World's cholera vaccine stash is empty-but relief is on its way.

A dramatic shortage of the oral vaccine may ease in the years ahead as more companies enter the market.

India plans to invigorate science with hefty new funding agency.

Proposed National Research Foundation would spend $6 billion over 5 years, but is drawing mixed reactions.

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum.

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions-hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950)-directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆hp and ∆nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

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.

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.

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.

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.

Simultaneous lipid biosynthesis and recovery for oleaginous yeast Yarrowia lipolytica.

BACKGROUND: Recent trends in bioprocessing have underlined the significance of lignocellulosic biomass conversions for biofuel production. These conversions demand at least 90% energy upgradation of cellulosic sugars to generate renewable drop-in biofuel precursors (Heff/C ~ 2). Chemical methods fail to achieve this without substantial loss of carbon; whereas, oleaginous biological systems propose a greener upgradation route by producing oil from sugars with 30% theoretical yields. However, these oleaginous systems cannot compete with the commercial volumes of vegetable oils in terms of overall oil yields and productivities. One of the significant challenges in the commercial exploitation of these microbial oils lies in the inefficient recovery of the produced oil. This issue has been addressed using highly selective oil capturing agents (OCA), which allow a concomitant microbial oil production and in situ oil recovery process. RESULTS: Adsorbent-based oil capturing agents were employed for simultaneous in situ oil recovery in the fermentative production broths. Yarrowia lipolytica, a model oleaginous yeast, was milked incessantly for oil production over 380 h in a media comprising of glucose as a sole carbon and nutrient source. This was achieved by continuous online capture of extracellular oil from the aqueous media and also the cell surface, by fluidizing the fermentation broth over an adsorbent bed of oil capturing agents (OCA). A consistent oil yield of 0.33 g per g of glucose consumed, corresponding to theoretical oil yield over glucose, was achieved using this approach. While the incorporation of the OCA increased the oil content up to 89% with complete substrate consumptions, it also caused an overall process integration. CONCLUSION: The nondisruptive oil capture mediated by an OCA helped in accomplishing a trade-off between microbial oil production and its recovery. This strategy helped in realizing theoretically efficient sugar-to-oil bioconversions in a continuous production process. The process, therefore, endorses a sustainable production of molecular drop-in equivalents through oleaginous yeasts, representing as an absolute microbial oil factory.

Utilization of a Modified Phage E Protein Lysis System Accounts for Increased Biomass in Salmonella Gallinarum Ghosts.

A major limiting issue of bacterial ghost technology involves the stable maintenance of Phix174 lysis gene E expression. Unwanted leaky expression of gene E in the absence of induction temperature results in reduced biomass production of host bacterium, consequently leading to the lower yield of bacterial ghost. To mitigate the leaky expression status of lysis gene E, we utilized a novel E-lysis system in which gene E is located between sense λpR promoter with a CI857 regulator and antisense ParaBAD promoter with the AraC regulator. In the presence of L-arabinose at 28 C, unwanted transcription of lysis gene E from λpR promoter is repressed by a simultaneous transcription event from ParaBAD promoter by means of anti-sense RNA-mediated inhibition. Tight repression of lysis gene E in the absence of induction temperature resulted in higher bacterial cell number in culture suspension and, consequently, higher production of Salmonella Gallinarum (SG) ghost biomass. The safety and protective efficacy of the SG ghost vaccine were further examined in chickens. All of the immunized chickens showed significantly higher mucosal and systemic antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response. Vaccination of chickens with SG ghost preparation offered efficient protection against wild-type SG challenge.