Non-axenic Cultures Research Articles

Air–dust-borne associations of phototrophic and hydrocarbon-utilizing microorganisms: promising consortia in volatile hydrocarbon bioremediation

Aquatic and terrestrial associations of phototrophic and heterotrophic microorganisms active in hydrocarbon bioremediation have been described earlier. The question arises: do similar consortia also occur in the atmosphere? Dust samples at the height of 15m were collected from Kuwait City air, and analyzed microbiologically for phototrophic and heterotrophic hydrocarbon-utilizing microorganisms, which were subsequently characterized according to their 16S rRNA gene sequences. The hydrocarbon utilization potential of the heterotrophs alone, and in association with the phototrophic partners, was measured quantitatively. The chlorophyte Gloeotila sp. and the two cyanobacteria Nostoc commune and Leptolyngbya thermalis were found associated with dust, and (for comparison) the cynobacteria Leptolyngbya sp. and Acaryochloris sp. were isolated from coastal water. All phototrophic cultures harbored oil vapor-utilizing bacteria in the magnitude of 10(5)g(-1). Each phototrophic culture had its unique oil-utilizing bacteria; however, the bacterial composition in Leptolyngbya cultures from air and water was similar. The hydrocarbon-utilizing bacteria were affiliated with Acinetobacter sp., Aeromonas caviae, Alcanivorax jadensis, Bacillus asahii, Bacillus pumilus, Marinobacter aquaeolei, Paenibacillus sp., and Stenotrophomonas maltophilia. The nonaxenic cultures, when used as inocula in batch cultures, attenuated crude oil in light and dark, and in the presence of antibiotics and absence of nitrogenous compounds. Aqueous and diethyl ether extracts from the phototrophic cultures enhanced the growth of the pertinent oil-utilizing bacteria in batch cultures, with oil vapor as a sole carbon source. It was concluded that the airborne microbial associations may be effective in bioremediating atmospheric hydrocarbon pollutants in situ. Like the aquatic and terrestrial habitats, the atmosphere contains dust-borne associations of phototrophic and heterotrophic hydrocarbon-utilizing bacteria that are active in hydrocarbon attenuation.

Anaerobic digestibility of Scenedesmus obliquus and Phaeodactylum tricornutum under mesophilic and thermophilic conditions

Two types of non-axenic algal cultures, one dominated by the freshwater microalgae Scenedesmus obliquus and the other by the marine microalgae Phaeodactylum tricornutum, were cultivated in two types of simple photobioreactor systems . The production rates, expressed on dry matter (DM) basis, were in the order of 0.12 and 0.18 g DM L −1 d −1 for S. obliquus and P. tricornutum respectively. The biogas potential of algal biomass was assessed by performing standardized batch digestion as well as digestion in a hybrid flow-through reactor (combining a sludge blanket and a carrier bed), the latter under mesophilic and thermophilic conditions. Biomethane potential assays revealed the ultimate methane yield ( B 0) of P. tricornutum biomass to be about a factor of 1.5 higher than that of S. obliquus biomass, i.e. 0.36 and 0.24 L CH 4 g −1 volatile solids (VS) added respectively. For S. obliquus biomass, the hybrid flow-through reactor tests operated at volumetric organic loading rate (Bv) of 2.8 gVS L −1 d −1 indicated low conversion efficiencies ranging between 26–31% at a hydraulic retention time (HRT) of 2.2 days for mesophilic and thermophilic conditions respectively. When digesting P. tricornutum at a Bv of 1.9 gVS L −1 d −1 at either mesophilic or thermophilic conditions and at an HRT of 2.2 days, an overall conversion efficiency of about 50% was obtained. This work indicated that the hydrolysis of the algae cells is limiting the anaerobic processing of intensively grown S. obliquus and P. tricornutum biomass.

