Lytic Characteristics and Identification of Two Alga-lysing Bacterial Strains

Lytic Characteristics and Identification of Two Alga-lysing Bacterial StrainsBeaulieu, S. E., M. R. Sengco, and D. M. Anderson, 2005. Using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs. Harmful Algae . (4): 123-138…     

Mapping the resting cysts of dinoflagellate Alexandrium catenella along the coast of Qinhuangdao,China

Dinoflagellate Alexandrium catenella is a cosmopolitan bloom-forming species with complex life cycle, the formation and germination of resting cysts are critical for its bloom dynamics. In the coastal waters of Qinhuangdao, A. catenella has been identified as the major causative agent for paralytic shellfish poisoning, but there is little knowledge concerning its resting cysts in this region. In this study, three surveys were carried out along the coast of Qinhuangdao from 2020 to 2021 to map the distribution of A. catenella resting cysts, using a quantitative PCR (qPCR) assay specific for A. catenella. The resting cysts were detected in surface sediments during all the three surveys, and their distribution patterns were similar. High abundance of resting cysts (maximum 1 300 cysts/g sediment (wet weight)) were found in a region (119.62°E–119.99°E, 39.67°N–39.98°N) northeast to the coastal waters of Qinhuangdao, where surface sediments were mainly composed of clay and silt (percentage above 50%). Prior to the formation of the A. catenella bloom in March 2021, the abundance of A. catenella vegetative cells in seawater had extremely significant positive correlation with the abundance of resting cysts in surface sediments, reflecting the important role of resting cysts in the initiation of A. catenella blooms. As far as we know, this is the first report on the distribution of A. catenella cysts along the coast of Qinhuangdao. The results will offer a sound basis for the future monitoring and mitigation of toxic A. catenella blooms and paralytic shellfish poisoning events in this region.

     

Effects of co-existing microalgae and grazers on the production of hemolytic toxins in Karenia mikimotoi

Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup is associated with harmful algal blooms in temperate and subtropical zones of the world. The hemolytic substances produced by K. mikimotoi are thought to cause mortality in fishes and invertebrates. We evaluated the composition of the hemolytic toxin produced by K. mikimotoi cultured in the laboratory using thin-layer chromatography.In addition, we evaluated the effect of co-occuring algae (Prorocentrum donghaiense and Alexandrium tamarense) and the cladoceran grazer Moina mongolica on hemolytic toxin production in K. mikimotoi. The hemolytic toxins from K. mikimotoi were a mixture of 2 liposaccharides and 1 lipid. Waterborne clues from P. donghaiense and A. tamarense inhibited the growth of K. mikimotoi but increased the production of hemolytic toxins. Conversely, K. mikimotoi strongly inhibited the growth of caged P. donghaiense and A. tamarense. In addition, the ingestion of K. mikimotoi by M. mongolica induced the production of hemolytic toxins in K. mikimotoi. Taken together, our results suggest that the presence of other microalgae and grazers may be as important as environmental factors for controlling the production of hemolytic substances. K. mikimotoi secreted allelochemicals other than unstable fatty acids with hemolytic activity. The production of hemolytic toxins in dinoflagellates was not only dependent on resource availability, but also on the risk of predation. Hemolytic toxins likely play an important role as chemical deterrents secreted by K. mikimotoi.     

Effect of Nitrogen and Phosphorus on Tissue Nutrition and Biomass of Freshwater Wetland Plant in Sanjiang Plain, Northeast China

1 Introduction It has been reported that habitat nutrient availability frequently limited plant growth and determined species dominance and abundance in natural communities (Miao et al., 2000). Nutrient availability is also a main regulator of aquatic primary production. Human-induced nutrient enrichment results in die-back of native vegetation and alteration of species dominance in various aquatic eco- systems (Miao et al., 2000; Green and Galatowitsch, 2002). Particularly, nutrient enrichm…     

Sewage pollution in Negril, Jamaica: effects on nutrition and ecology of coral reef macroalgae

Coral reefs in the Negril Marine Park (NMP), Jamaica, have been increasingly impacted by nutrient pollution and macroalgal blooms following decades of intensive development as a major tourist destination. A baseline survey of DIN and SRP concentrations, C:N:P and stable nitrogen isotope ratios (δ15N) of abundant reef macroalgae on shallow and deep reefs of the NMP in 1998 showed strong P-limitation and evidence of increasing sewage pollution. In 1999, a sewage collection and treatment project began divertin...     

