Transcriptomics aids in uncovering the metabolic shifts and molecular machinery of Schizochytrium limacinum during biotransformation of hydrophobic substrates to docosahexaenoic acid.

BACKGROUND: Biotransformation of waste oil into value-added nutraceuticals provides a sustainable strategy. Thraustochytrids are heterotrophic marine protists and promising producers of omega (ω) fatty acids. Although the metabolic routes for the assimilation of hydrophilic carbon substrates such as glucose are known for these microbes, the mechanisms employed for the conversion of hydrophobic substrates are not well established. Here, thraustochytrid Schizochytrium limacinum SR21 was investigated for its ability to convert oils (commercial oils with varying fatty acid composition and waste cooking oil) into ω-3 fatty acid; docosahexaenoic acid (DHA). RESULTS: Within 72 h SR21 consumed ~ 90% of the oils resulting in enhanced biomass (7.5 g L- 1) which was 2-fold higher as compared to glucose. Statistical analysis highlights C16 fatty acids as important precursors of DHA biosynthesis. Transcriptomic data indicated the upregulation of multiple lipases, predicted to possess signal peptides for secretory, membrane-anchored and cytoplasmic localization. Additionally, transcripts encoding for mitochondrial and peroxisomal ß-oxidation along with acyl-carnitine transporters were abundant for oil substrates that allowed complete degradation of fatty acids to acetyl CoA. Further, low levels of oxidative biomarkers (H2O2, malondialdehyde) and antioxidants were determined for hydrophobic substrates, suggesting that SR21 efficiently mitigates the metabolic load and diverts the acetyl CoA towards energy generation and DHA accumulation. CONCLUSIONS: The findings of this study contribute to uncovering the route of assimilation of oil substrates by SR21. The thraustochytrid employs an intricate crosstalk among the extracellular and intracellular molecular machinery favoring energy generation. The conversion of hydrophobic substrates to DHA can be further improved using synthetic biology tools, thereby providing a unique platform for the sustainable recycling of waste oil substrates.

Nutrient removal and biomass production of marine microalgae cultured in recirculating aquaculture systems (RAS) water with low phosphate concentration.

This study was conducted to investigate the feasibility of microalgal biomass production and nutrient removal from recirculating aquaculture systems (RAS) water (RASW) with low phosphate concentration. For this purpose, Nannochloropsis oculata, Pavlova gyrans, Tetraselmis suecica, Phaeodactylum tricornutum, and their consortium were cultivated in RASW and RASW supplemented with vitamins (+V). Among them, N. oculata showed the maximum biomass production of 0.4 g/L in RASW. Vitamins supplementation significantly increased the growth of T. suecica from 0.16 g/L in RASW to 0.33 g/L in RASW + V. Additionally, T. suecica showed the highest nitrate (NO3-N) removal efficiency of 80.88 ± 2.08 % in RASW and 83.82 ± 2.08 % in RASW + V. Accordingly, T. suecica was selected for scaling up study of microalgal cultivation in RASW and RASW supplemented with nitrate (RASW + N) in 4-L airlift photobioreactors. Nitrate supplementation enhanced the growth of T. suecica up to 2.2-fold (day 15). The fatty acid nutritional indices in T. suecica cultivated in RASW and RASW + N showed optimal polyunsaturated fatty acids (PUFAs)/saturated fatty acid (SFAs), omega-6 fatty acid (n-6)/omega-3 fatty acid (n-3), indices of atherogenicity (IA), and thrombogenicity (IT)). Overall, the findings of this study revealed that despite low phosphate concentration, marine microalgae can grow in RASW and relatively reduce the concentration of nitrate. Furthermore, the microalgal biomass cultivated in RASW consisting of pigments and optimal fatty acid nutritional profile can be used as fish feed, thus contributing to a circular bioeconomy.

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach.

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.

An approach for dairy wastewater remediation using mixture of microalgae and biodiesel production for sustainable transportation.