The buffer composition impacts the hydrogen production and the microbial community composition in non-axenic cultures

Abstract Hydrogen obtained from biomass via dark fermentation is considered a sustainable and clean energy carrier. Batch fermentations with cheese whey powder were performed to assess total hydrogen production ( H max ), volumetric hydrogen production rate (VHPR), maximum lactose consumption ( S max ), maximum lactose consumption rate ( R max,S ), hydrogen molar yield (HMY) and the bacterial species present using two mineral media formulation (A, B). The highest VHPR was 304.8 cm 3 dm −3 h −1 and the HMY was 1.8 mol mol −1 . Medium B yielded around twice the VHPR than the attained with medium A, but HMY only had a slight increment with the use of medium B. The values reached for S max (17.3 g dm −3 ) , H max (4.863 dm 3 ) and R max,S (2.7 g dm −3 h −1 ) were also enhanced with medium B. Results suggest that butyrate levels and lower pH are the reasons for diminished hydrogen production with medium A. The microbial communities were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Only one band was observed in the experiments with medium A, the sequence retrieved from this band presented a closest relative match to the sequence from Citrobacter freundii JCM (100% identity); whereas for medium B, three bands were detected. Sequences from these bands presented high homology to sequences from Clostridium perfringens W11 (95% identity), uncultured Lachnospiraceae bacterium clone MS146A1 E12 (100% identity) and Enterobacter cloacae GH1 (100% identity). From the results obtained it is clear that the formulation of culture media had a strong effect on hydrogen production, kinetics and also on the microbial diversity.

Culturing of stoneworts and submersed angiosperms with phosphate uptake exclusively from an artificial sediment

Summary 1. We aimed to demonstrate reproducible nutrition and growth of macrophytes in non-axenic laboratory cultures preventing growth of phytoplankton and epiphytes. 2. Macrophyte shoot segments were planted in a mixture of commercial acid-washed silica sand with crystalline tricalcium phosphate, and this artificial sediment was covered with a layer of pure silica sand. The liquid mineral media used did not contain phosphorus but were rich in all other nutrient elements. A CO2 reservoir provided sustainable CO2 supply to macrophyte cultures by gas diffusion through a polyethylene membrane. 3. Chara hispida, Chara tomentosa, Chara baltica, Elodea canadensis, Potamogeton pectinatus and Zanichellia palustris could be cultivated for long term without medium exchange and aeration. Microalgae growth was prevented by the absence of phosphate in the water column. Mobilisation of tricalcium phosphate and phosphate uptake by the rhizoids of C. hispida enabled sustainable rapid shoot growth and increased the concentration of inorganic phosphate in the shoot dry weight by five to six times in comparison with plants cultivated on pure silica sand. A significant growth support from tricalcium phosphate was also observed for E. canadensis, but the rate of phosphate uptake by the roots was not sufficient to maintain a storage pool of inorganic phosphate (Pi) in the growing shoots of this plant. 4. Membrane-controlled CO2 supply from a reservoir and artificial sediments like the one described provide attractive options for the laboratory culture of macrophytes.

First report of saxitoxin production by a species of the freshwater benthic cyanobacterium, Scytonema Agardh

Saxitoxins or paralytic shellfish poisons (PSP) are neurotoxins produced by some species of freshwater cyanobacteria and marine dinoflagellates. Samples collected from the metaphyton of a drinking-water supply’s pre-treatment reservoir and a small eutrophic lake in New Zealand returned positive results when screened using a Jellett PSP Rapid Test Kit. The dominant species in the sample was identified as Scytonema cf. crispum. A non-axenic clonal culture (UCFS10) was isolated from the lake. The partial 16S rRNA gene sequence shared only a 91% or less sequence similarity with other Scytonema species, indicating that it is unlikely that this genus is monophyletic and that further in-depth phylogenetic re-evaluation is required. The sxtA gene, which is known to be involved in saxitoxin production, was detected in UCFS10. Saxitoxin concentrations were determined from the lake samples and from UCFS10 using pre-column oxidation high performance liquid chromatography with fluorescence detection. Saxitoxin was the only variant detected and this was found at concentrations of 65.6 μg g−1 dry weight in the lake sample and 119.4 μg g−1 dry weight or 1.3 pg cell−1 in UCFS10. This is the first confirmation of a saxitoxin-producing species in New Zealand and the first report of saxitoxin production by a species of Scytonema.

