Proteomics Reveals Damaging Effect of Alpha-Cypermethrin Exposure in a Non-Target Freshwater Microalga Chlorella sp. NC-MKM.

Alpha-cypermethrin, a pyrethroid pesticide, is frequently used on crops to prevent insect attacks. However, occasionally, due to drift, leaching, or with rainwater, it enters the aquatic environment and poses a serious threat to the growth of non-target aquatic organisms. In the current study, we were interested in investigating the damaging effect of alpha-cypermethrin on a local freshwater non-target green alga Chlorella sp. NC-MKM in terms of its protein levels. This was achieved by exposing Chlorella sp. NC-MKM to an EC50 concentration of alpha-cypermethrin for 1 day, followed by the two-dimensional (2-D) gel electrophoresis and MALDI-TOF MS. Fifty-three proteins, which had showed significant differential accumulation (> 1.5 fold, P < 0.05) after exposure to alpha-cypermethrin, were considered as differentially accumulated proteins (DAPs). These DAPs were further divided into several functional categories, and the expressions of each in control and treatment samples were compared. Comparison revealed that alpha-cypermethrin exposure affects the accumulation of proteins related with photosynthesis, stress response, carbohydrate metabolism, signal transduction and transporters, translation, transcription, cell division, lipid metabolism, amino acid and nucleotide biosynthesis, secondary metabolites production, and post-translational modification, and thus rendered the tested algal isolate sensitive toward this pesticide. The overall findings of this research thus offer a fundamental understanding of the possible mechanism of action of the insecticide alpha-cypermethrin on the microalga Chlorella sp. NC-MKM and also suggest potential biomarkers for the investigation of pesticide exposed microalgae.

Proteomics analysis reveals several metabolic alterations in cyanobacterium Anabaena sp. NC-K1 in response to alpha-cypermethrin exposure.

In the current study, the effect of the EC50 and LC90 concentrations of pyrethroid insecticide alpha-cypermethrin to cyanobacteria Anabaena sp. NC-K1 was investigated at different time exposures (1st day, 4th day and 7th day) with reference to growth, photosynthetic pigments, oxidative damage and antioxidant defence system. Superoxide dismutase (1.38-fold), peroxidase (5.04) and proline content (2.27-fold) were enhanced compared to the control. After performing 2D gel electrophoresis at 1st day EC50 exposure, where appropriate differences in the biochemical and physiological parameters were observed, 22 differentially accumulated proteins (20 upregulated and 2 downregulated) were selected for mass spectrometry. Out of 42 proteins identified, 20 upregulated protein spots were classified into twelve categories according to their metabolic functions. Proteins related to photosynthesis (phycobilisome rod-core linker polypeptide, rubisco), stress responses (Hsp70, Hsp40, catalase family peroxidase), translation (elongation factor Tu) and amino acid biosynthesis and metabolism (3-phosphoshikimate 1-carboxyvinyl transferase) were significantly upregulated. Additionally, proteins involved in transcription and DNA repair (Snf-2 histone linker phd ring helicase, RNA polymerase sigma factor RpoD and Holliday junction ATP-dependent DNA helicase RuvA) were considerably upregulated. Upregulation of these proteins against pesticide stress presumably maintained the photosynthesis, energy metabolism, carbohydrate metabolism, transport and signalling proteins, transcription, translation and DNA repair. Additionally, these proteins might involve in sufficient detoxification of ROS and play a crucial role in damage removal and repair of oxidized proteins, lipids and nucleic acids. Taken together, Anabaena sp. NC-K1 responded towards alpha-cypermethrin stress via modulating its proteome to maintain its cellular metabolism and homeostasis.

Exogenous addition of indole acetic acid and kinetin under nitrogen-limited medium enhances lipid yield and expression of glycerol-3-phosphate acyltransferase & diacylglycerol acyltransferase genes in indigenous microalgae: A potential approach for biodiesel production.

In the present study, a combination of phytohormones (indole acetic acid and kinetin) was augmented in nitrogen-limited medium to achieve higher biomass and lipid yield in Graesiella emersonii NC-M1 and Chlorophyta sp. NC-M5. This condition was recorded with a 2.3- and 2.5-fold increase in biomass and lipid yield for Graesiella emersonii NC-M1 compared to the nitrogen-limited condition. Also, this condition showed a 1.6- and 1.08-fold increase in lipid yield and neutral lipid compared to the standard condition. Phytohormones addition also reduced oxidative damage caused by nitrogen-limitation and enhanced monounsaturated fatty acid content. Further, a 5.2- and 3.17-fold enhance in expression level of GPAT and DGAT genes were noticed under nitrogen-limited medium supplemented with phytohormones compared to control.

