Volatile organic compound emissions from Microcystis aeruginosa under different phosphorus sources and concentrations

In eutrophicated water, cyanobacteria massively grow and release an abundance of volatile organic compounds (VOCs) that contribute to the water odor. To uncover the effects of different phosphorus (P) nutrients on the formation of cyanobacteria VOCs and water odor, the cell growth and VOC emissions of Microcystis aeruginosa were investigated under different P nutrient conditions. Among K₂HPO₄, Na₄P₂O₇, and (NaPO₃)₆, K₂HPO₄ showed the largest increase in cell density, while a reduction in P concentration decreased the cell density. There were 26, 23 and 22 compounds in M. aeruginosa VOCs with K₂HPO₄, Na₄P₂O₇ and (NaPO₃)₆ as the sole P source, respectively, including sulfocompounds, terpenoids, benzenes, hydrocarbons, alcohols, aldehydes, and esters. Non‐P markedly promoted the VOC emission, and six additional compounds were observed: α‐pinene, 1‐phenyl‐1‐butanone, 1H‐1‐ethylidene‐indene, 2,6,10‐trimethyl‐tetradecane, 2‐ethyl‐hexanal, and acetic acid 2‐ethylhexyl ester. It can be deduced that cyanobacteria release different VOC blends using various P forms in eutrophicated waters, and the reduction of P amount promotes VOC emission and increases the water odor.     

Isolate-specific conidiation in Trichoderma in response to different nitrogen sources

A characteristic feature of Trichoderma is the production of concentric rings of conidia in response to alternating light/dark conditions and a single ring of conidia in response to a single burst of light. In this study, conidiation was investigated in four biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens) and one isolate from the mushroom pathogen species, T. pleuroticola. All five isolates produced concentric conidial rings under alternating light/dark conditions on potato-dextrose agar (PDA), however, in response to a 15min burst of blue light, only T. asperellum and T. virens produced a clearly defined conidial ring. Both T. pleuroticola and T. hamatum photoconidiated in a disk-like fashion and T.?atroviride produced a broken ring with a partially filled in appearance. In the presence of primary nitrogen, T. asperellum and T. pleuroticola conidiated in a disk, whereas, when grown in the presence of secondary nitrogen, a ring of conidia was produced. Primary nitrogen promoted photoconidiation and competency to conidiate in response to light appeared dependent on the nitrogen catabolite repression state of the cell. Mycelial injury was also investigated in the same five isolates of Trichoderma on PDA and under different nitrogen statuses. For the first time, we report that conidiation in response to injury is differentially regulated in different isolates/species of Trichoderma.     

Increased algicidal activity of Aeromonas veronii in response to Microcystis aeruginosa: interspecies crosstalk and secondary metabolites synergism

Toxic Microcystis spp. blooms constitute a serious threat to water quality worldwide. Aeromonas veronii was isolated from Microcystis sp. colonies collected in Lake Kinneret. Spent Aeromonas media inhibits the growth of Microcystis aeruginosa MGK isolated from Lake Kinneret. The inhibition was much stronger when Aeromonas growth medium contained spent media from MGK suggesting that Aeromonas recognized its presence and produced secondary metabolites that inhibit Microcystis growth. Fractionations of the crude extract and analyses of the active fractions identified several secondary metabolites including lumichrome in Aeromonas media. Application of lumichrome at concentrations as low as 4 nM severely inhibited Microcystis growth. Inactivation of aviH in the lumichrome biosynthetic pathway altered the lumichrome level in Aeromonas and the extent of MGK growth inhibition. Conversely, the initial lag in Aeromonas growth was significantly longer when provided with Microcystis spent media but Aeromonas was able to resume normal growth. The longer was pre-exposure to Microcystis spent media the shorter was the lag phase in Aeromonas growth indicating the presence of, and acclimation to, secondary MGK metabolite(s) the nature of which was not revealed. Our study may help to control toxic Microcystis blooms taking advantage of chemical languages used in the interspecies communication.     

