Freshwater Diatom Nitzschia Palea Research Articles

Isolation and cultivation of freshwater diatom Nitzschia palea HY1 for increasing biomass and fucoxanthin production

Diatoms, a type of microalgae distributed worldwide, have been identified as potential sources of biomass, lipids, and high-value compounds. While marine diatoms have been extensively studied, the potential of freshwater diatoms still needs to be explored. In this study, a novel strain of freshwater diatom was isolated from the Jungnangcheon stream located in Seoul, Republic of Korea (37°33'08.0" N, 127°02'40.0" E). This newly isolated strain was classified through phylogenetic analysis, and its morphology was investigated using light and electron microscopy; it was named Nitzschia palea HY1. N. palea HY1 grown in freshwater media (FDM) produced higher biomass (0.68 g L<sup>-1</sup>) and fucoxanthin production (9.19 mg L<sup>-1</sup>) than in conventional diatom media. Furthermore, increasing the bicarbonate concentration from 2 to 10 mM enhanced the maximum biomass and fucoxanthin production in FDM by 2.7 fold and 1.5 fold, respectively. Remarkably, the introduction of aeration to the modified FDM (MFDM) led to a substantial increase in the maximum biomass and fucoxanthin production of N. palea HY1, exhibiting 3.8-fold and 4.1-fold enhancement, respectively, compared to FDM alone. These findings suggest that optimizing the cultivation of N. palea HY1 using MFDM could provide an alternative to marine sources for fucoxanthin production.

Characterization and in vitro anticancer potential of exopolysaccharide extracted from a freshwater diatom Nitzschia palea (Kütz.) W.Sm. 1856

Diatoms are photoautotrophic microalgae classified under class Bacillariophyceae, engulfed by hard silicate frustules, which give mechanical support and protection from bacterial infections. They exude polysaccharides extracellularly that help them with their gliding motion (locomotion). However, the bioactivity of such compounds was least explored from freshwater diatoms. In the present study, a single species of pennate diatom identified as Nitzschia palea was isolated and molecularly characterized by 18S rRNA smaller subunit gene (partial) sequencing and submitted to GenBank NCBI and accession number retrieved as ON360983. Based on logarithmic growth curve analysis, the exponential phase was obtained from 3rd to 4th day of diatom culture. The exopolysaccharide was extracted by the hot-water extraction method, and characterized by FT-IR. The total yield of exopolysaccharide from Nitzschia palea was estimated as 1.56 mg in 100 mL of culture after 7 days of incubation. The estimated carbohydrate content was 51.35 µg/100 µL. The monosaccharide constituents were determined by acid hydrolysis of exopolysaccharide, silylation (derivatization), followed by GC–MS analysis and tabulated. The extracted exopolysaccharide was evaluated for its anti-cancer potential against the Human Adenocarcinoma lung cancer cell line (A549) and the estimated IC50 value was 62.64 µg/mL. Acridine orange staining assay and DNA fragmentation assay also confirmed the apoptotic activity of exopolysaccharide derived from the diatom Nitzschia palea.

Development of diatom entrapped alginate beads and application of immobilized cells in aquaculture

The objective of this study was entrapment of freshwater diatom Nitzschia palea in a Ca-alginate hydrogel bead through the gelation method. The study further aimed at remediating aquaculture water and its efficient utilization for diatom productivity and futuristic aquaculture applications. Results demonstrate that entrapped diatom in Ca-alginate hydrogel bead showed no swelling behavior​ and they were quite stable during analysis. The maximum cell density was 71.4 × 105 cell mL −1 which was 3.5 times higher than the cell density achieved in free cell culture. The maximum nitrate, phosphate, ammonia removal efficiency was 86.93 ± 3.46%, 57.51 ± 3.8%, 63.83 ± 1.18% which signifies the efficiency of diatom entrapped Ca-alginate hydrogel bead in removing the nutrient load from the aquaculture water. The entrapped diatom cells were quite successful in controlling the water hardness (52.33 ± 2.51%) to a significant level. This work demonstrates that diatom entrapped in Ca-alginate hydrogel bead is a cost-effective method in aquaculture water remediation which can be a long-lasting solution towards sustainability and diverse aquaculture applications.

