Perumal Varalakshmi

Characterization and fatty acid profiling in two fresh water microalgae for biodiesel production: Lipid enhancement methods and media optimization using response surface methodology

Two fresh water microalgae, Coelastrella sp. M-60 and Micractinium sp. M-13 were investigated in this study for their potential of biodiesel production. For increasing biomass and lipid production, these microalgae were subjected to nutrient starvation (nitrogen, phosphorous, iron), salinity stress and nutrient supplementation with sugarcane industry effluent, citric acid, glucose and vitamin B12. The lipid productivity obtained from the isolates Coelastrella sp. M-60 (13.9 ± 0.4 mg/L/day) and Micractinium sp. M-13 (11.1 ± 0.2 mg/L/day) was maximum in salinity stress. The media supplemented with all the four nutrients yielded higher lipid productivity than the control. The response surface methodology (RSM) was employed to evaluate the effect of sugarcane industry effluent and citric acid on growth and lipid yield. Fatty acid profile of Coelastrella sp. M-60 and Micractinium sp. M-13 were composed of C-14, C-16:0, C-18:0, C-18:1 and C-18:2 and their fuel properties were also in accordance with international standards.

Biodegradation and utilization of dimethylformamide by biofilm forming Paracoccus sp. strains MKU1 and MKU2

Two bacterial strains capable of degrading N,N-dimethylformamide (DMF) were isolated from the effluent and sludge samples of textile and tyre industries. The 16S rRNA gene analysis revealed that bacterial strains belonged to the genera Paracoccus and named as Paracoccus sp. MKU1 and Paracoccus sp. MKU2. The DMF degradation experiments conducted at a DMF concentration of 1% v/v and HPLC analysis revealed that MKU1 and MKU2 degraded 55% and 46% of DMF after 120 h of growth. Biofilm quantification by microtiter plate assay revealed that both the bacterial isolates can form efficient biofilm on during DMF utilization. The presence of secondary carbon sources influenced the DMF degradation and biofilm formation where highest biofilm formation was observed in the presence of acetate and enhanced the DMF degradation to a maximum of 86.59% with MKU1 whereas glucose and acetate enhanced DMF degradation by MKU2 to a maximum of 82.7% and 80% respectively.

Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production

Lipid from microalgae is one of the putative oil resources to facilitate the biodiesel production during this era of energy dissipation and environmental pollution. In this study, the key parameters such as biomass productivity, lipid productivity and lipid content were evaluated at the early stationary phase of Chlamydomonas reinhardtii, CC1010 cultivated in nutrient starved (nitrogen, phosphorous), glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 supplementation (0.001%, 0.002% and 0.003%) in Tris-Acetate-Phosphate (TAP) medium. The lipid content in nitrogen starved media was 61% which is 2.34 folds higher than nutrient sufficient TAP medium. Glucose supplementation has lead to proportional increase in biomass productivity with the increasing concentration of glucose whereas vitamin B12 supplementations had not shown any influence in lipid and biomass production. Further, fatty acid methyl ester (FAME) profiling of C. reinhardtii, CC 1010 has revealed more than 80% of total SFA (saturated fatty acid) and MUFA (mono unsaturated fatty acid) content. Quality checking parameters of biodiesel like cetane number, saponification value, iodine number and degree of unsaturation were analyzed and the biodiesel fuel properties were found to be appropriate as per the international standards, EN 14214 and ASTM D6751. Conclusively, among all the treatments, nitrogen starvation with 0.1% glucose supplementation had yielded high lipid content in C. reinhardtii, CC 1010.

Green renewable energy production from Myxosarcina sp.: media optimization and assessment of biodiesel fuel properties

Renewable energy resources, like biomass from plants and algae, have gained more interest for biodiesel production as an energy source to reduce the consumption of fossil fuels and elevated global warming. In this study, Myxosarcina sp., a unicellular cyanobacterium, was evaluated for higher biomass and lipid production via the supplementation of sugar industrial waste (SIW) and sodium chloride (NaCl), and medium optimization with response surface methodology (RSM) for biodiesel production. The outcome of the findings was that greater biomass and lipid productivities of 28.5 ± 2.4 (1.2 fold) and 3.4 ± 0.2 (1.3 fold) mg L−1 per day were observed in BG-11 medium supplemented with SIW when compared with the control (24 ± 1 and 2.6 ± 0.4 mg L−1 per day). However, a higher lipid content 20.6 ± 1.8% (1.5 fold) was achieved with RSM optimized medium, including NaCl at 0.8 M, SIW at 2 mL L−1, sodium nitrate (NaNO3) at 2.5 g L−1 and magnesium sulphate (MgSO4) at 0.075 g L−1, than in the control (13.6 ± 1.4%). Fatty acid characterization by GC-MS analysis revealed that Myxosarcina sp. yielded 39 ± 5.3% of saturated fatty acids (SFA) and 61 ± 5.3% of monounsaturated fatty acid (MUFA) and its biodiesel fuel properties, evaluated using empirical equations, showed that almost all the properties calculated were in compliance with the national and international biodiesel standards EN 14214 (Europe), ASTM D6751-02 (US) and IS 15607 (India). Thus, Myxosarcina sp. can be utilized as the environmentally friendly biodiesel feedstock for high quality biodiesel production in the current scenario to meet escalating energy demand.

