Bioproduction of yeast single cell oil with acute oral toxicity study intended for edible oil application.

Human nutrition and health rely on edible oils. Global demand for edible oils is expanding, necessitating the discovery of new natural oil sources subjected to adequate quality and safety evaluation. However, in contrast to other agricultural products, India's edible oil supply is surprisingly dependent on imports. The microbial oil is generated by fermentation of oleaginous yeast Rhodotorula mucilaginosa IIPL32 MTCC 25056 using biodiesel plant byproduct crude glycerol as a fermentable carbon source. Enriched with monounsaturated fatty acid, nutritional indices mapping based on the fatty acid composition of the yeast SCO, suggested its plausible use as an edible oil blend. In the present study, acute toxicity evaluation of the yeast SCO in C57BL/6 mice has been performed by randomly dividing the animals into 5 groups with 50, 300, 2000, and 5000 mg/Kg yeast SCO dosage, respectively, and predicted the median lethal dose (LD50). Detailed blood biochemistry and kidney and liver histopathology analyses were also reported. The functions of the liver enzymes were also evaluated to check and confirm the anticipated toxicity. To determine cell viability and in vitro biocompatibility, the 3T3-L1 cell line and haemolysis tests were performed. The results suggested the plausible use of yeast SCO as an edible oil blend due to its non-toxic nature in mice models.

Efficient valorization of feather waste by Bacillus cereus IIPK35 for concomitant production of antioxidant keratin hydrolysate and milk-clotting metallo-serine keratinase.

Keratinase production by Bacillus cereus IIPK35 was investigated under solid-state fermentation (SSF) and the maximum titer of 648.28 U/gds was revealed. Feather hydrolysates obtained from SSF exhibited paramount antioxidant properties in ABTS [2,2'-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid], FRAP [Ferric ion reducing antioxidant power], and DPPH [2,2,-Diphenyl-1-picrylhydrazyl] assay. The keratinase was purified up to homogeneity have a molecular weight of 42 kDa, and showed its stability between pH 6.5-10.0 and temperature 35-60 °C with optimum enzyme activity at pH 9.0 and 55 °C. The catalytic indices viz. Km of 9.8 mg/ml and Vmax of 307.7 µmol/min for keratin were determined. Besides keratin, the enzyme displayed broad and proteolytic activity towards other proteinaceous substrates such as casein, skim milk, gelatin, and bovine serum albumin. Pure keratinase activity was stimulated in presence of Ca2+ and Mg2+ ions, while it was strongly inhibited by both iodoacetamide and EDTA, indicating it to be a metallo-serine protease in nature. Circular dichroism study endorses the structural stability of the secondary structure at the said range of pH and temperature. The IIPK35 keratinase is non-cytotoxic in nature, shows remarkable storage stability and is stable in presence of Tween 80, Triton X 100, and sodium sulfite. Furthermore, it showed excellent milk clotting potential (107.6 Soxhlet Unit), suggesting its usefulness as an alternative milk clotting agent in the dairy industry. This study unlocks a new gateway for keratinase investigation in SSF using chicken feathers as substrate and biochemical and biophysical characterization of keratinase for better understanding and implication in industrial applications.

Lipids of Rhodotorula mucilaginosa IIPL32 with biodiesel potential: Oil yield, fatty acid profile, fuel properties.

This study analyzes the single cell oil (SCO), fatty acid profile, and biodiesel fuel properties of the yeast Rhodotorula mucilaginosa IIPL32 grown on the pentose fraction of acid pre-treated sugarcane bagasse as a carbon source. The yeast biomass from nitrogen limiting culture conditions (15.3 g L-1 ) was able to give the SCO yield of 0.17 g g-1 of xylose consumed. Acid digestion, cryo-pulverization, direct in situ transesterification, and microwave assisted techniques were evaluated in comparison to the Soxhlet extraction for the total intracellular yeast lipid recovery. The significant differences were observed among the SCO yield of different methods and the in situ transesterification stood out most for effective yeast lipid recovery generating 97.23 mg lipid as FAME per gram dry biomass. The method was fast and consumed lesser solvent with greater FAME yield while accessing most cellular fatty acids present. The yeast lipids showed the major presence of monounsaturated fatty esters (35-55%; 18:1, 16:1) suitable for better ignition quality, oxidative stability, and cold-flow properties of the biodiesel. Analyzed fuel properties (density, kinematic viscosity, cetane number) of the yeast oil were in good agreement with international biodiesel standards. The sugarcane bagasse-derived xylose and the consolidated comparative assessment of lab scale SCO recovery methods highlight the necessity for careful substrate choice and validation of analytical method in yeast oil research. The use of less toxic co-solvents together with solvent recovery and recycling would help improve process economics for sustainable production of biodiesel from the hemicellulosic fraction of cheap renewable sources.