Physiological response and removal ability of freshwater diatom Nitzschia palea to two organophosphorus pesticides

ABSTRACT Organophosphorus pesticides, one of the most extensively used organic chemicals to control several insect pests, making them threaten the aquatic creatures. Here, we analysed the response mechanism of Nitzschia palea to insecticides (acephate and trichlorfon) exposure, through physiological, biochemical, morphological changes and the removal ability. Results showed that the growth of N. palea was inhibited by trichlorfon, with the cell density was significantly decreased at 7 days of exposure. Although the diatom growth was hardly inhibited after acephate treatment, the diatom valve significantly abnormal morphology be found at 50 mg L−1 acephate treatment after 7 days. Meanwhile, these insecticides had no negative effect on the chlorophyll a content of N. palea. In addition, acephate and trichlorfon induced the activities of superoxide dismutase (SOD) and catalase (CAT) in the diatom, which play a defensive role against various environmental stresses. For further analysis revealed that the content of malondialdehyde (MDA) and extracellular polysaccharides (EPS) increased on exposure to both insecticides. N. palea also promoted 24.44–32.75% removal of acephate. The biochemical changes in N. palea on exposure to acephate and trichlorfon indicate that the diatom undergoes an adaptive change in response to the insecticides-induced oxidative stress.

Probing the Adhesion of the Common Freshwater Diatom Nitzschia palea at Nanoscale.

Freshwater biofilms play an essential ecological role, but they also adversely affect human activities through undesirable biofouling of artificial submerged structures. They form complex aggregates of microorganisms that colonize any type of substratum. In phototrophic biofilms, diatoms dominate in biomass and produce copious amount of extracellular polymeric substances (EPSs), making them efficient early colonizers. Therefore, a better understanding of diatoms adhesive properties is essential to develop new anti-biofouling strategies. In this context, we used atomic force microscopy (AFM) to decipher the topography and adhesive mechanisms of the common freshwater diatom Nitzschia palea. Images taken in physiological conditions revealed typical ultrastructural features with a few nanometers resolution. Using single-cell force spectroscopy, we showed that N. palea strongly adheres to hydrophobic surfaces as compared to hydrophilic ones. Chemical force spectroscopy with hydrophobic tips further confirmed that the adhesion is governed by surface-associated hydrophobic EPS distributed in clusters at the frustule surface, and mostly composed of (glyco)-lipids as revealed by Raman spectroscopy. Collectively, our results demonstrate that AFM-based nanoscopy, combined with Raman spectroscopy, is a powerful tool to provide new insights into the adhesion mechanisms of diatoms.

Differential cell viability in Nitzschia palea on exposure to different organic and inorganic environmental effluents

This research is focused to report the effects of various effluents on the cell viability and density of diatoms. Experiments have been performed on fresh water diatom Nitzschia palea with seven effluents, to check the cell viability and density using spectroscopic and microscopic techniques. On exposing the diatom cells to crude sewage water (40% and 60% dilution), there is approximately 10 times elevation in cell density without loss in the viability of cells, up to 25 days. On the other hand, individual exposure to β propranolol drugs resulted in a total loss of cell viability and cell destruction with only 10% of growth rate. Further, using sodium metasilicate as effluent, the diatom population dropped about 30%–60% on increasing concentration. Besides physiological stress, these effluents have also direct influence on the lipid production in diatoms. The objective of this research is to provide the monitoring report of different environmental effluents on water quality as well as their influence on lipid biosynthesis and also their impact over the diatom’s health which acts as a biological sensor for water ecosystem.

Volatile and phenolic compounds in freshwater diatom Nitzschia palea as a potential oxidative damage protective and anti-inflammatory source

