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.

In today’s bio-based industry, lipase-catalyzed processes hold eminent commercial worth, yet their use is restricted owing to low yields, high production cost, inconsistent reproducibility, and poor performance in native form. Cloning and expression of multiple lipase genes in various systems have been investigated in order to produce enzymes for the food and detergent industries more cheaply since the development of recombinant DNA technology. The identification of novel lipases is still hampered by the rather difficult expression of these enzymes. The expression of lipases still requires a case-to case optimization. However, the unbiased choice of the appropriate promoter system and host for a specific protein of interest remains difficult. Here, we concisely expressed TLIP (Lipase from Thermomyces lanuginosus; mainly used in the detergent industry) in the frequently used conventional and alternative host systems, with their unique features, along with different promoters (T5, T7, aprE and hp4d) to produce recombinant products. Screening of expression was done among both prokaryotic ( Escherichia coli, Bacillus subtilis) and eukaryotic ( Yarrowia lipolytica) hosts consisting of both intracellular and secretory expression. E. coli (BL21 Shuffle) and Y. lipolytica (extracellular) were found to be the best expression systems for lipase 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.

In efforts to replace petro-derived polyethylene terephthalate (PET) with a ‘green’ equivalent, technologies are being developed and scaled up for biobased production of the two components of PET viz. monoethylene glycol (MEG) and terephthalic acid. Herein, we report work on single-pot catalytic hydrogenolysis of cellulose isolated from lignocellulosic biomass into polyols with the major fraction being monoethylene glycol. A combination of silica-alumina-supported nickel (NSA) and bulk tungsten-based catalyst (HW) was investigated for selective conversion of cellulose to MEG. While raw rice straw showed negligible conversion, the two-step pretreated rice straw performed similar to pure cellulose. Ethylene glycol in up to 60 % yield was obtained over 0.15%NSA-0.2%HW within minutes at a hydrogen pressure lower than reported thus far for similar catalysts. Product buildup was seen to have no effect on the catalyst activity and a fed-batch approach could generate product stream in high concentrations with MEG as a single major product.

Catechol is an industrially relevant chemical with myriad applications. Its production via chemical route suffers from several drawbacks the major being a non-green and nonselective route. Currently, bio-based products using biocatalyst are gaining attention due to the growing environmental and health hazards concerns over the use of petroleum-derived feedstock. Lignocellulosic biomass serves as a promising feedstock. Lignin valorization is the demand of the current scenario which is complicated task by its complexity, heterogeneity and diversity of lignin structures posing limitations toward lignin valorization via chemical routes. There are several microorganisms that possess the ability to metabolize lignin monomers via their central metabolic pathways and this paves the way to the synthesis of a number of products. Pseudomonas putida KT2440 is one such organism and was chosen for genetic manipulations for catechol biosynthesis using lignin-derived model compounds and biomass hydrolysate stream comprising of various lignin monomers. Catechol production was engineered by diverting various lignin monomers and addressing the identified metabolic bottlenecks particularly vanillic acid accumulation toward catechol biosynthesis. The engineered strain could convert the model lignin monomers as well as monomers in the biomass hydrolysates to catechol and vanillic acid in more than 60% and 90% molar yields, respectively.

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.

Biobased chemicals are gaining popularity and market in attempts to mitigate the deteriorating environmental and sustainability issues. Components of renewable agricultural and forest biomass residues are projected to serve as abundant precursors to synthesis of expanding range of products. Agroindustrial wastes comprises of several phenolic compounds associated with lignin via ether linkages such as ferulic acid, p-coumaric, syringic acid and vanillin. These aromatic chemicals have myriad industrial applications. In this study, p-coumaric acid and ferulic acid were found to be two major components in corn bran derived lignin hydrolysate. Engineered Pseudomonas putida KT2440 was constructed and found to convert p-coumaric acid and vanillic acid to protocatechuic acid in >90% and >50% yields, respectively. Engineering the strain included deletion of the gene encoding protocatechuate 3,4-dioxygenase, and overexpression of vanillate-O-demethylase gene from Acinetobacter sp. ADP1.

Capreomycin (CMN) is a second-line aminoglycoside antibiotic for treating multi-drug resistant tuberculosis. Produced as a complex broth in microbial bioreactors, the downstream purification of CMN traditionally requires multiple steps. Current work aims to purify CMN as mixed isomers in a single step by preparative macroporous cation-exchange chromatography in >98% purity in >90% yield. Selection of the adsorbent best suited for CMN purification was guided by modeling the molecular interactions, followed by wet-lab static and dynamic adsorption studies that validated the simulation results. Among the evaluated adsorbents (both weak and strong cation resins), the weak cationic Macro-Prep CM showed a favorable combination of selective binding and elution of CMN. Solute loading and elution conditions were further optimized to produce the desired quality of CMN. This single-step method was integrated with a continuous simulated bed reverse-phase desalting chromatography to produce 99% pure salt-free CMN. The proposed method provided productivity of about 2 g product/L resin/h of CMN on Macro-Prep CM; and 0.7 g product/L adsorbent/h on SMB. The study provides for a robust and scalable process for the industrial-scale production of purified capreomycin.

