Diversity of virus-like agents killing Microcystis aeruginosa in a hyper-eutrophic pond

We demonstrated the existence of the diversity of virus-likeagents killing Microcystis aeruginosa in a shallow hyper-eutrophicpond in Japan, but without specific virus isolation, once inOctober and twice in December 2001. The pond water was treatedby chloroform, filtered throughout GF-75 filter (0.3 µm)and then incubated with axenic M. aeruginosa for 7 days. Thecell density of M. aeruginosa decreased 10-fold in the courseof the incubation. Results suggested that the active agent forcell death was virus-like, based upon its size fraction (from30 kDa to 0.3 µm), sensitivity on heat-treatment, andevidence of protein protection of DNA during our extractionprocedure. From our results of pulsed field gel electrophoresis(PFGE) analysis, two or four different-sized DNA bands appearedfrom the culture lysate in each experiment. In total, 10 different-sizedbands (10–90 kb) were observed from the three sample dates,suggesting that multiple virus-like agents killing M. aeruginosaexisted in this pond. To our knowledge, this is the first reportof the existence of virus-like agents with different genomicsize that kill M. aeruginosa.     

Influence of Cultivation Parameters on Growth and Microcystin Production of Microcystis aeruginosa (Cyanophyceae) Isolated from Lake Chao (China)

Microcystis aeruginosa isolated in 2005 from the shallow eutrophic Lake Chao (Anhui, China) was investigated in terms of growth parameters and microcystin production under varying nutrient concentrations (P, N) and pH values (abiotic factors) as well as under the influence of spent medium of the non-toxic cyanobacterium Synechocystis sp. (biotic factors). Stimulating effects on growth were observed at the alkaline pH value (10.5), whereas toxin production was significantly increased under phosphate-P limitation (0.6 mg L⁻¹ medium). Within a broad range of nitrate–N concentrations (41.2–247.2 mg L⁻¹ medium), no significant influence on cell growth and microcystin production was observed; however, N-starvation resulted in a typical decrease of growth and toxicity. In addition, cryopreservation of M. aeruginosa evidenced the decrease of toxin production by time-dependent exposure with the cryoprotectant dimethyl sulfoxide under thawing conditions without affecting the growth of the cyanobacterial cells.     

Growth response of Microcystis spp. to iron enrichment in different regions of Lake Taihu, China

Iron (Fe) is an essential micronutrient for algal growth and can be a potential limiting nutrient in aquatic system, especially regions that exhibits nitrogen (N) limitation. Using short-term nutrient addition bioassays, we evaluated the potential role that iron might play in modifying the response of Microcystis spp. to the anthropogenic phosphorus (P) and N enrichment in hypereutrophic Lake Taihu, the third largest freshwater lake in China. Three nutrient enrichment experiments involving additions of N (as NO₃ ^?) and P (as PO₄ ^3?) with and without Fe were conducted during 2009?C2010 in Meiliang Bay, a region characterized by summer cyanobacterial (Microcystis spp.) blooms, and East Taihu, a region largely free of cyanobacterial blooms and dominated by macrophytes. In Meiliang Bay, Fe addition alone did not significantly increase Microcystis spp. biomass. However, Fe addition occasionally increased the stimulatory effect of N and P additions on Microcystis spp., indicating that Fe was not a primary limiting nutrient for Microcystis spp. growth. Occasionally Fe was co-limiting with N and P in this region. In East Taihu, the addition of Fe alone significantly stimulated Microcystis spp. growth, while addition of N and/or P had no effects on growth, indicating that Fe was a primary limiting nutrient in East Taihu. The combined addition of Fe and N resulted in a growth response similar to Fe alone, while combined addition of Fe and P yielded greater biomass increases than the addition of Fe alone. This indicated that in East Taihu, N was not limiting and Fe and P supplies facilitated Microcystis spp. growth. These results reflect differential availabilities and limitations of N, P, and Fe in distinct regions of Taihu. The potential role of Fe in eutrophication dynamics of large, regionally complex lakes like Taihu requires further attention.     