Ethanol fermentation from molasses at high temperature by thermotolerant yeast Kluyveromyces sp. IIPE453 and energy assessment for recovery.

High temperature ethanol fermentation from sugarcane molasses B using thermophilic Crabtree-positive yeast Kluyveromyces sp. IIPE453 was carried out in batch bioreactor system. Strain was found to have a maximum specific ethanol productivity of 0.688 g/g/h with 92 % theoretical ethanol yield. Aeration and initial sugar concentration were tuning parameters to regulate metabolic pathways of the strain for either cell mass or higher ethanol production during growth with an optimum sugar to cell ratio 33:1 requisite for fermentation. An assessment of ethanol recovery from fermentation broth via simulation study illustrated that distillation-based conventional recovery was significantly better in terms of energy efficiency and overall mass recovery in comparison to coupled solvent extraction-azeotropic distillation technique for the same.

Water footprint and wastewater quality assessment of yeast single cell oil production: Gate to gate approach for industrial water sustainability.

Effective water resource utilization and sustainability for industrial operations is a growing concern. With increased industrial water demand, abstraction and water quality changes are rising. In India, distilleries generate more than 40.4 billion litres of effluent daily within the fermentation industry. Water, a public good with market and opportunity costs, needs effective mapping and management. Emerging distillery processes such as yeast lipid fermentation, if developed along with water sustainability, could aid in advancing water resource management. In the scope of this idea, the present study focuses on assessing the water footprint and water quality mapping for Rhodotorula mucilaginosa IIPL32 lipid production using crude glycerol, a by-product of the biodiesel industry. The assessment was based on primary data generated during the 500 L plant scale operation. The process's blue water footprint was assessed by applying a chain-summation approach, and the grey water requirement was determined by measuring water quality parameters for the effluent streams. The process's net blue and grey water footprint were estimated to be 3.87 and 23.66 m3 water/kg of lipid, respectively. Water quality index ratings were identified for all the respective water streams within the processing system, and human risk factors were estimated. The results suggested proper treatment of the spent broth, whereas the secondary effluent stream from cleaning operations could be reutilized within the system. Quality mapping also suggested that the effluent's high organic and mineral load can be processed for water and material recovery, which may significantly reduce the process's grey water and pollution load.

Time-resolved transcriptomic profile of oleaginous yeast Rhodotorula mucilaginosa during lipid and carotenoids accumulation on glycerol.

The study reports the exploration of the transcriptome landscape of the red oleaginous yeast Rhodotorula mucilaginosa IIPL32 coinciding with the fermentation kinetics of the yeast cultivated in a two-stage fermentation process to exploit the time-series approach to get the complete transcripts picture and reveal the persuasive genes for fatty acid and terpenoid synthesis. The finding displayed the molecular drivers with more than 2-fold upregulation in the nitrogen-limited stage than in the nitrogen-excess stage. The rate-limiting diphosphomevalonate decarboxylase, acetylCoA-citrate lyase, and acetyl-CoA C-acetyltransferase were significant in controlling the metabolic flux in the synthesis of reduced compounds, and acetoacetyl-CoA synthase, 3-ketoacyl-acyl carrier-protein reductase, and ß-subunit enoyl reductase catalyze the key starting steps of lipids or terpenoid synthesis. The last two catalyze essential reduction steps in fatty acid synthesis. These enzymes would be the prime targets for the metabolic engineering of the oleaginous yeast for enhanced fatty acids and terpenoid production.

Enhanced remediation of polyaromatic hydrocarbon using agro-industrial waste for biofuel production and environmental pollution mitigation.