Hemocompatible glutaminase free L-asparaginase from marine Bacillus tequilensis PV9W with anticancer potential modulating p53 expression

Bacillus tequilensis PV9W, a marine bacterial isolate obtained from Gulf of Mannar, Rameswaram, India, produced glutaminase free L-asparaginase which was purified to homogeneity with a significant increase (13 fold) in specific activity. The apparent Km (0.045 ± 0.013 mM) and Vmax (7.465 ± 0.372 μmol ml−1 min−1) values of this purified L-asparaginase was identified and it was found that the maximum activity of the L-asparaginase was at a pH 8.5 and a temperature of 35 °C. The enzyme is a mixed α/β protein and the influence of different effectors were documented. The purified L-asparaginase had effective acrylamide degradation activity (6 IU per ml) and cytotoxic activity against HeLa cell lines with an IC50 of 0.036 ± 0.009 IU per ml. Furthermore, the purified enzyme showed p53 dependent G2 arrest in HeLa cells analyzed by FACS and was hemocompatible. Thus, this study highlights the marine isolate PV9W as a potential source for glutaminase free L-asparaginase with industrial as well as pharmaceutical applications. This study also paves a way for a possible therapeutic drug with the least amount of side effects.

Industrial effluent as a substrate for glutaminase free L-asparaginase production from Pseudomonas plecoglossicida strain RS1; media optimization, enzyme purification and its characterization

Glutaminase free L-asparaginase is a vital enzyme because of its anticancer potential. A potent bacterium isolated from a marine environment which produces glutaminase free L-asparaginase using M-9 medium with L-asparagine, was identified as Pseudomonas plecoglossicida RS1 by 16S rRNA gene sequencing. Statistical modeling was employed to optimize the medium using sugar cane industry effluent as the sole substrate for L-asparaginase production. The enzyme activity of L-asparaginase was higher with M-9 medium containing 0.8% effluent (3.25 ± 0.12 IU mL−1) compared to M-9 medium containing 0.3% L-asparagine (0.73 ± 0.08 IU mL−1). The apparent Km and Vmax of the purified L-asparaginase was 2.25 ± 0.61 mM and 8.9 ± 0.81 IU mL−1 min−1 respectively and the optimal activity of L-asparaginase was at pH 8.5 and 55 °C. This study highlights the use of industrial effluent as an alternate to L-asparagine for the production of L-asparaginase and how to improve the cost effectiveness of this enzyme.

Novel nonsense mutation (p.Ile411Metfs*12) in the SLC19A2 gene causing Thiamine Responsive Megaloblastic Anemia in an Indian patient.

Thiamine-responsive megaloblastic anemia (TRMA), an autosomal recessive disorder, is caused by mutations in SLC19A2 gene encodes a high affinity thiamine transporter (THTR-1). The occurrence of TRMA is diagnosed by megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Here, we report a female TRMA patient of Indian descent born to 4th degree consanguineous parents presented with retinitis pigmentosa and vision impairment, who had a novel homozygous mutation (c.1232delT/ter422; p.Ile411Metfs*12) in 5th exon of SLC19A2 gene that causes premature termination of hTHTR-1. PROSITE analysis predicted to abrogate GPCRs family-1 signature motif in the variant by this mutation c.1232delT/ter422, suggesting uncharacteristic rhodopsin function leading to cause RP clinically. Thiamine transport activity by the clinical variant was severely inhibited than wild-type THTR-1. Confocal imaging had shown that the variant p.I411Mfs*12 is targeted to the cell membrane and showed no discrepancy in membrane expression than wild-type. Our findings are the first report, to the best of our knowledge, on this novel nonsense mutation of hTHTR-1 causing TRMA in an Indian patient through functionally impaired thiamine transporter activity.