Rapid and high yield conversion of xylose to ethanol remains a signi cant bottleneck in the cost-effective production of ethanol using mixed sugars derived from lignocellulosic biomass (LBM). The present study attempts to circumvent this by separate continuous fermentation of glucose and xylose using high cell densities of a Saccharomyces cerevisiae mutant (ICT-1) and a Scheffersomyces stipitis mutant (M1CD), respectively with the help of external micro ltration membrane assisted cell recycle. Different cell densities and aeration rates for xylose fermentation were studied for optimizing continuous fermentation. Consistent high ethanol yields and productivities of 0.46 g/g and 5.19 g/L/h with glucose; and 0.38 g/g and 1.62 g/L/h with xylose; were achieved in simple media. This provided an average ethanol yield of 0.44 g/g on combined sugars, and average productivity of 3.4 g/L/h which is higher than typical molasses-based batch ethanol fermentation. The study thus highlights the potential of high cell density recycle strategy as an effective approach for separate ethanol fermentation of LBM derived sugars.

Development of preparative methods for the isolation of chiral molecules has been considered challenging by conventional unit operations due to their identical physical and chemical properties. This has evolved chiral stationary phases for the separation of chiral components using chromatography technique. However, separation method using chiral adsorbents requires high pressure, are expensive, and have low productivity. Generation of bulk quantities purified nebivolols using the available high pressure chiral separation methods is impractical and operating cost-intensive. Thus, there is a need to develop economical methods using nonchiral adsorbents for the purification of nebivolols or similar active ingredients. The present work demonstrates a unique and scalable tandem two-column method for the separation of isomers of nebivolol using inexpensive reverse phase adsorbents. The first column of the scheme causes removal of charged and nonisomeric impurities whereas tandem operation of second column increases resolution of d-nebivolol and l-nebivolol. The maximization of separation due to tandem operation of second column causes enhancement of the throughput of the process. The developed preparative process produces >98% purity of both d-nebivolol and l-nebivolol with overall loading capacity of 56 g (L of adsorbent)-1 and productivity of 20 g L-1 day-1 .

The microalgal cell wall breakage has been identified as complex phenomenon which is highly dependent on the nature and composition of cell wall.A detailed analysis of plastids and their function requires the breaking open of cell without any damage to cellular components.To develop a rapid and universal methodology for cell wall breakage, liquid nitrogen crushing, sonication, enzymatic lysis, and homogenization procedures were applied to various microalgal species.Homogenization-based procedure for the isolation of intact chloroplast was found to be universal for all algal species under the study.The isolated chloroplasts were subjected to chloroplast integrity analysis.The intact chloroplast exhibited a positive maximum quantum yield and F v / F m values ranging from 0.1 to 0.4 as measured by pulse amplitude modulation fluorometry and was found to be suitable for further downstream applications such as isolation of protein-pigment complexes involved in photosynthetic O 2 evolution.The developed methodology is a quick and efficient technique for the isolation of intact chloroplasts across different genera of microalgae by employing minor changes in the base protocol as a species-specific characteristic.

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.

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the vanAB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95 +/- 1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87 +/- 0.38% molar yield. With fermentation time of less than 24 h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.

