emulsify hydrophobic compounds in water. These compounds are biodegradable, eco-friendly, and find applications in various industries. RESULTS: Thirteen yeasts were isolated from different sources in Alexandria, Egypt, and evaluated for their potential to produce intracellular bioemulsifiers. One yeast, isolated from a local market in Egypt, showed the highest emulsification index (EI24) value. Through 26S rRNA sequencing, this yeast was identified as Saccharomyces cerevisiae strain MYN04. The growth kinetics of the isolate were studied, and after 36 h of incubation, the highest yield of cell dry weight (CDW) was obtained at 3.17 g/L, with an EI24 of 55.6%. Experimental designs were used to investigate the effects of culture parameters on maximizing bioemulsifier SC04 production and CDW. The study achieved a maximum EI24 of 79.0 ± 2.0%. Furthermore, the crude bioemulsifier was precipitated with 50% ethanol and purified using Sephadex G-75 gel filtration chromatography. Bioemulsifier SC04 was found to consist of 27.1% carbohydrates and 72.9% proteins. Structural determination of purified bioemulsifier SC04 was carried out using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance spectroscopy (NMR). FTIR spectroscopy revealed characteristic bands associated with carboxyl and hydroxyl groups of carbohydrates, as well as amine groups of proteins. HPLC analysis of monosaccharide composition detected the presence of mannose, galactose, and glucose. Physicochemical characterization of the fraction after gel filtration indicated that bioemulsifier SC04 is a high molecular weight protein-oligosaccharide complex. This bioemulsifier demonstrated stability at different pH values, temperatures, and salinities. At a concentration of 0.5 mg/mL, it exhibited 51.8% scavenging of DPPH radicals. Furthermore, in vitro cytotoxicity evaluation using the MTT assay revealed a noncytotoxic effect of SC04 against normal epithelial kidney cell lines. CONCLUSIONS: This study presents a new eco-friendly bioemulsifier, named SC04, which exhibits significant emulsifying ability, antioxidant and anticancer properties, and stabilizing properties. These findings suggest that SC04 is a promising candidate for applications in the food, pharmaceutical, and industrial sectors.

Semidry acid hydrolysis of cellulose sustained by autoclaving for production of reducing sugars for bacterial biohydrogen generation from various cellulose feedstock.

Cellulosic biowastes are one of the cheapest and most abundant renewable organic materials on earth that can be, subsequent to hydrolysis, utilized as an organic carbon source for several fermentation biotechnologies. This study was devoted to explore a semidry acid hydrolysis of cellulose for decreasing the cost and ionic strength of the hydrolysate. For semidry acid hydrolysis, cellulose was just wetted with HCl (0 to 7 M) and subjected to autoclaving. The optimum molar concentration of HCl and period of autoclaving for semidry acid hydrolysis of cellulose were 6 M and 50 min respectively. Subsequent to the semidry acid hydrolysis with a minimum volume of 6 M HCl sustained by autoclaving, the hydrolysate was diluted with distilled water and neutralized with NaOH (0.5 M). The reducing sugars produced from the semidry acid hydrolysis of cellulose was further used for dark fermentation biohydrogen production by Escherichia coli as a representative of most hydrogen producing eubacteria which cannot utilize non-hydrolyzed cellulose. An isolated E. coli TFYM was used where this bacterium was morphologically and biochemically characterized and further identified by phylogenetic 16S rRNA encoding gene sequence analysis. The reducing sugars produced by semidry acid hydrolysis could be efficiently utilized by E. coli producing 0.4 mol H2 mol-1 hexose with a maximum rate of hydrogen gas production of 23.3 ml H2 h-1 L-1 and an estimated hydrogen yield of 20.5 (L H2 kg-1 dry biomass). The cheap cellulosic biowastes of wheat bran, sawdust and sugarcane bagasse could be hydrolyzed by semidry acid hydrolysis where the estimated hydrogen yield per kg of its dry biomass were 36, 18 and 32 (L H2 kg-1 dry biomass) respectively indicating a good feasibility of hydrogen production from reducing sugars prepared by semidry acid hydrolysis of these cellulosic biowastes. Semidry acid hydrolysis could also be effectively used for hydrolyzing non-cellulosic polysaccharides of dry cyanobacterial biomass. The described semidry acid hydrolysis of cellulosic biowastes in this study might be applicable not only for bacterial biohydrogen production but also for various hydrolyzed cellulose-based fermentation biotechnologies.

Characterization of an efficient extracellular cyanophycinase and its encoding cphEStrept. gene from Streptomyces pratensis strain YSM.