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non-heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro-oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark-mediated nitrogenase in the absence of DCMU nor light-mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non-axenic cultures of L. nodulosa. RT-PCR demonstrated that this gene was expressed only in non-axenic cultures. Western blotting showed that the Fe-protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.

The Effect of Initial Cell and Nutrient Concentrations on the Growth and Biomass Production of Outdoor Cultures ofDunaliellasp.

The final concentrations of non-axenic outdoor mass cultures of Dunaliella sp. grown for two or three days in f/2 and 2f media were found to be dependent on the initial cell concentration, since the cultures started with 80 × 103 cells ml-1 gave better yields than those started with 40 × 103 cells ml-1. There was a correlation between nutrient availability and cell yields: the two and three days-old cultures started with 40 × 103 cells ml-1 and grown in medium f/2 had lower concentrations than those grown in 2f medium, whereas in cultures with 80 × 103 cells ml-1 inoculum the yield was significantly higher only after three days. The lowest dry biomass yields after two and three days were with the inoculum of 40 × 103 cells ml-1 and medium f/2.

Marine-derived fungi from Kappaphycus alvarezii and K. striatum as potential causative agents of ice-ice disease in farmed seaweeds

Ice-ice disease in cultivated seaweeds is often associated with environmental stress and infection by pathogenic marine bacteria. No studies have associated the disease with marine fungi. Our study aimed to isolate marine fungi from cultivated Kappaphycus species and assess their ability to induce the disease. Kappaphycus alvarezii and K. striatum were collected from Calatagan, Batangas, Philippines. Following washing with sterile artificial seawater and inoculation into culture media, 18 morphospecies of marine-derived fungi (MDF) were isolated. Fungal diversity (Hss2.4) in infected seaweeds was higher than in healthy specimens. K. striatum (orange variety) had the highest incidence of MDF with 67 isolates, while K. striatum (green variety) had the lowest incidence with only 17 isolates. The ability of MDF to produce carrageenolytic and cellulolytic enzymes and utilize algal components was also tested. Of the 18 MDF selected, three had carrageenolytic activity and ten had cellulolytic activity. Most isolates utilized carrageenan, agar, and cellulose. Among the 10 MDF assayed for their ability to induce ice-ice disease, three isolates (Aspergillus ochraceus, A. terreus and Phoma sp.) induced ice-ice disease symptoms (thallus bleaching) in healthy, non-axenic cultures of K. alvarezii.

Recycling and Uptake of Si(OH) 4 when Protozoan Grazers Feed on Diatoms

Herbivory of microzooplankton is an emerging key factor of diatom mortality in the ocean. As part of the microbial loop, protozoan grazers also feed on bacteria that accelerate the degradation of diatom detritus. The potentially pivotal effect of microzooplankton grazing on Si(OH)(4) recycling was investigated with cultures of single-celled diatoms, Thalassiosira pseudonana and Chaetoceros gracilis, and heterotrophic protozoans, the dinoflagellate Oxyrrhis marina and the ciliate Strombidium sp. Both grazers ingested diatoms and the bacteria in the non-axenic cultures. C. gracilis, whose frustule is "armed" with setae, was less suitable as a prey than T. pseudonana. Ingestion rates of T. pseudonana were comparable for O. marina and Strombidium, but the dinoflagellate produced two orders of magnitude more detrital bSiO(2) than the ciliate, due to the higher abundance reached by O. marina. Total net release of Si(OH)(4) was lower in the grazing treatments compared to the control possibly due to the reduced bacterial growth by microzooplankton bacterivory, and to the transient protection of detrital bSiO(2) in discarded feeding vacuoles. Over the first 24h, microzooplankton grazing even led to enhanced uptake of Si(OH)(4) by diatoms, confirming the potential of grazing to influence the silicification of diatom frustules. Subsequently however, the Si dynamics in bottles with grazers turned rapidly from net uptake to net Si(OH)(4) release. Protozoan grazers hence tie Si(OH)(4) recycling into the microbial loop by producing detrital bSiO(2).

