Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.     

Membrane effects of toxins isolated from a cyanobacterium, Cylindrospermopsis raciborskii, on identified molluscan neurones

The effect of anatoxin (ANTX), the crude extract (AlgTX) and purified fraction (F1) isolated from cyanobacterium C. raciborskii was studied on the neurones of two snail species. ANTX and AlgTX exerted excitatory, inhibitory and biphasic effects on the spontaneous activity of identified neurones. Both ANTX and AlgTX elicited an inward current, which could be decreased by curare or amiloride. On the contrary, F1 had no direct effect on the spontaneous activity; it was not able to induce conductance changes of the neuronal membrane, but it did antagonise the acetylcholine (ACh)-induced inward current. We concluded that ANTX affects the neuronal membrane of neurones acting on ACh receptors. The AlgTX had similar effects, and therefore the extract of C. raciborskii may contain an ANTX-like component. The purified fraction prolonged and decreased the ACh-elicited response, but had no direct membrane effect. We suggest, therefore, that both AlgTX and the purified fraction F1 interact with the ACh receptor, but they have different binding sites on the neuronal ACh receptor-ion channel complex. The possible neurotoxic effects of the C. raciborskii extract and F1 are demonstrated for the first time; the molecular mechanism of their action, however, remains to be elucidated.     

The cyanobacterium Cylindrospermopsis raciborskii is facilitated by copepod selective grazing

Blooms of the toxin-producing cyanobacterium Cylindrospermopsis raciborskii occur in tropical and subtropical lakes during spring-summer but the mechanisms behind bloom formation are unclear. This study tests the hypothesis that C. raciborskii accumulations in freshwater systems are facilitated by selective copepod grazing. Prey selection was examined in a series of experiments with C. raciborskii and the green alga, Chlamydomonas reinhardtii, as well as within natural phytoplankton assemblages. Clearance rates of the copepod Boeckella sp. on a C. raciborskii diet were 2–4 times lower than that of a common cladoceran Ceriodaphnia sp. when both grazers had prey choice. More C. raciborskii was cleared by Boeckella sp. when in mixed natural phytoplankton assemblages, but the clearance rate declined when nutrient replete C. reinhardtii was added, demonstrating that when alternate “high quality” algae were present, so did C. raciborskii consumption. The clearance rates of Boeckella sp. on two toxic C. raciborskii strains were significantly lower than on a non-toxic strain, and on C. raciborskii with low cellular P content. When we tested the grazing preference of a copepod dominated mixed zooplankton community on C. raciborskii during the early bloom period, clearance rates were relatively low (0.05–0.20 ml individual⁻¹ h⁻¹), and decreased significantly as the proportion of C. raciborskii increased above 5%. These results suggest that C. raciborskii persistence could be promoted by copepods preferentially grazing on other algae, with significant loss of top-down control as C. raciborskii abundance increases.     

Interspecific allelopathy in cyanobacteria: Cylindrospermopsin and Cylindrospermopsis raciborskii effect on the growth and metabolism of Microcystis aeruginosa

The biological role of cyanobacteria secondary metabolites is relatively unknown although several possible hypotheses have been discussed. In the following study the effect of cylindrospermopsin (CYN) and metabolites of non-CYN producing Cylindrospermopsis raciborskii strain on growth, alkaline phosphatase (ALP) activity and microcystin-LR (MC-LR) production in Microcystis aeruginosa was evaluated. Higher concentrations of CYN (10 and 50 μg L⁻¹) induced toxicity effects demonstrated by significant growth inhibition and M. aeruginosa cell necrosis. Lower concentrations of CYN (1 and 5 μg L⁻¹) slightly decreased growth rates but significantly up-regulated ALP activity. Moreover, under all studied CYN concentrations MC-LR production strongly decreased. Spent C. raciborskii medium mimicked the CYN action by inducing strong inhibition of M. aeruginosa growth and MC-LR production and through up-regulation of ALP activity. On the other hand, spent M. aeruginosa medium did not affect C. raciborskii growth and no alterations in ALP activity were observed. Co-culturing of these two species resulted in an increase of C. raciborskii contribution at the expense of M. aeruginosa. From the results we conclude that CYN can be involved in interspecific competition in cyanobacteria and that non-CYN producing C. raciborskii strains may produce a hitherto unknown bioactive compound(s) which can mimic CYN action.     

