Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineered C. reinhardtii, establishing it as a one -cell two -wells biorefinery.

The Haematococcus pluvialis is a recognized source of natural astaxanthin. The realm of astaxanthin bioprocessing is emerging with advanced techniques of cultivation and astaxanthin extraction. The present study addresses constraint of H. pluvialis cultivation under the Indian tropical environment for improved astaxanthin production, followed by purification of extracted astaxanthin. The chemical -based extraction using DMSO, yielded 163 mg g-1 of astaxanthin. Further, alkaline saponification was performed with 0.015 M NaOH at 25 degrees C to de-esterify astaxanthin esters. Considering the prerequisite of food and feed applications, a process was developed for the removal of non -GRAS solvent (DMSO) using adsorptive chromatography. Based on static adsorption and desorption studies of the resins, Amberlite XAD 1180 N was selected for column chromatography. Purification by column chromatographic study yielded DMSO-free 97.3 mg g-1 of astaxanthin, resulting in 80.8 % recovery. Stability studies confirmed 92 % retention of purified astaxanthin with 85 % antioxidant activity at 4 degrees C over 90 days.

Marine macroalgae - next-generation renewable feedstock is a potential solution to the growing need for sustainable future resources, with seaweed protein to cater to the ever-increasing demand of feeding the fastgrowing population. The conventional cultivation methods for marine macroalgae are impeded by low productivity, seasonal constraints, and climate change issues, necessitating improved cultivation strategies. The present study demonstrates the use of tank-based (T-PBR) and vertical (V-PBR) outdoor photobioreactors for free-floating substrate-independent land-based cultivation of filamentous green seaweed Enteromorpha prolifera. Optimization of the stocking density (SD) for seasonal variation was carried out to attain maximal biomass productivity. The study demonstrated biomass production with average annual productivity of 480 g fw m-2 d-1 in V-PBR and 413 g fw m- 2 d-1 in T-PBR. The season-specific SD, carbon dioxide supplementation (5%), and nutrient supply management ensured a year-round consistent supply of E. prolifera biomass. In spite of seasonal variation in biomass quality and quantity, an annual average content of 24% protein and 51% carbohydrate was observed. The present work substantiates the advancement of land-based seaweed cultivation for sustainable vegan protein production.

In order to improve the potential of cyanobacterial cell factories, Synechococcus sp. PCC7002 was engineered as 'one cell-two wells bio-refinery', for ethylene ('heterologous' hydrocarbon) and carotenoids ('natural' metabolites) production, and demonstrating its outdoor performance. Although the cultures showed better production outdoor, they experienced multiple collapses during scale-up. Hence, flux balance analysis was performed which predicted higher ethylene production with increase in carbon input under outdoor light conditions. Furthermore, FBA predicted that ethylene production will not increase beyond a threshold carbon input flux, owing to limitations on ribulose-1,5-bisphosphate regeneration. Hence, a bicarbonate-supplementation strategy was devised. Cultures grown outdoor at optimal bicarbonate concentration (20 g/L) resulted in improved growth (0.141/h) and ethylene productivity (1.88 mL/L.h) for > 10 days, with enhanced carotenoid titres (40.4 mg/L). In a 100 L air-lift photo-bioreactor; cultures exhibited efficient ethylene (2.464 mL/L.h) and biomass (0.3 g/L.d) productivities, and carotenoids titres (64.4 mg/L), establishing a significant step towards commercialization.

The present study conceived a novel technology - 'MARiNE photobioreactor (PBR): MacroAlgae Remediates Nutrients for Energy in PBR'. The technology targets the simultaneous treatment of desalination effluent and municipal liquid waste. The present study showcases the proof of concept wherein Ulva lactuca, a marine green macroalga, well-reported for its pollutant scavenging properties, has been explored for the synchronized treatment of two effluents, namely desalination effluent and municipal liquid waste and making it safe for marine discharge. A total nutrient removal of 94.1%, 80.4%, and 100% for COD, TN, and TP was achieved at an optimum HRT of 12 h with the highest biomass productivity of 340 g DW m(-2) d(-1). The study demonstrates MARiNE PBR to be a biologically cohesive strategy for rapid MLW remediation. A presumptive proposal for biorefinery of the generated biomass which enables recycling of the captured nutrients from the effluents is also proposed.

