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.

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.

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.

Production, purification, characterization and over-expression of xylanases from actinomycetes.

Xylanases are a group of depolymerizing enzymes often used for the hydrolysis of xylan (present in hemicellulose) to monomeric sugars and comprise endo-xylanases (EC 3.2.1.8) and beta-xylosidases (EC 3.2.1.37). They often act in synergy with other enzymes for complete hydrolysis of hemicellulose. Xylanases find several industrial applications, for example in food and feed industries, paper and pulp industries and more recently have acquired a great role in biomass to biofuels program. Bacteria and fungi can best produce xylanases. Recent developments in rDNA technology have resulted in molecular cloning and expression of xylanases in heterologous and homologous hosts. In view of significance of the actinomycetes for the production of biotechnological products, attempts have been made in recent years to explore them for the production of industrial enzymes, including xylanses, aiming to find the enzyme with novel features. This review provides the state-of-art information and developments on the xylanases from actinomycetes, presenting the production, purification, characterization and over-expression from various actinomycetes cultures.

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.

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.

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.

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.

Mycobacterium tuberculosis expresses ftsE gene through multiple transcripts.

Bacterial FtsE gene codes for the ATP-binding protein, FtsE, which in complex with the transmembrane protein, FtsX, participates in diverse cellular processes. Therefore, regulated expression of FtsE and FtsX might be critical to the human pathogen, Mycobacterium tuberculosis, under stress conditions. Although ftsX gene of M. tuberculosis (MtftsX) is known to be transcribed from a promoter inside the upstream gene, ftsE, the transcriptional status of ftsE gene of M. tuberculosis (MtftsE) remains unknown. Therefore, the authors initiated transcriptional analyses of MtftsE, using total RNA from M. tuberculosis cells that were grown under stress conditions, which the pathogen is exposed to, in granuloma in tuberculosis patients. Primer extension experiments showed the presence of putative transcripts, T1, T2, T3, and T4. T1 originated from the intergenic region between the upstream gene, MRA_3135, and MtftsE. T2 and T3 were found initiated from within MRA_3135. T4 was transcribed from a region upstream of MRA_3135. RT-PCR confirmed co-transcription of MRA_3135 and MtftsE. The cloned putative promoter regions for T1, T2, and T3 elicited transcriptional activity in Mycobacterium smegmatis transformants. T1, T2, and T3, but no new transcript, were present in the M. tuberculosis cells that were grown under the stress conditions, which the pathogen is exposed to in granuloma in tuberculosis patients. It showed lack of modulation of MtftsE transcripts under the stress conditions tested, indicating that ftsE may not have a stress response-specific function in M. tuberculosis.

Mycobacterium tuberculosis FtsZ requires at least one arginine residue at the C-terminal end for polymerization in vitro.

We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (MtFtsZ) have any role in MtFtsZ polymerization in vitro. MtFtsZ-deltaC1, which lacks C-terminal extreme Arg residue (underlined in the C-terminal extreme stretch of 13 residues, DDDDVDVPPFMRR), but retaining the penultimate Arg residue (DDDDVDVPPFMR), polymerizes like full-length MtFtsZ in vitro. However, MtFtsZ-deltaC2 that lacks both the Arg residues at the C-terminus (DDDDVDVPPFM), neither polymerizes at pH 6.5 nor forms even single- or double-stranded filaments at pH 7.7 in the presence of 10 mM CaCl(2). Neither replacement of the penultimate Arg residue, in the C-terminal Arg deletion mutant DDDDVDVPPFMR, with Lys or His or Ala or Asp (DDDDVDVPPFMK/H/A/D) enabled polymerization. Although MtFtsZ-deltaC2 showed secondary and tertiary structural changes, which might have affected polymerization, GTPase activity of MtFtsZ-deltaC2 was comparable to that of MtFtsZ. These data suggest that MtFtsZ requires an Arg residue as the extreme C-terminal residue for polymerization in vitro. The polypeptide segment containing C-terminal 67 residues, whose coordinates were absent from MtFtsZ crystal structure, was modeled on tubulin and MtFtsZ dimers. Possibilities for the influence of the C-terminal Arg residues on the stability of the dimer and thereby on MtFtsZ polymerization have been discussed.

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.

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%).

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.

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.

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.

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).

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.