Comparative studies on phosphate utilization of two bloom-forming Microcystis spp. (cyanobacteria) isolated from Lake Taihu (China)

Microcystis is a common freshwater bloom-dominating cyanobacterial genus. However, the crucial factors that affect the seasonal succession and extent of dominance of different species remain largely unknown. This study investigated inorganic phosphorus (P) uptake, growth, and P utilization of two dominant Microcystis species from Lake Taihu (China) at different P concentrations and temperatures. Compared with Microcystis wesenbergii, Microcystis flos-aquae had higher maximum uptake rate and luxury storage coefficient. However, M. flos-aquae also had a higher P demand for growth. Under P-rich conditions (64.5 to 174.5 μM), M. flos-aquae had a higher growth rate (0.16 to 0.21 d^?1) than M. wesenbergii (0.06 to 0.19 d^?1) at 22 to 30 °C. M. wesenbergii, with higher phosphate affinity, had a lower P demand to sustain its growth, and yielded a higher growth rate of 0.10 d^?1 at low P concentration (6.5 μM) at 34 °C, whereas M. flos-aquae had a lower growth rate of 0.03 d^?1. Therefore, M. flos-aquae was dominant in late spring under P-rich conditions, whereas M. wesenbergii prevailed in hot summer after M. flos-aquae decreased under P-limited conditions. These results agree well with their succession patterns in the field. Overall, succession and dominance of M. flos-aquae and M. wesenbergii in Lake Taihu are regulated by P concentration and water temperature.     

Morphospecies and genospecies of Microcystis during blooms in eutrophic Lake Taihu (China) in autumn

Microcystis colonies collected in a hypertrophic lake (Lake Taihu, China) in October and November 2012 were divided into five subsamples according to colony size (<75, 75–150, 150–300, 300–500, and >500 μm). All the subsamples collected in November were dominated with Microcystis ichthyoblabe and the percentages of M. ichthyoblabe exceeded 83%. The percentages of Microcystis aeruginosa of the subsamples in >500 μm class collected in October was 93.5%. For the sample collected in October, the percentage of M. ichthyoblabe was more than 58% in <75, 75–150, 150–300 μm classes. The 16S rDNA as well as some polysaccharide biosynthesis-related genes were analyzed to understand the phylogeny of Microcystis species. There was no variant site presented in each Microcystis subsample but a single nucleotide polymorphism (SNP) was found in 16S rDNA alignment tested using MSR1 in subsamples between the two months in the current study. Our results also showed that samples collected in two months can by divided into two parts by the phylogenetic analysis using two polysaccharide biosynthesis-related genes (espL and TagH). All the results suggested that 16S rDNA was valuable to identify seasonal succession of Microcystis genospecies and the diversity of Microcystis morphospecies would be explained by these polysaccharide biosynthesis-related genes.     

Biodegradation of microcystin-LR and-RR by a novel microcystin-degrading bacterium isolated from Lake Taihu

Microcystin-LR (MC-LR) and microcystin-RR (MC-RR) are the two most common microcystins (MCs) present in fresh water posing a direct threat to public health because of their hepatotoxicity. A novel MC-degrading bacterium designated MC-LTH1 capable of degrading MC-LR and -RR was isolated, and the degradation rates and mechanisms of MC-LR and -RR for this bacterium were investigated. The bacterium was identified as Bordetella sp. and shown to possess a homologous mlrA gene responsible for degrading MCs. To the best of our knowledge, this is the first report of mlrA gene detection in Bordetella species. MC-LR and -RR were completely degraded separately at rates of 0.31 mg/(L h) and 0.17 mg/(L h). However, the degradation rates of MC-LR and -RR decreased surprisingly to 0.27 mg/(L h) and 0.12 mg/(L h), respectively, when both of them were simultaneously present. Degradation products were identified by high performance liquid chromatography coupled with time-of-flight mass spectrometry. Adda (m/z 332.2215, C₂₀H₂₉NO₃) commonly known as a final product of MC degradation by isolated bacteria was detected as an intermediate in this study. Linearized MC-LR (m/z 1013.5638, C₄₉H₇₆N₁₀O₁₃), linearized MC-RR (m/z 1056.4970, C₄₉H₇₇N₁₃O₁₃), and tetrapeptide (m/z 615.3394, C₃₂H₄₆N₄O₈) were also detected as intermediates. These results indicate that the bacterial strain MC-LTH1 is quite efficient for the detoxification of MC-LR and MC-RR, and possesses significant bioremediation potential.     