Assessing amino acid uptake by phototrophic nanoflagellates in nonaxenic cultures using flow cytometric sorting

Biologically available concentrations of individual dissolved amino acids in the open ocean are generally <1 nM. Despite this, the microbial turnover of amino acids is usually measured in hours indicating high demand. It is thought that the majority of uptake is due to bacterioplankton, although protists, particularly phototrophic protists, are also expected to take up amino acids. In order to assess the ability of protists to compete with prokaryotes for amino acids at subnanomolar concentrations, we examined the direct uptake of (3)H-leucine by phototrophic nanoflagellates (prasinophytes, pelagophytes and trebouxiophytes) and by associated bacteria using flow cytometric cell sorting. In contrast to (3)H-leucine-assimilating bacterial copopulations, none of the six studied nanoflagellates showed measurable direct uptake of (3)H-leucine, suggesting that the studied phototrophic protists were unable to utilize dissolved (3)H-leucine at natural oceanic concentrations. More practically, the flow-sorting technique allowed rapid and unequivocal differentiation of organic nitrogen uptake between prokaryotic cells and eukaryotic cells in mixed microbial populations, reducing the need to establish and maintain axenic algal cultures.

UREASE GENE SEQUENCES FROM ALGAE AND HETEROTROPHIC BACTERIA IN AXENIC AND NONAXENIC PHYTOPLANKTON CULTURES(1).

While urea has long been recognized as an important form of nitrogen in planktonic ecosystems, very little is known about how many or which phytoplankton and bacteria can use urea as a nitrogen source. We developed a method, targeting the gene encoding urease, for the direct detection and identification of ureolytic organisms and tested it on seven axenic phytoplankton cultures (three diatoms, two prymnesiophytes, a eustigmatophyte, and a pelagophyte) and on three nonaxenic Aureococcus anophagefferens Hargraves et Sieburth cultures (CCMP1784 and two CCMP1708 cultures from different laboratories). The urease amplicon sequences from axenic phytoplankton cultures were consistent with genomic data in the three species for which both were available. Seven of 12 phytoplankton species have one or more introns in the amplified region of their urease gene(s). The 63 urease amplicons that were cloned and sequenced from nonaxenic A. anophagefferens cultures grouped into 17 distinct sequence types. Eleven types were related to α-Proteobacteria, including three types likely belonging to the genus Roseovarius. Four types were related to γ-Proteobacteria, including two likely belonging to the genus Marinobacter, and two types were related to β-Proteobacteria. Terminal restriction fragment length polymorphism (TRFLP) analyses suggested that the sequenced amplicons represented approximately half of the diversity of bacterial urease genes present in the nonaxenic cultures. While many of the bacterial urease sequence types were apparently lab- or culture-specific, others were found in all three nonaxenic cultures, suggesting the possibility of specific relationships between these bacteria and A. anophagefferens.

Molecular Identification of T4 and T5 Genotypes in Isolates from Acanthamoeba Keratitis Patients

Acanthamoeba keratitis (AK) is a rare but sight-threatening ocular infection. Outbreaks have been associated with contaminated water and contact lens wear. The epidemiology and pathology may be associated with unique genotypes. We determined the Rns genotype for 37 clinical isolates from 23 patients presenting at the University of Miami Bascom Palmer Eye Institute with confirmed AK infections in 2006 to 2008. The genus-specific ASA.S1 amplicon allowed for rapid genotyping of the nonaxenic cultures. Of the 37 isolates, 36 were of the T4 genotype. Within this group, 13 unique diagnostic fragment 3 sequences were identified, 3 of which were not in GenBank. The 37th isolate was a T5, the first in the United States and second worldwide to be found in AK. For five patients with isolates from the cornea and contact lens/case, identical sequences within each patient cluster were observed, confirming the link between contact lens contamination and AK infection. Genotyping is an important tool in the epidemiological study of AK. In this study, it allowed for the detection of new strains and provided an etiological link between source and infection. Additionally, it can allow for accurate categorizing of physiological differences, such as strain virulence, between isolates and clades.