Background: Nitzschia palea is a freshwater diatom species (Bacillariophyceae), is easy to cultivate, and is a primary producer of organic matters in aquatic environments. Materials and Methods: In this study, proximate composition in axenic culture of N. palea was determined, phenolic content and volatile compounds were determined for methanol and ethyl acetate extracts. Both the extracts were evaluated for eight different in vitro antioxidant and free radical scavenging activities. Macromolecular damage prevention properties were determined by electrophoretic methods, and antihemolytic activity was validated by lactate dehydrogenase assay, atomic force microscope, and scanning electron microscope image analysis. Results: Both the extracts showed antioxidant, macromolecular damage prevention, and antihemolytic properties. Among the two extracts, ethyl acetate extract showed high activity compared to methanol extract. Based on this result, ethyl acetate extract was evaluated for in vitro anti-inflammatory properties using RAW 264.7 cells. The extract showed IC50 value of 50.73 μg/mL and inhibition of inflammatory cytokines such as nitric oxide, tumor necrosis factor-alpha, and prostaglandin E2. The observed activity was correlated with identified important metabolites such as butyl isobutyl phthalate, pristane, and squalene of methanol extracts. Similar co-relation was observed for 7,9-di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione, methyl palmitate, hentriacontane, and dibutyl phthalate were of ethyl acetate extract. Conclusion: The study concludes that N. palea has potentiality to isolate pharmacologically active metabolites using advanced chromatographic techniques, which can be useful in combating oxidative stress-related inflammatory diseases.

Sensitive vs. tolerant Nitzschia palea (Kützing) W. Smith strains to atrazine: a biochemical perspective

Organic contaminants, and herbicides in particular, represent a risk for aquatic ecosystems. The primary target of herbicides are producers, the base of food webs, but frequently they end up far from the application point affecting non-target species. Its presence can work as sub-lethal stimulus, which sort the genetic and phenotypic differences within a species. Intraspecific variation allows adaptation to changes in the environment but also to new niches due to variations in species' sensitivity and biochemical response to a certain chemical. A better understanding of these variations can lead to the development of improved strategies for ecosystem protection. This research aimed to compare a sensitive and a tolerant strain of the freshwater diatom Nitzschia palea to atrazine. Strains were exposed to three concentrations within their tolerance range, during 96 h. The activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione-S-transferases and glutathione peroxidases was determined. In addition, chlorophylls a and c, carotenoids, reduced glutathione, proteins and lipid peroxidation were quantified. Both strains displayed different strategies to deal with atrazine toxicity: while the sensitive strain decreased the oxidative stress, increasing the activity of antioxidant enzymes such as superoxide dismutase, the tolerant strain invested in conjugation pathways and carotenoids' maintenance.

Combined toxicity of arsenite and dimethylarsenic acid on the freshwater diatom Nitzschia palea.

The toxicity and bioavailability of single arsenic species have been widely investigated, however, the biological effects of mixed arsenic species co-existing in natural waters still remain unknown. The objective of this work was to discern the adverse effects of combined arsenite (As(III)) and dimethylarsenic acid (DMA) on diatom Nitzschia palea. The combined ecotoxicity of As(III) and DMA on N. palea was observed to be time-dependent and showed dose-effect relation. The toxicity of DMA and As(III) mixture was higher than individual DMA or As(III) when the As(III) concentration was in the range of 0.085-0.316 mg L-1. As the As(III) concentration increased from 0.487 to 0.858 mg L-1, the antagonistic effect was found, which could be due to the higher thiols contents in the thiol-containing proteins (e.g., frustulins, silaffins and other glycoproteins). The content of malondialdehyde (MDA) in the treatment of mixed arsenic species was found to be at the same level compared to the As-free control after 72 h of exposure, indicating that the co-toxicity of As(III) and DMA on diatom frustules was not significant. Furthermore, the increase of frustule formation rate in the mixture of EC50 As(III)-EC10 DMA at 72 h exposure time indicated that the damaged diatom cell walls was likely repaired gradually. The results from this study suggested that the effects of co-existed arsenic species were concentration-specific and should be considered in the risk assessment of arsenic and development of water quality criteria for the protection of aquatic ecosystems.