In the present study, attention has been paid to the development of economically feasible strategies for enhanced remediation of anthracene and its conversion into biofuels. The strategies developed (B1, B2, B3, and B4) include bagasse and lipid-producing strain Rhodotorula mucilagenosa IIPL32 synthesizing surface active metabolites. The results indicate the highest production of surface-active metabolites in strategies B2, B3, and B4 along with a maximum biodegradation rate. GC-MS analysis affirmed the conversion of anthracene into phthalic acid in all the strategies. Biofuel quality of the lipid produced by the strain showed higher cetane number and improved cold flow property indicating the efficiency of the developed strategies for the production of commercial grade biodiesel. Furthermore, the phytotoxicity study of the spent wash revealed that 50% and 75% diluted spent wash were non-toxic and can be employed for ferti-irrigation. Thus, the study signifies the development of an economically feasible process that can be commercially employed in biofuel industries.

An insight into omics analysis and metabolic pathway engineering of lignin-degrading enzymes for enhanced lignin valorization.

Lignin, a highly heterogeneous polymer of lignocellulosic biomass, is intricately associated with cellulose and hemicellulose, responsible for its strength and rigidity. Lignin decomposition is carried out through certain enzymes derived from microorganisms to promote the hydrolysis of lignin. Analyzing multi-omics data helps to emphasize the probable value of fungal-produced enzymes to degrade the lignocellulosic material, which provides them an advantage in their ecological niches. This review focuses on lignin biodegrading microorganisms and associated ligninolytic enzymes, including lignin peroxidase, manganese peroxidase, versatile peroxidase, laccase, and dye-decolorizing peroxidase. Further, enzymatic catalysis, lignin biodegradation mechanisms, vital factors responsible for lignin modification and degradation, and the design and selection of practical metabolic pathways are also discussed. Highlights were made on metabolic pathway engineering, different aspects of omics analyses, and its scope and applications to ligninase enzymes. Finally, the advantages and essential steps of successfully applying metabolic engineering and its path forward have been addressed.

Remediation and detoxification of oil contaminated marine intertidal sites through lipopeptide assisted washing strategy: An experimental and kinetic validation approach.

This paper presents a tightly coupled experimental and kinetic approach for efficient remediation of oil spill from contaminated marine intertidal zone surface through a methodical strategy that deals with biosurfactant mediated washing strategy. The study deals with production, optimization and characterization of lipopeptide biosurfactant from Bacillus subtilis T1 and its application in remediation of oil contaminants from mimic model system of various marine intertidal zone i.e. woodland-Group1, saltmarsh-Group2, mangrove-Group3 and mudflats-Group4. Results demonstrates enhanced washing performance with oil desorption rate of 35 % in Group 4, 17.22 %, 15.6 % and 11 % in Group 3, 2 and 1 along with bio surfactant recovery rate of 41 %, 48.7 %, 51.71 % and 50.3 % respectively. Further, the washing strategy was efficient in soil detoxification with highest rate in Group 4. The kinetic validation depicts good match among experimental data and Lagergren pseudo second order data.

Characterization of the de-oiled yeast biomass for plausible value mapping in a biorefinery perspective.

Oleaginous yeast fermentation process has gained attention for yeast single cell oil production. However, after lipid extraction, the leftover de-oiled yeast biomass has not been investigated in detail for its suitability for thermochemical conversion. To understand the structural and morphological changes, the comparative characterization of yeast and de-oiled yeast biomass before and post lipid extraction is necessary. The present study investigates the characteristics of an oleaginous yeast Rhodotorula mucilaginosa IIPL32's de-oiled biomass for its potential utilization. FTIR, XRD, SEM, EDX, XRF, and TGA analysis were performed to understand the biomass properties. Increased surface area and structural changes were observed in de-oiled yeast biomass with an increase in crystallinity, indicating chitosan availability. Maximum thermal degradation temperature was reduced to 260 °C for de-oiled yeast biomass from 300 °C for dried yeast after lipid extraction. The findings favored de-oiled yeast biomass for multiple applications that merit further detailed investigation with different thermochemical interventions.

Pyrolysis of de-oiled yeast biomass of Rhodotorula mucilaginosa IIPL32: Kinetics and thermodynamic parameters using thermogravimetric analysis.