Modulation of lipid content and lipid profile by supplementation of iron, zinc, and molybdenum in indigenous microalgae.

The effects of iron (Fe), zinc (Zn), and molybdenum (Mo) on the biomass yield, lipid content, lipid yield, and fatty acid composition of Chlorella sp. NC-MKM, Graesiella emersonii NC-M1, Scenedesmus acutus NC-M2, and Chlorophyta sp. NC-M5 were studied. Among them, G. emersonii NC-M1 recorded the highest percentage increase in lipid content (140.3%) and neutral lipid (50.9%) under Zn-supplemented condition compared to the control. Also, it showed a 105% and 41.88% increase in lipid yield and neutral lipid under Fe-supplemented condition compared to the control. However, Chlorella sp. NC-MKM recorded an elevation in lipid yield (70.3% rise) and neutral lipid (24.32% rise) compared to the control in Mo-supplemented condition. The enhanced production of reactive oxygen species (ROS) and antioxidant enzyme (SOD and POD) under Fe-, Zn-, and Mo-supplemented condition supports the lipid accumulation. FAME analysis showed that the overall percentage of SFA and MUFA increased after the addition of Fe, Zn, and Mo in a culture medium compared to the control which is vital for a good-quality biodiesel. Further, biodiesel properties derived from FAMEs such as CN, SV, IV, CFPP, OS, υ, ρ, and HHV were found in accordance with biodiesel standard.

Molecular characterization of freshwater microalgae and nutritional exploration to enhance their lipid yield.

Biomass and lipid yield under nutrient depleted and supplemented conditions of N and Mg in two freshwater isolates, Chlorella sp. NC-MKM and Scenedesmus acutus NC-M2 from Meghalaya were investigated for biodiesel production. Both the strains, Chlorella sp. NC-MKM and Scenedesmus acutus NC-M2 are oleaginous in nature having lipid content of 40.2 ± 1.4 and 37.3 ± 2.6% DCW, respectively. The significant increase (92.8%) in lipid content was found in N-depleted condition while an increase (46.65%) in biomass yield was observed under Mg-supplemented condition in Chlorella sp. NC-MKM. Studying the interactive effects of nutrient depletion and supplementation, combination of N-depleted and Mg-supplemented condition was selected for further investigation to check enhanced lipid yield in Chlorella sp. NC-MKM. The results showed a significant increase in biomass yield, lipid yield and lipid content (30, 66.8, and 28.66%, respectively). Under this condition, accumulation of neutral lipid was also enhanced (47.17% M2 gated cells) compared to control (21.37% M2 gated cells). Further, FAMEs revealed that the relative percentage of saturated and mono-unsaturated fatty acids increased (66.16%) in Chlorella sp. NC-MKM compared to control that improves biodiesel properties.

Purification, biochemical characterization and structural modelling of alkali-stable ß-1,4-xylan xylanohydrolase from Aspergillus fumigatus R1 isolated from soil.

BACKGROUND: Aspergillus fumigatus R1 produced xylanase under submerged fermentation which degrades the complex hemicelluloses contained in agricultural substrates. Xylanases have gained considerable attention because of their tremendous applications in industries. The purpose of our study was to purify xylanase and study its biochemical properties. We have predicted the secondary structure of purified xylanase and evaluated its active site residues and substrate binding sites based on the global and local structural similarity. RESULTS: Various microorganisms were isolated from Puducherry soil and screened by Congo-red test. The best isolate was identified to be Aspergillus fumigatus R1. The production kinetics showed the highest xylanase production (208 IU/ml) by this organism in 96 h using 1 % rice bran as the only carbon source. The purification of extracellular xylanase was carried out by fractional ammonium sulphate precipitation (30-55 %), followed by extensive dialysis and Bio-Gel P-60 Gel-filtration chromatography. The enzyme was purified 58.10 folds with a specific activity of 38196.22 IU/mg. The biochemical characterization of the pure enzyme was carried out for its optimum pH and temperature (5.0 and 50(0)C), pH and temperature stability, molecular mass (Mr) (24.5 kDa) and pI (6.29). The complete sequence of protein was obtained by mass spectrometry analysis. Apparent Km and Vmax values of the xylanase for birchwood xylan were 11.66 mg/ml and 87.6 µmol min(-1) mg(-1) respectively. CONCLUSION: Purified xylanase was analyzed by mass-spectrometry which revealed 2 unique peptides. Xylanase under current study showed significant production using agricultural residues and a broad range of pH stability in the alkaline region. Xylanase produced by Aspergillus fumigatus R1 could serve as the enzyme of choice in industries.