Until now, no enzymes were described that hydrolyze cyanophycin granular protein (CGP) from a species of the genus Streptomyces. An isolate able to hydrolyze CGP was identified as Streptomyces pratensis strain YSM. The CGPase from S. pratensis strain YSM had an optimum activity at 42 °C and pH 8.5, and was able to degrade CGP at a rate of 12 ±â€¯0.3 µg/mL min. Additionally, this CGPase hydrolyzes water-soluble CGP significantly faster than water-insoluble CGP. The molecular mass of CGPase subunits from S. pratensis strain YSM as determined by SDS-PAGE was about 43 kDa, and the enzyme was entirely inhibited by serine-protease inhibitors. The CGPase coding gene (cphEStrept.) was amplified from genomic DNA using primers designed form consensus sequence of putative CGPase sequences. The cphEStrept. was 1427 bp encoding a CGPase of 420 amino acids that showed about 44% and 22% similarities to CGPase from Pseudomonas anguilliseptica BI and Synechocystis sp. PCC 6803, respectively. The catalytic triad and serine-protease residues (GXSXG) were identified in the CphEStrept. sequence. Dipeptides and tetrapeptides were identified as hydrolysis products. Biotechnological exploitation of S. pratensis strain YSM for CGPase production might have an advantage due to the reduction of separation costs and its ability to degrade CGP in phosphate buffer saline using actively growing or resting cells.

Impact of nanoparticles on transcriptional regulation of catabolic genes of petroleum hydrocarbon-degrading bacteria in contaminated soil microcosms.

This study was conducted to determine what effects nanoparticles (NPs) like TiO2 , ZnO, and Ag may pose on natural attenuation processes of petroleum hydrocarbons in contaminated soils. The solid NPs used were identified using x-ray diffraction technique and their average size was certified as 18.2, 16.9, and 18.3 nm for Ag-NPs, ZnO-NPs, and TiO2 -NPs, respectively. NPs in soil microcosms behave differently where it was dissolved as in case of Ag-NPs, partially dissolved as in ZnO-NPs or changed into other crystalline phase as in TiO2 -NPs. In this investigation, catabolic gene encoding catechol 2,3 dioxygenase (C23DO) was selected specifically as biomarker for monitoring hydrocarbon biodegradation potential by measuring its transcripts by RT-qPCR. TiO2 -NPs amended microcosms showed almost no change in C23DO expression profile or bacterial community which were dominated by Bacillus sp., Mycobacterium sp., Microbacterium sp., Clostridium sp., beside uncultured bacteria, including uncultured proteobacteria, Thauera sp. and Clostridia. XRD pattern suggested that TiO2 -NPs in microcosms were changed into other non-inhibitory crystalline phase, consequently, showing the maximum degradation profile for most low molecular weight oil fractions and partially for the high molecular weight ones. Increasing ZnO-NPs concentration in microcosms resulted in a reduction in the expression of C23DO with a concomitant slight deteriorative effect on bacterial populations ending up with elimination of Clostridium sp., Thauera sp., and uncultured proteobacteria. The oil-degradation efficiency was reduced compared to TiO2 -NPs amended microcosms. In microcosms, Ag-NPs were not detected in the crystalline form but were available in the ionic form that inhibited most bacterial populations and resulted in a limited degradation profile of oil, specifically the low molecular weight fractions. Ag-NPs amended microcosms showed a significant reduction (80%) in C23DO gene expression and a detrimental effect on bacterial populations including key players like Mycobacterium sp., Microbacterium sp., and Thauera sp. involved in the biodegradation of petroleum hydrocarbons.

The novel oleaginous bacterium Sphingomonas sp. EGY1 DSM 29616: a value added platform for renewable biodiesel.

Oleaginous microorganisms are regarded as efficient, renewable cell factories for lipid biosynthesis, a biodiesel precursor, to overwhelm the cosmopolitan energy crisis with affordable investment capital costs. Present research highlights production and characterization of lipids by a newly isolated oleaginous bacterium, Sphingomonas sp. EGY1 DSM 29616 through an eco-friendly approach. Only sweet whey [42.1% (v/v)] in tap water was efficiently used as a growth medium and lipid production medium to encourage cell growth and trigger lipid accumulation simultaneously. Cultivation of Sphingomonas sp. EGY1 DSM 29616 in shake flasks resulted in the accumulation of 8.5 g L-1 lipids inside the cells after 36 h at 30 °C. Triglycerides of C16:C18 saturated and unsaturated fatty acids showed a similar pattern to tripalmitin or triolein; deduced from gas chromatography (GC), thin layer chromatography (TLC), and Matrix-assisted laser desorption/ionization time-of-flight-mass spectra analysis (MALDI-TOF-MS) analyses. Batch cultivation 2.5 L in a laboratory scale fermenter led to 13.8 g L-1 accumulated lipids after 34 h at 30 °C. Present data would underpin the potential of Sphingomonas sp. EGY1 DSM 29616 as a novel renewable cell factory for biosynthesis of biodiesel.

Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

Pilot-scale production of fatty acid ethyl esters by an engineered Escherichia coli strain harboring the p(Microdiesel) plasmid.

Fatty acid ethyl esters (FAEEs) were produced in this study by the use of an engineered Escherichia coli p(Microdiesel) strain. Four fed-batch pilot scale cultivations were carried out by first using glycerol as sole carbon source for biomass production before glucose and oleic acid were added as carbon sources. Cultivations yielded a cell density of up to 61 +/- 3.1 g of cell dry mass (CDM) per liter and a maximal FAEE content of 25.4% +/- 1.1% (wt/wt) of CDM.

Large-scale production of poly(3-hydroxyoctanoic acid) by Pseudomonas putida GPo1 and a simplified downstream process.

The suitability of Pseudomonas putida GPo1 for large-scale cultivation and production of poly(3-hydroxyoctanoate) (PHO) was investigated in this study. Three fed-batch cultivations of P. putida GPo1 at the 350- or 400-liter scale in a bioreactor with a capacity of 650 liters were done in mineral salts medium containing initially 20 mM sodium octanoate as the carbon source. The feeding solution included ammonium octanoate, which was fed at a relatively low concentration to promote PHO accumulation under nitrogen-limited conditions. During cultivation, the pH was regulated by addition of NaOH, NH(4)OH, or octanoic acid, which was used as an additional carbon source. Partial O(2) pressure (pO(2)) was adjusted to 20 to 40% by controlling the airflow and stirrer speed. Under the optimized conditions, P. putida GPo1 was able to grow to cell densities as high as 18, 37, and 53 g cells (dry mass) (CDM) per liter containing 49, 55, and 60% (wt/wt) of PHO, respectively. The resulting 40 kg CDM from these three cultivations was used directly for extraction of PHO. Three different methods of extraction of PHO were applied. From these, only acetone extraction showed better performance and resulted in 94% recovery of the PHO contents of cells. A novel mixture of precipitation solvents composed of 70% (vol/vol) methanol and 70% (vol/vol) ethanol was identified in this study. The ratio of PHO concentrate to the mixture was 0.2:1 (vol/vol) and allowed complete precipitation of PHO as white flakes. However, at a ratio of 1:1 (vol/vol) of the solvent mixture to PHO concentrate, a highly purified PHO was obtained. Precipitation yielded a dough-like polymeric material which was cast into thin layers and then shredded into small strips to allow evaporation of the remaining solvents. Gas chromatographic analysis revealed a purity of about 99% +/- 0.2% (wt/wt) of the polymer, which consisted mainly of 3-hydroxyoctanoic acid (96 mol%).

Engineering the genotype of Acinetobacter sp. strain ADP1 to enhance biosynthesis of cyanophycin.

To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. DeltaastA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.

Protamylasse, a residual compound of industrial starch production, provides a suitable medium for large-scale cyanophycin production.

Protamylasse is a residual compound occurring during the industrial production of starch from potatoes. It contains a variety of nutrients and all necessary minerals and could be used as a carbon, nitrogen, and energy source for the growth of bacteria and also for cyanophycin (CGP) biosynthesis. Media containing protamylasse as the sole compound diluted only in water were therefore examined for their suitability in CGP production. Among various bacterial strains investigated in this study, a recombinant strain of Escherichia coli DH1 harboring plasmid pMa/c5-914::cphA6803, which carries the cyanophycin synthetase structural gene (cphA) from Synechocystis sp. strain PCC6803, was found to be most suitable. Various cultivation conditions for high CGP contents were first optimized in shake flask cultures. The optimized conditions were then successfully applied to 30- and 500-liter fermentation scales in stirred tank reactors. A maximum CGP content of 28% (wt/wt) CGP per cell dry matter was obtained in 6% (vol/vol) protamylasse medium at an initial pH of 7.0 within a cultivation period of only 24 h. The CGP contents obtained with this recombinant strain employing protamylasse medium were higher than those obtained with the same strain cultivated in mineral salts medium or in expensive commercial complex media such as Luria-Bertani or Terrific broth. It was shown that most amino acids present in the protamylasse medium were almost completely utilized by the cells during cultivation. Exceptions were alanine, tryptophan, tyrosine, and most interestingly, arginine. Furthermore, CGP was easily isolated from protamylasse-grown cells by applying the acid extraction method. The CGP exhibited a molecular mass of about 26 to 30 kDa and was composed of 50% (mol/mol) aspartate, 46% (mol/mol) arginine, and 4% (mol/mol) lysine. The use of cheap residual protamylasse could contribute in establishing an economically and also ecologically feasible process for the biotechnological production of CGP.

Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1.

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30 degrees C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 microg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::OmegaKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.