Optimized quantification of fatty acids in complex media using advanced analytical techniques.

RATIONALE: Various medium formulations contain essential fatty acids at concentrations ranging from 10 to 100 mg/L. Accurate and precise lipid measurement in media is crucial for monitoring media quality and conducting studies on lipids in the context of cell culture. This study employed two-dimensional gas chromatography (GC × GC) analyses to offer enhanced resolution, sensitivity, and separation performance compared to GC. METHODS: Quantification of fatty acid methyl esters (FAMEs) in a medium was conducted using GC × GC combined with a high-resolution mass spectrometer and flame ionization detector, considering potential interference from nonionic surfactant Tween 80, which was precipitated and removed by optimizing the concentration of cobalt thiocyanate (CTA) solution during pretreatment. This advanced analytical approach enabled identification of cis and trans isomers of identical molecular weights and determination of the location and number of double bonds in the same carbon number structure. RESULTS: Our analysis identified 36 FAMEs within the C6-C24 region, and a 5% CTA solution was optimal for efficient removal of Tween 80 during lipid extraction. Additionally, this advanced method minimized FAME contamination and loss during pretreatment, thereby significantly reducing the sample volume required to detect trace levels of FAMEs. This improvement led to a fatty acid recovery rate of 106% while maintaining the average relative standard deviation for the target FAMEs of about 3%. CONCLUSIONS: Our research paves the way for future investigation into medium quality control and the role of fatty acids in cell culture. This offers the possibility for economical and effective trace quantification of fatty acids in complex media.

Whole genome sequencing of a novel chitinolytic Streptomyces sp. RB7AG reveals it's chitosan production potential: optimization of the process through Taguchi experimental design.

This study reports about the De novo whole genome sequencing and analysis of a bacterial isolate Streptomyces sp. Strain. RB7AG, isolated from the sediments of Chilika Lake, Odisha, India. The genome report in this paper highlights a size of 7,708,681 bp and a GC content of 72.3%. It also consists of 7274 coding sequence, 66 tRNA and 4 rRNA. Furthermore, carbohydrate active enzyme analysis revealed that the strain RB7AG has 127 glycoside hydrolase family genes, which is well known for hydrolysis of glycosidic bond in complex sugars. Thus, exploiting these microorganisms for the production of chitosan can be an appropriate waste disposal method of choice. Chitosan being an important biomolecule that has various industrial applications. Hence, the study also sought to improve the culture conditions for the Streptomyces sp. strain RB7AG for generation, recovery, and characterization of chitosan. Utilizing the isolate, various low-cost nitrogen sources, including peptone, yeast extract, ammonium chloride, urea along with pH, media, metal ions and surfactant were assessed for chitosan synthesis. In this context, traditional methods such as One Factor One Time are more time consuming and expensive too. The current work aims to establish a methodology to optimize the degradation of chitin by the chitinolytic Streptomyces sp. strain RB7AG, isolated from lake sediment for the production of chitosan. More than one factor was considered at the time of experiments, and the knowledge was integrated into Taguchi statistical design to determine the contribution of the most important factors required to achieve the desired end product i.e. chitosan. Highest chitosan production (2.188 µg/ml) was observed in MSM media, 1.0% NaCl (w/v), 0.5% Yeast extract, 1% Ca2+ and 0.1% Tween 80 at pH 9. The whole genome analysis of RB7AG would help in determining the mechanism involved in the breakdown activity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03613-z.

Phage-tail-like bacteriocins as a biomedical platform to counter anti-microbial resistant pathogens.

Phage Tail Like bacteriocins (PTLBs) has been an area of interest in the last couple of years owing to their varied application against multi-drug resistant (MDR), anti-microbial resistant (AMR) pathogens and their evolutionary link with the dsDNA virus and bacteriophages. PTLBs are defective phages derived from Myoviridae and Siphoviridae phages, PTLBs are distinguished into R-type (Rigid type) characterized by a non-flexible contractile nanotube resembling Myoviridae phage contractile tails, and F-type (Flexible type) with a flexible non-contractile rod-like structure similar to Siphoviridae phages. In this review, we have discussed the structural association, mechanism, and characterization of PTLBs. Moreover, we have elucidated the symbiotic biological function and application of PTLBs against MDR and XDR pathogens and highlighted the evolutionary role of PTLBs. The difficulties that must be overcome to implement PTLBs clinically are also discussed. It is imperative that these issues be addressed by academics in future studies before being implemented in clinical settings. This article is novel in its way as it will not only provide us with a gateway that acts as a novel strategy for scholars to mitigate and control the uprising issue of AMR pathogens but also promote the development of clinical studies for PTLBs.