Investigation of a toxic water-bloom of Microcystis aeruginosa (Cyanophyceae) in Lake Akersvatn,Norway

During the summer and fall of 1984 and 1985, the eutrophic Lake Akersvatn in south-eastern Norway, used as reserve drinking water reservoir, was found to produce heavy water-blooms of the colonial blue-green alga Microcystis aeruginosa. Samples of the water-bloom were found to be toxic using the mouse bioassay. No toxin was found free in the water as detected by HPLC and mouse bioassay. The toxic cells (minimum lethal dose 8–15 mg/kg body weight in mice) and purified toxin (minimum lethal dose 50 μg/kg body weight in mice) showed signs of poisoning in laboratory rats and mice identical to that of other hepatotoxin-producing M. aeruginosa blooms and strains reported from other parts of the world. The toxin has chemical properties similar to the cyclic heptapeptide reported for a South African M. aeruginosa toxin. The toxin from Lake Akersvatn bloom material has a molecular weight of 994. The toxic bloom of M. aeruginosa persisted from August to November in 1984 and reappeared in July of 1985. While water from Lake Akersvatn was not used for municipal water supply during this period, the presence of toxic blue-green algae in a drinking water reservoir indicates the need to develop monitoring and detection methods for toxic cells and toxin(s).     

Phylogenetic Inference of Colony Isolates Comprising Seasonal Microcystis Blooms in Lake Taihu, China

Blooms of the toxin-producing cyanobacterium, Microcystis spp., are an increasingly prevalent water quality problem and health hazard worldwide. China's third largest lake, Lake Taihu, has been experiencing progressively more severe Microcystis blooms over the past three decades. In 2009 and 2010, individual Microcystis colonies, consisting of four different morphospecies, were isolated and genotyped using a whole-cell multiplex PCR assay. The 16S-23S rDNA-ITS sequences were aligned based on Bayesian inference and indicated that one morphospecies was genetically unique (Microcystis wesenbergii) and three were indistinguishable (Microcystis aeruginosa, Microcystis flos-aquae, and Microcystis ichthyoblabe). Microcystin (mcyB) genes were detected intermittently in two of the morphospecies while the other two morphospecies lacked the mcyB gene in all samples. Water temperature was found to influence bloom formation and morphotype prevalence, and chlorophyll a and temperature were positively and significantly correlated with microcystin concentration. Cooler water temperatures promoted toxigenic strains of Microcystis. Wind appeared to influence the distribution of morphotypes across the lake, with M. aeruginosa and M. ichthyoblabe being more susceptible to wind stress than M. wesenbergii and M. flos-aquae. The results of this study indicated that the blooms were composed of a variety of Microcystis morphospecies, with more genotypes observed than can be attributed to individual morphotypes. We conclude that morphology is not a reliable indicator of toxigenicity in Lake Taihu, and caution should be exercised when the M. aeruginosa morphotype is present because it is capable of producing MC-LR, the most toxic microcystin isoform.     

Characterization of the bacterial community composition in a hypoxic zone induced by Microcystis blooms in Lake Taihu, China

Cyanobacterial blooms have become more frequent as a result of eutrophication in lakes. The accumulation and breakdown of huge cyanobacterial biomasses often cause hypoxia in lakes. However, little is known about microbial diversity in these areas. In this study, we characterized the bacterial community composition of a Microcystis-bloom-induced hypoxic area in Lake Taihu, which is a large, shallow lake, by analysing terminal restriction fragment length polymorphisms of 16S ribosomal RNA genes and clone libraries generated from selected samples. Bacterial samples were collected at different sites within the hypoxic zone at different times during the development of hypoxia. The results showed that the composition of both free-living and particle-attached bacterial communities in the water column varied spatially and temporally and that these variations were largely related to changes in the concentrations of dissolved oxygen and ions in the water column. Sequences affiliated with Clostridium were predominantly found at the onset of hypoxia, whereas members of the LD12 cluster were detected at the posthypoxia stage; Desulfovibrio and Comamonadaceae dominated throughout the hypoxic event. We speculate that these organisms may be associated with the decomposition of Microcystis biomass and the production of volatile organic compounds; however, their specific function in Microcystis-bloom-induced hypoxia warrants further study.     