Growth and temperature‐related phenotypic plasticity in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is an invasive and potentially toxic cyanobacterium, which has recently spread worldwide, mainly because of its tolerance to a wide range of climatic conditions. C. raciborskii is able to change several traits in response to environmental changes and its morphology is also affected by these changes (especially in nutrients). We also expected temperature to affect the morphology of this cyanobacterium. We examined the growth and morphology of C. raciborskii at different temperatures and compared laboratory results to the morphology of this cyanobacterium in situ. As expected, growth rates increased with temperature. In addition, a high carrying capacity at 32°C suggests that this cyanobacterium is able to form more dense blooms at high temperatures. Fragile trichomes and low growth rates were observed at 12°C. An increase in the growth rate related to temperature resulted in a decrease in trichome length, with shorter trichomes at 32°C. The same pattern was observed in wild populations of C. raciborskii in a tropical reservoir, where shorter trichomes were observed in warmer months, when biomass was highest. This species' high ability to adapt to different environmental conditions throughout the year (i.e., nutrients, temperature) may have provided it with an additional advantage to increase its perennial blooms, mainly in tropical regions.     

Growth optimization of the invasive cyanobacterium Cylindrospermopsis raciborskii in response to phosphate fluctuations

The role of ecophysiological traits in the success and expansion of the toxic cyanobacterium Cylindrospermopsis raciborskii is still under debate. One key factor appears to be the high physiological flexibility of this organism when obtaining limiting resources. Recent studies have found that filamentous bloom-forming cyanobacteria are able to optimize their growth by adjusting phosphate uptake during fluctuating nutrient conditions. We investigated the growth response of two phosphate-deficient C. raciborskii isolates (MVCC19 from Uruguay and CCMP1973 from USA) to short-term fluctuations in phosphate supply. These isolates were exposed to five phosphate concentrations which were provided in two supply modes: a single pulse (SingleP) versus the same amount divided in 10 pulses (TenP), with one pulse applied every 6 min. Morphological traits and changes in chlorophyll a and phycocyanin fluorescence were also evaluated. Growth rates of CCMP1973 and MVCC19 almost doubled and tripled, respectively, when exposed to multiple rather than single phosphate additions. Different growth rates were observed with the same total added resource, thus contradicting the classical model of dependence of growth rate on absolute external concentration. Phosphate-deficient C. raciborskii showed a remarkable physiological flexibility in adapting to phosphate availability on a timescale from minutes to hours. The TenP mode provided an extension of phosphate exposure time that allowed the energetic optimization of uptake and growth. The morphological plasticity of the species in response to phosphate supply mode was also shown by differences in trichome length and individual size. Our results are the first evidence of growth optimization of phosphate-deficient C. raciborskii to short-term nutrient fluctuations, revealing its physiological flexibility. This adaptive behaviour may help to explain the invasive success of this diazotrophic cyanobacterium in a wide range of aquatic ecosystems where phosphorus is frequently the limiting resource.     

Effects of light history on primary productivity in a phytoplankton community dominated by the toxic cyanobacterium Cylindrospermopsis raciborskii

1. The effects of instantaneous irradiance and short‐term light history on primary production were determined for samples from a subtropical water reservoir dominated by the toxic cyanobacterium Cylindrospermopsis raciborskii. ¹⁴C‐bicarbonate uptake incubations were conducted on water samples from the reservoir, for irradiance (photosynthetically active radiation) ranging from 0 to 1654 μmol quanta m⁻² s⁻¹. Prior to the ¹⁴C incubations, cells were pre‐treated at irradiance levels ranging from 0 to 1006 μmol quanta m⁻² s⁻¹. 2. The average irradiance experienced by cells during the 2–2.5 h pre‐treatment incubations affected the productivity–irradiance (P–I) parameters: exposure to high light in pre‐treatment conditions caused a substantial decrease in maximum rate of primary production P_max and the photoinhibition parameter β when compared to cells pre‐treated in the dark. 3. While the data collected in this study were not sufficient to develop a full dynamic model of C. raciborskii productivity, P_max and β were modelled as a function of pre‐treatment irradiance, and these models were applied to predict the rate of primary production as a function of both instantaneous and historical irradiance. The results indicated that while cells with a history of exposure to high irradiance will be the most productive in high irradiance, production rates will be highest overall for dark‐acclimated cells in moderate irradiance. 4. Our results may explain why optically‐deep mixing favours C. raciborskii. If the mixing depth z_m exceeds the euphotic depth z_eu, cells will be dark‐acclimated, which will increase their rate of production when they are circulated through the euphotic zone. These results also predict that production rates will be higher during morning hours than for the same irradiance in the afternoon, which is consistent with other phytoplankton studies. 5. Since the rate of production of C. raciborskii‐dominated systems cannot be described by a single P–I curve, accurate estimates of production rates will require measurements over the daily light cycle.     

