Green synthesis, characterization and functional validation of bio-transformed selenium nanoparticles.

Selenium, an essential micronutrient with potent anticancer and antioxidant properties, the inorganic form of selenium is highly toxic, while organic and elemental nanoforms are more bioavailable and less toxic and have gained attention owing to their dietary and clinical relevance. This study aims to optimize conditions for the biosynthesis and production of elemental selenium nanoparticles for selenium supplements using marine microalgae, Nannochloropsis oceanica CASA CC201. The 10 mM precursor solution treated with 1 % of the algal extract (10:1 ratio of precursor and algal extract, respectively) was shown to be the optimal concentration for synthesizing highly stable selenium nanoparticles with a size of 183 nm and a zeta potential of -38.5 mV. AFM and TEM analysis suggest that the spherical-shaped nanoparticles with smooth surfaces were polydispersely distributed. The nanoparticles are well characterized using various analytical and advanced techniques, including Raman spectroscopy and X-ray photoelectron spectroscopy. FT-IR analyses reveal the presence of microalgae proteins and peptides as stabilizing and fabricating agents of Se-NPs to further understand the mode of bioreduction. The synthesized elemental nanoform (Se0) has been validated for its biological functions, showing enhanced radical scavenging activity (74 % in a concentration-dependent manner). Subsequently, algal-mediated selenite reduction and nanoparticle synthesis is an eco-friendly, non-toxic, and sustainable method for the large-scale production of highly stable Se-NPs for niche applications as dietary and feed supplements.

Sustainable microalgal cultivation in poultry slaughterhouse wastewater for biorefinery products and pollutant removal.

Microalgae's ability to grow in poultry slaughterhouse wastewater (PSHWW) is attracting interest for low-cost biomass production and wastewater treatment. In this study, PSHWW is evaluated by the cultivation of Chlorella sp. andNeochloris sp. for biomass,bioproducts, and nutrient removal. Results showed that Neochloris sp.produced the maximum of 1.4 g L-1 biomass and 38% lipids compared toChlorella sp. (1.3 g L-1 and 36%). The maximum carotenoids, proteins, and carbohydrates obtained from Neochloris sp. are 38 mg/g DW, 41.7%, and 29%, respectively. COD, nitrite, and phosphate removal efficiencies of 96.8%, 95%, and 79%, respectively, by Neochloris sp. and 89%, 93.5%, and 64.5%, respectively, by Chlorella sp. FTIR confirms the role of functional groups in pollutant absorption by microalgae. The predominant fatty acids found were C16, C18, C18:1, C18:2, C18:3, C20:5, and C22:6. The research demonstrated that microalgae can be used for the treatment of wastewater, nutraceuticals, food additives, and biofuels.

Comparative study of different catalysts mediated FAME conversion from macroalga Padina tetrastromatica biomass and hydrothermal liquefaction facilitated bio-oil production.

Marine macroalgae offer an endurable source of renewable biomass, which do not require cultivable area, fertilizers for cultivation for bioproducts production. In this study, marine brown macroalga Padina tetrastromatica as an alternate sustainable feedstock for the production of liquid fuels. Padina tetrastromatica biomass was collected from Mandapam; the coastal region of Rameswaram, Tamil Nadu, India. and the algal oil was extracted using sequential extractions using various solvents. Petroleum ether (PE) and dichloromethane (DCM) solvent fractions were found to have high lipids and further utilized for biodiesel production, wherein four different heterogeneous nanocatalysts (TiO2, Bio-Fe, GO, and MgO) and commercial homogeneous catalysts (HCl and NaOH) were employed for the transesterification. High fatty acid methyl ester (FAME) recovery (92.3%) was achieved from TiO2 mediated transesterification than the other conventional catalysts. Further, the conversion of algal biomass into bio-oil and by-products was carried out using hydrothermal liquefaction (HTL). Subsequently, the compounds were characterized by FT-IR and GC-MS analysis. The quality parameters of liquid biofuels were examined and they are in accordance with the international fuel standards. Thus, brown macroalga Padina tetrastromatica may be considered as an alternate feedstock for biofuel and other bioproducts production and TiO2 would be a suitable catalyst for the conversion of FAME.

Enhancement of lipid accumulation in microalga Desmodesmus sp. VV2: Response Surface Methodology and Artificial Neural Network modeling for biodiesel production.