A consolidated bioprocessing (CBP), where lignocellulose is converted into the desired product(s) in a single fermentative step without the addition of expensive degradative enzymes, represents the ideal solution of renewable routes to chemicals and fuels. Members of the genus Geobacillus are able to grow at elevated temperatures and are able to utilise a wide range of oligosaccharides derived from lignocellulose. This makes them ideally suited to the development of CBP. In this study, we engineered Geobacillus thermoglucosidasius NCIMB 11955 to utilise lignocellulosic biomass, in the form of nitric acid/ammonia treated wheat straw to which expensive hydrolytic enzymes had not been added. Two different strains, BZ9 and BZ10, were generated by integrating the cglT (β-1,4-glucosidase) gene from Thermoanaerobacter brockii into the genome, and localising genes encoding different cellulolytic enzymes on autonomous plasmids. The plasmid of strain BZ10 carried a synthetic cellulosomal operon comprising the celA (Endoglucanase A) gene from Clostridium thermocellum and cel6B (Exoglucanase) from Thermobifida fusca; whereas, strain BZ9 contained a plasmid encoding the celA (multidomain cellulase) gene from Caldicellulosiruptor bescii. All of the genes were successfully expressed, and their encoded products secreted in a functionally active form, as evidenced by their detection in culture supernatants by Western blotting and enzymatic assay. In the case of the C. bescii CelA enzyme, this is one of the first times that the heterologous production of this multi-functional enzyme has been achieved in a heterologous host. Both strains (BZ9 and BZ10) exhibited improved growth on pre-treated wheat straw, achieving a higher final OD600 and producing greater numbers of viable cells. To demonstrate that cellulosic ethanol can be produced directly from lignocellulosic biomass by a single organism, we established our consortium of hydrolytic enzymes in a previously engineered ethanologenic G. thermoglucosidasius strain, LS242. We observed approximately twofold and 1.6-fold increase in ethanol production in the recombinant G. thermoglucosidasius equivalent to BZ9 and BZ10, respectively, compared to G. thermoglucosidasius LS242 strain at 24 h of growth. We engineered G. thermoglucosidasius to utilise a real-world lignocellulosic biomass substrate and demonstrated that cellulosic ethanol can be produced directly from lignocellulosic biomass in one step. Direct conversion of biomass into desired products represents a new paradigm for CBP, offering the potential for carbon neutral, cost-effective production of sustainable chemicals and fuels.

In the present study, we report a reverse-phase high-performance liquid chromatography (RP-HPLC) method for separation of the regio-isomers of Glyceryl MonoRicinoleate (GMR) identified using position specificity of lipases. The approaches explored to identify these regio-isomers include LC-mass spectrometry, UV spectroscopy, and selective hydrolysis with lipases. A distinct UV absorption spectrum and λmax values for each isomer were noted, and mass spectral analysis further revealed their molecular weight. Lastly, the purified regio-isomers were subjected to hydrolysis with two distinctive regio-specific lipases to identified as sn-2 and sn-1(3) GMR. The current methodology of using analytic tool and enzyme specificity provides a useful platform for identifying regio-isomers for structured lipid synthesis.

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 work presents detailed life cycle assessment (LCA) of a novel process to produce ethanol from rice straw in India. The process has been successfully demonstrated and proposed to be scaled-up, and detailed LCA of that process is the key novel contribution of this work. Cradle-to-gate system boundary is considered, which includes rice farming, transportation, and processing at the biorefinery. 1 l of ethanol is used as the functional unit. The process data are based on the demonstration-scale plant as well as the scale-up plant of 100 kilo litres per day being designed based on the same process. The life cycle inventory data are taken from the Ecoinvent® database. OpenLCA 1.6 is used to develop the LCA model, and impact assessment is performed using ILCD 2011 midpoint indicators. The GWP was 2.82 kg of CO2 eq. per liter of ethanol using economic impact allocation. Electricity contributed 86% of the total impact, and use of hydroelectricity reduced the impact to 0.07 kg of CO2 eq. per liter of ethanol. If additional benefits due to this process are considered, the impact reduced to − 0.392 kg of CO2 eq. per liter of ethanol indicating considerable relative reduction in the GWP. Without allocation and implementing system expansion, the impact was 3.35 kg of CO2 eq. per liter of ethanol. The energy return on investment was 1.59, indicating that the process was net energy positive. The lower bound on the life cycle water use was 507.4 l per liter of ethanol. The integrated nature of the process producing various value-added chemicals provided significant benefits from the perspective of environmental impacts.

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.

Microwave assisted synthesis of 5-hydroxymethylfurfural (5-HMF) over sulfonic acid anchored solid acid catalysts in isopropyl alcohol (IPA) offers specific advantages. Comparison of catalysts with different backbones stipulates a profound effect on the selectivity of fructose dehydration in IPA. The catalyst with an aliphatic backbone has a higher yield than the catalyst with a styrenic backbone. Of the tested catalyst, the DBT-ICT-Centre for Energy Biosciences, Institute of Chemical Technology Catalyst #2 (DICAT-2) in IPA resulted in 85% yield with >95% conversion in 120 s. The modeling analysis depicted the characteristic role of the aliphatic backbone for obtaining higher selectivity in IPA. Moreover, the use of IPA is ecologically preferred and it also facilitates product, solvent, and catalyst separation. The structural and morphological characterization with a recyclability study for five consecutive runs showed consistency in yield and conversion. The turnover number (TON), turnover frequency (TOF), E-factor, and process mass intensity (PMI) indicate an environmentally safer and industrially viable process.

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.