Direct contact between Pseudo-nitzschia multiseries and bacteria is necessary for the diatom to produce a high level of domoic acid

Pseudo-nitzschia multiseries loses most of its ability to produce domoic acid when it is cultured axenically. However, domoic acid production recovers when bacteria from the original culture are reintroduced to axenic cultures, indicating a bacterial association with domoic acid production in this species. In order to determine the role of bacteria in domoic acid production, an axenic strain prepared from a nonaxenic strain of P. multiseries was inoculated into media in cellophane tubes, which were then placed in a bottle containing the original nonaxenic culture. Both strains showed characteristic domoic acid production similar to that previously observed. Domoic acid level in the cells of the axenic strain in the tubes was much smaller than that of the cells outside the tubes. These results show that direct contact between living bacteria and P. multiseries is necessary for producing high levels of domoic acid in P. multiseries.

The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays

Although single-species laboratory toxicity tests with microalgae are sensitive and highly reproducible, they lack environmental realism. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter algal sensitivity to contaminants, which are not mimicked in laboratory toxicity tests. This study investigated the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth-rate inhibition bioassays. Four species of microalgae were used, two isolates of each; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchneriella subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72-h (IC50)) was not significantly different for the axenic and non-axenic strains of N. closterium, P. subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46 μg Cu L −1, while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208 μg Cu L −1. However, when the bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rRNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE), it was found that bacteria were actually present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria, and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The “axenic” cultures contained less bacterial phylotypes than the non-axenic cultures, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment.

P‐affinity measurements of specific osmotroph populations using cell‐sorting flow cytometry

To elucidate the role that the marine microbes play in global nutrient cycling, it is necessary to recognize how various phyto‐ and bacterioplankton groups compete for limiting nutrients. Specific phosphate affinity describes an organism's ability to harvest phosphate at low concentrations from the surrounding water. For the first time, we have taken advantage of cell‐sorting flow cytometry in combination with radio‐labeled phosphorus to measure this feature of specific osmotrophic groups in natural communities. Specific phosphate affinities for Synechococcus spp. and picoeukaryotes were measured using live, unstained cells. The results were always lower than theoretical calculated maximum values, corresponding well with observations of P‐deficiency, or sub‐optimal P supply for the osmotroph community, at the time of investigation. Fixing and staining cells before flow sorting offers the advantage of better separation of phytoplankton and showed high sorting reproducibility when applied to nonaxenic Synechococcus cultures. A subsequent investigation of P‐leakage from isotopically labeled, fixed, stained cells in nonaxenic cultures of Synechococcus showed that it was only slightly larger than the loss of 17% found when uptake of new label was stopped with adding “cold” phosphate. Possible applications of the currently developed methodology for population specific P affinity measurements by flow sorting are discussed.

Metabolic Engineering of ω3-Very Long Chain Polyunsaturated Fatty Acid Production by an Exclusively Acyl-CoA-dependent Pathway

omega3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Delta6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds.

Bacterial involvement in determining domoic acid levels in Pseudo-nitzschia multiseries cultures

This study examining factors contributing to the production or elimination of domoic acid (DA) in cultures of Pseudo-nitzschia multiseries showed that in axenic cultures doubling the silicate concentrations increased growth, but not DA levels. DA concentration for axenic cultures was increased by the addition of gluconolactone (GlcA), especially in cultures with increased silicon. In non-axenic cultures, there were similar increases in growth with increased silicon, but a reduction of DA production in the presence of GlcA. Detailed examinations confirmed these findings and also showed that in non-axenic cultures, glucose alone resulted in a reduction of DA while a combination of glucose with gluconolactone resulted in a complete elimination of DA. Subsequent trials with axenic P. multiseries cultures showed that additions of DA or DA plus glucose introduced at the early stationary growth phase and incubated for 5 d had no impact on DA concentrations. In contrast, a 6 d incubation of the associated bacteria separated from the non-axenic diatom cultures showed reduc- tions of added DA concentrations ranging from 46 to 72%, depending upon co-additives. The diatom does not use extracellular DA present in surrounding culture medium whereas bacteria associated with the diatom can utilize DA readily. Reductions in the production of DA by aging P. multiseries cultures appear to be the result of changing balances over time among bacteria associated with the diatom. These data coupled with results from other studies indicate that the amount of DA measured in P. multiseries cultures is a result of competitive interaction, i.e. a function of the diatom's produc- tion rate versus the extra-cellular utilization of DA by associated bacteria.