Microalgae are the most attractive renewable energy sources for the production of biofuels because of their luxurious growth and lipid accumulation ability in diverse nutritional conditions. In the present study, Desmodesmus sp. VV2, an indigenous microalga, was evaluated for its biodiesel potential using Response Surface Methodology (RSM) to improve the lipid accumulation with the combination of nutrients stress NaNO3 starvation, CaCl2 depletion, and supplementation of magnesium oxide nanoparticles (MgO). Among different stress conditions, 57.6% lipid content was achieved from RSM optimized media. Owing to this, RSM results were also validated by the Artificial Neural Network (ANN) with 11 training algorithms and it is found that RSM was more significant. In addition, the saturated fatty acid (SFA) content was noticeably increased in RSM optimized media (95.8%) while compared with control. Further, the highest total FAME content 97.21% was also achieved in cells grown in RSM optimized media. Biodiesel quality parameters were analyzed and found that they are in accordance with international standards. Thus, this study suggesting that the fatty acid profile of Desmodesmus sp. VV2 attained under optimized media conditions would be suitable for biodiesel production for future energy demand.

Statistical optimization and formulation of microalga cultivation medium for improved omega 3 fatty acid production.

Microalgae are considered a rich source of high-value metabolites with an array of nutraceutical and pharmaceutical applications. Different strategies have been developed for cultivating microalgae at large-scale photobioreactors but high cost and low productivity are the major hurdles. Optimizing the composition of media for the cultivation of microalgae to induce biomass production and high-value metabolite accumulation has been considered as an important factor for sustainable product development. In this study, the effect of plant growth regulators together with basal microalgal cultivation medium on biomass, total lipid, and EPA production was studied using the Plackett-Burman model and Response surface methodology. The traditional one-factor-at-a-time optimization approach is laborious, time-consuming, and requires more experiments which makes the process and analysis more difficult. The Designed PB model was found to be significant for biomass (396 mg/L), lipid (254 mg/L), and EPA (5.6%) production with a P value < 0.05. The major objective of this study is to formulate a medium for EPA production without compromising the growth properties. Further, we had formulated a new media using RSM to achieve the goal and the significant variables selected were NaNO3, NaH2PO4, and IAA and was found to be significant with 16.72% EPA production with a biomass production of 893 mg/L with a P value < 0.05. The formulated medium can be used in large-scale cultivation systems which can enhance biomass production as well as the omega 3 fatty acid production in marine microalgae Nannochloropsis oceanica. Supplementary Information: The online version contains supplementary material available at 10.1007/s43393-021-00069-1.

Structural elucidation of selenocysteine insertion machinery of microalgal selenoprotein T and its transcriptional analysis.

Essential trace element selenium in association with selenoproteins, which is found in almost all organisms except higher plants and fungi, is involved in various biological functions. Advancement in the field of whole genome sequencing and data analyzing bioinformatic tools led to the accumulation of genome information of organisms. However, selenoproteins are unique and it needs specialized genomics tool for its identification as well as characterization. In this study, the presence of selenoprotein T (SelT) from Scenedesmus quadricauda was shown for the first time with experimental evidence and compared with SelT of marine microalgae Nannochloropsis oceanica. Along with SelT, all the associated machineries required to synthesize the selenoproteins were also identified. Also, the present study tried to explicate the evolutionary relatedness of SelT of these two organisms with other known bacteria and eukaryotes. Transcript level analysis in S. quadricauda under endoplasmic reticulum stress showed a 1.2 ± 0.28-fold increase in SelT expression. Thus, it provided the first experimental evidence on SelT expression from microalgae.

Targeted Metabolomic and Biochemical Changes During Nitrogen Stress Mediated Lipid Accumulation in Scenedesmus quadricauda CASA CC202.

Scenedesmus quadricauda CASA CC202, a potent freshwater microalga is being used as a biofuel feedstock, which accumulates 2.27 fold lipid during nitrogen stress induction. Upon nitrogen starvation, S. quadricauda undergoes biochemical and metabolic changes that perturb the cell to cope up the stress condition. The nitrogen stress-induced biochemical changes in mitochondrion exhibits due to the oxidative stress-induced Reactive Oxygen species (ROS) generation at high membrane potential (Δψm). The predominant ROS generated during nitrogen starvation was H2O2, OH-, O2⋅- and to suppress them, scavenging enzymes such as peroxidase and catalase increased to about 23.16 and 0.79 U/ml as compared to control (20.2, 0.19 U/ml). The targeted metabolic analysis showed, stress-related non-proteinogenic amino acids and energy equivalents elevated during the initial hours of nitrogen starvation. The nitrogen stress-triggered biochemical and metabolic changes along with other cellular events eventually lead to lipid accumulation in S. quadricauda.

