Acid-assisted oil extraction directly from thraustochytrids fermentation broth and its energy assessment for docosahexaenoic acid-enriched oil production.

Thraustochytrids are the most prominent source of polyunsaturated fatty acids, specifically docosahexaenoic acid (DHA). Downstream processing constitutes a significant fraction of total production cost and thus needs judicious optimization. Currently, hazardous solvent-based extraction methods are used to extract oil from the dry or wet thraustochytrids cell mass. The process is also highly energy-intensive due to involvement of dewatering and drying as unit operations. Current work devised an energy-efficient acid-assisted extraction (AAE) methodology to overcome dry and wet biomass-based extraction limitations. AAE recovered 91 % of total oil with 35-40 % PUFA from the direct fermentation broth, eliminating the need for dewatering and drying of fermentation broth/cell biomass. The current work also presents an all-inclusive comparison of the energy assessment of oil extraction from dry and AAE method. AAE produced PUFA enriched oil with a total energy consumption of 210 MJ/kg, which was four times lower than that of conventional dry cell extraction methodology.

Heterologous mannitol-1-phosphate dehydrogenase gene over-expression in Parachlorella kessleri for enhanced microalgal biomass productivity.

BACKGROUND: Microalgae have tremendous potential in CO2 sequestration, bioenergy, biofuels, wastewater treatment, and high-value metabolites production. However, large-scale production of microalgae is hampered due to photo-inhibition in outdoor cultivation. Mannitol, as an osmolyte, is known to relieve the stress produced under different abiotic stress conditions during the growth of a photosynthetic organism. RESULTS: In the present study, Mannitol-1-phosphate 5-dehydrogenase (Mt1D) was over-expressed to study the effect of mannitol over-production in Parachlorella kessleri under high-light induced stress. Over-expression of Mt1D led to 65% increased mannitol content in the transformed P. kessleri compared to that of wild type. Mannitol transformant demonstrated > 20-fold reduction in reactive oxygen species generation and 15% higher biomass productivity when grown in outdoor cultivation with high-light irradiance of 1200 µmol photons m-2 s-1. CONCLUSIONS: The current study establishes that a higher mannitol concentration provides stress shielding and leads to better acclimatization of transgenic microalgae against high-light generated stress. It also led to reduced ROS generation and improved growth of microalga under study. Thus, overexpression of the Mt1D gene in microalgae can be a suitable strategy to combat high-light stress.

Co-cultivation of Phaeodactylum tricornutum and Aurantiochytrium limacinum for polyunsaturated omega-3 fatty acids production.

Marine protist Aurantiochytrium limacinum produces docosahexaenoic acid (DHA) as main polyunsaturated fatty acid (PUFA) and lacks any monounsaturated fatty acids (MUFA), while eicosapentaenoic acid (EPA) and MUFA's are produced by Phaeodactylum tricornutum. The marine diatom P. tricornutum was co-cultured with A.limacinum to match the EPA:DHA ratio of fish oil. Modulation in initial cell density ratio overcame the dominance of A.limacinum during co-cultivation and led to regulated proliferation of both species. Media engineering with nitrate and glycerol concentration yielded 2:1 (56.44: 30.11) mg g-1 and 1:1 (47.43: 49.61) mg g-1 EPA: DHA ratio. The oil and biomass obtained from co-cultivation comprised of MUFA's such as palmitoleic acid (2.65 mg g-1) and oleic acid (1.25 mg g-1) along with pigments like fucoxanthin (367.18 µg g-1), ß-carotene (8.98 µg g-1) and astaxanthin (0.77 µg g-1). Thus, co-cultivation of P. tricornutum with A. limacinum represented a unique strategy towards achieving desired fatty acid composition.

Prebiotic activity of paramylon isolated from heterotrophically grown Euglena gracilis.