The increasing demand for natural resources has highlighted the need to search for unutilized carbon resource that satisfy the demand and pose a minor threat to the environment. Yeast is a microbe with large industrial applications, and the biomass leftover after fermentation needs utilization for achieving increased efficiency. De-oiled yeast biomass (DYB), the residue after yeast lipid extraction, has not yet been evaluated for its potential application in the pyrolysis process. The present study was performed to understand its detailed pyrolysis kinetics. The observed activation energy (87-216 KJ/mol), random nucleation mechanism, pre-exponential factor (7.87 × 1031-3.24 × 1031/min), and thermodynamic profile showed the DYB pyrolysis process to be feasible. .

Understanding the molecular interactions of inhibitors against Bla1 beta-lactamase towards unraveling the mechanism of antimicrobial resistance.

This study evaluated the inhibitory potential of various beta-lactamase inhibitors such as mechanism-based inhibitors (MBIs), carbapenems, monobactam, and non-beta-lactam inhibitors against Bla1, a class-A beta-lactamase encoded by Bacillus anthracis. The binding potential of different inhibitors was estimated using competitive kinetic assay, isothermal titration calorimetry, and Biolayer interferometry. We observed that tazobactam has better inhibition among other MBIs with a characteristics inhibition dissociation constant of 0.51 ± 0.13 µM. Avibactam was also identified as good inhibitor with an inhibition efficiency of 0.6 ± 0.04 µM. All the MBIs (KD = 1.90E-04 M, 2.05E-05 M, 3.55E-04 M for clavulanate, sulbactam and tazobactam) showed significantly better binding potential than carbapenems (KD = 1.02E-03 M, 2.74E-03 M, 1.24E-03 M for ertapenem, imipenem and biapenem respectively). Molecular dynamics simulations were carried out using Bla1-inhibitor complexes to understand the dynamics and stability. The minimum inhibitory concentration (MIC) was carried out by taking various substrates and inhibitors, and later it was followed by cell viability assay. Together, our study helps develop a proper understanding of Bla1 beta-lactamase and its interaction with inhibitory molecules. This study would facilitate comprehending the catalytic divergence of beta-lactamases and the newly emergent resistant strains, focusing on the new generation of therapeutics being less prone to antimicrobial resistance.

Variations in the SDN Loop of Class A Beta-Lactamases: A Study of the Molecular Mechanism of BlaC (Mycobacterium tuberculosis) to Alter the Stability and Catalytic Activity Towards Antibiotic Resistance of MBIs.

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for an immediate search for novel treatment strategies. Recently, BlaC, the principal beta-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. BlaC belongs to Ambler class A, which is generally susceptible to the beta-lactamase inhibitors currently used in clinics: tazobactam, sulbactam, and clavulanate. Alterations at Ser130 in conserved SDN loop confer resistance to mechanism-based inhibitors (MBIs) commonly observed in various clinical isolates. The absence of clinical evidence of S130G conversion in M. tuberculosis draws our attention to build laboratory mutants of S130G and S130A of BlaC. The study involving steady state, inhibition kinetics, and fluorescence microscopy shows the emergence of resistance against MBIs to the mutants expressing S130G and S130A. To understand the molecular reasoning behind the unavailability of such mutation in real life, we have used circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), molecular dynamics (MD) simulation, and stability-based enzyme activity to compare the stability and dynamic behaviors of native and S130G/A mutant form of BlaC. A significant decrease in melting temperature (BlaC T M 60°C, S130A T M 50°C, and S130G T M 45°C), kinetic instability at higher temperature, and comparative dynamic instability correlate the fact that resistance to beta-lactam/beta-lactamase inhibitor combinations will likely not arise from the structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be potentially applied as a part of a successful treatment regimen against M. tuberculosis.

Effect of utilization of crude glycerol as substrate on fatty acid composition of an oleaginous yeast Rhodotorula mucilagenosa IIPL32: Assessment of nutritional indices.