Stress hormones mediated lipid accumulation and modulation of specific fatty acids in Nannochloropsis oceanica CASA CC201.

The aim of this study is to analyze the effect of two plant growth regulators on selective modulation of nutraceutically important fatty acids. Exogenous application of methyl jasmonate (MeJA) promoted microalgal growth compared to control. Treatment with 10 ppm salicylic acid (SA) induced significantly higher lipid production of 475 mg/L (2.2 fold). Interestingly treatment with higher doses of MeJA promoted monounsaturated fatty acid production, particularly oleic acid (C18:1) at early stationary growth phase, while treatment with SA induces essential omega 3 fatty acid production (EPA, C20:5). This significant modification of fatty acid compositions was correlated with the oxidative stress in terms of total reactive oxygen species production and endogenous growth hormone levels. Taken together, the results indicated that treatment with stress associated plant hormones significantly increased high value metabolite accumulation specifically MUFA and PUFA production by modulating stress mechanisms and endogenous growth hormone levels.

Lipase of Pseudomonas guariconesis as an additive in laundry detergents and transesterification biocatalysts.

A newly isolated culture, Pseudomonas guariconesis, is reported for the first time for lipase production. Various process parameters affecting enzyme production were optimized through statistical design experiments. The Plackett-Burman experimental design was used for screening 10 parameters for lipase production, which was further optimized using the central composite design of response surface methodology. Maximum lipase activity of 220 U/ml was obtained after 24 h of incubation in shake-flask cultures with an inoculum concentration of 0.6% v/v, incubation temperature of 30°C, and medium pH 9.0. Castor oil (0.5% v/v) was used as the inducer for lipase production. The enzyme was found to be compatible with five different commercial detergents, indicating its potential to be used in detergent formulations. It also acted as a biocatalyst in a transesterification process. The alkaline enzyme was found to be stable in the presence of bleaching agents, metal ions, and organic solvents as well.

Omega-3-rich Isochrysis sp. biomass enhances brain docosahexaenoic acid levels and improves serum lipid profile and antioxidant status in Wistar rats.

BACKGROUND: Isochrysis sp. is a marine microalga, rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The potential use of its biomass as an alternative source of polyunsaturated fatty acids (PUFAs) has not been studied in animal models. Male albino Wistar rats were divided into three groups and treated for 28 days. The rats were fed with (1) standard chow (control group), (2) microalgal biomass rich in EPA and DHA along with standard chow (microalga group), and (3) fish oil that contains equivalent amounts of EPA and DHA along with standard chow (fish oil group). After intervention, biochemical indices, histopathological indices, relative mRNA expression of PUFA genes, antioxidant genes, inflammatory markers, and the fatty acid profile of major tissues were studied. RESULTS: Animals treated with microalgal biomass showed significantly increased serum HDL levels (P < 0.05) and reduced oxidative stress markers with a concomitant decrease in urea and creatinine levels. Oral supplementation of microalgal biomass did not show any toxicity or damage in any major organs. The mRNA expression of PUFA genes was significantly downregulated (P < 0.05) and antioxidant genes were upregulated. Furthermore, the mRNA expression of pro-inflammatory markers was significantly downregulated (P < 0.05) and anti-inflammatory markers were upregulated. Oral supplementation of microalgal biomass improved DHA status in brain and liver. CONCLUSION: The present study demonstrated that Isochrysis sp. can be used as a safe, alternative food supplement for ω-3 fatty acids. © 2019 Society of Chemical Industry.

Batch Sedimentation Studies for Freshwater Green Alga Scenedesmus abundans Using Combination of Flocculants.

Microalga is the only feedstock that has the theoretical potential to completely replace the energy requirements derived from fossil fuels. However, commercialization of this potential source for fuel applications is hampered due to many technical challenges with harvesting of biomass being the most energy intensive process among them. The fresh water microalgal species, Scenedesmus abundans, has been widely recognized as a potential feedstock for production of biodiesel (Mandotra et al., 2014). The present work deals with sedimentation of algal biomass using extracted chitosan and natural bentonite clay powder as flocculant. The effect of flocculant combination and different factors such as temperature, pH, and concentration of algal biomass on sedimentation rates has been analyzed. A high flocculation efficiency of 76.22 ± 7.81% was obtained at an algal biomass concentration of 1 ± 0.05 g/L for a settling time of 1 h at 50 ± 5°C with a settling velocity of 103.2 ± 0.6 cm/h and a maximum surface conductivity of 2,260 ± 2 µS/cm using an optimal design in response surface methodology (RSM). Biopolymer flocculant such as chitosan exhibited better adsorption property along with bentonite clay powder that reduced the settling time significantly.

