Phylogenetic classification of heterotrophic bacteria associated with filamentous marine cyanobacteria in culture

Fifty-one heterotrophic bacterial strains were isolated from the marine cyanobacterial cultures of heterocystous Nodularia harveyana strain Bo53 and non-heterocystous Oscillatoria brevis strain Bo10. Fluorescence in situ hybridisation and fingerprinting methods were used for a preliminary taxonomical classification of 44 of the 51 isolates. The strains obtained from Bo53 were mostly Alphaproteobacteria (10/24), followed by Bacteroidetes (7/24), and Gammaproteobacteria (3/24). The affiliation of the isolates originating from Bo10 was dominated by Alphaproteobacteria (8/20) and Bacteroidetes (7/20), followed by Gammaproteobacteria (3/20). The 16S rRNA genes of four selected isolates were sequenced. A red-coloured bacterium from Bo53 grouped with the alphaproteobacterial genus Porphyrobacter, while the other three strains, obtained from Bo10, belonged to the alphaproteobacterial genera Roseobacter (pink) and Rhodobacter (colourless), and to the genus Muricauda (yellow) of Bacteroidetes. The findings indicated that the aerobic anoxygenic phototroph Porphyrobacter and its relatives only occurred in Bo10 culture, whereas members of the Roseobacter clade and the Bacteroidetes bacterium Muricauda sp. seemed to be more ubiquitous.     

Anatoxin-a synthetase gene cluster of the cyanobacterium Anabaena sp. strain 37 and molecular methods to detect potential producers

Cyanobacterial mass occurrences are common in fresh and brackish waters. They pose a threat to water users due to toxins frequently produced by the cyanobacterial species present. Anatoxin-a and homoanatoxin-a are neurotoxins synthesized by various cyanobacteria, e.g., Anabaena, Oscillatoria, and Aphanizomenon. The biosynthesis of these toxins and the genes involved in anatoxin production were recently described for Oscillatoria sp. strain PCC 6506 (A. Méjean et al., J. Am. Chem. Soc. 131:7512-7513, 2009). In this study, we identified the anatoxin synthetase gene cluster (anaA to anaG and orf1; 29 kb) in Anabaena sp. strain 37. The gene (81.6% to 89.2%) and amino acid (78.8% to 86.9%) sequences were highly similar to those of Oscillatoria sp. PCC 6506, while the organization of the genes differed. Molecular detection methods for potential anatoxin-a and homoanatoxin-a producers of the genera Anabaena, Aphanizomenon, and Oscillatoria were developed by designing primers to recognize the anaC gene. Anabaena and Oscillatoria anaC genes were specifically identified in several cyanobacterial strains by PCR. Restriction fragment length polymorphism (RFLP) analysis of the anaC amplicons enabled simultaneous identification of three producer genera: Anabaena, Oscillatoria, and Aphanizomenon. The molecular methods developed in this study revealed the presence of both Anabaena and Oscillatoria as potential anatoxin producers in Finnish fresh waters and the Baltic Sea; they could be applied for surveys of these neurotoxin producers in other aquatic environments.     

Microcystin production and ecological physiology of Caribbean black band disease cyanobacteria

Molecular studies of black band disease (BBD), a coral disease found on tropical and subtropical reefs worldwide, have shown that one 16S rRNA gene sequence is ubiquitous. This sequence has been reported to be a member of the cyanobacterial genus Oscillatoria. In this study, extracts of two cultured laboratory strains of BBD Oscillatoria, and for comparison two strains of BBD Geitlerinema, all isolated from reefs of the wider Caribbean, were analysed using Ultra-Performance Liquid Chromatography-Tandem Quad Mass Spectrometry (UPLC-MS/MS). The cyanotoxin microcystin-LR (MC-LR) was found in all strains, and one Geitlerinema strain additionally produced MC-YR. Growth experiments that monitored toxin production using enzyme-linked immunosorbent assay (ELISA) showed that BBD Oscillatoria produced yields of MC-LR equivalent (0.02-0.04 mg g(-1)) independent of biomass and culture conditions (varying temperature, pH, light and organic carbon). This pattern is different from BBD Geitlerinema, which increased production of MC-LR equivalent in the presence of organic carbon in the light and dark and at a relatively lower temperature. These results indicate that different species and strains of BBD cyanobacteria, which can occur in the same BBD infection, may contribute to BBD pathobiology by producing different toxins and different amounts of toxin at different stages in the disease process. This is the first detailed study of laboratory cultures of the ubiquitous BBD cyanobacterium Oscillatoria sp. isolated from Caribbean reefs.     

