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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.

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

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.

Asterarcys is a resilient microalgal species which can sustain high light intensities and have high biomass and lipid productivity under mixotrophy. These attributes make it a potential candidate to produce biodiesel and biofuels. However, no genetic information is currently available for this species. Here, we report the chloroplast genome sequence of Asterarcys sp. and compare it with the chloroplast genome of closely related chlorophyceaen microalgae. The chloroplast genome of Asterarcys sp. is present as a ~111 kb circular molecule with 88 genes and a coding density of 50.28%. The 111,776 bp genome shows a quadripartite structure with two 6297 bp long inverted repeats separating single copy regions (SSC) of almost equal sizes. The small and compact genome of Asterarcys shows similarity in gene structure and organization with that of its closest relative Scenedesmus obliquus. It reinforces the compact nature of chloroplast genomes of Sphaeropleales as compared to that of inflated genomes in Chlamydomonadales. The genome shows a biased distribution of genes with 50 of the protein-coding genes encoded from one strand and 15 from the opposite strand. This biased distribution of genes is likewise to that of Scenedesmus obliquus. The phylogenetic tree based on protein-coding genes from chlorophycean species places Asterarcys close to Scenedesmus. The chloroplast genome information of Asterarcys will help understand the phylogeny of Sphaeropleales and Chlorophyceae. In the present study, we also report a simple and effective method for isolation of Asterarcys sp. chloroplast DNA of high quality and purity which is an essential prerequisite for efficient genome sequencing.

In plants, a mutation in the Orange (Or) gene is known to enhance the accumulation of carotenoids in comparison to the wild-type (WT) allele. We investigated the effect of a point mutation in the Or gene of Brassica oleracea on its protein structure via in silico analysis. Subsequently we studied the effect of the Or mutation on carotenoid regulation in Chlamydomonas reinhardtii. The Or mutation in Brassica oleracea—mBoOr (R88H)—results in a small β-sheet in place of the α-helix that is present in the WT protein, and was found to be associated with enhanced carotenoid content when screened in Escherichia coli. The expression of the equivalent mutant from C. reinhardtii—mCrOr (R106H)—did not result in any change in protein structure and exhibited lower levels of carotenoid in E. coli. An in silico analysis of microalgal genomes revealed the presence of Or homologues in several microalgae from the class Trebouxiphyceae, while C. reinhardtii was the only representative of the Chlorophyceae. The heterologous expression of mBoOr in C. reinhardtii resulted in a 1.7-fold increase in carotenoid content, as well as an increase in cell size and the appearance of multicellularity in C. reinhardtii, which was evidenced by the presence of both orange and green transgenic cells grouping together as a population. A palmelloid formation of the orange and green cells impaired the motility of the transgenic population, resulting in flocculation. This study establishes mBoOr expression as a promising strategy for enhancing carotenoid content in microalgae.

Suspension culture of Azadirachta indica produces the biopesticide azadirachtin. Some elicitors (salicylic acid, chitosan, jasmonic acid, methyl jasmonate, yeast extract and yeast extract carbohydrate fraction) at different concentrations were added in shake flask suspension culture of A. indica. Chitosan, salicylic acid and jasmonic acid stimulated the highest increase in azadirachtin content, which ranged from 2 to 3-fold greater than the control. The combined effect of these elicitor(s) on azadirachtin content was then studied by Response Surface Methodology. A synergistic effect of these elicitor(s) on azadirachtin production resulted in 5-fold higher azadirachtin production (15.9 mg/g DCW versus 3.2 mg/g in control cultures). Exposure time studies with elicitor(s) addition on 8th day revealed that highest azadirachtin accumulation reached after 48 h of combined addition of elicitor(s) (17.4 mg/g). Cultivation of A. indica cells was also carried out with combined (statistically optimized) elicitors addition on 8th day in Stirred Tank Bioreactor. This led to more than 3-fold greater azadirachtin accumulation (161.1 mg/l) as opposed to control bioreactor with no elicitor addition (50 mg/l) in 10 days of cultivation period. The present study not only identifies the elicitor(s) and their respective concentrations for enhanced azadirachtin synthesis but also establishes the role of combined elicitors to improve secondary metabolite production of plant cell cultures more efficiently.