Few Layer Graphene sticking by biofilm of freshwater diatom Nitzschia palea as a mitigation to its ecotoxicity

Carbon-based nanoparticles such as graphene have many applications leading to their industrial production. Few-Layer Graphene (FLG) is thus likely to be found in the environment, and especially in rivers. In this study, the effect of FLG on the photosynthetic benthic diatom Nitzschia palea was assessed making distinction between the impact of a direct contact with FLG and a shading effect of FLG on diatoms. Growth inhibition of diatoms exposed to FLG at 50 mg L−1 was observed at 48 h of exposure associated with an increase in diatoms mortality. At 144 h, the growth rate was recovered. However, in shading condition, at 48 h of FLG exposure, a persistent growth inhibition was observed at 50 mg L−1. Microscopic observations and a monitoring of FLG concentration in the medium allowed to conclude that exo-polymeric substances (EPS), naturally secreted by N. palea, strongly interact with FLG, sticking nanoparticles at the bottom of wells. Our results highlight the potential mechanisms of clarification of the water column by diatoms biofilms, by sticking FLG even at high concentration. Overall, these results suggest that one potential toxicity process of graphene could be a combination of direct and shading effect leading to a strong interaction between biofilm and nanoparticles.

Intraspecific differences in cadmium tolerance of Nitzschia palea (Kützing) W. Smith: a biochemical approach.

Intraspecific variability occurs in all types of organisms and is a driving force to speciation, conferring genotypic and phenotypic differences that enable adaptive responses to sub-lethal stimuli such as exposure to pollutants (including cadmium, Cd). Thus, differences in biochemical parameters are expected among isolates of the same species. Studying the extent of these differences throughout a stress range, will provide information for the development of approaches to mitigate habitat contamination. This work was designed to identify possible differences in Cd tolerance of five isolates of the freshwater diatom Nitzschia palea from different sampling sites. Each isolate was exposed to five increasing Cd concentrations during 10days. Growth inhibition was assessed and intracellular accumulation of Cd was quantified. Superoxide dismutase and catalase activities were determined. Glutathione as well as lipid peroxidation (LPO) and intracellular protein content were quantified. The results obtained identified intraspecific differences among isolates. These differences were associated with different approaches of coping with Cd stress. Higher intracellular Cd concentrations induced lower tolerance in isolates, since antioxidant mechanisms were unable to fight effectively against higher oxidative stress. Reversely, lower intracellular accumulation of Cd induced lower oxidative damage and allowed cells to better tolerate exposure to Cd. LPO emerged as an excellent marker of oxidative stress in N. palea and its use can differentiate isolates according to their tolerance.

Multi-walled carbon nanotubes, natural organic matter, and the benthic diatom Nitzschia palea: “A sticky story”

Different effects of multi-walled carbon nanotubes (MWCNTs) on the freshwater diatom Nitzschia palea were examined. MWCNTs used in this study (MWCNT) were dispersed either by sonication without (MWCNTsonicated) or with a realistic concentration (10 mg L−1) of Natural Organic Matter (MWCNT+NOM). A pocket-size device was designed to distinguish shading effect (using MWCNT suspensions as external filters) from total exposure effect of MWCNTsonicated and MWCNT+NOM on benthic algae. This study demonstrates that cell division was strongly inhibited after a 48 h exposure to MWCNT+NOM compared to MWCNTsonicated. This device did not yield a quantifiable contribution of shading to growth inhibition of MWCNTsonicated and below 10 mg L−1 of MWCNT+NOM. In all cases, neither lethal effects nor drops in photosynthetic quantum yield were observed. After a 6-d exposure, a complete growth recovery was observed for all conditions except at the highest concentration of MWCNT+NOM. Different microscopic approaches using carbohydrates markers revealed the strong affinity between MWCNT and extracellular polymeric substances (EPS) produced by N. palea. These seem to constitute a defensive mechanism against MWCNT.

Efficiency of cadmium chelation by phytochelatins in Nitzschia palea (Kützing) W. Smith

Phytochelatins (PCs) are thiol-rich peptides, enzymatically synthesized by plants and algae under exposure to certain metals (Cd, Pb, Zn, Ag, As, Cu). Due to their ability to bind metal ions, they play an important role in the cellular detoxification, forming stable metal-PC complexes that minimize the intracellular deleterious effects of metals. The aim of the present work was to evaluate the efficiency of PC-Cd chelation in the freshwater diatom Nitzschia palea under 0, 0.1 and 0.2mgCdL(-1), which induced different levels of oxidative stress. This objective was accomplished by the isolation of PC-Cd complexes through size exclusion chromatography. Two peaks were identified, corresponding to high molecular weight (HMW) and low molecular weight (LMW) complexes. In each of the complexes, Cd was quantified by inductively coupled plasma-mass spectrometry, thiol composition was determined by HPLC analysis and the efficiency of Cd chelation calculated by -SH/Cd ratios in HMW and LMW complexes at both Cd concentrations. Results showed that the majority of intracellular Cd was complexed with PCs (75.2-91.2%). PCs-binding efficiency in this diatom species was higher at HMW than at LMW complexes and enhanced with the increase of Cd concentration exposure. Our work evidenced the important role of PCs as the main intracellular tolerance mechanism in this species. The efficiency increase of Cd-PC binding is related to the increment of PCs synthesis and to the number of Cd ions coordinately bonded to -SH groups in LMW and HMW complexes.

