Perspectives of intracellular polymers in functional evaluation of the microbes for EBPR

To substantiate and interpret the performance of the Enhanced Biological Phosphorus Removal (EBPR) processes with simultaneous nitrogen removal in five full-scale sequencing batch reactors (SBR) systems (with or without pre-anoxic/anaerobic selector) across India, conventional microscopic examinations were performed. Regular examining and cyclic behavior evaluation studies specified that these systems worked for EBPR with effectiveness depending on the wastewater quality and operational steadiness. Treatment with Neisser stain for identifying polyphosphates (poly-P) and Sudan black B stain for observing poly-β-hydroxybutyrates (PHB) granules showed that the enriched biomass of the SBR plants was very diverse concerning morphology, residing populations of traditional rod-shaped PAOs, tetrad (or Sarcina-like cells) forming organisms (submitted as TFOs instead of GAOs), diplococci-shaped cells, and staphylococci-like clustered populations (CC), including few filaments which correlate well with biochemical processes undergoing in SBR plants. SBR plants with readily biodegradable chemical oxygen demand (rbCOD) fraction in COD > 16% and rbCOD/TP ∼10–20 in Varanasi, Mumbai, and Gurgaon, respectively, have performed for >20% EBPR (∼77.8%, ∼76.6%, and ∼84.8% TP removal, respectively) as well as >85% Simultaneous Nitrification and Denitrification (SND). This study can open novel dimensions for optimization by relating microscopic observations (qualitative examination) with the processes undergoing in the plants under varied physicochemical parameters.

Polyhydroxyalkanoate Recovery From Newly Screened Bacillus Sp. LPPI-81 Using Various Methods of Extraction From Loktak Lake Sediment Sample

Nowadays the conventional plastic wastes are very challenging to environments and its production cost also creates an economic crisis due to petrochemical-based plastic. In order to solve this problem, the current studies were aimed at screening and characterizing these PHA producing isolates and evaluating the suitability of some carbon source for newly screened PHA producing isolates. Some carbon sources such as D-fructose, glucose, molasses, D-ribose and sucrose were evaluated for PHA production. Data were analyzed using SPSS version 20. The 16SrRNA gene sequence of these isolates was performed. This newly isolated taxa was related to Bacillus species. It was designed as Bacillus sp. LPPI-18 and affiliated Bacillus cereus ATCC 14577T (AE01687) (99.10%). Paenibacillus sp. 172 (AF273740.1) was used as an out-group. Bacillus sp. LPPI-18 is a gram-positive, rod-shaped, endospore former, and citrate test positive. This isolate showed positive for amylase, catalase, pectinase, and protease test. They produced intracellular PHA granules when this isolate was stained with Sudan Black B (SBB) and Nile Blue A (NBA) preliminary and specific staining dyes, respectively. Both Temperature and pH used to affect PHA productivity. Bacteria are able to reserve PHA in the form of granules during stress conditions. This isolate produces only when supplied with carbon sources. More PHA contents (PCs) were obtained from glucose, molasses, and D-fructose. In this regard, the maximum mean value of PC was obtained from glucose (40.55±0.7%) and the minimum was obtained from D-Ribose (12.4±1.4%). Great variations (p≤0.05) of PCs were observed among glucose & sucrose, molasses & sucrose and D-fructose & sucrose carbon sources for PHA productivity (PP) of Cell Dry Weight (CDW) g/L. After extraction, PHA film was produced for this typical isolate using glucose as a sole carbon source. Fourier transform infrared spectrum was performed for this isolate and showed the feature of polyester at 1719.64 to 1721.16 wavelength for these extracted samples. The peak of fingerprinting (band of carboxylic acid group) at this wave-length is a characteristic feature of PHB and corresponds to the ester functional group (C=O).

Acoustic Characterization Of Sudan Black Nanobubbles For Multimodal Ultrasound & Photoacoustic Imaging

Dyed, shelled, nanobubbles (NBs) have recently been proposed as contrast agents for multimodal ultrasound-photoacoustic (US-PA) imaging. However, changes to the shell composition due to the presence of the dye can modify the response of bubbles to ultrasound. In this work, the effects of the dye, Sudan Black B, on a formulation of lipid shelled NBs are studied. Formulations were produced with increasing concentration of Sudan Black B. The size and concentration of activated NBs were tested. The surface tension of bulk lipid solution was measured using pendant drop tensiometry and activated bubble solutions were measured for single bubble and bubble population response to incident ultrasound. While results show no statistically significant effect of Sudan Black concentration on bubble concentration or size, surface tension increased linearly with dye concentration to a maximum increase of 13%. With the addition of Sudan Black B, single bubble experiments show an increase in the contribution of bubble growth signals, a decrease in contribution of nonlinear signals, and a decrease in bubble destruction. The results presented in this work indicate that the presence of Sudan Black B in the lipid shell of a nanobubble may increase the shell permeability impacting stability of the bubble population and their potential for multimodal US-PA imaging.

Reduction of autofluorescence in whole adult worms of Schistosoma japonicum for immunofluorescence assay

Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.