Isolation, culture and characterization of a new strain of Euglena gracilis

A new strain of Euglena gracilis Klebs has been isolated from a highly polluted river; it was named MAT. Strain growth in different culture media was evaluated under heterotrophic and autotrophic conditions. Total lipid, sugar, protein and chlorophyll a production were studied. Results obtained for MAT were compared with data obtained for a UTEX Culture Collection strain. Likewise, cells from both strains were bleached using streptomycin, and grown in the same media used for green samples. Both MAT and UTEX showed clear differences in their biochemical composition and growth rate depending on the media used. They also exhibited different growth patterns. E. gracilis medium proved to be the best culture environment for both strains either in autotrophic or heterotrophic conditions. Results show that basal contents of lipids, sugars, proteins and chlorophyll a vary depending on the strain, and thus values obtained for one strain do not apply to another. Moreover, strain origin may have an influence on the mechanisms of adaptation or defense developed by each sample.     

Characterization of trophic changes and a functional oxidative pentose phosphate pathway in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Cyanobacteria have a tremendous activity to adapt to environmental changes of their growth conditions. In this study, Synechocystis sp. PCC 6803 was used as a model organism to focus on the alternatives of cyanobacterial energy metabolism. Glucose oxidation in Synechocystis sp. PCC6803 was studied by inactivation of slr1843, encoding glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of the oxidative pentose phosphate pathway (OPPP). The resulting zwf strain was not capable of glucose supported heterotrophic growth. Growth under autotrophy and under mixotrophy was similar to that of the wild-type strain, even though oxygen evolution and uptake rates of the mutant were decreased in the presence of glucose. The organic acids citrate and succinate supported photoheterotrophic growth of both WT and zwf. Proteome analysis of soluble and membrane fractions allowed identification of four growth condition-dependent proteins, pentose-5-phosphate 3-epimerase (slr1622), inorganic pyrophosphatase (sll0807), hypothetical protein (slr2032) and ammonium/methylammonium permease (sll0108) revealing details of maintenance of the cellular carbon/nitrogen/phosphate balance under different modes of growth.     

Isolation of cyanobacteria from the aquatic fern,Azolla

A procedure has been developed to isolate cyanobacteria from the aquatic fern Azolla. The method is based upon recovery of cyanobacterial bundles from digests of plants and use of this material as a massive inoculum for nitrogen-free media, followed by prolonged incubation in light. The procedure appears to select for those cells capable of growth in vitro. Isolated cyanobacteria were found to resemble Anabaena sp. morphologically but were capable of heterotrophic growth and had high nitrogenase activity when grown on fructose in the dark.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo Ice Shelf, Antarctica

This study investigated the diversity of cyanobacterial mat communities of three meltwater ponds--Fresh, Orange and Salt Ponds, south of Bratina Island, McMurdo Ice Shelf, Antarctica. A combined morphological and genetic approach using clone libraries was used to investigate the influence of salinity on cyanobacterial diversity within these ecosystems without prior cultivation or isolation of cyanobacteria. We were able to identify 22 phylotypes belonging to Phormidium sp., Oscillatoria sp. and Lyngbya sp. In addition, we identified Antarctic Nostoc sp., Nodularia sp. and Anabaena sp. from the clone libraries. Fresh (17 phylotypes) and Orange (nine phylotypes) Ponds showed a similar diversity in contrast to that of the hypersaline Salt Pond (five phylotypes), where the diversity within cyanobacterial mats was reduced. Using the comparison of identified phylotypes with existing Antarctic sequence data, it was possible to gain further insight into the different levels of distribution of phylotypes identified in the investigated cyanobacterial mat communities of McMurdo Ice Shelf.     

Adaptation to sulfide and to the underwater light field in three cyanobacterial isolates from Lake Arcas (Spain)