Effect of nitrogen to phosphorus ratios on cell proliferation in marine micro algae

The ratio of nitrogen/phosphorus (N/P) is known to affect cell proliferation of some marine micro algae. We evaluated the effect of N/P ratios on the proliferation and succession of phytoplankton using five marine micro algae species. We used two sources of nitrogen, NH₄Cl (N₁) and urea (N₂), and a single source of phosphorous, NaH₂PO₄(P). The optimal N/P ratio that differed among the five species was affected by the source of nitrogen, being as follows (N₁/P, N₂/P in order): Thalassiosira sp. (30/1, 20/1), Heterosigma akashiwo (30/1, 30/1), Chroomonas salina (20/1, 30/1), Chaetoceros gracilis (40/1, 60/1), and Alexandrium sp. (10/1, 30/1). Thus, the source of nitrogen must be considered when analyzing the N/P ratio. Our results provide insight for predicting phytoplankton succession in coastal waters and may be used to forecast the potential risk of harmful algal blooms.     

The selection of alkaline protease-producing yeasts fi＇om marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains (HN3.11, N11b, YF04C and HN4.9) capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N11b was 10.0. The optimal temperature for protease activity was 45°C for strains HN3.11, N11b, and YF04C, and 50°C for strain HN4.9. After digestion of shrimp (Penaeus vannamei) protein and spirulina (Arthospira platensis) protein with the four crude alkaline proteases, the filtrate from spirulina (Arthrospira platensis) powder digested by the crude alkaline protease of strain HN3.11 was found to have the highest antioxidant activity (61.4%) and the highest angiotensin I converting enzyme (ACE)-inhibitory activities (68.4%). The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.     

Effects of nitrogen and phosphorus on the growth of <Emphasis Type="Italic">Levanderina fissa</Emphasis>: How it blooms in Pearl River Estuary

Effects of nitrogen (N) and phosphorus (P) from different sources and at different concentrations on the growth of Levanderina fissa (= Gyrodinium instriatum) were studied in laboratory conditions. The findings might explain the recurrent blooms of this species in Pearl River Estuary, China. Results showed that nutrient limitation significantly inhibited the growth of L. fissa. The values of specific growth rate (μ _max) and half-saturation nutrient concentration (K _S) were 0.37 divisions/d and 8.49 μmol L^?1 for N, and 0.39 divisions/d and 1.99 μmol L^?1 for P, respectively. Based on K _S values, dissolved inorganic N level in PRE was sufficient to support the high proliferation of L. fissa, while dissolved inorganic P concentration was far lower than the minimum requirement for its effective growth. L. fissa was not able to utilize dissolved organic N (DON) compounds such as urea, amino acids, and uric acid. However, it grew well by using a wide variety of dissolved organic P (DOP) sources like nucleotides, glycerophosphate, and 4-nitrophenylphosphate. The results from this study suggested that the ability in DOP utilization of L. fissa offers this species a competitive advantage in phytoplankton communities. The high level and continuous supply of DIN, enrichment of DOP, together with warm climate and low salinity in the Pearl River Estuary provided a suitable nutrient niche for the growth of L. fissa, and resulted in the recurrent blooms in the estuary.     

The decline process and major pathways of Microcystis bloom in Taihu Lake, China

Eutrophication has become a serious concern in many lakes, resulting in cyanobacterial blooms. However, the mechanism and pathways of cyanobacteria decline are less understood. To identify and define the growth and decline of Microcystis blooms in Taihu Lake of China, and to illuminate the destination of surface floating blooms, we investigated the biomass distribution and variations in colony size, morphology, and floating velocity from October 2008 to September 2009. The results showed that the Microcystis bloom declined in response to biomass decrease, colony disaggregation, buoyancy reduction, and increased phytoplankton biodiversity, and these indicative parameters could be applied for recognition of the development phases of the bloom. Three major decline pathways were proposed to describe the bloom decline process, colony disaggregation (Pathway Ⅰ), colony settlement (Pathway Ⅱ), and cell lysis in colonies (Pathway Ⅲ). We proposed a strategy to define the occurrence and decline of Microcystis blooms, to evaluate the survival state under different stress conditions, and to indicate the efficiency of controlling countermeasures against algal blooms.     