Paramylon from Euglena gracilis is an insoluble crystalline ß-1,3-glucan which have pharmaceutical and nutraceuticals applications. The present study aims to check the prebiotic potential of paramylon derived from heterotrophically grown E. gracilis in bioreactor. The Paramylon was extracted using sodium dodecyl sulfate from E. gracilis biomass. The Fourier Transform-Infra Red spectroscopy and scanning electron microscopy demonstrated the isolated paramylon to be equivalent to that of analytical standard. The prebiotic activity of E. gracilis cell extract and isolated paramylon was studied. E. gracilis cell extract as well as isolated paramylon led to cell number enhancement of Lacfid (Lactobacillus) strain exhibiting the prebiotic activities.

Bioconversion of waste acid oil to docosahexaenoic acid by integration of "ex novo'' and "de novo'' fermentation in Aurantiochytrium limacinum.

Thraustochytrids have predominantly been grown on hydrophilic substrates i.e. by "de novo" fermentation. The fatty acid composition of thraustochytrids oil in "de novo" mode is enriched in saturated palmitic acid and polyunsaturated docosahexaenoic acid. The "ex novo" fermentation of a novel Aurantiochytrium limacinum ICTSG-17 with waste acid oil altered the fatty acid composition of produced oil. This led to increased total unsaturated fatty acids (TUFA) and concomitant decrease in the total saturated fatty acids (TSFA) resulting in higher TUFA/TSFA ratio. However, cell growth and DHA content in "ex novo" were lower than that of "de novo" fermentation. Integration of "de novo" and "ex novo" fermentation modes were devised to attain high biomass and lipids enriched in DHA. Sequential "de novo"-"ex novo" fermentation resulted in ~20 g/L biomass and ~40% DHA content and higher TUFA/TSFA ratio as compared to that of "de novo" mode.

2,3-Butanediol production using soy-based nitrogen source and fermentation process evaluation by a novel isolate of Bacillus licheniformis BL1.

2,3-Butanediol (2,3-BDO) has varied applications in chemical, pharmaceutical, & food industry. Microorganisms belonging to Klebsiella, Enterobacter & Serratia genera are well-known producers of 2,3-BDO. However, they have limited usage in industrial-scale owing to their pathogenic nature. A nonpathogenic soil isolate identified as Bacillus licheniformis (BL1) was thus investigated for 2,3-BDO production. Soy flakes, soy flour, defatted soy, and soybean meal-based hydrolysates replaced yeast extract and peptone as nitrogen sources. Defatted soy flakes and soybean meal hydrolysate led to an equivalent 2,3-BDO yield and productivity as compared to that of Yeast Extract and peptone. The pH and oxygen variation influenced the proportion of various products of the mixed acid-butanediol pathway. Further, the batch mode fermentation with soy hydrolysate and optimized process parameter resulted in 2,3-BDO titer, yield and productivity of 11.06 g/L, 0.43 g/g and 0.48 g/L h respectively. Glucose concentration above 5% was inhibitory and led to reduction in the specific growth rate of BL1 in batch cultivation. Intermittent glucose feeding in fed-batch mode overcame this substrate limitation resulting in increased titers (49.8 g/L) and productivity (0.62 g/L h). Modified medium containing soy hydrolysate as nitrogen source with fermentation process optimization resulted in 67% decrease in medium cost for 2,3-BDO production.

Isolation and optimization of a novel thraustochytrid strain for DHA rich and astaxanthin comprising biomass as aquafeed supplement.

A marine organism, belonging to the Thraustochytrids family was isolated from mangroves of Mumbai, India. The isolated strain was identified as Aurantiochytrium limacinum by internal transcribed spacer sequence analysis. Optimization of process parameters yielded 14.47 g/L dry cell weight containing 55-58% oil in 3.5 days' cultivation on glucose, yeast extract, and peptone in the bioreactor. Docosahexaenoic acid (DHA) was found to be the dominant long-chain polyunsaturated fatty acid, accounting for 32-35% of total fatty acid content. The process parameter was tweaked to simultaneously synthesize astaxanthin along with DHA. The concurrent synthesis of DHA and astaxanthin-containing biomass establishes the isolated strain as a perfect choice for aquafeed. Accession number: NCBI accession number MN046792.