The tolerance of cyanobacterium Cylindrospermopsis raciborskii to low-temperature photo-inhibition affected by the induction of polyunsaturated fatty acid synthesis

Acyl-lipid desaturation introduces double bonds (unsaturated bonds) at specifically defined positions of fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. Desaturation patterns of the glycerolipids of Cylindrospermopsis raciborskii, a filamentous cyanobacterium, were determined in cells grown at 35 degrees C and 25 degrees C. The lowering of the growth temperature from 35 degrees C to 25 degrees C resulted in a considerable accumulation of polyunsaturated octadecanoic fatty acids in all lipid classes. The tolerance to low-temperature photo-inhibition of the C. raciborskii cells grown at 25 degrees C and 35 degrees C was also compared. The lower growth temperature increased the tolerance of C. raciborskii cells. These results strengthen the importance of polyunsaturated glycerolipids in the tolerance to environmental stresses and may give a physiological explanation for the determinative role of C. raciborskii in algal blooming in Lake Balaton (Hungary).     

Elevated CO2 causes changes in the photosynthetic apparatus of a toxic cyanobacterium,Cylindrospermopsis raciborskii

We studied the physiological acclimation of growth, photosynthesis and CO₂-concentrating mechanism (CCM) in Cylindrospermopsis raciborskii exposed to low (present day; L-CO₂) and high (1300 ppm; H-CO₂) pCO₂. Results showed that under H-CO₂ the cell specific division rate (μ_c) was higher and the CO₂- and light-saturated photosynthetic rates (V_max and P_max) doubled. The cells’ photosynthetic affinity for CO₂ (K_0.5CO₂) was halved compared to L-CO₂ cultures. However, no significant differences were found in dark respiration rates (R_d), pigment composition and light harvesting efficiency (α). In H-CO₂ cells, non-photochemical quenching (NPQ), associated with state transitions of the electron transport chain (ETC), was negligible. Simultaneously, a reorganisation of PSII features including antenna connectivity (J_conPSIIα), heterogeneity (PSIIα/β) and effective absorption cross sectional area (σPSIIα/β) was observed. In relation to different activities of the CCM, our findings suggest that for cells grown under H-CO₂: (1) there is down-regulation of CCM activity; (2) the ability of cells to use the harvested light energy is altered; (3) the occurrence of state transitions is likely to be associated with changes of electron flow (cyclic vs linear) through the ETC; (4) changes in PSII characteristics are important in regulating state transitions.     

Nitrogen fixation by the diazotroph Cylindrospermopsis raciborskii (Cyanophyceae)

Nitrogen fixation has been proposed as a mechanism that allows the diazotrophic cyanobacterium, Cylindrospermopsis raciborskii, to bloom in nitrogen‐limited freshwater systems. However, it is unclear whether dinitrogen fixation (N₂ fixation) can supplement available dissolved inorganic nitrogen (DIN) for growth, or only provides minimum nitrogen (N) for cell maintenance under DIN deplete conditions. Additionally, the rate at which cells can switch between DIN use and N₂ fixation is unknown. This study investigated N₂ fixation under a range of nitrate concentrations. Cultures were grown with pretreatments of nitrate replete (single dose 941 μmol  · L^?1) and N‐free conditions and then either received a single dose of 941 μmol  · L^?1 (N941), 118 μmol  · L^?1 (N118) or 0 N. Heterocysts appeared from days 3 to 5 when treatments of high were transferred to N free media (N941:N0), and from day 5 in N941 transferred to N118 treatments. Conversely, transferring cells from N0 to N941 resulted in heterocysts being discarded from day 3 and day 5 for N0:N118. Heterocyst appearance correlated with a detectable rate of N₂ fixation and up‐regulation of nifH gene expression, the discard of heterocysts occurred after sequential reduction of nifH expression and N₂ fixation. Nitrate uptake rates were not affected by pretreatment, suggesting no regulation or saturation of this uptake pathway. These data demonstrate that for C. raciborskii, N₂ fixation is regulated by the production or discard of heterocysts. In conclusion, this study has shown that N₂ fixation only provides enough N to support relatively low growth under N‐limited conditions, and does not supplement available nitrate to increase growth rates.     