Adult-Onset Neuronal Ceroid Lipofuscinosis in a Shikoku Inu

A two-year-and-eleven-month-old male Shikoku Inu was referred for evaluation of progressive gait abnormality that had begun three months prior. Neurological examination revealed ventral flexion of the neck, a wide-based stance in the hindlimb, wide excursions of the head from side to side, tremor in all four limbs, hypermetria in all four limbs, proprioceptive deficits in all four limbs, reduced patellar reflex in both hindlimbs, and postural vertical nystagmus. Later, behavioral and cognitive dysfunction, ataxia, and visual deficits slowly progressed. Magnetic resonance imaging revealed symmetrical progressive atrophy of the whole brain and cervical spinal cord. Bilateral retinal degeneration was observed, and both flush and flicker electroretinograms were bilaterally non-recordable at the age of five years and eight months, and the dog was euthanized. Histopathologically, faint-to-moderate deposition of light-brown pigments was frequently observed in the cytoplasm of neurons throughout the cerebrum, cerebellum, and nuclei of the brainstem. The pigments were positive for Luxol fast blue, periodic acid–Schiff, and Sudan black B, and exhibited autofluorescence. Electron microscopic examination revealed the accumulation of membranous material deposition in the neuronal cytoplasm. Small foci of pigment-containing macrophages were frequently observed around the capillary vessels. Based on these clinical and pathological findings, the animal was diagnosed with adult-onset neuronal ceroid lipofuscinosis.

Morphological, cytochemical and ultrastructural aspects of blood cells in freshwater stingray species in the middle Rio Negro basin of Amazonian Brazil

In the present work, we examined the morphology, dimensions, cytochemical staining reactions and ultrastructure of blood cells from three freshwater stingray species, Potamotrygon wallacei, Potamotrygon motoro and Paratrygon aiereba, living in the waters of the middle Rio Negro basin (Barcelos, Amazonas, Brazil). We identified erythrocytes, erythroblasts, thrombocytes and four types of leukocytes (basophils, heterophils, lymphocytes and monocytes) in the blood of these stingray species. In all the freshwater stingray species studied, the shapes and dimensions of these cells were similar to those of marine elasmobranchs. Positive PAS staining occurred in heterophils and thrombocytes, and weak staining occurred in lymphocytes and monocytes, while metachromasia only occurred in basophils. Positive Sudan Black B staining was observed in thrombocytes and lymphocytes, and weak staining occurred in heterophils. Basophils and heterophils were the only cells with positive bromophenol blue staining, while no peroxidase staining was observed in any of the four leukocyte types. This is the first study to establish the dimensions and cytochemical staining profiles of blood cells in Amazonian stingray species. Because these elasmobranch species are exported as ornamental fish to countries worldwide, this study can contribute to establishing standards for blood constituents that may be helpful in assessing the health and welfare of these fish in artificial systems.

Lipolytic Activities of Bacteria and Fungi Isolated from Soil Samples

This study was carried out at the Department of Microbiology, Microbiology Laboratory, Ado-Ekiti State University, Ekiti State, Nigeria between July, 2018 to March, 2019. Due to the diverse biotechnological importance of lipases as a biocatalytic enzyme, extracellular production of microbial lipases has to gain lots of interest. This study, therefore, focused on the physicochemical parameters of lipase producing microorganisms from different soil samples. Microorganisms were isolated from four different soil samples using Nutrient Agar (NA) and Potato Dextrose Agar (PDA). The isolates were identified and characterized. Production, an assay for Lipase enzymes, purification, the effect of pH, Temperature and metal ion was investigated. The isolates were culturally, morphologically and biochemically characterized. Two of the bacteria strains (Bacillus sp. and Staphylococcus sp.) and four fungi (Fusarium sp., Aspergillus fumigatus, Aspergillus niger, and Trichophyton sp.) isolates were able to produce lipid using Sudan Black B Fat staining techniques. Fusarium sp. isolated from dumpsite soil had the highest specific lipase activity (21.16 µmol/min/ml) while Bacillus sp. isolated from red oil spill soil had the highest lipase activity (0.59 µmol/min/mg). The specific activity of partially purified lipase for Fusarium sp. was 2.39 µmol/min/mg while Bacillus sp. had a specific activity of 2.46 µmol/min/mg. 30oC - 50oC, pH 7.0 to 9.0 and KCl2 (139.672%) supported the highest production of lipase by the Bacillus sp. and Fusarium sp. This study demonstrated that the Bacillus sp. produced a high amount of lipase activity followed by Fusarium sp. Extensive and persistent screening for new microorganisms and their lipolytic activities will help to provide faster ways to solve most environmental soil pollution.

Reduction of Autofluorescence in Whole Adult Worms of Schistosoma Japonicum for Immunofluorescence Assay

Background Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. Methods In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. Results By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. Conclusions We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.

Isolation of Two Bacterial Species from Argan Soil in Morocco Associated with Polyhydroxybutyrate (PHB) Accumulation: Current Potential and Future Prospects for the Bio-Based Polymer Production

The environmental issues caused by the impacts of synthetic plastics use and derived wastes are arising the attention to bio-based plastics, natural polymers produced from renewable resources, including agricultural, industrial, and domestic wastes. Bio-based plastics represent a potential alternative to petroleum-based materials, due to the insufficient availability of fossil resources in the future and the affordable low cost of renewable ones that might be consumed for the biopolymer synthesis. Among the polyhydroxyalkanoates (PHA), the polyhydroxybutyrate (PHB) biopolymer has been synthesized and characterized with great interest due to its wide range of industrial applications. Currently, a wide number of bacterial species from soil, activated sludge, wastewater, industrial wastes, and compost have been identified as PHB producers. This work has the purpose of isolating and characterizing PHB-producing bacteria from the agricultural soil samples of Argania spinosa in the south region of Morocco where the plant species is endemic and preserved. During this research, four heat-resistant bacterial strains have been isolated. Among them, two species have been identified as endospore forming bacteria following the Schaffer-Fulton staining method with Malachite green and the Methylene blue method. Black intracellular granules have been appreciated in microscopy at 100× for both strains after staining with Sudan black B. The morphological and biochemical analyses of the isolates, including sugar fermentation and antibiotic susceptibility tests, preliminarily identified the strains 1B and 2D1 belonging to the genus Serratia and Proteus, respectively.