Abstract: Three strains of cyanobacteria isolated from karstic Lake Arcas were tested for photosynthetic adaptations to soluble sulfide. One of them, AO11, was identified as Oscillatoria cf. ornata , and forms dense populations in the sulfide-rich anoxic hypolimnion of this lake. This cyanobacterium was able to perform sulfide-dependent anoxygenic photosynthesis and its oxygenic photosynthesis was relatively insensitive to sulfide. The other strains studied were AP1 and AO21, identified respectively as Pseudanabaena sp. and Oscillatoria cf. tenuis , populations of which were present only in epilimnetic waters at low population densities. Pseudanabaena sp. also carried out anoxygenic photosynthesis, but oxygenic photosynthesis was totally inhibited by 0.5 mM sulfide. Oscillatoria cf. tenuis lost most of its oxygenic photosynthetic capacity when submitted to 0.1 mM sulfide and anoxygenic photosynthesis accounted for less than 20% of sulfide-free controls. In addition to different photosynthetic capabilities, the three cyanobacteria exhibited differences in light-harvesting photosynthetic accessory pigments. Pigment analysis of cultures grown under different light conditions showed the capacity of Oscillatoria cf. ornata AO11 to produce phycoerythrin under low light intensity or under predominantly green light, while neither Pseudanabaena sp. AP1 nor Oscillatoria cf. tenuis AO21 produced this pigment. The complementary chromatic adaptation of Oscillatoria cf. ornata correlates well with its summertime distribution under the dim light field of the hypolimnion. The distribution and abundance of specific cyanobacterial populations in Lake Arcas can thus be explained by the interplay of light regime and presence of sulfide as some of the most determinant ecological parameters.     

The glycine decarboxylase complex is not essential for the cyanobacterium Synechocystis sp. strain PCC 6803

In order to investigate the metabolic importance of glycine decarboxylase (GDC) in cyanobacteria, mutants were generated defective in the genes encoding GDC subunits and the serine hydroxymethyl-transferase (SHMT). It was possible to mutate the genes for GDC subunits P, T, or H protein in the cyanobacterial model strain Synechocystis sp. PCC 6803, indicating that GDC is not necessary for cell viability under standard conditions. In contrast, the SHMT coding gene was found to be essential. Almost no changes in growth, pigmentation, or photosynthesis were detected in the GDC subunit mutants, regardless of whether or not they were cultivated at ambient or high CO2 concentrations. The mutation of GDC led to an increased glycine/serine ratio in the mutant cells. Furthermore, supplementation of the medium with low glycine concentrations was toxic for the mutants but not for wild type cells. Conditions stimulating photorespiration in plants, such as low CO2 concentrations, did not induce but decrease the expression of the GDC and SHMT genes in Synechocystis. It appears that, in contrast to heterotrophic bacteria and plants, GDC is dispensable for Synechocystis and possibly other cyanobacteria.     

The Effects of Cyanobacterial Exudates on Bacterial Growth and Biodegradation of Organic Contaminants

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.     

Partial characterization of extracellular polysaccharides from cyanobacteria

Four cyanobacterial strains, Cyanothece sp., Oscillatoria sp., Nostoc sp. and Nostoc carneum were studied for physico-chemical characterization of extracellular polysaccharide (EPS) secreted during the controlled growth condition. Hydrolyzed EPSs showed the compositional involvement of four sugar moieties viz. mannose, glucose, xylose and ribose in varying combinations. Infrared spectra of EPSs showed a specific absorbance of O-H stretching at 3448-3400 cm(-1), asymmetrical-symmetrical C-H stretching at 2924 and 2854 cm(-1) and a bending vibration of C-H at 1400-1380 cm(-1). Absorbance at 1259 and 1140 cm(-1) with Cyanothece sp. EPS, indicated the presence of sulfur containing functional group. Thermal gravimetric analysis and differential scanning calorimetric analysis confirmed the polysaccharides thermal stability as high as around 250 degrees C. In the presence of 0.1 M NaCl aqueous solution, the intrinsic viscosity of polysaccharides from Oscillatoria sp. and Nostoc sp. decreased 1.6 fold, whereas, 3-5 fold reduction in intrinsic viscosity was observed with commercially available guar and xanthan gum.     

The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin

In cyanobacteria, cytochrome c6 and plastocyanin are able to replace each other as redox carriers in the photosynthetic and respiratory electron transport chains with the synthesis of one or another protein being regulated by the copper concentration in the culture medium. However, the presence of a third unidentified electron carrier has been suggested. To address this point, we have constructed two deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803, each variant lacking either the petE or petJ gene, which respectively codes for the copper or heme protein. The photoautotrophic and heterotrophic growth rate of the two mutants in copper-free and copper-supplemented medium as well as their photosystem I reduction kinetics in vivo were compared with those of wild-type cells. The two mutant strains grow at equivalent rates and show similar in vivo photosystem I reduction kinetics as wild-type cells when cultured in media that allow the expression of just one of the two electron donor proteins, but their ability to grow and reduce photosystem I is much lower when neither cytochrome c6 nor plastocyanin is expressed. These findings indicate that the normal functioning of the cyanobacterial photosynthetic and respiratory chains obligatorily depends on the presence of either cytochrome c6 or plastocyanin.     