Cyanobacterial research is impeded by the substantial discrepancies between laboratory studies and outdoor performances, despite successful demonstrations of genetically engineered strains for array of compounds. Therefore, evaluation of adaptive responses is necessary to achieve outdoor scale-up cultivation of cyanobacteria. Under current study, cyanobacterium Synechococcus elongatus PCC7942 engineered for ethylene biosynthesis, was gradually acclimatised, ensuring sustained and progressive transition from laboratory to outdoor conditions. Bubble size of 4.9 +/- 0.2 mm and air-flow rate of 0.05 vvm in BG11 supplemented with 5 g/L bicarbonate giving mass transfer coefficient (K(L)a) of 10.48 h(-1) yielded highest specific growth rate (0.24 h(-1)) with the transformants. At the 100 L photobioreactor scale, ethylene productivity of 1.5 mL.L-1.h(-1) was achieved. A comprehensive investigation on photosynthetic responses of the transformants adapted to the outdoor conditions exhibited interesting photosynthetic electron transport regulations, involving antenna density modulation in response to diurnal and dynamic light transitions, indicating successful transition.

Conventional off-shore and on-shore cultivation methods for marine macroalgae are both inadequate to pitch macroalgae as scalable renewable feedstock that can be grown across all coastal locations. With on-shore cultivation likely to be sustainable and preferred over eco-damaging open seas cultivation, new reactor systems need to be developed for on-shore cultivation of seaweeds at scale. The present work is an attempt to use the indigenously designed vertical multi-tubular air-lift photobioreactor system to grow Ulva lactuca through the entire year under natural conditions. Optimized operation of the 1000 L photobioreactor assembly demonstrated a year-round averaged productivity of 0.87 kg m- 2.d- 1 (fresh weight) implying 1800 ton.ha- 1.y- 1 feedstock production. Carbon dioxide supplementation (5%), optimized circulation velocity (0.25-0.35 m/s), and managing nitrogen supply (17 ppm), under natural light intensities (500-1400 mu mol m- 2.s- 1) provided a year-round sustained and continuous production of Ulva lactuca biomass. The photobioreactor system designed as a modular, linearly scalable, and resilient system operates with low land and water footprints, and gives a multi-fold increase in renewable feedstock production compared to the conventional sea-based and other on-shore tank-based practices. For the video summary of this article, see the file in the supplemental data.

Exploiting solar energy for growing algal biomass in waters enriched with farm manures is a holistic method of waste management. The proposed cultivation strategy termed SAR'CENA ('Synergistic Algal Refinery for Circular Economy using Nutrient Analogues), involves integrated cultivation of microalga, Scenedesmus obliquus and marine macroalga, Ulva lactuca in litter to harness biorefinery products. From various litters tested, poultry litter manure (PLM) was most amenable for growth. The microalga yielded 410 +/- 6.2 g.DW m(-2) d(-1) of biomass with total nitrogen (TN) concentration of 70 mg.L-1 in the media, while the macroalgae yielded 334 +/- 9.9 g DW m(-2) d(-1) of biomass with TN concentration of 17.5 mg.L-1. The nutrient uptake efficiency was observed to be > 60% with uncompromised biomass composition. Thus, SAR'CENA is projected as an ideal farming solution incorporating efficient waste management and feedstock generation thereby establishing a circular economy towards clean energy.

Microalgal bioremediation is currently being venerated for its potential in municipal liquid waste (MLW) treatment. Algae-based water treatment with retention time competitive to the conventional water treatment processes is a challenge. The present study investigated the role of algal biomass concentration to improve treatment efficiency to reduce the time required for water treatment. Eighty percent removal of pollutants (in terms of chemical oxygen demand (COD), ammonia, phosphate and fecal coliforms) was obtained in 12 hours at a biomass concentration of 1 gL−1. Further, continuous treatment of MLW using membrane-assisted photobioreactor was established. The treatment led to >95% removal of ammonia, >75% removal of COD and 100% removal of fecal coliforms within 12 hours. Physiological assessment of the algal culture showed that the cells did not manifest symptoms of stress throughout the reactor cycle, a consequence of continuous availability of the nutrients, maintaining the culture in continuous growth state.

Open Raceway Ponds (RWP) are at present the most used large-scale reactors for microalgae culture. RWPs are extensively applied technology for algae mass cultivation although the scientific design of these ponds remains a major hurdle in this field. An erroneous design result in the presence of dead zones where the fluid flow is sluggish and non-uniform velocity throughout the pond actualized negative impact on algae growth, further these designs are energy inefficient. A dominant component of energy loss is the energy required to circulate the fluid around the raceway, particularly at the 180° bends. This paper investigates effects of various ratio of channel length to width (L/W) and position of side entry axial flow impeller (distance from the bottom of the tank) on the hydrodynamics in RWP. To curtail the dead zone, power consumption, shear stress and enhance surface renewal, the different designs of RWPs with flow deflectors and different types of central baffles were investigated by using Computational Fluid Dynamics (CFD). The CFD model was validated through Particle Image Velocimetry (PIV) tests. A feasible move headed for energy optimization and thus reduction in operational cost can be established through a better understanding of the mixing phenomena by CFD simulations.