Growth and phosphate uptake kinetics of the cyanobacterium,Cylindrospermopsis raciborskii (Cyanophyceae) in throughflow cultures

• 1 Cylindrospermopsis raciborskii occupies a rapidly expanding geographical area. Its invasive success challenges eutrophication control in many lakes. To understand better the load‐dependent behaviour of this nitrogen fixing cyanobacterium under in situ conditions, we studied P‐dependent growth of a C. raciborskii strain under continuous and pulsed P supply.
• 2 The Droop model reasonably described P‐dependent growth in the continuously supplied chemostats. Large P pulses, however, caused a delay in growth and cells subject to P pulses grew slower than their counterparts with the same P quota supplied continuously.
• 3 The kinetics of P uptake indicated that C. raciborskii is opportunistic with respect to P. Its high excess P storage capacity after a saturating P pulse (Q_ex=95 µg P [mg C]^‐1) and P‐specific uptake capacity (U_max = V_max/Q_P=150–1200) are indicative of storage adaptation. At the same time, the affinity of the P uptake system (U_max/K = 800–4000) is also high.
• 4 Rate of leakage exceeded that of the steady state net P uptake by one to two orders of magnitude. Growth affinity of C. raciborskii (µ_max/K_µ≈ 20) was relatively low, presumably due to the substantial leakage.
• 5 The dynamics of the particular water body determine which trait contributes most to competitive success of C. raciborskii. In deep lakes with vertical nutrient gradients, the cyanobacterium may rely primarily on its high P storage capacity, which is coupled to a lack of short‐term feedback inhibition and efficient buoyancy regulation. In lakes without such gradients, high P uptake affinity may be vitally important.

     

Palaeolimnological evidence for the independent evolution of neighbouring terminal lakes,the Murray Darling Basin,Australia

Phytoplankton composition and production are highly unpredictable within an estuary, due to the high variability of forcing factors, such as freshwater flow, salinity, nutrients and light. The Guadiana estuary has shown sharp inter-annual differences in freshwater flow, related to variable precipitation, which is expected to affect nutrient loadings, light availability and phytoplankton succession. Water retention due to dam construction will further enhance changes in river flow and ecosystem dynamics. The main goal of the present study was to describe and relate phytoplankton succession and environmental conditions, namely nutrients and light, in the Guadiana upper estuary (south-western Iberian Peninsula), a dam regulated temperate estuary. From March 2004 to October 2005, water samples were collected in three stations along a longitudinal transect covering the upper estuary. Several water variables were determined and phytoplankton composition was studied through inverted and epifluorescence microscopy. A typical freshwater phytoplankton succession was observed, from a diatom spring bloom to cyanobacteria dominance in the summer, and a second diatom bloom in the autumn. Neither nutrients nor light availability seemed to be related to the observed succession, especially the seasonal variation of diatom abundance. During summer, nutrient concentrations (especially Si) were high and non-limiting, whilst light was available in the mixing layer. However, diatoms were present in low numbers. Grazing pressure was probably responsible for the regulation of diatom seasonal succession in the Guadiana upper estuary, which should be addressed in future studies. Handling editor: K. Martens     

Effects of saxitoxin- and non-saxitoxin-producing strains of the cyanobacterium Cylindrospermopsis raciborskii on the fitness of temperate and tropical cladocerans

Life-table experiments were performed with two strains of Cylindrospermopsis raciborskii, a saxitoxin-producing strain (T3) and a non-saxitoxin-producing strain (NPLP-1) combined with the green algae Ankistrodesmus falcatus, aiming to detect effects on the reproduction of three cladocerans species: Daphnia pulex, Daphnia gessneri and Moina micrura. Survivorship, age at first reproduction, clutch size and population growth rate during 12–15 days were recorded. Cladocerans showed different susceptibility to saxitoxin-producing strain (T3), with D. pulex and M. micrura being negatively affected, followed by and D. gessneri, which did not show any decrease in fitness and performed even better than the controls with this strain. A reverse response was found in experiments with the non-saxitoxin-producing strain (NPLP-1): while D. pulex and M. micrura had an increase, D. gessneri showed a significant decrease in fitness, suggesting that this strain may produce some bioactive compound. The contrasting responses of the cladoceran species to saxitoxins and non-saxitoxin-producing strains of C. raciborskii suggest that the presence of this cyanobacterium may be a selective factor in determining the composition of zooplankton communities.     