Biochemical characterization of cyanobacterial extracellular polymers (EPS) from modern marine stromatolites (Bahamas)

A range of biochemical characterizations were used to examine the extracellular polymeric secretions (EPS) of two cyanobacteria, Synechocystis sp. and Oscillatoria sp., isolated from marine stromatolites, Bahamas. Two-dimensional gel electrophoresis was successfully used to fractionate proteins in EPS. The results suggest that cyanobacterial EPS is composed of a network of macromolecules having different biochemical properties, which may contribute to extracellular functions.     

Chromium-resistant bacteria and cyanobacteria: impact on Cr(VI) reduction potential and plant growth

Two chromium-resistant bacterial strains, Bacillus cereus S-6 and Ochrobactrum intermedium CrT-1, and two cyanobacterial strains, Oscillatoria sp. and Synechocystis sp., were used in this study. At initial chromate concentrations of 300 and 600 microg K2CrO4 mL(-1), and an inoculum size of 9.6 x 10(7) cells mL(-1), B. cereus S-6 completely reduced Cr(VI), while O. intermedium CrT-1 reduced Cr(VI) by 98% and 70%, respectively after 96 h. At 100 microg K2CrO4 mL(-1), Synechocystis sp. MK(S) and Oscillatoria sp. BJ2 reduced 62.1% and 39.9% of Cr(VI), respectively, at 30 degrees C and pH 8. Application of hexavalent chromate salts adversely affected wheat seedling growth and anatomical characters. However, bacterial inoculation alleviated the toxic effects, as reflected by significant improvements in growth as well as anatomical parameters. Cyanobacterial strains also led to some enhancement of various growth parameters in wheat seedlings.     

Development and evaluation of an organically certifiable growth medium for cultivation of cyanobacteria

On-farm cultivation of phototrophic, N-fixing cyanobacteria, grown in raceway ponds, could provide organic farmers an alternative N source to meet crop fertilizer needs. The application of cyanobacterial fertilizer in certified organic agriculture requires a growth medium of certified organic ingredients. This study compared growth and N-fixation of cyanobacteria cultured in two media, Allen and Arnon (AA) and a growth medium of organic-approved ingredients (RB) developed by the authors. A xenic culture of Anabaena sp. was grown for 2 weeks in the laboratory. The RB medium had significantly lower concentrations of P, Fe, B, Zn, and Cu than the AA medium. Cyanobacteria grown in RB had significantly greater exponential growth rate but significantly lower net total Kjeldahl nitrogen (TKN) than those grown in AA. In a follow-up replicated field study, the xenic culture of Anabaena sp. was inoculated into raceways aerated by paddle wheels. There was no significant difference in exponential or linear growth rate between the two treatments though the RB medium had lower concentrations of P, Co, Zn, and B than the AA medium. The lack of difference could be the result of an overarching limiting factor evident in both treatments such as light or C depletion or that the lower nutrient concentrations in RB were still sufficient for growth and N-fixation. There was no difference in net TKN between the two treatments, suggesting similar rates of N-fixation. Since bone meal contributed trace amounts of N to the RB media, it is possible that maximal N-fixation was not achieved. However, RB medium was able to support growth similar to that of the AA medium in raceway cultivation.     

<Emphasis Type="Italic">Scenedesmus</Emphasis> sp. cultivation using commercial-grade ammonium sources

The use of commercial-grade nutrients such as agricultural fertilizers is important for commercial microalgae cultivation, and this is particularly the case for biofuel production which is associated with low added value. Nitrogen is a very important macronutrient in microalgae cultivation, and ammonium sources are cheaper than nitrate sources. However, the growth response and cellular composition can be altered by the different nutrient sources. In the study reported here, we investigated the effects of different ammonium doses and commercial-grade macronutrients from agricultural fertilizers on the growth of Scenedesmus sp. BR003, a promising genus for biofuel production. Five growth media were developed using fertilizers and evaluated during Scenedesmus sp. cultivation under autotrophic conditions. The growth media differed in terms of their composition and concentration of macronutrients. We found that all commercial-grade media supported equal or higher cell concentrations, dry weight, water-soluble proteins, neutral carbohydrates, and total lipid production compared to the conventional BG11 medium. However, the commercial-grade growth medium with the highest ammonium content affected the coenobium pattern of Scenedesmus sp. BR003. Commercial-grade nutrient sources were a low-cost alternative to improve the growth of Scenedesmus sp. BR003. The different fertilizers also allowed for manipulation of microalgae chemical composition and phenotypic plasticity to target traits of commercial interest. Our results demonstrate the potential of using ammonium from agricultural fertilizers as a nitrogen source in combination with other commercial-grade macronutrients sources. In addition, this work demonstrates the ability of a robust Scenedesmus strain to grow in media of different compositions, even when a high dosage of ammonium was used.     