Diversity and community structure of eukaryotic microalgae in surface sediments in the central Bohai Sea,China, based on a metabarcoding approach

Sediment samples were collected at 17 stations in the central Bohai Sea, China, and the diversity and community structure of eukaryotic microalgae were assessed by metabarcoding the V4 region of 18S rDNA. A total of 930 operational taxonomic units (OTUs) were detected for microeukaryotes, including 98 algal OTUs. The algal communities comprised 42 genera belonging to 19 classes of six phyla, and they were dominated by chrysophytes and dinoflagellates. Dinoflagellates were also the most diverse microalgal group. The nano-sized dinoflagellates Biecheleria halophila and Azadinium trinitatum occurred abundantly in the study area; however, they have not been reported previously, as they may be overlooked or misidentified in light microscopy. Many pico-sized chlorophytes were detected in the sediment samples. Sixteen of the detected OTUs were assigned to potentially harmful and/or bloom-forming microalgae, suggesting some potential risks of harmful algal blooms in the central Bohai Sea. The capacity of metabarcoding to detect morphologically cryptic and small species makes this method a sufficiently sensitive means of detection for assessing eukaryotic microalgae in sediments.

     

Intraspecific diversity of Aureobasidium pullulans strains from different marine environments

Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureobasidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc, A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A. pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.     

Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.     

Expression of organophosphorus-degradation gene (opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

Genetic engineering in filamentous N₂-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene (opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P _psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N₂-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.     

Screening for lipophilic marine toxins and their potential producers in coastal waters of Weihai in autumn, 2020

Lipophilic marine toxins (LMTs) produced by some microalgae in the sea could accumulate in shellfish and pose potential threats to the health of seafood consumers. Phytoplankton and shellfish samples were collected from coastal waters of Weihai in Shandong Peninsula, China in autumn, 2020, and screened for lipophilic marine toxins and their potential producers using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and high throughput sequencing of partial DNA (V4 region of the 18S rRNA gene) extracted from phytoplankton. Pectenotoxin-2 (PTX2), trace amounts of azaspiracid (AZA1 or AZA40), and 13-desmethyl spirolide C (13-DesMe-C) were detected in phytoplankton samples, while PTX2 and gymnodimine (GYM) were detected in shellfish samples. The toxin content in shellfish samples was much lower than the regulatory limit or values reported previously. Results suggest that lipophilic marine toxins should have low risk in coastal waters of Weihai in autumn. Based on the data of high throughput sequencing, the OTUs were assigned to 5 identified species of Alexandrium, including A. ostenfeldii capable of producing 13-DesMe-C and GYM. Two OTUs were found closely related to the toxic species in genus Dinophysis, but it is impossible to assign them to any identified species due to the low resolving power of the V4 region for Dinophysis. The OTUs could not be assigned to any identified species in the genus Azadinium, suggesting the existence of unidentified species in this region.

     

Contribution of trimethylamine N-oxide on the growth and pressure tolerance of deep-sea bacteria

Trimethylamine N-oxide (TMAO) is widely dispersed in marine environments and plays an important role in the biogeochemical cycle of nitrogen. Diverse marine bacteria utilize TMAO as carbon and nitrogen sources or as electron acceptor in anaerobic respiration. Alteration of respiratory component according to the pressure is a common trait of deep-sea bacteria. Deep-sea bacteria from different genera harbor high hydrostatic pressure (HHP) inducible TMAO reductases that are assumed to be constitutively expressed in the deep-sea piezosphere and facilitating quick reaction to TMAO released from fish which is a potential nutrient for bacterial growth. However, whether deep-sea bacteria universally employ this strategy remains unknown. In this study, 237 bacterial strains affiliated to 23 genera of Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria were isolated from seawater, sediment or amphipods collected at different depths. The pressure tolerance and the utilization of TMAO were examined in 74 strains. The results demonstrated no apparent correlation between the depth where the bacteria inhabit and their pressure tolerance, regarding to our samples. Several deep-sea strains from the genera of Alteromonas, Halomonas, Marinobacter, Photobacterium, and Vibrio showed capacity of TMAO utilization, but none of the isolated Acinebacter, Bacillus, Brevundimonas, Muricauda, Novosphingobium, Rheinheimera, Sphingobium and Stenotrophomonas did, indicating the utilization of TMAO is a species-specific feature. Furthermore, we noticed that the ability of TMAO utilization varied among strains of the same species. TMAO has greater impact on the growth of deep-sea isolates of Vibrio neocaledonicus than shallow-water isolates. Taken together, the results describe for the first time the TMAO utilization in deep-sea bacterial strains, and expand our understanding of the physiological characteristic of marine bacteria.