不同氮、磷浓度对铜绿微囊藻生长、光合及产毒的影响

对一株从野外分离得到的铜绿微囊藻产毒株进行分批培养,在不同的氮磷条件下研究其生长、光合荧光及毒素含量的变化。结果表明:正磷酸盐浓度不变时,铵氮浓度的改变对铜绿微囊藻的生长有明显影响。叶绿素a(Chl.a)含量在铵氮浓度为1.83-18.3mg/L时明显较大;微囊藻毒素(包括MC-LR和MC-RR)的含量在铵氮浓度为1.83mg/L时达到最大;当铵氮浓度为0-1.83mg/L时,随着铵氮浓度升高,可变荧光FV和MC的产量均增大,同时MC异构体的种类增多;铵氮浓度过大对M.aeruginosa的生长、生理和产毒均有抑制作用。在另一组实验中,即铵氮浓度不变而正磷酸盐浓度增大时,Chl.a含量呈总体下降的趋势,并且与FV/Fm呈显著正相关关系(P<0.01,r=0.97),MC(MC-LR和MC-RR)的含量在正磷酸盐浓度小于0.56mg/L时明显升高,MC-LR与FV/Fm呈显著正相关关系(P<0.01,r=0.967)。       

黑暗条件下不同氮源对铜绿微囊藻(Microcystis aeruginosa)生长和pH的影响

在黑暗条件下,利用不同形态的氮源(硝酸盐氮,氨氮,有机氮和硝酸盐氮,有机氮)培养蓝藻水华优势种铜绿微囊藻(Microcystis aeruginosa),分析其氮代谢和对水体pH的影响.研究结果表明,在不同氮源的培养液中铜绿微囊藻密度在最初的24 h内出现波动,之后下降.培养液中pH值在试验最初的24 h显著下降,之后趋于稳定,在硝态氮培养液中pH值下降最大,从8.18下降到7.19,其反硝化作用产生的NO-2浓度也最大.不同氮源培养液中总氮含量都有所下降,以混合氮源培养液中总氮减少量最大,说明化合态氮经过反硝化作用生成了氮气并溢出培养液,因此,在黑夜条件下藻华水体中存在反硝化作用.     

Buoyant density changes in the cyanobacterium Microcystis aeruginosa due to changes in the cellular carbohydrate content

Abstract The cyanobacterium Microcystis aeruginosa was grown in light-limited chemostat cultures with various light—dark rhythms providing a total periodicity length of 24 h. The buoyant density of the cells changed in parallel with the carbohydrate content. Short incubation experiments with different light intensities, and experiments with the inhibitors iodoacetic acid and arsenate, showed that the buoyant density changes were due to variations in the cellular carbohydrate content. It seems likely that the low dark-growth yield on carbohydrate, which had been stored during the light period, served to facilitate buoyancy changes.     

Mechanism of photosynthetic response in Microcystis aeruginosa PCC7806 to low inorganic phosphorus

Photosynthetic response of Microcystis aeruginosa PCC7806 to different concentrations of phosphorus supply was studied so as to elucidate if the declining process of Microcystis bloom under freshwater ecosystem is related to soluble reactive phosphorus (SRP) decrease in water volume. Growth rate of M. aeruginosa PCC7806 was significantly reduced under P-deficient conditions, and its photosynthetic activity in terms of rETR_max (maximum electron transport rate) decreased significantly after 48 h growth, while it kept elevating and reached to a relative stable value when supplied with rich phosphorus of 0.6 mg/L. With the increasing actinic irradiance along the rapid light curves of M. aeruginosa PCC7806 cultured under low-phosphorus level, qP (photochemical quenching) and rETR (relative electron transport rate) decreased greatly, and the increase in qN (non-photochemical quenching) and Φ_PS (actual photochemical efficiency of PSII) was obviously inhibited. The affinity of M. aeruginosa PCC7806 to inorganic carbon was reduced evidently in 0.02 mg/L P compared with in 0.6 mg/L P. When P was reduced from 0.6 to 0.02 mg/L, the decreasing rate of rETR_max (77%) was significantly greater than that of photosynthetic carbon assimilation (22%), which indicated that down-regulation of CO₂ affinity caused by P-deficiency was, but not the only reason that resulted in the decline of photosynthetic efficiency. Instantaneous low-temperature significantly limited rETR_max under rich-P condition but had no effect on it when P was insufficient, and 1% ethanol could enhance rETR_max at low-P level but did not influence it at rich-P level. These two results proved that the decrease in thylakoid membrane fluidity caused by P-deficiency was another important reason that results in the decline of photosynthetic efficiency of M. aruginosa PCC7806.     