Exploration of genomic and functional features of chitinolytic bacterium Streptomyces chilikensis RC1830, isolated from Chilika Lake, India.

Streptomyces chilikensis RC1830 was previously isolated as a novel chitinolytic streptomycete from Chilika Lake, Odisha, India. The strain RC1830 is a representative member of the soil-dwelling, filamentous Streptomyces group that produces the majority of natural antibiotics and secondary metabolites. The objective of this work was to assess the chitin degradation ability and whole-genome sequence of Streptomyces chilikensis RC1830. TLC analysis of the fermentation product revealed that strain RC1830 can convert shrimp shell colloidal chitin to N-acetylated chitooligosaccharides (N-AcCOS). A genome-wide investigation of RC1830 was also carried out to investigate the genetic basis for chitin breakdown. The result showed that the RC1830 genome possesses a chromosome with 7,121,774 bp (73.2% GC). The genome consists of 6807 coding sequences, 69 tRNA, and 3 rRNA genes. Furthermore, carbohydrate-active enzyme (CAZyme) analysis revealed that RC1830 has 89 glycoside hydrolase family genes, which could modulate the enzymes involved in the degradation of chitin ultimately producing industrially important COS. The whole-genome information of RC1830 could emphasize the mechanism involved in the RC1830's chitin breakdown activity, endowing RC1830 with a promising alternative for COS production. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03184-5.

Phage delivered CRISPR-Cas system to combat multidrug-resistant pathogens in gut microbiome.

The Host-microbiome interactions that exist inside the gut microbiota operate in a synergistic and abnormal manner. Additionally, the normal homeostasis and functioning of gut microbiota are frequently disrupted by the intervention of Multi-Drug Resistant (MDR) pathogens. CRISPR-Cas (CRISPR-associated protein with clustered regularly interspersed short palindromic repeats) recognized as a prokaryotic immune system has emerged as an effective genome-editing tool to edit and delete specific microbial genes for the expulsion of bacteria through bactericidal action. In this review, we demonstrate many functioning CRISPR-Cas systems against the anti-microbial resistance of multiple pathogens, which infiltrate the gastrointestinal tract. Moreover, we discuss the advancement in the development of a phage-delivered CRISPR-Cas system for killing a gut MDR pathogen. We also discuss a combinatorial approach to use bacteriophage as a delivery system for the CRISPR-Cas gene for targeting a pathogenic community in the gut microbiome to resensitize the drug sensitivity. Finally, we discuss engineered phage as a plausible potential option for the CRISPR-Cas system for pathogenic killing and improvement of the efficacy of the system.

A simple and rapid colorimetric method for the estimation of chitosan produced by microbial degradation of chitin waste.

Chitin is one of the most abundant biopolymers present in the environment. Chitosan being its major derivative can be obtained by hydrolysis of chitin, especially by microbial degradation. Estimation of resulting chitosan produced by chitin degradation is crucial to the process. Usefulness of the method of Badawy (Badawy, 2012) for estimation of chitosan is limited by interference resulting from susceptibility to variation in the pH of the sample and thiobarbituric acid. This work presents an improvement of the method proposed by Badawy for colorimetric determination of chitosan by using 3, 5-Dinitrosalicylic acid (DNSA) reagent instead of thiobarbituric acid, after one step depolymerization and deamination of chitosan with sodium nitrite (NaNO2). Eventually colorimetric estimation was carried out at 540 nm. With the use of DNSA reagent, the limitation of thiobarbituric acid are overcome. This method is easy, cost effective, and sensitive for quantitative determination of chitosan. This new improved method was applied for evaluation and quantification of chitosan produced by microbial degradation of chitin waste by different novel Streptomyces strains.