Selective degradation of the recalcitrant cell wall of Scenedesmus quadricauda CASA CC202.

MAIN CONCLUSION: An eco-friendly cell wall digestion strategy was developed to enhance the availability of nutritionally important bio molecules of edible microalgae and exploit them for cloning, transformation, and expression of therapeutic proteins. Microalgae are the source for many nutritionally important bioactive compounds and potential drugs. Even though edible microalgae are rich in nutraceutical, bioavailability of all these molecules is very less due to their rigid recalcitrant cell wall. For example, the cell wall of Scenedesmus quadricauda CASA CC202 is made up of three layers comprising of rigid outer pectin and inner cellulosic layer separated by a thin middle layer. In the present investigation, a comprehensive method has been developed for the selective degradation of S. quadricauda CASA CC202 cell wall, by employing both mechanical and enzymatic treatments. The efficiency of cell wall removal was evaluated by measuring total reducing sugar (TRS), tannic acid-ferric chloride staining, calcoflour white staining, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) analysis. It was confirmed that the yield of TRS increased from 129.82 mg/g in 14 h from pectinase treatment alone to 352.44 mg/g by combined sonication and enzymatic treatment within 12 h. As a result, the combination method was found to be effective for the selective degradation of S. quadricauda CASA CC202 cell wall. This study will form a base for our future works, where this will help to enhance the digestibility and availability of nutraceutically important proteins.

Improved biomass and lipid production in Synechocystis sp. NN using industrial wastes and nano-catalyst coupled transesterification for biodiesel production.

In this study, the improved biomass (1.6 folds) and lipid (1.3 folds) productivities in Synechocystis sp. NN using agro-industrial wastes supplementation through hybrid response surface methodology-genetic algorithm (RSM-GA) for cost-effective methodologies for biodiesel production was achieved. Besides, efficient harvesting in Synechocystis sp. NN was achieved by electroflocculation (flocculation efficiency 97.8±1.2%) in 10min when compared to other methods. Furthermore, different pretreatment methods were employed for lipid extraction and maximum lipid content of 19.3±0.2% by Synechocystis sp. NN was attained by ultrasonication than microwave and liquid nitrogen assisted pretreatment methods. The highest FAME (fatty acid methyl ester) conversion of 36.5±8.3mg FAME/g biomass was obtained using titanium oxide as heterogeneous nano-catalyst coupled whole-cell transesterification based method. Conclusively, Synechocystis sp. NN may be used as a biodiesel feedstock and its fuel production can be enriched by hybrid RSM-GA and nano-catalyst technologies.

Selective enrichment of Eicosapentaenoic acid (20:5n-3) in N. oceanica CASA CC201 by natural auxin supplementation.

The present study aims to evaluate the effect of different concentration of natural auxin, Indole-3 acetic acid (IAA) on growth, lipid yield, PUFA and EPA accumulation in Nannochloropsis oceanica CASA CC201. It was observed that the, treatment with 10ppm concentration of IAA resulted in high cell number 579.5×106cells/ml than the control (215.5×106cells/ml). Treatment with IAA at a concentration of 40ppm gives the highest cellular lipid accumulation of 60.9% DCW than the control 31.05% DCW). Lipid yield is also found to be increased by the addition of 40ppm IAA (319.5mg/L) compared with the control (121.5mg/L). EPA percentage is increased to 10.76% by the addition of 40ppm IAA compared to the control (1.87%).

Influence of abscisic acid on growth, biomass and lipid yield of Scenedesmus quadricauda under nitrogen starved condition.

Scenedesmus quadricauda, accumulated more lipid but with a drastic reduction in biomass yield during nitrogen starvation. Abscisic acid (ABA) being a stress responsible hormone, its effect on growth and biomass with sustainable lipid yield during nitrogen depletion was studied. The result revealed that the ABA level shoots up at 24h (27.21pmol/L) during the onset of nitrogen starvation followed by a sharp decline. The external supplemented ABA showed a positive effect on growth pattern (38×10(6)cells/ml) at a lower concentration. The dry biomass yield is also increasing up to 2.1 fold compared to nitrogen deficient S. quadricauda. The lipid content sustains in 1 and 2µM concentration of ABA under nitrogen-deficient condition. The fatty acid composition of ABA treated S. quadricauda cultures with respect to nitrogen-starved cells showed 11.17% increment in saturated fatty acid content, the desired lipid composition for biofuel application.