The use of partial cox1, rbcL and LSU rDNA sequences for phylogenetics and species identification within the Nitzschia palea species complex (Bacillariophyceae)

Cox1 has been suggested as a barcode marker for diatoms but it has not been tested intensively in any group of closely related species outside Sellaphora. We evaluated the use of cox1, LSU and rbcL for phylogeny and identification in the taxonomically problematic but ecologically important freshwater diatom Nitzschia palea, for which LSU rDNA sequences, mating and morphological data had already been published, and for which DNA and/or clonal isolates were still available; some new isolates were added. The previous and new information concur in suggesting that N. palea is a complex of several or many species. Where cox1 sequences could be obtained, they were more variable than LSU and rbcL and discriminated between lineages that differed in their morphologies, mating compatibilities, LSU sequences or rbcL sequences. Repeated failures for some strains to recover cox1 sequences from DNA that yielded LSU and rbcL suggest that cox1 will be impractical as a universal barcode marker in diatoms until better primers are designed. LSU and rbcL, though less variable than cox1, can be sequenced reliably in N. palea and, together, seem to show sufficient discrimination to be worth further consideration for species recognition.

Sensitivity of biochemical markers to evaluate cadmium stress in the freshwater diatom Nitzschia palea (Kützing) W. Smith

Human activities have been increasing the cadmium levels in soils and waters, disturbing many organisms in the primary trophic levels such as microalgae. Toxic metal pollution is a focus point of serious concern and the examination and monitoring water quality are becoming essential procedures. Diatoms are important bioindicators to monitor the metal concentrations in diverse habitats. The present study was planned to determine the biochemical mechanisms used by freshwater diatoms to cope with cadmium stress and to identify biomarkers of metal stress. For this, Nitzschia palea (Kützing) W. Smith was grown under different concentrations of Cd (0.01–0.1 mg l −1) and the IC 50 determined. Three concentrations (0.1, 0.2 and 0.3 mg Cd l −1) and a control (no cadmium) were used to undergo the experimental assays which allowed the determination of cadmium accumulation and several biochemical markers currently used to assess metal stress. N. palea was sensitive to cadmium, as the IC 50 calculated was 0.0276 mg Cd l −1. Cadmium accumulation increased sharply and was mainly associated to the frustule. Total protein content increased with cadmium exposure, inducing increases and decreases in polypeptide expression, indicating an attempt of N. palea cells to adjust to the new prevailing conditions induced by metal stress. In order to cope with cadmium stress, cells induced the synthesis of chelating molecules such as phytochelatins (PCs). The enzymatic (SOD and CAT) and non-enzymatic (glutathione and proline) ROS scavenging mechanisms were also induced. Our results indicate the existence of diverse metal stress-mediated mechanisms in order to lessen metal damages to the cell. PCs showed to be a suitable biomarker of metal stress; besides being metal specific and concentration respondent it also allows to infer about the level of stress imposed to cells, constituting a useful tool to complement the evaluation of diatom communities when accessing aquatic metal toxicity.

Effect of Sulphanilamides on the Growth of Micro-organisms in Presence and Absence of p-Aminobenzoic Acid

LAST year I presented some results of experiments on the antagonism between p-aminobenzoic acid and sulphanilamides (sulphanilamide, sulphapyridine and sulphathiazole) in autotrophic organisms1. These experiments were carried out with two strains of the freshwater diatom Nitzschia palea var. debilis (Kg.) Grun. I also made experiments on the inhibitory action of sulphanilamide on the growth of N. communis Rabenhorst, N. Kutzingiana var. exilis Grun. (f. multiplex2) and further strains of N. palea var. debilis.