Earlier and warmer springs increase cyanobacterial (Microcystis spp.) blooms in subtropical Lake Taihu,China

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Recruitment of Total Phytoplankton,Chlorophytes and Cyanobacteria from Lake Sediments Recorded by Photosynthetic Pigments in a Large,Shallow Lake (Lake Taihu,China)

Recruitment of total phytoplankton, chlorophytes and cyanobacteria from lake sediments to the water column was studied using photosynthetic pigments at one site (1.5 m) in Lake Taihu, a large shallow lake in China. Samples were taken weekly from the migration traps installed on the bottom from March to May 2004. Abundance of total phytoplankton, chlorophytes and cyanobacteria were represented by Chlorophyll (Chl) a, b, and phycocyanin (PC), respectively. Over the three months, total phytoplankton, chlorophytes, and cyanobacteria corresponding to 48.9%, 68.9% and 316.2% of their initial concentrations in surface sediments were recruited in Lake Taihu. However, compared with their increase in pelagic abundance over the same period, the recruitment accounted for a rather small inoculum. Accompanying the recruitment, total phytoplankton and chlorophytes declined and cyanobacteria increased in the upper 0–2 cm sediments; colonies of Microcystis aeruginosa in the water column enlarged from small size with several cells to large colonies with hundreds of cells. Thus, overwintering and subsequent growth renewal of pelagic phytoplankton merits further study and comparison with benthic survival and recruitment. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects on water quality following water transfer in Lake Taihu,China

To improve lake water quality, two experimental water transfers were conducted in winter–spring 2002 and summer–fall 2003 in Lake Taihu, a large shallow lake in China. Both observed data and estimated nutrient concentration with the elimination of effect from natural factors were used in this research to assess the spatial and temporal variations of water quality improvement induced by the two transfers. Clear improvement of water quality associated with deduction of TN, TP, and chlorophyll a (Chl-a) concentration was observed in many areas of the lake during the two water transfers. The over all reduction in TP concentration was notable in Southwest Zone, Centre Zone, and Dongtaihu Bay during the 2002 transfer, and was more pronounced in Meiliang Bay and Southwest Zone during the 2003 transfer period. However, the reduction in TN and Chl-a concentration was relatively weak. Results indicate a less impressive improvement of water quality from water transfer in large lakes than in small ones as the effectiveness of water transfer in large lakes is generally limited by large size, complex boundaries, and the difficulty of finding proper water source to be transferred. The comparison of observed and estimated water transfer effectiveness suggests a greater improvement of water quality derived from water transfer than appeared from the observation.     

Ecological impacts of water transfers on Lake Taihu from the Yangtze River,China

As an ecosystem responds to external environmental changes slowly due to its reorganization compared with the water quality and it has great influence on lake ecosystems, it is indispensable to evaluate ecological impacts from water transfers for the security of the water source in Lake Taihu. Evaluations were conducted by comparing the indicators exergy (Ex), structural exergy (Ex_st) and phytoplankton buffer capacity (β_((TP)/(Phy))) changes after water transfers from 2002 to 2007 with those reference statuses where the water transfers were not implemented from 1998 to 2001. Besides those three ecological indicators, affiliated indicators such as ratio of zooplankton biomass to phytoplankton biomass, diversity index and trophic state index were also involved in the evaluation. The results showed the water transfers altered the ecosystem status and had positive effects on Lake Taihu and most of its sub-zones, such as Gonghu Bay, Northwest Zone, Southwest Zone and Centre Zone in the post-transfer period. The seasonable trends indicated that the ecosystem health with environmental influences excluded in November and February was better than that in May and August during the water transfers from 2002 to 2007 and there were significant differences in the ecosystem health of the first and second stages during water transfers. The ecosystem health in May and August was better than in November and February during the first stage of the water transfers (from 2002 to 2004), while the opposite obtained during the second stage from 2005 to 2007 in Dongtaihu Bay and East Epigeal Zone. On the whole, water transfers serve to deter algal congregation in Lake Taihu.     