Growth and photosynthetic response of the cyanobacterium Microcystis aeruginosa in relation to photoperiodicity and irradiance

Growth and photosynthetic characteristics, P _max (maximum light-saturated oxygen production rate) and (photosynthetic affinity), of Microcystis aeruginosa were studied in continuous cultures under a range of photoperiod lengths and growth irradiances. Microcystis showed a low specific maintenance rate constant and a high growth affinity for light (typical cyanobacterial features), but required a dark period to obtain maximum growth rate. P _max and per unit dry weight increased, as did pigment content, when less light became available. By regulation in and P _max (crucial in light-limiting and high-light conditions, respectively) this buoyant species can flourish in low light, but also in high-light environments which may arise when buoyancy is lost.The two different types of light conditions affected growth, and photosynthesis, in different ways. One needs thus to discriminate between photoperiod- and irradiance-limitation, which restricts the utility of simple algal growth models. It was emphasized that photosynthetic adaptation patterns of light-limited species may resemble short-term nutrient uptake kinetics of nutrient-limited organisms.With prior knowledge of the growth limitation, we were able to assess the growth rate of a natural population of Microcystis from its photosynthetic response and from data of laboratory cultures of a known physiological state.     

Intraspecific variability in response to phosphorus depleted conditions in the cyanobacteria Microcystis aeruginosa and Raphidiopsis raciborskii

Phosphorus loading plays an important role in the occurrence of cyanobacterial blooms and understanding how this nutrient affects the physiology of cyanobacteria is imperative to manage these phenomena. Microcystis aeruginosa and Raphidiopsis raciborskii are cyanobacterial species that form potentially toxic blooms in freshwater ecosystems worldwide. Blooms comprise numerous strains with high trait variability, which can contribute to the widespread distribution of these species. Here, we explored the intraspecific variability in response to phosphorus depleted conditions (P-) testing five strains of each species. Strains could be differentiated by cell volume or genetic profiles except for those of the same species, sampling location and date, though these presented differences in their response to (P-). Although differently affected by (P-) over 10 days, all strains were able to grow and maintain photosynthetic activity. For most M. aeruginosa and R. raciborskii strains growth rates were not significantly different comparing (P+) and (P-) conditions. After ten days in (P-), only one M. aeruginosa strain and two R. raciborskii strains showed reduction in biovolume yield as compared to (P+) but in most strains chlorophyll-a concentrations were lower in (P-) than in (P+). Reduced photosystem II efficiency was found for only one R. raciborskii strain while all M. aeruginosa strains were affected. Only two M. aeruginosa and one R. raciborskii strain increased alkaline phosphatase activity under (P-) as compared to (P+). Variation in P-uptake was also observed but comparison among strains yielded homogeneous groups comprised of representatives of both species. Comparing the response of each species as a whole, the (P-) condition affected growth rate, biovolume yield and chlorophyll yield. However, these parameters revealed variation among strains of the same species to the extent that differences between M. aeruginosa and R. raciborskii were not significant. Taken together, these results do not support the idea that R. raciborskii, as a species, can withstand phosphorus limitation better than M. aeruginosa and also point that the level of intraspecific variation may preclude generalizations based on studies that use only one or few strains.     

