Investigating the mechanism of interfacial tension reduction through the combination of low-salinity water and bacteria.

In the enhanced oil recovery (EOR) process, interfacial tension (IFT) has become a crucial factor because of its impact on the recovery of residual oil. The use of surfactants and biosurfactants can reduce IFT and enhance oil recovery by decreasing it. Asphaltene in crude oil has the structural ability to act as a surface-active material. In microbial-enhanced oil recovery (MEOR), biosurfactant production, even in small amounts, is a significant mechanism that reduces IFT. This study aimed to investigate fluid/fluid interaction by combining low biosurfactant values and low-salinity water using NaCl, MgCl2, and CaCl2 salts at concentrations of 0, 1000, and 5000 ppm, along with Geobacillus stearothermophilus. By evaluating the IFT, this study investigated different percentages of 0, 1, and 5 wt.% of varying asphaltene with aqueous bulk containing low-salinity water and its combination with bacteria. The results indicated G. Stearothermophilus led to the formation of biosurfactants, resulting in a reduction in IFT for both acidic and basic asphaltene. Moreover, the interaction between asphaltene and G. Stearothermophilus with higher asphaltene percentages showed a decrease in IFT under both acidic and basic conditions. Additionally, the study found that the interaction between acidic asphaltene and G. stearothermophilus, in the presence of CaCl2, NaCl, and MgCl2 salts, resulted in a higher formation of biosurfactants and intrinsic surfactants at the interface of the two phases, in contrast to the interaction involving basic asphaltene. These findings emphasize the dependence of the interactions between asphaltene and G. Stearothermophilus, salt, and bacteria on the specific type and concentration of asphaltene.

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLIGnGn and AFLISia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.

Heterologous production of hyaluronic acid in Nicotiana tabacum hairy roots expressing a human hyaluronan synthase 2.

Hyaluronic acid (HA), a unique polysaccharide with excellent Physico-chemical properties, is broadly used in pharmaceutical, biomedical, and cosmetic fields. It is widely present in all vertebrates, certain bacterial strains, and even viruses while it is not found in plants, fungi, and insects. HA is naturally synthesized by a class of integral membrane proteins called Hyaluronic acid synthase (HAS). Thus far, industrial production of HA is carried out based on either extraction from animal sources or large-scale microbial fermentation. The major drawbacks to using these systems are contamination with pathogens and microbial toxins. Recently, the production of HA through recombinant systems has received considerable attention. Plants are eco-friendly ideal expression systems for biopharmaceuticals production. In this study, the optimized human hyaluronic acid synthase2 (hHAS2) sequence was transformed into Nicotiana tabacum using Agrobacterium rhizogenes. The highest rhHAS2 concentration of 65.72 ng/kg (wet weight) in transgenic tobacco hairy roots was measured by the human HAS2 ELISA kit. The HA production in the transgenic hairy roots was verified by scanning electron microscope (SEM) and quantified by the HA ELISA kit. The DPPH radical scavenging activity of HA with the highest concentration of 0.56 g/kg (wet weight) showed a maximum activity of 46%. Gel Permeation Chromatography (GPC) analyses revealed the high molecular weight HA (HMW-HA) with about > 0.8 MDa.

Isopentenyl Transferase (IPT) Gene Transfer to Perennial Ryegrass Through Sonication-Assisted Agrobacterium-Mediated Transformation (SAAT), Vacuum and Heat Treatment.

The successful introduction of isopentenyl transferase (IPT) gene into perennial ryegrass, cultivars Numan and Grassland using Agrobacterium tumefaciens via three explants (callus, seed and meristem tip) under three individual experiment was evaluated. In the first experiment, the calli were inoculated with LBA4404 Agrobacterium strain under vacuum, heat and in combination of both at 42 °C for 5 min followed by vacuum treatment (390 mm Hg pressure) for 15 min. Sonication-assisted Agrobacterium-mediated transformation (SAAT) was applied for seed and meristem tip transformation of perennial ryegrass for the first time. Results showed positive effects of heat treatment on transformation efficiency during Agro-infection in both cultivars. However, heat shock treatment was more effective in 'Grassland' than 'Numan' (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of 'Grassland' and 'Numan' based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in 'Grassland' and 'Numan' under SAAT method reached to 37.5% and 16.65%, respectively. Results of these experiments revealed that LBA4404 strain was more efficient than GV3101 in transformation of both perennial ryegrass cultivars. The DNA-blot analysis confirmed that a single T-DNA copy of the IPT gene was integrated into the genomic DNA of the positive transgenic T0 plants which obtained from callus and meristem tip explants of 'Grassland' after heat and SAAT treatment, respectively. Because monocots are not the host of Agrobacterium tumefaciens, this novel protocol can be used in further experiments on genetic transformation of perennial ryegrass cultivars.

Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification.

BACKGROUND: Lactoferricin (LFcin) is a strong cationic peptide released from the N-terminus of lactoferrin by gastric pepsin digestion. LFcin has some important properties, including high antimicrobial activity. To date, lactoferricins have been isolated and characterised from various animal species, but not from camel. The aim of this study was to characterise and express recombinant camel lactoferricin (LFcinC) in Pichia pastoris and investigate its antimicrobial activity. RESULTS: After methanol induction, LFcinC was expressed and secreted into a culture broth medium and the results determined by concentrated supernatant culture medium showed high antimicrobial activity against the following microorganisms: Escherichia coli PTCC 1330 (ATCC 8739), Staphylococcus aureus PTCC 1112 (ATCC 6538), Pseudomonas aeruginosa PTCC 1074 (ATCC 9027), Bacillus subtilis PTCC 1023 (ATCC 6633), and Candida albicans PTCC 5027 (ATCC 10231). Thermal stability was clarified with antibacterial activity against Escherichia coli PTCC 1330 (ATCC 8739). CONCLUSION: Results confirmed that camel lactoferricin had suitable antimicrobial activity and its production by Pichia pastoris can be used for recombinant production.

Statistical screening of medium components for recombinant production of Pseudomonas aeruginosa ATCC 9027 rhamnolipids by nonpathogenic cell factory Pseudomonas putida KT2440.

Rhamnolipids (RLs) produced by the opportunistic human pathogen Pseudomonas aeruginosa are considered as potential candidates for the next generation of surfactants. Large-scale production of RLs depends on progress in strain engineering, medium design, operating strategies, and purification procedures. In this work, the rhlAB genes extracted from a mono_RLs_producing strain of P. aeruginosa (ATCC 9027) were introduced to an appropriate safety host Pseudomonas putida KT2440. The capability of the recombinant strain was evaluated in various media. As a prerequisite for optimal medium design, a set of 32 experiments was performed in two steps for screening a number of macro-nutritional compounds. In the experiments, a two-level fractional factorial design resolution IV was followed by a two-level full factorial one. By means of this approach, it was observed that glycerol, yeast extract, and peptone have significant positive influence on recombinant RLs production while the yeast extract/peptone two-factor and glycerol/yeast extract/peptone three-factor interactions have considerable negative effects. A wide range of variation from 0 to 570 mg/l was obtained for RLs production during the screening experiments indicating the importance of medium optimization. The results point out the opportunity for possible higher yields of RLs through further screening, mixture/combined mixture designs, and high-cell-density cultivations.