Integration of continuous-high cell density-fed-batch fermentation for Aurantiochytrium limacinum for simultaneous high biomass, lipids and docosahexaenoic acid production.

Docosahexaenoic acid (DHA) rich oil or biomass is currently being produced by fermentation of thraustochytrids by repeated fed-batch. Continuous cultivation has not been successful for DHA production because of excess carbon and limited nitrogen conditions requirement. The present study describes an alternative integrative fermentation strategy to simultaneously produce high cell density, lipids and DHA in continuous mode for Aurantiochytrium limacinum. The high cell density system (≥120 g/L DCW basis) on carbon feeding led to DHA productivity of 0.508 g/L.h on poultry waste based medium with a process time of 48-54 h. The strategy integrates the advantages of repeated fed-batch for high cell densities and DHA content in continuous cultivation.

Organic waste streams as feedstock for the production of high volume-low value products.

Valorisation of organic wastes to produce industrially relevant commodity products is a sustainable, cost-effective and viable alternative providing a green platform for chemical production while simultaneously leading to waste disposal management. In the present study, organic wastes such as agricultural residue-derived sugars, oilseed meals, poultry waste and molasses were used for substituting expensive organic fermentation medium components. Moorella thermoacetica and Aurantiochytrium limacinum were adapted on these waste-derived hydrolysates to produce high volume-low value products such as bio-acetic acid (80% theoretical yields) and oil-rich fish/animal feed (more than 85% dry cell weight as compared with conventional nutrient sources) respectively. Use of these waste-derived nutrients led to ~ 75% and ~ 90% reduction in media cost for acetic acid and oil-rich biomass production respectively as compared with that of traditionally used high-priced medium components. The strategy will assist in the cost reduction for high volume-low value products while also ensuring waste recovery.

Metabolic Engineering of Chlamydomonas reinhardtii for Enhanced ß-Carotene and Lutein Production.

The metabolic engineering of Chlamydomonas reinhardtii, one of the fastest-growing microalgae, is a potential alternative for enhanced carotenoid productivity. CrtYB (phytoene-ß-carotene synthase - PBS) gene from red yeast Xanthophyllomyces dendrorhous encodes for a bifunctional enzyme that harbours both phytoene synthase (psy) and lycopene cyclization (lcyb) activities. Heterologous expression of this bifunctional PBS gene led to 38% enhancement in ß-carotene along with 60% increase in the lutein yields under low light conditions of 75 µmol photons m-2 s-1. Short Duration-High Light induction strategy led to overall 72% and 83% increase in ß-carotene and lutein yield reaching up to 22.8 mg g-1 and 8.9 mg g-1, respectively. This is the first report of expression of heterologous bifunctional PBS gene resulting in simultaneous enhancement in ß-carotene and lutein content in phototrophic engineered cells. Graphical Abstract.

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation.

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54 ± 0.023 g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48 hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74 ± 0.06 g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.

Role of Lipids in Retinal Vascular and Macular Disorders.

Retinal diseases are significant by increasing problem in every part of the world. While excellent treatment has emerged for various retinal diseases, treatment for early disease is lacking due to an incomplete understanding of all molecular events. With aging, there is a striking accumulation of neutral lipids in Bruch's membrane. These neutral lipids leads to the creation of a lipid wall at the same locations where drusen and basal linear deposit, pathognomonic lesions of Age-related macular degeneration, subsequently form. High lipid levels are also known to cause endothelial dysfunction, an important factor in the pathogenesis of Diabetic Retinopathy. Various studies suggest that 20 % of Retinal Vascular Occlusion is connected to hyperlipidemia. Biochemical studies have implicated mutation in gene encoding ABCA4, a lipid transporter in pathogenesis of Stargardt disease. This article reviews how systemic and local production of lipids might contribute to the pathogenesis of above retinal disorders.