The aim of this work is remediation of dairy wastewater (DWW) for biodiesel feedstock production using poly-microalgae cultures of four microalgae namely Chlorella minutissima (C. minutissima), Scenedesmus abundans (S. abundans), Nostoc muscorum (N. muscorum) and Spirulina sp. The poly-microalgae cultures were prepared as C. minutissima + N. muscorum (CN), C. minutissima + N. muscorum + Spirulina sp. (CNSS) and S. abundans + N. muscorum + Spirulina sp. (SNSS). Poly-microalgae culture CNSS cultivated on 70% DWW achieved 75.16, 61.37, 58.76, 84.48 and 84.58%, removals of biological oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP), and suspended solids (SS), respectively, at 12:12 h photoperiod that resulted into total biomass and lipid yield of 3.47 ± 0.07 g/L and 496.32± 0.065 mg/L. However, maximum biomass and lipid yields of 5.76 ± 0.06 and 1152.37 ± 0.065 mg/L were achieved by poly-microalgae culture CNSS cultivated on 70% DWW + 10 g/L of glucose at 18:6 h photoperiod. Fatty acid methyl ester (FAME) analysis shown presence of C14:0 (myristic acid) C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:0 (stearic acid), C18:2 (linoleic acid) and C18:3 (linolenic acid), it indicates that the lipids produced from poly-microalgae cultures are suitable for biodiesel production. Thus, poly-microalgae cultures could be more efficient than mono-microalgae cultures in the remediation of DWW and for biodiesel feedstock production.

Pyrolysis of sewage sludge for sustainable biofuels and value-added biochar production.

The study deals with the pyrolysis of sewage sludge to produce eco-friendly and sustainable fuels along with value-added biochar products. The experiments were conducted in a fixed-bed cylindrical glass reactor in the temperature range of 250-700 °C and achieved the product yield of 22.4 wt% bio-oil, 18.9 wt % pyrolysis gases, and 58.7 wt% biochar at 500 °C optimum temperature. The chemical composition of bio-oil was investigated by gas chromatograph-mass spectroscopy and fourier transformation infrared techniques. The ASTM standard procedures were used to assess the fuel qualities of bio-oil, and they were found to be satisfactory. Bio-oil has a greater H/C ratio (3.49) and a lower O/C ratio (1.10), indicating that it is suitable for engine use. The gas chromatographic analysis of pyrolysis gases confirmed the presence of 41.16 wt % combustible gases, making it suitable for use in spark-ignition engines. X-ray fluorescence analysis of biochar showed that it had a good amount of carbon, nitrogen, phosphorus, and potassium along with some micro-and macro-nutrient which proves its potential to utilize as organic manure in the agriculture sector. In addition, the data obtained from the TGA analysis during the pyrolysis of sewage sludge was applied to calculate kinetic parameters via the Coats-Redfern method.

Biosynthesis of Nutraceutical Fatty Acids by the Oleaginous Marine Microalgae Phaeodactylum tricornutum Utilizing Hydrolysates from Organosolv-Pretreated Birch and Spruce Biomass.

Polyunsaturated fatty acids (PUFAs) are essential for human function, however they have to be provided through the diet. As their production from fish oil is environmentally unsustainable, there is demand for new sources of PUFAs. The aim of the present work was to establish the microalgal platform to produce nutraceutical-value PUFAs from forest biomass. To this end, the growth of Phaeodactylum tricornutum on birch and spruce hydrolysates was compared to autotrophic cultivation and glucose synthetic media. Total lipid generated by P. tricornutum grown mixotrophically on glucose, birch, and spruce hydrolysates was 1.21, 1.26, and 1.29 g/L, respectively. The highest eicosapentaenoic acid (EPA) production (256 mg/L) and productivity (19.69 mg/L/d) were observed on spruce hydrolysates. These values were considerably higher than those obtained from the cultivation without glucose (79.80 mg/L and 6.14 mg/L/d, respectively) and also from the photoautotrophic cultivation (26.86 mg/L and 2.44 mg/L/d, respectively). To the best of our knowledge, this is the first report describing the use of forest biomass as raw material for EPA and docosapentaenoic acid (DHA) production.

An Overview of Current Pretreatment Methods Used to Improve Lipid Extraction from Oleaginous Micro-Organisms.