Characterization of a model cyanobacterium Synechocystis sp. PCC 6803 autotrophic growth in a flat‐panel photobioreactor

We characterized the photoautotrophic growth of glucose‐tolerant Synechocystis sp. PCC 6803 in a flat‐panel photobioreactor running on a semicontinuous regime under various lights, temperatures, and influx carbon dioxide concentrations. The maximum reached growth rate was 0.135 h^?1, which corresponds to a doubling time of 5.13 h—a growth speed never reported for Synechocystis before. Saturating red light intensity for the strain was 220–360 μmol(photons) m^?2 s^?1, and we did not observe any photoinhibition up to 660 μmol(photons) m^?2 s^?1. Synechocystis was able to grow under red light only; however, photons of wavelengths 405–585 and 670–700 nm further improved its growth. Optimal growth temperature was 35°C. Below 32°C, the growth rates decreased linearly with temperature coefficient (Q₁₀) 1.70. Semicontinuous cultivation is known to be efficient for growth characterization and optimization. However, the assumption of correct growth rates calculation—culture exponential growth—is often not fulfilled. The semicontinuous setup in this study was operated as a turbidostat. Accurate online OD measurements with high time‐resolution allowed fast and reliable growth rates determination. Repeating diluting frequencies (up to 18 dilutions per day) were essential for rapid growth stability evaluation. The presented setup provides improvement to previously published semicontinuous characterization strategies by decreasing experimental time requirements and maintaining the culture in exponential growth phase throughout the entire characterization procedure.     

Cylindrospermopsin production and release by the potentially invasive cyanobacterium Aphanizomenon ovalisporum under temperature and light gradients

The growth rates, production and release of the potent cytotoxin cylindrospermopsin (CYN) were studied in batch and semi-continuous cultures of Aphanizomenon ovalisporum (Cyanobacteria; Nostocaceae) strains UAM 289 and UAM 290 from Spain, over a gradient of temperatures (10–40 °C) and irradiances (15–340 μE m⁻² s⁻¹). This species grew in temperatures ranging from 15 °C to 35 °C as well as under all irradiances assayed. The growth rates ranged from 0.08 d⁻¹ to 0.35 d⁻¹, and the maximum growth was recorded above 30 °C and at 60 μE m⁻² s⁻¹. CYN was produced under all conditions where net growth occurred. Total CYN reached up to 6.4 μg mg⁻¹ dry weight, 2.4 μg mm⁻³ biovolume, 190.6 fg cell⁻¹ and 0.5 μg μg⁻¹ chlorophyll a. Although CYN concentrations varied only 1.9-fold within the 15–30 °C range, a drastic 25-fold decrease was observed at 35 °C. The irradiance induced up to 4-fold variations, with maximum total CYN measured at 60 μE m⁻² s⁻¹. An elevated extracellular CYN share ranging from 20% to 35% was observed during the exponential growth phase in most experiments, with extreme temperatures (15 and 35 °C) being related to the highest release (63% and 58%, respectively) and without remarkable influence of irradiance. Growth did not have a direct influence on either CYN production or release throughout the entire range of experimental conditions. Our study demonstrates a strong and stable production and release of CYN by A. ovalisporum along field-realistic gradients of temperature and light, thus becoming a predictive tool useful for the management of water bodies potentially affected by this ecologically plastic cyanobacterium.     

Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir

Cylindrospermopsis raciborskii, a tropical blooming species of cyanobacterium (blue-green alga), was isolated from the domestic water supply reservoir on Palm Island, a continental island off the tropical northeast coast of Australia. This species, not previously known to be toxic, was shown to be severely hepatotoxic for mice. The 50% lethal dose at 24 h after injection was found to be 64 +/- 5 mg of freeze-dried culture per kg of mouse. The principal lesion produced was centrilobular to massive hepatocyte necrosis, but various degrees of injury were also seen in the kidneys, adrenal glands, lungs, and intestine. The possible implication of this finding in relation to an incident of hepatoenteritis in humans living on the island is discussed.     

Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir.

Cylindrospermopsis raciborskii, a tropical blooming species of cyanobacterium (blue-green alga), was isolated from the domestic water supply reservoir on Palm Island, a continental island off the tropical northeast coast of Australia. This species, not previously known to be toxic, was shown to be severely hepatotoxic for mice. The 50% lethal dose at 24 h after injection was found to be 64 +/- 5 mg of freeze-dried culture per kg of mouse. The principal lesion produced was centrilobular to massive hepatocyte necrosis, but various degrees of injury were also seen in the kidneys, adrenal glands, lungs, and intestine. The possible implication of this finding in relation to an incident of hepatoenteritis in humans living on the island is discussed.     

Zooplankton community structure, micro-zooplankton grazing impact, and seston energy content in the St. Johns river system, Florida as influenced by the toxic cyanobacterium Cylindrospermopsis raciborskii

Zooplankton can influence the phytoplankton community through preferential grazing. In turn, nuisance cyanobacteria may affect zooplankton community structure by allowing certain species to out-compete others. We examined zooplankton-phytoplankton interactions, micro-zooplankton (< 200 m) grazing, and biochemical components of the seston in the St. Johns River System (SJR), Florida in the presence and absence of the toxin-producing cyanobacterium Cylindrospermopsis raciborskii. We tested whether this cyanobacterium would cause a decrease in the size structure of the zooplankton community and postulated a resultant decline in the metabolic energy and carbon available to higher consumers (i.e. fish). When numbers of C. raciborskii were low or undetectable, zooplankton were more diverse and were comprised of larger species. Rotifers were the dominant zooplankton, and their numbers relative to other zooplankton increased as C. raciborskii concentrations increased. Micro-zooplankton grazing was higher in times of C. raciborskii abundance, suggesting competitive and predatory exclusion by larger zooplankton in times of higher phytoplankton diversity. Total caloric content of the seston was higher in times of C. raciborskii abundance. However, essential fatty acids and phosphorus may be lacking in the seston, or nutrients may potentially be sequestered by the cyanobacteria and remain as organic matter in the water column. In such cases, higher trophic levels would not be able to obtain optimal energy requirements. Overall, there was a greater impact of micro-grazers on phytoplankton in the presence of C. raciborskii and apparent negative effects on the larger zooplankton species, suggesting a potential for changes in zooplankton and higher trophic level community structure.     

Random Forest model and TRIX used in combination to assess and diagnose the trophic status of Bizerte Lagoon,southern Mediterranean

A combined multimetric trophic index (TRIX) and the Random Forest (RF) model were used to characterize the trophic status of Bizerte Lagoon. The RF model was used to build a predictive model of chlorophyll a using physicochemical variables (nitrite, nitrate, ammonium, phosphate, oxygen, temperature and salinity) as predictors. The approach is based on physicochemical and biological parameters measured in samples collected twice weekly from January to December 2012 at one representative sampling station located at the lagoon center.The observed TRIX values vary from 5.18 to 6.12, reflecting waters ranging from moderate to poor quality with a high trophic level. The results of the RF model show that the most important predictor of chlorophyll a variation appears to be dissolved oxygen, followed by nitrate and temperature, suggesting a reduced model for this variable. The link between chlorophyll a and observed variables was also studied using numerical models, analyzing the linear statistical correlations and a recently proposed non linear model, the Random Forest. Both methods highlight a high correlation between chlorophyll a and dissolved oxygen as opposed to chlorophyll a and nitrate.     

Production of optically pure d-lactate from CO2 by blocking the PHB and acetate pathways and expressing d-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803

Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. d-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. PCC 6803 represents a promising host for biosynthesis of d-lactate from CO₂ as it only contains d-lactate dehydrogenase. The production of d-lactate from CO₂ by an engineered strain of Synechocystis sp. PCC 6803 with overexpressing d-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce d-lactate from CO₂. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetyl-CoA node, and introducing a more efficient d-lactate dehydrogenase into Synechocystis sp. PCC 6803. The engineered strain of Synechocystis sp. PCC 6803 was capable of producing 1.06 g/L of d-lactate from CO₂. This alternative strategy for the production of optically pure d-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO₂ by using engineered cyanobacteria.