Comparison of Serum Apolipoproteins and Traditional Lipids in Eyes with Diabetic Retinopathy in Indian Population: A Case Series.

PURPOSE: To review the associations of serum lipoproteins and apolipoproteins with diabetic retinopathy (DR). MATERIALS AND METHODS: This was a cross-sectional study of patients of DR. DR was graded according to modified Airlie House Classification system. Traditional serum lipids (total, low-density lipoprotein [LDL], and high-density lipoprotein [HDL] cholesterol and triglycerides [TGs]), apolipoprotein AI (apoAI), apolipoprotein B (apoB), and apoB-to-apoAI ratios were assessed. The analysis of variance test was applied. P < 0.05 was considered statistically significant, and P < 0.001 was considered highly significant. RESULTS: Twenty-four patients with DR were evaluated. Serum apoAI was statistically significantly associated with a reduced likelihood of having more severe DR levels (P < 0.001). Increasing levels of apoB (P < 0.001) and apoB-to-apoAI ratio (P < 0.001) were statistically significantly associated with increasing DR severity levels. The association of total (P > 0.05) and LDL cholesterol (P > 0.05) with severity of DR was not statistically significant. HDL cholesterol levels were inversely associated with the severity of DR (P < 0.05). TGs were also statistically significantly associated with the severity of DR (P < 0.05). CONCLUSION: Serum apolipoprotein levels may be stronger biomarkers of DR than traditional lipids.

Cloning, expression, and purification of Chlamydomonas reinhardtii CC-503 sedoheptulose 1,7-bisphosphatase in Escherichia coli.

Sedoheptulose 1,7-bisphosphatase (SBPase), a nuclear-encoded chloroplastic enzyme, is an important rate-limiting enzyme of the carbon fixation cycle (Calvin cycle). SBPase is unique to only photosynthetic organisms and is involved in the regeneration of ribulose-1,5-bisphosphate. SBPases from several sources have been studied for their induction and regulation. However, SBPase from Chlamydomonas reinhardtii CC-503, the widely studied model microalga, has not been isolated and functionally confirmed to date. In this study, the full-length cDNA for SBPase was isolated from C. reinhardtii CC-503 using anchored oligo(dT)24VGN primer for reverse transcription. The SBPase cDNA was cloned into pET28a expression vector for the production of 6X His-tagged protein in Escherichia coli BL21 (DE3) strain. Although initially most of the enzyme was obtained as insoluble protein aggregates, solubilization of protein was improved by optimization of protein induction with respect to growth temperature and isopropyl ß-D-1-thiogalactopyranoside concentrations. The induced protein was purified by immobilized metal affinity chromatography using nickel-nitrilotriacetic acid resin in a phosphate-free buffer leading to an accurate SBPase activity measurement. The present study demonstrates, for the first time, successful cloning of C. reinhardtii CC-503 SBPase in E. coli leading to the expression of a functionally active enzyme.

Trehalose phosphate synthase overexpression in Parachlorella kessleri improves growth and photosynthetic performance under high light conditions.

Parachlorella kessleri is a promising oil-bearing marine alga which shows decreased growth under high light stress. Osmolytes are known to relieve stress by protecting the cell membrane, proteins, and enzymes. Enhanced production of osmolyte (trehalose) was thus used to relieve stress in P. kessleri by overexpression of trehalose phosphate synthase (TPS) gene. Transformed P. kessleri was grown under different light regimes to study the effect of trehalose overproduction on growth. Study of one of the TPS transformants showed increased trehalose as well as increased biomass and decreased pigments, reactive oxygen species, and lipid peroxidation of cell membrane. The improved photosynthetic performance of the transformant was also signified by pulse-amplitude-modulated fluorometric analysis. All of these factors reveal improved stress tolerance under high light conditions by increased trehalose accumulation due to TPS overexpression in P. kessleri.

Normal Macular Thickness in Healthy Indian Eyes Using Spectral Domain Optical Coherence Tomography.