Microbial oils, obtained from oleaginous microorganisms are an emerging source of commercially valuable chemicals ranging from pharmaceuticals to the petroleum industry. In petroleum biorefineries, the microbial biomass has become a sustainable source of renewable biofuels. Biodiesel is mainly produced from oils obtained from oleaginous microorganisms involving various upstream and downstream processes, such as cultivation, harvesting, lipid extraction, and transesterification. Among them, lipid extraction is a crucial step for the process and it represents an important bottleneck for the commercial scale production of biodiesel. Lipids are synthesized in the cellular compartment of oleaginous microorganisms in the form of lipid droplets, so it is necessary to disrupt the cells prior to lipid extraction in order to improve the extraction yields. Various mechanical, chemical and physicochemical pretreatment methods are employed to disintegrate the cellular membrane of oleaginous microorganisms. The objective of the present review article is to evaluate the various pretreatment methods for efficient lipid extraction from the oleaginous cellular biomass available to date, as well as to discuss their advantages and disadvantages, including their effect on the lipid yield. The discussed mechanical pretreatment methods are oil expeller, bead milling, ultrasonication, microwave, high-speed and high-pressure homogenizer, laser, autoclaving, pulsed electric field, and non-mechanical methods, such as enzymatic treatment, including various emerging cell disruption techniques.

Valorization of Brewers' Spent Grain for the Production of Lipids by Oleaginous Yeast.

Brewers' spent grain (BSG) accounts for 85% of the total amount of by-products generated by the brewing industries. BSG is a lignocellulosic biomass that is rich in proteins, lipids, minerals, and vitamins. In the present study, BSG was subjected to pretreatment by two different methods (microwave assisted alkaline pretreatment and organosolv) and was evaluated for the liberation of glucose and xylose during enzymatic saccharification trials. The highest amount of glucose (46.45 ± 1.43 g/L) and xylose (25.15 ± 1.36 g/L) were observed after enzymatic saccharification of the organosolv pretreated BSG. The glucose and xylose yield for the microwave assisted alkaline pretreated BSG were 34.86 ± 1.27 g/L and 16.54 ± 2.1 g/L, respectively. The hydrolysates from the organosolv pretreated BSG were used as substrate for the cultivation of the oleaginous yeast Rhodosporidium toruloides, aiming to produce microbial lipids. The yeast synthesized as high as 18.44 ± 0.96 g/L of cell dry weight and 10.41 ± 0.34 g/L lipids (lipid content of 56.45 ± 0.76%) when cultivated on BSG hydrolysate with a C/N ratio of 500. The cell dry weight, total lipid concentration and lipid content were higher compared to the results obtained when grown on synthetic media containing glucose, xylose or mixture of glucose and xylose. To the best of our knowledge, this is the first report using hydrolysates of organosolv pretreated BSG for the growth and lipid production of oleaginous yeast in literature. The lipid profile of this oleaginous yeast showed similar fatty acid contents to vegetable oils, which can result in good biodiesel properties of the produced biodiesel.

Patterns of Piscirickettsia salmonis load in susceptible and resistant families of Salmo salar.

The pathogen Piscirickettsia salmonis produces a systemic aggressive infection that involves several organs and tissues in salmonids. In spite of the great economic losses caused by this pathogen in the Atlantic salmon (Salmo salar) industry, very little is known about the resistance mechanisms of the host to this pathogen. In this paper, for the first time, we aimed to identify the bacterial load in head kidney and muscle of Atlantic salmon exhibiting differential familiar mortality. Furthermore, in order to assess the patterns of gene expression of immune related genes in susceptible and resistant families, a set of candidate genes was evaluated using deep sequencing of the transcriptome. The results showed that the bacterial load was significantly lower in resistant fish, when compared with the susceptible individuals. Based on the candidate genes analysis, we infer that the resistant hosts triggered up-regulation of specific genes (such as for example the LysC), which may explain a decrease in the bacterial load in head kidney, while the susceptible fish presented an exacerbated innate response, which is unable to exert an effective response against the bacteria. Interestingly, we found a higher bacterial load in muscle when compared with head kidney. We argue that this is possible due to the availability of an additional source of iron in muscle. Besides, the results show that the resistant fish could not be a likely reservoir of the bacteria.

Ameliorating microalgal OMEGA production using omics platforms.