Riboflavin Responsive Mitochondrial Dysfunction in Neurodegenerative Diseases

Mitochondria are the repository for various metabolites involved in diverse energy-generating processes, like the TCA cycle, oxidative phosphorylation, and metabolism of amino acids, fatty acids, and nucleotides, which rely significantly on flavoenzymes, such as oxidases, reductases, and dehydrogenases. Flavoenzymes are functionally dependent on biologically active flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), which are derived from the dietary component riboflavin, a water soluble vitamin. Riboflavin regulates the structure and function of flavoenzymes through its cofactors FMN and FAD and, thus, protects the cells from oxidative stress and apoptosis. Hence, it is not surprising that any disturbance in riboflavin metabolism and absorption of this vitamin may have consequences on cellular FAD and FMN levels, resulting in mitochondrial dysfunction by reduced energy levels, leading to riboflavin associated disorders, like cataracts, neurodegenerative and cardiovascular diseases, etc. Furthermore, mutations in either nuclear or mitochondrial DNA encoding for flavoenzymes and flavin transporters significantly contribute to the development of various neurological disorders. Moreover, recent studies have evidenced that riboflavin supplementation remarkably improved the clinical symptoms, as well as the biochemical abnormalities, in patients with neuronopathies, like Brown-Vialetto-Van-Laere syndrome (BVVLS) and Fazio-Londe disease. This review presents an updated outlook on the cellular and molecular mechanisms of neurodegenerative disorders in which riboflavin deficiency leads to dysfunction in mitochondrial energy metabolism, and also highlights the significance of riboflavin supplementation in aforementioned disease conditions. Thus, the outcome of this critical assessment may exemplify a new avenue to enhance the understanding of possible mechanisms in the progression of neurodegenerative diseases and may provide new rational approaches of disease surveillance and treatment.

Enhanced Extracellular Polysaccharide Production and Self-Sustainable Electricity Generation for PAMFCs by Scenedesmus sp. SB1

In this study, a freshwater microalga, Scenedesmus sp. SB1, was isolated, purified, and identified by its internal transcribed spacer region (ITS1-5.8S-ITS2). Media optimization through the Plackett-Burman Design and response surface methodology (RSM) showed a maximum exopolysaccharide (EPS) production of 48 mg/L (1.8-fold higher than that for unoptimized media). Characterization using gas chromatography-mass spectrometry, Fourier transform infrared, X-ray diffraction, and thermogravimetric analysis reveals that the EPS is a sulfated pectin polysaccharide with a crystallinity index of 15.2% and prompt thermal stability. Furthermore, the photoelectrogenic activity of Scenedesmus sp. SB1 inoculated in BG-11 and RSM-optimized BG-11 (ROBG-11) media was tested by cyclic voltammogram studies, revealing the potential of the inoculated strain in ROBG-11 toward photosynthetic algal microbial fuel cells over normal BG-11. To the best of our knowledge, functional group characterization, physical and thermal property and media optimization for EPS production by RSM and electrogenic activity studies are reported for the first time in Scenedesmus sp. SB1.

SLC52A2 [p.P141T] and SLC52A3 [p.N21S] causing Brown-Vialetto-Van Laere Syndrome in an Indian patient: First genetically proven case with mutations in two riboflavin transporters.

BACKGROUND Brown-Vialetto-Van Laere Syndrome (BVVLS), a rare neurological disorder characterized by bulbar palsies and sensorineural deafness, is mainly associated with defective riboflavin transporters encoded by the SLC52A2 and SLC52A3 genes. METHODS Here we present a 16-year-old BVVLS patient belonging to a five generation consanguineous family from Indian ethnicity with two homozygous missense mutations viz., c.421C>A [p.P141T] in SLC52A2 and c.62A>G [p.N21S] in SLC52A3. RESULTS Functional characterization based on 3H-riboflavin uptake assay and live-cell confocal imaging revealed that the effect of mutation c.421C>A [p.P141T] identified in SLC52A2 had a slight reduction in riboflavin uptake; on the other hand, the c.62A>G [p.N21S] identified in SLC52A3 showed a drastic reduction in riboflavin uptake, which appeared to be due to impaired trafficking and membrane targeting of the hRFVT-3 protein. CONCLUSIONS This is the first report presenting mutations in both riboflavin transporters hRFVT-2 and hRFVT-3 in the same BVVLS patient. Also, c.62A>G [p.N21S] in SLC52A3 appears to contribute more to the disease phenotype in this patient than c.421C>A [p.P141T] in SLC52A2.