黑暗限气条件下铜绿微囊藻细胞死亡的形态结构和生理生化变化

【目的】研究铜绿微囊藻细胞死亡过程中形态和生理生化变化,探讨蓝藻细胞死亡机制。【方法】通过黑暗限气处理模拟水华爆发后期水体环境,在处理后不同时间取样,对藻液的OD值,溶氧含量和pH值进行监测,使用透射电镜对细胞形态结构变化进行观察,通过胱天蛋白酶(Cysteine-dependent aspartate specificprotease,Caspase)活性检测、活性氧含量测定、末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记(Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling,TUNEL)染色和琼脂糖凝胶电泳对处理后藻细胞的死亡生理进行研究。【结果】黑暗限气处理后,藻培养液pH值和溶解氧含量下降,处理12 h后藻液开始变黄,48 h后藻细胞全部死亡。电镜观察结果表明,藻细胞在黑暗限气处理所导致的死亡过程中出现空泡和类囊体、核糖体等内部结构解体但细胞壁仍保持完整等现象。活性氧含量和caspase活性检测表明,在藻细胞死亡过程中活性氧含量和caspase活性上升。TUNEL染色和琼脂糖凝胶电泳分析发现,藻细胞在死亡过程中DNA发生断裂和降解。【结论】铜绿微囊藻细胞在黑暗和限气处理中表现出和真核生物细胞程序性死亡相类似的死亡特征,这说明细胞死亡机制是保守的,原核细胞和真核细胞一样具有程序性死亡机制。     

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a complex response to changes in nitrogen supply

The extent to which individual plants utilise nitrate and ammonium, the two principal nitrogen sources in the rhizosphere, is variable and many species require a balance between the two forms for optimal growth. The effects of nitrate and ammonium on gene expression, enzyme activity and metabolite composition have been documented extensively with the aim of understanding the way in which plant cells respond to the different forms of nitrogen, but ultimately the impact of these changes on the organisation and operation of the central metabolic network can only be addressed by analysing the fluxes supported by the network. Accordingly steady‐state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown in Murashige and Skoog and ammonium‐free media, treatments that influenced growth and biomass composition. Fluxes through the central metabolic network were deduced from the redistribution of label into metabolic intermediates and end products observed when cells were labelled with [1‐¹³C]‐, [2‐¹³C]‐ or [¹³C₆]glucose, in tandem with ¹⁴C‐measurements of the net accumulation of biomass. Analysis of the flux maps showed that: (i) flux through the oxidative pentose phosphate pathway varied independently of the reductant demand for biosynthesis, (ii) non‐plastidic processes made a significant and variable contribution to the provision of reducing power for the plastid, and (iii) the inclusion of ammonium in the growth medium increased cell maintenance costs, in agreement with the futile cycling model of ammonium toxicity. These conclusions highlight the complexity of the metabolic response to a change in nitrogen nutrition.     

Optical characteristics of the harmful dinoflagellate Alexandrium tamarense in response to different nitrogen sources

The chlorophyll a specific absorption coefficient a_ph^* and absorption ratio (a_ph ratio) of Alexandrium tamarense at five concentrations of nitrate, ammonium and urea over a range from 6 to 100 μM were examined. The experimental results were compared to two coastal diatom species and a prymnesiophyte to identify differences in absorption ratios. Cells exposed to increasing nitrate concentrations were characterized by an increase in a_ph^* at 443, 490, 510, 555 and 675 nm. However, ammonium and urea induced low a_ph^* values at their lowest and highest concentrations. The a_ph relative to 510 or 555 nm was constant regardless of the concentration of the N source, but dependent on the N source. Oxidized N induced a lower a_ph ratio than the reduced form. Comparisons of the a_ph ratio among taxonomic groups revealed significant differences. The a_ph ratio of A. tamarense was 20–30 and >50% lower than those of two diatoms and a prymnesiophyte, respectively. The a_ph ratio of the present study could assist in increasing the capability for detecting harmful species such as A. tamarense.     