Over the past decade, the focus on omega (ω)-3 fatty acids from microalgae has intensified due to their diverse health benefits. Bioprocess optimization has notably increased ω-3 fatty acid yields, yet understanding of the genetic architecture and metabolic pathways of high-yielding strains remains limited. Leveraging genomics, transcriptomics, proteomics, and metabolomics tools can provide vital system-level insights into native ω-3 fatty acid-producing microalgae, further boosting production. In this review, we explore 'omics' studies uncovering alternative pathways for ω-3 fatty acid synthesis and genome-wide regulation in response to cultivation parameters. We also emphasize potential targets to fine-tune in order to enhance yield. Despite progress, an integrated omics platform is essential to overcome current bottlenecks in optimizing the process for ω-3 fatty acid production from microalgae, advancing this crucial field.

A bioprocess engineering approach for the production of hydrocarbons and fatty acids from green microalga under high cobalt concentration as the feedstock of high-grade biofuels.

Botryococcus braunii, a colonial green microalga which is well-known for its capacity to synthesize hydrocarbons, has significant promise as a long-term source of feedstock for the generation of biofuels. However, cultivating and scaling up B. braunii using conventional aqua-suspended cultivation systems remains a challenge. In this study, we optimized medium components and light intensity to enhance lipid and hydrocarbon production in a multi-cultivator airlift photobioreactor. BBM 3N medium with 200 µmol/m2/s light intensity and a 16 h light-8 h dark regimen yielded the highest biomass productivity (110.00 ± 2.88 mg/L/day), as well as the highest lipid and hydrocarbon content. Cultivation in a flat-panel bioreactor resulted in significantly higher biomass productivity (129.11 ± 2.74 mg/L/day), lipid productivity (32.21 ± 1.31 mg/L/day), and hydrocarbon productivity (28.98 ± 2.08 mg/L/day) compared to cultivation in Erlenmeyer flasks and open 20-L raceway pond. It also exhibited 20.15 ± 1.03% of protein content including elevated levels of chlorophyll a, chlorophyll b, and carotenoids. This work is noteworthy since it is the first to describe fatty acid and hydrocarbon profiles of B. braunii during cobalt treatment. The study demonstrated that high cobalt concentrations (up to 5 mg/L of cobalt nitrate) during Botryococcus culture affected hydrocarbon synthesis, resulting in high amounts of n-alkadienes and trienes as well as lipids with elevated monounsaturated fatty acids concentration. Furthermore, pyrolysis experiments on microalgal green biomass and de-oiled biomass revealed the lipid and hydrocarbon compounds generated by the thermal degradation of B. braunii that facilitate extra economical value to this system.

Scanning electron microscope analysis to evaluate the effect of pretreatment with ozone and sodium hypochlorite on pits and fissures of primary teeth.

Introduction: The cornerstone of preventative therapy is made up of pit and fissure sealants and fluorides. Resin sealants have been shown to help reduce fissure decay in both primary and permanent teeth. Etching of primary molars is not effective due to prismless enamel, higher organic content, and diversity in fissure shape. Methods of pretreatment of pits and fissures have been hypothesized to promote microporosities in etched enamel and hence sealant adherence. Examining the etching pattern and surface roughness of the enamel surface can be used to gauge these. Objective: This study aimed to evaluate the effect of pretreatment with ozone gas and 5.25% sodium hypochlorite solution on pits and fissures of primary teeth to check for the etching pattern and surface roughness. Materials and Methods: Thirty noncarious primary molars were sectioned to retain the crowns, and randomly divided into three groups, sodium hypochlorite, ozone gas, and control. Each sample was pretreated with the agent, washed, followed by etching with 37% phosphoric acid. Samples were sectioned and subjected to scanning electron microscope analysis to evaluate the etching pattern and surface roughness. Results: Comparison of the etching pattern in three groups showed a statistically insignificant difference (P=0.364). Surface roughness was highest in the hypochlorite group followed by ozone and control which showed a statistically significant difference (P = 0.001). The surface area between the three groups showed a statistically insignificant difference. Conclusion: Sodium hypochlorite is a better pretreatment agent compared to ozone gas and acid etching alone. However, as all results were not statistically significant further research must be carried out to prove the effectiveness of these agents.

Clinical evaluation of feasibility and effectiveness using a virtual reality device during local anesthesia and extractions in pediatric patients.