     

Records of bulk organic matter and plant pigments in sediment of the “red-tide zone” adjacent to the Changjiang River estuary

Cultural eutrophication caused by nutrient over-enrichment in coastal waters will lead to a cascading set of ecosystem changes and deleterious ecological consequences,such as harmful algal blooms(HABs) and hypoxia.During the past two decades since the late 1990s,recurrent large-scale HABs(red tides)and an extensive hypoxic zone have been reported in the coastal waters adjacent to the Changjiang River estuary.To retrieve the history of eutrophication and its associated ecosystem changes,a sediment core was collected from the "red-tide zone" adjacent to the Changjiang River estuary.The core was dated using the~(210)Pb radioisotope and examined for multiple proxies,including organic carbon(OC),total nitrogen(TN),stable isotopes of C and N,and plant pigments.An apparent up-core increase of OC content was observed after the 1970s,accompanied by a rapid increase of TN.The concurrent enrichment of δ~(13)C and increase of the C/N ratio suggested the accumulation of organic matter derived from marine primary production during this stage.The accumulation of OC after the 1970 s well reflected the significant increase of primary production in the red-tide zone and probably the intensification of hypoxia as well.Plant pigments,including chlorophyll a,p-carotene,and diatoxanthin,showed similar patterns of variation to OC throughout the core,which further confirmed the important contribution of microalgae,particularly diatoms,to the deposited organic matter.Based on the variant profiles of the pigments representative of different microalgal groups,the potential changes of the phytoplankton community since the 1970s were discussed.     

Dynamics of above- and below-ground biomass and C, N, P accumulation in the alpine steppe of Northern Tibet

The temporal dynamics of the biomass, as well as the carbon (C), nitrogen (N), phosphorus (P) concentrations and accumulation contents, in aboveand below-ground vegetation components were determined in the alpine steppe vegetation of Northern Tibet during the growing season of 2010. The highest levels of total biomass (311.68 g m⁻²), total C (115.95 g m⁻²), total N (2.60 g m⁻²), and total P (0.90 g m⁻²) accumulation contents were obtained in August in 2010. Further, biomass and nutrient stocks in the below-ground components were higher than those of the above-ground components. The dominant species viz., Stipa purpurea and Carex moorcrofti had lower biomass and C, N, P accumulations than the companion species which including Oxytropis. spp., Artemisia capillaris Thunb., Aster tataricus L., and so on.     

Effect of CO2 enrichment on competition between Skeletonema costatum and Heterosigma akashiwo

We investigated the effect of CO₂ enrichment and initial inoculum density on competition between Skeletonema costatum and Heterosigma akashiwo, two common algae seen in algal blooms. The initial inoculum density (0.2×10⁴, 0.4×10⁴, 0.8×10⁴ cells/ml) had a significant effect on population growth and competition between H. akashiwo and S. costatum. The time required for population growth to reach the exponential growth phase and stationary phase decreased significantly as the initial density increased. When the two species were cultured together, S. costatum tended to outcompete H. akashiwo, especially when present at higher initial ratios. CO₂ enrichment (5 000 μl/L CO₂) increased the maximum population density and carrying capacity of H. akashiwo but decreased these measures for S. costatum. Thus, CO₂ enrichment favored the growth of H. akashiwo over S. costatum.     

Applications of immuno-magnetic bead and immunofluorescent flow cytometric techniques for the quantitative detection of HAB microalgae

Overthelastseveraldecades,harmfulalgalblooms(HABs)havebecomeaseriousenvironmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo is one of the most important HABs species in China. The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H. akashiwo cells. Five HABs species (Alexandrium catenella, Thalassiosira sp., Cryptomonas sp., Alexandrium tamarense and Symbiodinium sp.,) were used in this study to evaluate the analysis process we developed. A polyclonal antibody with high titers against H. akashiwo was obtained by injecting H. akashiwo cells into rabbits. Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H. akashiwo cells from the culture. Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs, and only 5.3%-12.5% of the four other HAB microalgae species were captured, indicating that the constructed IMBs combined specifically with the H. akashiwo cells. At the same time, flow cytometry (FCM) sorting was exploited to screen H. akashiwo cells after labeling with FITC conjugated polyclonal antibodies. Using the FCM technique, 91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes. These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species. These techniques, especially immuno-magnetic separation, have low associated cost, and are fast and simple processes compared with other techniques currently in use.     

Occurrence and diversity of <Emphasis Type="Italic">Pichia</Emphasis> spp. in marine environments

A total of 328 yeast strains from seawater, sediments, mud of salterns, the guts of marine fish and marine algae were obtained. The results of routine identification and molecular methods show that five yeast strains obtained in this study belonged to Pichia spp., including Pichia guilliermondii 1uv-small, Pichia ohmeri YF04d, Pichia fermentans YF12b, Pichia burtonii YF11A and Pichia anomala YF07b. Further studies revealed that Pichia anomala YF07b could produce killer toxin against pathogenic yeasts in crabs while Pichia guilliermondii 1uv-small could produce high activity of extracellular inulinase. It is advisable to test if Pichia ohmeri YF04d obtained in this study is related to central-venous-catheter-associated infection.