青草沙水库中一株铜绿微囊藻抑藻细菌的作用研究

【目的】为了研究青草沙水库中土著微生物对藻类生长的抑制作用,从水库水体中筛选出对藻类有抑制作用的细菌并研究其对铜绿微囊藻的抑制效果。【方法】通过对水库水体中的细菌进行划线分离和筛选,挑选出一株对铜绿微囊藻生长有较好抑制作用的菌株CL。考察其对铜绿微囊藻的抑制效果及不同培养时间和菌液浓度对抑藻效果的影响,并对菌株进行16S rDNA序列分析。【结果】实验菌液浓度为4.5×108CFU/mL 8.4×108CFU/mL时,细菌对铜绿微囊藻的抑制率可达45.4%。抑藻效果随培养时间先增后降,在静置培养第6天抑藻效果达到最大。该菌经过16S rDNA序列分析,属于黄单胞菌科的寡养单胞菌。【结论】从青草沙水库中筛选出了对铜绿微囊藻有抑制作用的土著细菌寡养单胞菌,对青草沙水体铜绿微囊藻的控制具有一定的潜在应用价值。     

Microcystins derived from lysing Microcystis cells do not cause negative effects on crustacean zooplankton in Lake Taihu, China

Microcystins (MCs) have a toxic effect on crustacean zooplankton in the laboratory, but there is little or no unequivocal evidence in the literature of their lethal effects on crustacean zooplankton in the field. We used the natural microcystins extracted from Microcystis spp. to test if they could cause any negative effects on crustacean zooplankton. We conducted three experiments in enclosures with water from Lake Taihu, China, and microcystins derived by extraction from Microcystis spp. collected from the lake when the species was in bloom conditions. Initial concentrations of extracellular microcystins (EMCs = MC-RR + MC-LR + MC-YR) ranged from 9.7 to 44.9 μg/L in treatments with microcystin addition. Microcystin concentrations sharply decreased on second day in all the three experiments. EMCs at the end of the experiments varied from only 2.7 to 14.2 % of the levels at the start of the experiments. The dominant species of crustacean zooplankton in the lake were Bosmina longirotris, Ceriodaphnia cornuta, Mesocyclops spp., Limnoithona sinensis, Sinocalanus dorrii and Schmackeria inopinus. ANOVA analysis showed that the density and biomass of cladoceran and copepod did not significantly differ between treatments with microcystin addition and controls. Our results indicate that microcystins derived from lysing Microcystis do not cause any negative effects on crustacean zooplankton.     

松花湖铜绿微囊藻无菌株和单藻株生长因素的研究

用毛细滴管洗净法从松花湖分离纯化出铜绿微囊藻(Microcystisaeruginosa)的单藻株和无菌株,研究了温度、照度、N、P、Fe各种因素对单藻株和无菌株生长的影响。结果表明,各种因素对单藻株和无菌株最大比增长率的影响差别不大,单藻株和无菌株分别在温度33℃,照度5000Lx(2000Lx)、NO3-N浓度1.2mg·L-1,PO43-P浓度0.06mg·L-1,柠檬酸铁铁浓度0.05mg·L-1(0.04mg·L-1)时,达到最大比增长率。在一定浓度范围内,随着N、P、Fe浓度的增加,单藻株比无菌株的最大增长量高得多,可见附着细菌的存在,能促进铜绿微囊藻的生长。从松花湖湖水中N、P和Fe含量以及温度和照度的情况看,松花湖的某些区域已经基本具备了水华发生的条件。     