Metabolomic fluctuations in microalgal cells under mixotrophic growth regimes are scarcely understood. Our study explores these changes in an oleaginous microalga, Asteracys sp. when grown mixotrophically under high irradiances. Biomass production under autotrophic and mixotrophic regimes were studied at two light intensities (LL; 100 mu mol photonsm(-2) s(-1) and HL; 900 mu mol photonsm(-2) s(-1)) for Asteracys sp. Studies on supplementation of external carbon such as glucose (mixotrophic mode) under different light intensities has not been demonstrated in context with enhanced biomass production. In the present study, we have provided external source of carbon as glucose in LL and HL regimes and observed a marked influence of glucose assimilation on the overall machinery of the cell. Our qualitative metabolomics reveals a stress-like phenomenon in Asteracys sp. under HL with enhanced biomass. In overview, our data analysis highlights few metabolites such as trehalose, proline and a-tocopherol that showed significant changes in response to light as well as in growth regime. Our analysis predicts an interrelation cross talk between maltose and sucrose pathways within cells owing to possible alteration in starch degradation under the influence of light. However, mixotrophy with additional carbon supplementation in presence of light did not alter or enhance lipid biosynthesis. In conclusion, our hypothesis indicates modulation of metabolites in presence of glucose under high light would rather facilitate rearrangement in metabolic pathways for enhanced biomass production.

Cyanobacteria are photosynthetic prokaryotes. Owing to their efficient photosynthesis ability over other photoautotrophs, simple cell organization, and competency for molecular modification, they have been widely exploited as "green-factories" for production of value-added compounds like carotenoids. Carotenoids are industrially important fine chemicals used in food, pharmaceutical, and cosmetic products. Cyanobacteria, being photosynthetic, can naturally synthesize carotenoids as cellular antioxidants. However, successful demonstrations have been made by the researchers to improve their carotenoid content through genetic engineering. Cyanobacteria are considered low-input, high-output hosts, where such a pathway engineering approach leverages their significance for the commercial markets. This chapter focuses on two important aspects of cyanobacteria as a next-generation resource for biomanufacturing: the significance of carotenoids in cyanobacterial photosynthesis and synthetic biology attributes in the area of cyanobacterial genome editing and engineering them for improved carotenoid yields for commercial exploitation.

This study is the first to explore the influence of incident light intensity on the photosynthetic responses under mixotrophic growth of microalga Asteracys sp. When grown mixotrophically, there was an enhanced regulation of non-photochemical quenching (NPQ) of the excited state of chlorophyll (Chl) a within the cells in response to white cool fluorescent high light (HL; 600 µmol photons m−2 s−1). Simultaneous measurement of reactive oxygen species (ROS) production as malondialdehyde (MDA) and ascorbate peroxidase (APX), an ROS scavenger, showed improved management of stress within mixotrophic cells under HL. Despite the observed decrease in quantum yield of photosynthesis measured through the Chl a fluorescence transient, no reduction in biomass accumulation was observed under HL for mixotrophy. However, biomass loss owing to photoinhibition was observed in cells grown phototrophically under the same irradiance. The measurements of dark recovery of NPQ suggested that “state transitions” may be partly responsible for regulating overall photosynthesis in Asteracys sp. The partitioning of photochemical and non-photochemical processes to sustain HL stress was analysed. Collectively, this study proposes that mixotrophy using glucose leads to a change in the photosynthetic abilities of Asteracys sp. while enhancing the adaptability of the alga to high irradiances.

Ulva lactuca is regarded as a prospective energy crop for biorefinery owing to its affluent biochemical composition and high growth rate. In fast-growing macroalgae, biomass development strictly depends on external nitrogen pools. Additionally, nitrogen uptake rates and photosynthetic pigment content vary with type of nitrogen source and light conditions. However, the combined influence of nitrogen source and light intensity on photosynthesis is not widely studied. In present study, pale green phenotype of U. lactuca was obtained under high light (HL) condition when inorganic nitrogen (nitrate) in the media was substituted with organic nitrogen (urea). Further, pale green phenotype survived the saturating light intensities in contrast to the normal pigmented control which bleached in HL. Detailed analysis of biochemical composition and photosynthesis was performed to understand functional antenna size and photoprotection in pale green phenotype. Under HL, urea-grown cultures exhibited increased growth rate, carbohydrate and lipid content while substantial reduction in protein, chlorophyll content and PSII antenna size was observed. Further, in vivo slow and polyphasic chlorophyll a (Chl a) fluorescence studies revealed reduction in excitation pressure on PSII along with low non-photochemical quenching thus, transmitting most of the absorbed energy into photochemistry. The results obtained could be correlated to previous report on cultivation of U. lactuca through saturating summer intensities (1000 µmole photons m−2 s−1) in urea based: poultry litter extract (PLE). Having proved critical role of urea in conforming photoprotection, the application PLE was authenticated for futuristic, sustainable and year-round biomass cultivation.