Morphological and physiological changes in Microcystis aeruginosa as a result of interactions with heterotrophic bacteria

1. To reveal the role of aquatic heterotrophic bacteria in the process of development of Microcystis blooms in natural waters, we cocultured unicellular Microcystis aeruginosa with a natural Microcystis‐associated heterotrophic bacterial community. 2. Unicellular M. aeruginosa at different initial cell densities aggregated into colonies in the presence of heterotrophic bacteria, while axenic Microcystis continued to grow as single cells. The specific growth rate, the chl a content, the maximum electron transport rate (ETR_max) and the synthesis and secretion of extracellular polysaccharide (EPS) were higher in non‐axenic M. aeruginosa than in axenic M. aeruginosa after cell aggregation, whereas axenic and non‐axenic M. aeruginosa displayed the same physiological characteristic before aggregation. 3. Heterotrophic bacterial community composition was analysed by PCR–denaturing gradient gel electrophoresis (PCR–DGGE) fingerprinting. The biomass of heterotrophic bacteria strongly increased in the coinoculated cultures, but the DGGE banding patterns in coinoculated cultures were distinctly dissimilar to those in control cultures with only heterotrophic bacteria. Sequencing of DGGE bands suggested that Porphyrobacter, Flavobacteriaceae and one uncultured bacterium could be specialist bacteria responsible for the aggregation of M. aeruginosa. 4. The production of EPS in non‐axenic M. aeruginosa created microenvironments that probably served to link both cyanobacterial cells and their associated bacterial cells into mutually beneficial colonies. Microcystis colony formation facilitates the maintenance of high biomass for a long time, and the growth of heterotrophic bacteria was enhanced by EPS secretion from M. aeruginosa. 5. The results from our study suggest that natural heterotrophic bacterial communities have a role in the development of Microcystis blooms in natural waters. The mechanisms behind the changes of the bacterial community and interaction between cyanobacteria and heterotrophic bacteria need further investigations.     

Changes in extracellular polysaccharide content and morphology of Microcystis aeruginosa at different specific growth rates

The cyanobacterium Microcystis mainly exists in colonies under natural conditions but as single cells in typical laboratory cultures. Understanding the mechanism by which single cells form small and large colonies can provide a deeper insight into the life history of Microcystis and the mechanisms of Microcystis bloom formation. In this paper, Microcystis aeruginosa cultured under varying light intensities and temperatures exhibited different specific growth rates. Correlations were found between the specific growth rate, extracellular polysaccharide (EPS) content, and morphology of M. aeruginosa. Under low light intensities and temperatures, M. aeruginosa formed small colonies (maximum colony size approximately 100 μm) and exhibited low specific growth rates. By contrast, standard culture conditions yielded single or paired cells with high specific growth rates. Moreover, the EPS content decreased dramatically with increasing specific growth rate. A significant positive linear relationship was observed between the EPS content per cell and colony size. High EPS content and colony formation were associated with low specific growth rates. The specific growth rate in laboratory cultures was higher than the in situ growth rate under natural conditions. This result may explain why Microcystis normally exists as single cells or (more rarely) as paired cells in axenic laboratory cultures after long-term cultivation, but forms colonies under natural conditions.     

Investigations into nitrogen sources and supply in reclaimed colliery spoil

Summary Two trials were established to investigate the supply of nitrogen from ammonium and nitrate fertilizers, slow release nitrogen fertilizers, an organic nitrogen fertilizer and a legume, at two phosphate levels, to eight grass cultivars on colliery spoil. Spoil nitrogen supply and chemical characteristics and herbage dry matter and nitrogen yields were monitored for up to seven years. pH and conductivity fell at both sites. pH trends appeared to be independent of nitrogen treatment. Nitrogen in the ammonium form gave better yields than in the nitrate form when 125 kg N ha^–1 was supplied in a season but there was no difference when 62.5 kg N ha^–1 was applied. Slow release forms of nitrogen gave better yields more evenly distributed over the season than one application of ammonium sulphate per season. Once established white clover (Trifolium repens) plots had a more consistent nitrogen supply, more evenly distributed yield and better quality herbage than nitrogen fertilizer plots.Lolium perenne yielded poorly at low fertility.Festuca rubra andAgrostis castellana, although establishing slowly, yielded well under high and low fertility.