Toxicity evaluation of Meiliang Bay, Lake Taihu, China—a drinking water source

In this study, potential toxicity in organic pollutants in Meiliang Bay, Lake Taihu, a drinking water source, was investigated using the comet assay and zebrafish embryo test, and two control sites in Lake Changdang and the Changjiang (Yangtze) River were established. For the genotoxicity assay, results showed that organic extracts from water samples induce DNA damage on human lymphocytes and mouse testicular cells. A statistically significant difference (p < 0.01) was observed versus the solvent control, as shown by multiple comparisons at a dose of 100 ml tube⁻¹. The degree of DNA damage caused by Meiliang Bay water was most serious (human lymphocytes cells—184 arbitrary units (AU); 234 AU on mouse testicular cells). Organic extracts also affected zebrafish embryo development. Embryo coagulation, axis abnormality, slow absorbability of vitellicle, and multi-edema related to teratogenesis at 96 hpf were observed. In the high dose group, there was obvious edema in the hearts and vitellicles for most dysplastic embryos. Toxic potential in organic pollutants in drinking water sources from the Yangtze River and Lake Changdang were less serious than Meiliang Bay. Therefore, the drinking water source in Meiliang Bay was unsafe compared to the Changjiang River and Lake Changdang, and it is exigent that Meiliang Bay water quality should be ameliorated further.     

Pseudomonas aeruginosa UCBPP-PA14 a useful bacterium capable of lysing Microcystis aeruginosa cells and degrading microcystins

To identify a useful bacterium capable of controlling both Microcystis aeruginosa and microcystins (MCs), 30 strains of Pseudomonas were screened. Two of them (Pseudomonas aeruginosa UCBPP-PA14 and Pseudomonas putida KCCM 10464) could cause significant lysis of M. aeruginosa. PA14 exhibited higher degradation activity against microcystins than KCCM 10464, and hence, it was selected as the bacterium for further analysis. Following its introduction into M. aeruginosa culture (10⁵ cells mL^-1) at densities of 10⁷, 10⁵, and 10³ PA14 cells mL^-1, higher initial inoculations of PA14 removed correspondingly more M. aeruginosa cells (100%, 100%, and 92% at 15, 30, and 10?days, respectively) and degraded microcystin (extracellular MCs: 83.7%, 77.7%, and 51.6% at 30?days; total MCs: 91.0%, 86.9%, and 61.6% at 30?days, respectively). However, the activity of PA14 diminished when its density decreased to less than 10⁶ cells mL^-1. At three initial algal densities (10⁶, 10⁵, and 10³ cells mL^-1), PA14 at a density of 10⁵ cells mL^-1 easily and quickly removed algal cells (100%, 100%, and 97.3% at 8, 16, and 30?days, respectively). Host range assays showed that at lower initial PA14 inoculation (10⁵ cells mL^-1), the algicidal activity of PA14 was effective species-specifically on M. aeruginosa, while at higher initial inoculation (10⁷ cells mL^-1), a wider algicidal range regardless of the general taxonomical relationships was observed. These results indicate that inoculation with 10⁵ Pseudomonas aeruginosa PA14 cells mL^-1 into developing natural algal blooms can remove both M. aeruginosa and MCs without causing problems for other algae species.     

Growth interactions among blue-green (Anabaena Oscillarioides,Microcystis aeruginosa) and green (Chlorella sp.) algae

The growth interactions amongst the blue-green algal species Anabaena oscillarioides, Microcystis aeruginosa and the green alga, Chlorella sp. were studied both in mixed cultures and in filter cultures separated by a membrane filter in the two arms of an interaction U-tube. The role of nutrients especially phosphate upon the interaction has also been studied. Anabaena and Microcystis both inhibited the growth of Chlorella while Microcystis also inhibited the growth of Anabaena. The inhibitory effect of Microcystis was found to be dependent on high concentrations of the initial algal inocula and independent of the initial concentration of nutrients such as inorganic phosphate, indicating that the nature of the inhibition is probably due to the production of inhibitory extracellular products by Microcystis. On the other hand, the inhibitory effect of Anabaena on Chlorella is the consequence of nutrient competition with Anabaena competing more effectively for the available phosphate.