PURPOSE: Pediatric dentistry requires more than routine dental knowledge and skills in executing quality dental care. The quality of dental treatment is indirectly proportional to a child's anxiety and fear. The recent distraction technique, the use of virtual reality (VR) devices has intrigued many researcher's minds. Therefore, this study was conducted to assess the clinical feasibility and effectiveness of the VR device in reducing pain and anxiety in pediatric patients during mandibular primary molar extraction. METHODS: This research trial had 30 healthy children between the ages of 6 and 12 who required mandibular primary molar extraction. The subjects were divided into two groups using a simple randomization method. In the study group (n = 15), extraction was carried out using a VR device, whereas, in the control group (n = 15), extraction was carried out without the use of a VR device. Pre- and post-extraction anxiety levels were measured by Venham's picture test (VPT) and the heart rate. After the procedure, pain and behavior were evaluated by the Wong-Bakers Faces pain rating scale (WBS) and FLACC Scale respectively. RESULTS: The pre-extraction values of the VPT and heart rate showed no statistically significant difference. This indicates that participants of both groups had a similar level of anxiety at baseline. There was a statistically significant increase (p = 0.028) between pre- and post-heart rates in the control group, with no significant change in the study group. CONCLUSION: The use of a virtual reality device in children can reduce anxiety during primary molar extractions as evaluated by heart rate. Virtual reality devices might be an adjunct to high-quality dental care and to other behavior management methods. Definitive outcomes can be attained by encouraging studies overcoming the present limitations, and considering salivary biochemical indicators for the assessment of these devices.

Microalgal lutein: Advancements in production, extraction, market potential, and applications.

Lutein, a bioactive xanthophyll, has recently attracted significant attention for numerous health benefits, e.g., protection of eye health, macular degeneration, and acute and chronic syndromes etc. Microalgae have emerged as the best platform for high-value lutein production with high productivity, lutein content, and scale-up potential. Algal lutein possesses numerous bioactivities, hence widely used in pharmaceuticals, nutraceuticals, aquaculture, cosmetics, etc. This review highlights advances in upstream lutein production enhancement and feasible downstream extraction and cell disruption techniques for a large-scale lutein biorefinery. Besides bioprocess-related advances, possible solutions for existing production challenges in microalgae-based lutein biorefinery, market potential, and emerging commercial scopes of lutein and its potential health applications are also discussed. The key enzymes involved in the lutein biosynthesizing Methyl-Erythritol-phosphate (MEP) pathway have been briefly described. This review provides a comprehensive updates on lutein research advancements covering scalable upstream and downstream production strategies and potential applications for researchers and industrialists.

Unravelling Metagenomics Approach for Microbial Biofuel Production.

Renewable biofuels, such as biodiesel, bioethanol, and biobutanol, serve as long-term solutions to fossil fuel depletion. A sustainable approach feedstock for their production is plant biomass, which is degraded to sugars with the aid of microbes-derived enzymes, followed by microbial conversion of those sugars to biofuels. Considering their global demand, additional efforts have been made for their large-scale production, which is ultimately leading breakthrough research in biomass energy. Metagenomics is a powerful tool allowing for functional gene analysis and new enzyme discovery. Thus, the present article summarizes the revolutionary advances of metagenomics in the biofuel industry and enlightens the importance of unexplored habitats for novel gene or enzyme mining. Moreover, it also accentuates metagenomics potentials to explore uncultivable microbiomes as well as enzymes associated with them.

From Yeast to Biotechnology.

Yeasts are widely used in various sectors of biotechnology, from white (industrial) to red (medical) [...].

Microbial genetic engineering approach to replace shark livering for squalene.

Squalene is generally sourced from the liver oil of deep sea sharks (Squalus spp.), in which it accounts for 40-70% of liver mass. To meet the growing demand for squalene because of its beneficial effects for human health, three to six million deep sea sharks are slaughtered each year, profoundly endangering marine ecosystems. To overcome this unsustainable practice, microbial sources of squalene might offer a viable alternative to plant- or animal-based squalene, although only a few microorganisms have been found that are capable of synthesizing up to 30% squalene of dry biomass by native biosynthetic pathways. These squalene biosynthetic pathways, on the other hand, can be genetically manipulated to transform microorganisms into 'cellular factories' for squalene overproduction.

Improving the content of high value compounds in Nordic Desmodesmus microalgal strains.