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation--A Novel Role for NDK.

INTRODUCTION: Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. METHODS: Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. RESULTS: NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. CONCLUSION: Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.

Enhanced lipid accumulation and biomass yield of Scenedesmus quadricauda under nitrogen starved condition.

The experimental results revealed that the 2.27-fold lipid yield was enhanced in nitrogen-depleted condition (226 mg/L) when compared to nitrogen rich condition (99.33 mg/L). Since specific growth rate (SGR) is the single most criteria to decide the biomass yield of microalgae, SGR was analyzed. SGR of 0.33 day(-1) was observed with 0.47 doubling per day under nitrogen rich condition. However, low growth rate of 0.14 day(-1) with a doubling of 0.20 per day was recorded under nitrogen depleted condition. In accordance with SGR, the dry biomass yield was ranged from 0.055 ± 0.005 to 1.394 ± 0.010 g/L in the presence of nitrogen, indicating the essentiality of major nutrient in the growth medium. The enhanced lipid accumulation under nitrogen starved condition by Scenedesmus quadricauda was perhaps achieved by adopting adverse environmental condition and possible increment in cell size in terms of length/breadth ratio (1.11).

Mycobacterium tuberculosis cell division protein, FtsE, is an ATPase in dimeric form.

FtsE is one of the earliest cell division proteins that assembles along with FtsX at the mid-cell site during cell division in Escherichia coli. Both these proteins are highly conserved across diverse bacterial genera and are predicted to constitute an ABC transporter type complex, in which FtsE is predicted to bind ATP and hydrolyse it, and FtsX is predicted to be an integral membrane protein. We had earlier reported that the MtFtsE of the human pathogen, Mycobacterium tuberculosis, binds ATP and interacts with MtFtsX on the cell membrane of M. tuberculosis and E. coli. In this study, we demonstrate that MtFtsE is an ATPase, the active form of which is a dimer, wherein the participating monomers are held together by non-covalent interactions, with the Cys84 of each monomer present at the dimer interface. Under oxidising environment, the dimer gets stabilised by the formation of Cys84-Cys84 disulphide bond. While the recombinant MtFtsE forms a dimer on the membrane of E. coli, the native MtFtsE seems to be in a different conformation in the M. tuberculosis membrane. Although disulphide bridges were not observed on the cytoplasmic side (reducing environment) of the membrane, the two participating monomers could be isolated as dimers held together by non-covalent interactions. Taken together, these findings show that MtFtsE is an ATPase in the non-covalent dimer form, with the Cys84 of each monomer present in the reduced form at the dimer interface, without participating in the dimerisation or the catalytic activity of the protein.

Heat and SDS insensitive NDK dimers are largely stabilised by hydrophobic interaction to form functional hexamer in Mycobacterium smegmatis.

The primary structure and function of nucleoside diphosphate kinase (NDK), a substrate non-specific enzyme involved in the maintenance of nucleotide pools is also implicated to play pivotal roles in many other cellular processes. NDK is conserved from bacteria to human and forms a homotetramer or hexamer to exhibit its biological activity. However, the nature of the functional oligomeric form of the enzyme differs among different organisms. The functional form of NDKs from many bacterial systems, including that of the human pathogen, Mycobacterium tuberculosis (MtuNDK), is a hexamer, although some bacterial NDKs are tetrameric in nature. The present study addresses the oligomeric property of MsmNDK and how a dimer, the basic subunit of a functional hexamer, is stabilized by hydrogen bonds and hydrophobic interactions. Homology modeling was generated using the three-dimensional structure of MtuNDK as a template; the residues interacting at the monomer-monomer interface of MsmNDK were mapped. Using recombinant enzymes of wild type, catalytically inactive mutant, and monomer-monomer interactive mutants of MsmNDK, the stability of the dimer was verified under heat, SDS, low pH, and methanol. The predicted residues (Gln17, Ser24 and Glu27) were engaged in dimer formation, however the mutated proteins retained the ATPase and GTPase activity even after introducing single (MsmNDK- Q17A, MsmNDK-E27A, and MsmNDK-E27Q) and double (MsmNDK-E27A/Q17A) mutation. However, the monomer-monomer interaction could be abolished using methanol, indicating the stabilization of the monomer-monomer interaction by hydrophobic interaction.