Presence of previously undescribed bacterial taxa in non-axenic Chlorella cultures

We determined the bacterial community profile in non-axenic cultures of Chlorella (Chlorophyceae, Chlorophyta) isolated from soil. The bacterial composition at the phylum level was different from that of whole soil bacteria, but it was similar to that reported for non-axenic cultures of marine microalgae such as diatoms (Bacillariophyceae, Heterokontophyta). Expected novel bacteria, i.e. those which do not have close relatives among described species, were maintained in the cultures, and these bacteria were chiefly composed of members of the phylum Bacteroidetes. They may have been 'as-yet-uncultured' but in practice unintentionally been cultured in microalgal cultures. They could serve as good bioresources in various fields of biological and ecological studies.

Leptolyngbya nodulosa sp. nov. (Oscillatoriaceae), a subtropical marine cyanobacterium that produces a unique multicellular structure

Z. Li and J. Brand. 2007. Leptolyngbya nodulosa sp. nov. (Oscillatoriaceae), a subtropical marine cyanobacterium that produces a unique multicellular structure. Phycologia 46: 396–401. DOI: 10.2216/06-89.1A previously undescribed, filamentous, nonheterocystous cyanobacterium was isolated from the South China Sea. The nonbranching and nonmotile filaments had well-defined sheaths of variable thickness. Trichomes were 1.1–1.5 µm in diameter, while individual cells ranged from 1.2 to 2.4 µm in length under culturing conditions. Thylakoids within individual cells were peripheral and appeared to become confluent at one end of the cell. Cells did not contain gas vacuoles. Cultures grew under a wide range of conditions, including salinities ranging from freshwater to 60 ppt of sodium chloride. Nonaxenic cultures could be grown indefinitely (for at least 2 years) in media devoid of any combined nitrogen source but grew much more rapidly in the presence of ammonium or nitrate ions. A unique feature of this organism was the presence, under dim light, of well-defined multicellular structures (nodules), distributed at irregular intervals along the filaments. Nodules were highly variable in shape and size, with each nodule constructed of a region of a filament tightly folded and wrapped into a discrete unit and surrounded by a firm common sheath. The internal structure of cells within nodules appeared identical to the structures of cells occurring along other portions of the filament. Comparisons of 16S rDNA, the 16S–23S internal transcribed spacer (ITS) sequence, and morphological characteristics of this strain to corresponding features of other cyanobacteria indicate that it is a member of the genus Leptolyngbya. Differences in DNA sequences and isolation habitat of this organism, along with the occurrence of nodules, indicate that it is not closely related to the type species, L. boryana, and should be assigned a distinct specific epithet. We propose the name Leptolyngbya nodulosa for this organism.

Successful nodulation of Casuarina by Frankia in axenic conditions

In order to depict the fine interactions that lead to nodulation, absolute microbiological control of the symbiotic partners is required, i.e. the ability to obtain in vitro axenic nodulation, a condition that has never been fulfilled with the Casuarina-Frankia symbiosis. The effects of culture conditions on plant growth and nodule formation by Casuarina cunninghamiana were investigated. Axenic (capped tubes with different substrates), and nonaxenic cultures (Gibson tubes, pot cultures) were tested. In axenic conditions, C. cunninghamiana, inoculated with Frankia, had poor growth and did not form nodules at 6 weeks. Plants cultivated in Gibson tubes reached the four axillary shoots stage within 6 weeks and formed nodules 4 weeks after inoculation. Sand-pot cultures allowed us to relate the plant development stage at inoculation with nodulation. The sterile replacement of the cap by a plastic bag increased plant growth and enabled nodule formation 6 weeks after inoculation. The new system of plant culture allows the axenic nodule formation 6 weeks after inoculation. Nodulation behaviour is related to plant development and confinement. This axenic plant nodulation system is of major interest in analysing the roles of Frankia genes in nodulation pathways.