This work studied the use of crude glycerol obtained from biodiesel industry as substrate to generate yeast lipid from Rhodotorula mucilagenosa IIPL32 MTCC 25056. Crude glycerol is a low value by product obtained from biodiesel industry. Rhodotorula mucilagenosa IIPL32 MTCC 25056 was evaluated for its potential to produce lipid using crude glycerol as sole source of carbon. Under nitrogen limiting condition a lipid and biomass content of 5.6 g/L and19.7 g/L were obtained from crude glycerol. The fatty acid profile was found to be interestingly rich in oleic acid (61.88%), linoleic acid (16.17%) and linolenic acid (1.03%) comprising ~80% of MUFA and PUFA of total lipid. Further, evaluations were attempted to compare MUFA rich yeast lipid against different plant-borne edible oils commonly used in India. In this study, nutritional indices were calculated to check feasibility of using yeast oil as a plausible blend to edible oil.

An insight into the complete biophysical and biochemical characterization of novel class A beta-lactamase (Bla1) from Bacillus anthracis.

Bacillus anthracis, a potent pathogen of anthrax is becoming resistant to many beta-lactam antibiotics because of the expression of two chromosomally encoded beta-lactamases Bla1 and Bla2. Bla1 is a class A beta-lactamase whereas Bla2 is a Metallo beta-lactamase. In the current study, we have attempted in-detailed characterization of Bla1 beta-lactamase by taking interdisciplinary approaches. Our study includes structure and sequence comparison of this enzyme with other members of the class, to know the conservation pattern that includes active site residues, secondary structure, conserved fold, evolutionary relationships, etc. Dynamic characterizations of the enzyme, unfolding kinetics were determined with the help of Molecular dynamics simulation. Detailed enzyme stability and catalytic activity towards various physical (Temperature and pH), and chemical parameters (Urea, GnHCl) were performed. Together, our study helps to develop a proper understanding of this beta-lactamase by characterizing its biochemical, biophysical, dynamic, kinetic and thermodynamic properties. This would help contribute towards a better understanding of beta-lactamase based AMR emergence.

Connecting the dots: Advances in modern metabolomics and its application in yeast system.

History of metabolism originates with yeast making bread. In fact, study based on "Yeast" was so crucial in the development of the field of biochemistry that the word "enzyme" is derived from the Greek word meaning leavened (yeast). Yeast has always been a point of interest as a eukaryotic model system to demonstrate the metabolites and their function. In recent times their metabolites are widely studied to predict their role in various pathways. Many traditional and analytical techniques have been employed, but its study through metabolomics is of recent interest in research. The present review focuses on details about yeast metabolomics based on preliminary research on various analytical techniques along with computational approaches. The review also aimed to highlight machine learning and various inceptions of yeast metabolomics.

Scale-up strategy for yeast single cell oil production for Rhodotorula mucilagenosa IIPL32 from corn cob derived pentosan.

This work was aimed to strategically scale-up the yeast lipid production process using Reynolds number as a standard rheological parameter from 50 mL to 50 L scale. Oleaginous yeast Rhodotorula mucilaginosa IIPL32 was cultivated in xylose rich corncob hydrolysate. The fermentation process for growth and maturation was operated in fed-batch with two different C/N ratios of 40 and 60. The hydrodynamic parameters were used to standardize and represent the effect of rheology on the fermentation process. The growth pattern of the yeast was found similar in both shake flask and fermenter with the maximum growth observed at 48 h. The lipid yield increased from 0.4 g/L and 0.5 g/L to 1.3 g/L and 1.83 g/L for 50 mL to 50 L for C/N ratio 40 and 60 respectively. The increase in productivity during the growth phase and lipid accumulation during the maturation phase showed that the scale-up strategy was successful.

Xylitol Production from Lignocellulosic Pentosans: A Rational Strain Engineering Approach toward a Multiproduct Biorefinery.

Kluyveromyces marxianus IIPE453 can utilize biomass-derived fermentable sugars for xylitol and ethanol fermentation. In this study, the xylitol production in the native strain was improved by overexpression of endogenous d-xylose reductase gene. A suitable expression cassette harboring the gene of interest was constructed and incorporated in the native yeast. qPCR analysis demonstrated the 2.1-fold enhancement in d-xylose reductase transcript levels in the modified strain with 1.62-fold enhancement in overall xylitol yield without affecting its ethanol fermenting capacity. Material balance analysis on 2 kg of sugar cane bagasse-derived fermentable sugars illustrated an excess of 58.62 ± 0.15 g of xylitol production by transformed strain in comparison to the wild variety with similar ethanol yield. The modified strain can be suitably used as a single biocatalyst for multiproduct biorefinery application.

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans.

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.