Nordic Desmodesmus microalgal strains (2-6) and (RUC-2) were exposed to abiotic stress (light and salt) to enhance lipids and carotenoids. The biomass output of both strains increased by more than 50% during light stress of 800 µmol m-2 s-1 compared to control light. The biomass of Desmodesmus sp. (2-6) contained most lipids (15% of dry weight) and total carotenoids (16.6 mg g-1) when grown at moderate light stress (400 µmol m-2 s-1), which further could be enhanced up to 2.5-fold by salinity stress. Desmodesmus sp. (RUC-2) exhibited maximal lipid (26.5%) and carotenoid (43.8 mg L-1) content at light intensities of 400 and 100 µmol m-2 s-1, respectively. Salinity stress stimulated lipid accumulation by 39%. Nordic Desmodesmus strains therefore are not only able to tolerate stress conditions, but their biomass considerably improves under stress. These strains have high potential to be used in algal bio-factories on low-cost medium like Baltic seawater.

Biosynthesis of microalgal lipids, proteins, lutein, and carbohydrates using fish farming wastewater and forest biomass under photoautotrophic and heterotrophic cultivation.

Biorefineries enable the circular, sustainable, and economic use of waste resources if value-added products can be recovered from all the generated fractions at a large-scale. In the present studies the comparison and assessment for the production of value-added compounds (e.g., proteins, lutein, and lipids) by the microalga Chlorella sorokiniana grown under photoautotrophic or heterotrophic conditions was performed. Photoautotrophic cultivation generated little biomass and lipids, but abundant proteins (416.66 mg/gCDW) and lutein (6.40 mg/gCDW). Heterotrophic conditions using spruce hydrolysate as a carbon source favored biomass (8.71 g/L at C/N 20 and 8.28 g/L at C/N 60) and lipid synthesis (2.79 g/L at C/N 20 and 3.61 g/L at C/N 60) after 72 h of cultivation. Therefore, heterotrophic cultivation of microalgae using spruce hydrolysate instead of glucose offers a suitable biorefinery concept at large-scale for biodiesel-grade lipids production, whereas photoautotrophic bioreactors are recommended for sustainable protein and lutein biosynthesis.

Comparative Evaluation of the Remineralization Potential of Fluoride-containing Toothpaste, Honey Ginger Paste and Ozone. An In Vitro Study.

Introduction: A drop in pH of the oral cavity results in demineralization, which, if continued, leads to loss of minerals from tooth structure, resulting in dental caries. A goal of modern dentistry is to manage noncavitated caries lesions noninvasively through remineralization in an attempt to prevent disease progression. Materials and methods: A total of 40 extracted premolar teeth were selected for the study. The specimens were divided into four groups, group I, the control group; group II, remineralizing agent as fluoride toothpaste; group III, the treatment material as ginger and honey paste; and group IV, the treatment material as ozone oil. An initial reading of surface roughness and hardness was recorded for the group (control group). Repeated treatment has continued lasting 21 days. This saliva was changed each day. Following the lesion formation procedure, the surface microhardness was measured for all specimens. The parameters were 200 gm force for 15 seconds with a Vickers indenter and the roughness of the demineralized area of each specimen was obtained by using the surface roughness tester. Results: Surface roughness was checked by using a surface roughness tester. Before starting the pH cycle, the baseline value for the control group was calculated. The baseline value for the control group was calculated. The surface roughness average value for 10 samples is 0.555 µm and the average surface microhardness is 304 HV; the average surface roughness value for fluoride is 0.244 µm and the microhardness is 256 HV, 0.241 µm, and 271 HV value for honey-ginger paste. For ozone surface roughness average value is 0.238 µm and the surface microhardness average mean value is 253 HV. Conclusion: The future of dentistry will rely on the regeneration of tooth structure. There is no significant difference seen between each treatment group. Considering the adverse effect of fluoride, we can consider honey-ginger and ozone as good remineralizing agents for fluoride. How to cite this article: Kade KK, Chaudhary S, Shah R, et al. Comparative Evaluation of the Remineralization Potential of Fluoride-containing Toothpaste, Honey Ginger Paste and Ozone. An In Vitro Study. Int J Clin Pediatr Dent 2022;15(5):541-548.