Characteristics of Autotrophic and Heterotrophic Denitrification by the Strain Pseudomonas sp. H117

Strain H117 was isolated from the Tang Yu reservoir. Based on the phylogenetic characteristics, strain H117, which was identified as Pseudomonas sp. strain H117, had the capability to utilize bicarbonate and sodium acetate as a carbon source under anaerobic conditions. Furthermore, the strain could grow on both autotrophic and heterotrophic media, and could perform both autotrophic and heterotrophic denitrification in the medium. Response surface methodology analysis demonstrated that the maximum degradation ratio of nitrate-occurred under the following conditions in the autotrophic medium: initial pH of 6.00, C/N ratio of 4.68 and temperature of 31.33°C. The maximum degradation ratio of nitrate occurred under the following conditions in the heterotrophic medium: initial pH of 6.16, C/N ratio of 8.23 and temperature of 28.48°C. Finally, the denitrification performance of strain H117 was evaluated under the optimum conditions. These results suggest that strain H117 has potential applications for the bioremediation of polluted groundwater.     

A single cyanobacterial ribotype is associated with both red and black bands on diseased corals from Palau

Filamentous cyanobacteria forming red and black bands (black band disease, BBD) on 3 scleractinian corals from Palau were molecularly identified as belonging to a single ribotype. Red cyanobacterial mats sampled from infections on Pachyseris speciosa and a massive Porites sp. yielded red strains RMS1 and RMS2 respectively; the black cyanobacterial mat sampled from an infection on Montipora sp. yielded black strain BMS1. Following trials of a range of specialized media and culture conditions, 2 media, Grund and ASN-III, were identified as the best for successful isolation and culturing. Cultured cyanobacteria were examined under a light microscope to establish purity, color and morphological appearance. DNA extraction and partial sequencing of the 16S rDNA gene of both red and black cyanobacterial isolates demonstrated 100% sequence identity. These isolated strains were also found to have 99% sequence identity with an uncultured cyanobacterial strain previously identified by molecular techniques as belonging to a cyanobacterial ribotype associated with BBD-infected corals in the Caribbean. This is the first report of the successful isolation and culture of cyanobacterial strains derived from both red bands and BBD. Based on these findings, it is suggested that the classification of these 2 syndromes as separate coral diseases be postponed until further evidence is collected.     

beta-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria

In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against Oscillatoria amphibia NIES-361. One strain, C-979, identified as a Vibrio sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain. The purified very hydrophilic compound (16.4 mg) was determined to be beta-cyano-L-alanine (L-CNAla) by instrumental analyses and the application of the advanced Marfey method. L-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to L-CNAla at a concentration of 0.4 to 25 microg/ml. The effect of L-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed. CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance. The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria. This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium.     

Colony formations in a halotolerantBrevibacterium sp. JCM 6894 on solid medium with different pH values

Viable cells of a halotolerantBrevibacterium sp. JCM 6894 grown in a liquid medium with pH 7.1 were enumerated as the colony-forming cells on three kinds of agar media with different pH values. Unexpectedly they were lower at neutral pH rather than acidic or alkaline pH. This tendency was invariable regardless of the changes in the concentrations of nutrients in the agar medium as well as in the growth phases of the cells. From the comparison of cell growth between liquid and solid media with different pHs, we notified the importance of the pH changes in liquid medium accompanied with growth. Effects of salts and pH of the liquid medium on protonmotive force (Δp) was estimated from membrane potentials (ΔΨ) and proton gradients (ΔpH) of the strain JCM 6894. In the absence of salts, Δp of the strain JCM 6894 was the largest at neutral pH, which was conflicting with the result of cell viability. The addition of NaCl led to the reduction of Δp at acidic pH, mainly due to the dissipation of ΔΨ, which seems to be consistent with the lower numbers of colony formed at acidic pH in the presence of NaCl.