PURPOSE: To determine macular thickness and its variation with age and sex in healthy Indian eyes using Zeiss spectral domain optical coherence tomography (SD-OCT). DESIGN: A prospective, cross-sectional study. METHODS: Four hundred healthy adult subjects (≥18 years) underwent macular cube scanning using Zeiss SD-OCT. Macular thickness from all 9 regions of the Early Treatment Diabetic Retinopathy Study map was documented for each subject. Variations in macular thickness by age and sex were determined. RESULTS: The mean age of the subjects was 38.05 ± 12.13 (range, 18-78) years. The mean ages in men and women were 39.19 ± 12.16 and 37.13 ± 12.05 years, respectively (P > 0.05). The mean central subfield thickness (CST) of all subjects was 240.40 ± 18.26 µm, and mean macular thickness was 287.87 ± 18.07 µm. The mean CST in the right and left eyes were 240.40 ± 18.25 µm and 239.65 ± 17.73 µm, respectively (P = 0.55). Males were associated with greater mean CST and mean macular thickness compared with females (P < 0.05). There was an association of mean CST with sex (adjusted r = 0.095; P < 0.05) but not with age. Overall, the nasal quadrant was the thickest followed by the superior, inferior, and temporal subfields. CONCLUSIONS: This is the normative data for macular thickness in healthy Indian eyes using Zeiss SD-OCT. This will serve as a baseline for diagnosing and treating macular pathologies in Indian eyes because such data using Zeiss SD-OCT in Indian eyes have previously been lacking.

Recurrent Central Serous Chorioretinopathy with Dexamethasone Eye Drop used Nasally for Rhinitis.

Central serous chorioretinopathy (CSC) is characterized by serous retinal detachment at the posterior pole. Several factors have been implicated in the pathogenesis, and endogenous or exogenous corticosteroids are thought to play a major role. Here we present a case of a 35-year-old male with complaints of a dark circle in front of his right eye. Fundus examination, optical coherence tomography and fundus fluorescein angiography were performed. The patient was diagnosed with CSC. CSC resolved completely within seven weeks. Four weeks later the CSC recurred and spontaneously resolved over eight weeks. Overall, the patient had three additional recurrences of CSC in the same eye over the next year. A detailed history taking revealed the patient was using 0.1% dexamethasone eye drops nasally for recurrent rhinitis for few days prior to each episode of CSC. This indicates the strong correlation between steroids given by any route and the pathogenesis of CSC.

Susac's syndrome: first from India and youngest in the world.

A two and half year old female was admitted at the emergency room suffering from gradually worsening headache followed by nausea. The child presented with reduced level of consciousness and bilateral hypoacusis. The patient was lethargic. Ophthalmic examination showed branch retinal artery occlusion (BRAO). This finding was crucial to the diagnosis of Susac's syndrome (SS), a rare autoimmune disease characterized by, endotheliopathy of retina, encephalic tissues and cochlea. Magnetic resonance imaging of the brain also showed typical features. Thorough blood investigations did not reveal any other abnormality. Patient was treated with immunosuppressive to prevent her from developing severe sequelae of this disease. The child showed dramatic improvement in her systemic condition within 48 h of starting the treatment. This is the youngest ever and first case report from India.

Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri.

Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l(-1) hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne's to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species.

Central Serous Chorioretinopathy: A Review of the Literature.

Central serous chorioretinopathy (CSCR) is an incompletely understood multifactorial disease of those who are middle aged characterized by the collection of fluid between the retinal pigment epithelium and the neurosensory retina. The exact etiology of CSCR and the reason of its predominance in middle-aged males are still unknown. Many pharmacologic modalities are suggested for CSCR with no proven efficacy. So this article was written to give a review of the relevant and recent literature on CSCR and to summarize the etiology, clinical features, and diagnostic modalities for CSCR with special emphasis on the treatment options available and those that are still under trial and can be of help in the future to fasten the recovery and reduce the recurrences.