Heparosan is an unsulfated polysaccharide potentially important for its wide range of cosmetic and pharmaceutical applications, particularly as the precursor for the extensively used anticoagulant, heparin. Generally sourced from animals, commercially available heparin may encounter various immunological and contamination on risks. Thus, safe and sustainable microbial platforms could serve as an alternative heparin source. Synechococcus, due to their fast photoautotrophic growth, strong sugar phosphate metabolisms and generally regarded as safe (GRAS) nature, may serve as photo-biorefineries for manufacturing heparosan. In this study, we have synthesized an integrative plasmid pUPm48 for cloning galU and PmHS2 genes in Synechococcus elongatus PCC 7942. The engineered recombinants (pgp7942) exhibited significant production of heparosan under different culture conditions, where the products were present in both supernatant and cell biomass. The maximum yield of 0.7 +/- 0.2 mu g/g-DCW (dry cell weight) and a titer of 2.8 +/- 0.3 mu g/L was achieved by pgp7942 under shake flask and continuous light conditions. Large scale plastic-bag cultures with natural diurnal light exhibited heparosan production of 0.5 mu g/g-DCW with a titer of 0.44 mu g/L. The analysis also found PCC 7942 encodes a promiscuous uridyltransferase for UDP-glucose synthesis and naturally produces multiple glycosaminoglycans including chondroitin sulfate (CS). This study demonstrates for the first-time cyanobacteria as a promising photoautotrophic refinery for producing a high-value polysaccharide commonly from animals.

Biomethane Potential (BMP) of green macroalga Ulva lactuca post extraction of sap, ulvan and protein was analysed in batch studies. Extraction was performed in two different ways (individual and sequential) to understand the effect of removal of these components on methane yields. Both the treatments resulted in enhanced biomethane production in most of the treated residues, however, the highest methane yield of 408 +/- 20.02 ml CH4 g(-1) VS (70.93% of theoretical) was observed in sap and ulvan removed residue (batch VI). The methane production rates improved after both the treatments (0.15-0.28 day(-1) for untreated and treated batches). This corroborates well with the fact that high protein and sulphate content are major inhibitors in anaerobic digestion (AD) of U. lactuca and their removal leads to improved methane yields. Sequential extraction of value-added products prior to the AD process not only improves biomass amenability and respective methane yields but also makes the overall process more efficient and viable. (C) 2018 Elsevier Ltd. All rights reserved.

Cyanobacteria are globally recognized as potential photosynthetic platform cell factories for production of value-added chemicals. Carotenoids are industrially important fine chemicals used in food, pharmaceutical and health-care products. Zeaxanthin has emerged as a high value xanthophyll carotenoid that exhibits superior antioxidant properties over many other carotenoids. Traditionally sourced from plants like marigold flowers wherein, zeaxanthin co-occurs with lutein. Therefore, there is a need for isolated production system for zeaxanthin on account of difficulties in separation of zeaxanthin from lutein due to their very similar molecular structures. Synechococcus elongatus PCC 7942 (PCC 7942) is a cyanobacterium that is known to synthesize zeaxanthin as one of the predominant cellular carotenoids and does not possess pathway genes for lutein production. In order to construct a system capable of industrial production of zeaxanthin, we genetically modified PCC 7942 (Synechococcus 79R48) to improve beta-carotene flux towards zeaxanthin synthesis by cloning CrtR (beta-carotene oxygenase) gene from Synechococcus elongatus PCC 7002 through homologous recombination. This strategy effectively enhanced the yield (mg/g DCW) of zeaxanthin in the transformants. Moreover, to increase zeaxanthin titer (mg/L), an operon construct was synthesized where a heterologous gene GalP (Hexose-H+ symporter) from E. coli MG1655 was cloned downstream of CrtR (Synechococcus 79RG48) which facilitated inherently obligate photoautotrophic WT PCC 7942 cells to uptake extracellular glucose in transformants, thereby increasing biomass productivity. Autotrophically grown cells yielded 9.02 +/- 1.10 mg/g DCW of zeaxanthin while under mixotrophy the yield was 8.09 +/- 0.19 mg/g DCW, which was 2-fold improvement over the wild type. The volumetric productivities of the transformants were 1.18 +/- 0.17 mg/L.d and 1.8 +/- 0.06 mg/L.d under autotrophic and mixotrophic conditions, respectively while WT produced 0.58 +/- 0.02 mg/L.d. The work was successfully able to demonstrate that the modifications resulted in, enhanced production of lutein-free zeaxanthin in the cyanobacterial system.