Successful scale-up of Azadirachta indica suspension culture for azadirachtin production was done in stirred tank bioreactor with two different impellers. The kinetics of biomass accumulation, nutrient consumption and azadirachtin production of A. indica cell suspension culture were studied in a stirred tank bioreactor equipped with centrifugal impeller and compared with similar bioreactor with a setric impeller to investigate the role of O 2 transfer efficiency of centrifugal impeller bioreactor on overall culture metabolism. The maximum cell mass for centrifugal impeller bioreactor and stirred tank bioreactor (with setric impeller) were 18.7 and 15.5 g/L (by dry cell weight) and corresponding azadirachtin concentrations were 0.071 and 0.05 g/L, respectively. Glucose and phosphate were identified as the major growth-limiting nutrients during the bioreactor cultivation. The centrifugal impeller bioreactor demonstrated less shearing and improved O 2 transfer than the stirred tank bioreactor equipped with setric impeller with respect to biomass and azadirachtin production.

Azadirachtin, obtained from Azadirachta indica (neem) has emerged as wonderful natural biopesticide for pest control due to its efficacy, biodegradability and minimum side effects. To overcome constraints associated with its limited, variable and unreliable supply from natural trees, plant cell suspension culture of A. indica was developed (obtained from callus of seeds). The kinetics of cell growth/azadirachtin formation and substrate consumption of A. indica suspension culture was established in bioreactor with low shear setric impeller under optimized culture conditions (statistically optimized independently). The culture featured a growth of 15.5 and 0.05 g/l azadirachtin in 10 days of cultivation after which azadirachtin concentration declined. To prevent any inhibition caused by the excess feeding of substrate during fed-batch cultivation, inhibition of the carbon, nitrate and phosphate were separately established. Based on batch kinetics/inhibitory data a mathematical model was proposed and model parameters were evaluated. The model was extrapolated to fed-batch cultivation and nutrient feeding strategy (with respect to carbon, nitrate and phosphate) were computer simulated for a suitable fed-batch cultivation of A. indica cells to enhance growth and product formation. Fed-batch cultivation demonstrated a growth of 20.06 g/l biomass and 0.082 g/l of azadirachtin in 14 days of cultivation. A 30% increase in cell growth and 83% increase in azadirachtin production were obtained when a computer simulated feeding strategy was experimentally implemented in 3 l bioreactor.

Azadirachtin is one of the most widely used biopesticide originating from a plant source. Its production from plant cell cultivation was viewed to overcome constraints associated with its regular supply from the seed kernels. In order to select the effective carbon and nitrogen source, different concentrations of carbon (sucrose/glucose) and nitrogen (NO 3 − /NH 4 + ratio) were studied in A. indica suspension culture. Glucose turned out to be a better carbon source over sucrose yielding high biomass (6.32 g/L) and azadirachtin (11.12 mg/L) content. Nitrate alone as nitrogen source was favorable for both biomass and azadirachtin accumulation. Plackett–Burman design was adopted to select the most important nutrients influencing the growth and azadirachtin accumulation in suspension culture. After identifying effective nutrients, Central Composite Design (CCD) was used to develop mathematical model equations, study responses and establish the optimum concentrations of the key nutrients for higher growth and azadirachtin production. A maximum of 15.02 g/L biomass and 2.98 mg/g azadirachtin was produced using optimum nutrient concentrations representing 99 and 96% validity of the model prediction with respect to biomass and azadirachtin, respectively. This optimized media can be used for cultivation of A. indica cells in bioreactor for mass production of azadirachtin.

Case report: We document a previously unreported association of Vogt-Koyanagi-Harada (VKH) syndrome with intracranial meningioma. A female patient with diminished vision, exudative retinal detachment, and headache was diagnosed with VKH syndrome, more precisely a Harada form of disease with intracranial meningioma, on the basis of exudative retinal detachment, typical fundus fluorescein findings, and magnetic resonance imaging. With intravenous steroid therapy, visual acuity improved and the detachment settled within a week. At 3 months, the detachment recurred but improved after retreatment. At I year, the tumour was unchanged in size. Comments: VKH syndrome may be associated with intracranial meningioma that may affect the patient's overall morbidity or mortality.

The effect of major nutrients on growth and azadirachtin-related limonoids (AZRL) production in plant cell culture of Azadirachta indica (neem) was studied with the objective to increase the yield of AZRL, one of the major group of pesticidal compounds found in intact neem tees. We report the novel online monitoring of plant cell respiration activities in a new parallel shake flask measuring device. Results obtained using three standard plant cell culture media showed non-growth-associated production characteristics for AZRL. These findings were supported by the oxygen uptake rate data. Further investigations on AZRL production in a modified MS medium with different concentrations of nitrogen and phosphorus sources resulted in 0.25 mg × g(exp -1) dry weight of AZRL, compared to no detectable AZRL production in standard MS media. These characteristics suggest the necessity of a two-stage process for the production of AZRL in plant cell culture. Compared to the single-stage process, an almost twofold increase in the volumetric productivity of AZRL was achieved using the two-stage process.