UREASE GENE SEQUENCES FROM ALGAE AND HETEROTROPHIC BACTERIA IN AXENIC AND NONAXENIC PHYTOPLANKTON CULTURES1

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.     

KINETICS OF EXTRACELLULAR RELEASE IN AXENIC ALGAE AND IN MIXED ALGAL-BACTERIAL CULTURES: SIGNIFICANCE IN ESTIMATION OF TOTAL (GROSS) PHYTOPLANKTON EXCRETION RATES1

In axenic Chlorella pyrenoidosa Chick cultures, extracellular release was linear with time, but plateau-type curves were obtained in cultures with added bacteria. Initial rates of excretion were identical in both, systems. Kinetics of extracellular release in axenic Anabaena flos-aquae (Lyng.) Bréb. cultures were more complex than in Chlorella but the initial excretion rates were identical in axenic and mixed algal-bacterial cultures. In lakewater, extracellular release kinetics resemble the pattern in mixed Chlorella-bacteria cultures. An explanation is an initial lag in bacterial utilization of algal extracellular products. As a result, both in situ and in the laboratory, consecutive short, experiments give higher excretion rates than single long incubations. It is suggested that the former are close to total or gross extracellular release rates whereas the latter give net values, detecting only substances not, removed by heterotrophs.     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi

Nearly annual blooms of the marine dinoflagellate Karenia brevis, which initiate offshore on the West Florida Shelf in oligotrophic waters, cause widespread environmental and economic damage. The success of K. brevis as a bloom-former is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, although relatively little is known about the ability of K. brevis and the closely related Karenia mikimotoi to use a variety of organic sources of phosphorus, including phosphomonoesters, phosphodiesters, and phosphonates. Through a series of bioassays, this study characterized the ability of axenic and nonaxenic K. brevis and K. mikimotoi clones isolated from Florida waters to use a variety of organic phosphorus compounds as the sole source of phosphorus for growth, comparing this utilization to that of inorganic sources of phosphate. Differing abilities of axenic and nonaxenic K. brevis and K. mikimotoi cultures to use phosphorus from the compounds evaluated were documented. Specifically, growth of axenic cultures was greatest on inorganic phosphorus and was not supported on the phosphomonoester phytate, or generally on phosphodiesters or phosphonates. The nonaxenic cultures were able to use organic compounds that the axenic cultures were not able to use, often after lags in growth, highlighting a potential role of co-associated bacterial communities to transform nutrients to bioavailable forms. Given the ability of K. brevis and K. mikimotoi to use a diverse suite of inorganic and organic phosphorus, bloom mitigation strategies should consider all nutrient forms.     

Effect of associated bacteria on the growth and toxicity of Alexandrium catenella

Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na(+) channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.     

Effect of Associated Bacteria on the Growth and Toxicity of Alexandrium catenella

Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na⁺ channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.     

VOLATILE ORGANIC SULFIDES FROM FRESHWATER ALGAE1

Dimethyl sulfide (DMS) was found to be the principle biogenic sulfur compound in a freshwater environment. Endemic and non-endemic algae were cultured under axenic and nonaxenic conditions in defined media. Sulfur gas analysis of culture fluids indicated that members of the Cyanophyta were probable sources of DMS whereas representatives of the divisions Chlorophyta, Xanthophyta and Bacillariophyta apparently did not produce this compound. Comparison of gaseous contents of young and old nonaxenic cultures of filamentous algae in the divisions Chlorophyta and Xanthophyta showed DMS occurred only in aged cultures and was probably produced by bacteria utilizing substances from senescent algal cells. Data suggest that the composition of the algal community determines whether DMS is algal and/or bacterial in origin.     

Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish polymerase chain reaction (PCR) products of the cyanobacterial rRNA operons from those resulting from amplification of contaminating bacteria. Using this screening method the 16S rRNA genes of four nonaxenic filamentous cyanobacterial strains belonging to the generaLeptolyngbya andOscillatoria were cloned and sequenced. For the genusLeptolyngbya, the 16S rRNA sequence of the axenic strain PCC 73110 was also determined. Phylogenetic trees were constructed based on complete and partial sequences. The results show that the strainsLeptolyngbya foveolarum Komárek 1964/112,Leptolyngbya sp. VRUC 135 Albertano 1985/1, andLeptolyngbya boryanum PCC 73110 belong to the same cluster. StrainOscillatoria cf.corallinae SAG 8.92, which contains the rare photosynthetic pigment CU-phycoerythrin, is not closely related to other CU-phycoerythrin-containing cyanobacteria.Oscillatoria agardhii CYA 18, which is a representative of planktonicOscillatoria species that form toxic blooms in Norwegian inland waters, has no close relatives in the tree.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

The production of clonal and axenic cultures of microalgae using fluorescence-activated cell sorting

Unialgal cultures of the flagellate algae Cyanophora paradoxa, Haematococcus lacustris, Monomastix sp., Scherffelia dubia and Spermatozopsis similis which contained bacteria were sorted by flow cytometry to obtain axenic clonal cultures. The variables used for fluorescence-activated cell sorting (FACS) were chlorophyll autofluorescence, forward scatter and side scatter of the laser beam. To produce clonal cultures, a single cell was sorted into each culture flask. Depending on the species, about 20–30% of the sorted cultures grew successfully and at least 20% of these were axenic even if the numerical ratio betweeen bacteria and algae in the original cultures was as high as 300:1. FACS represents an effective and rapid method for the preparation of clonal and axenic cultures of microalgae.     

Relationship between axenic growth of Dictyostelium discoideum strains and their track morphology on substrates coated with gold particles

Amoebae of Dictyostelium discoideum produce tracks with two distinct morphologies on gold-coated coverslips. The wild-type strain and other strains that feed only by phagocytosis produced indistinct, fuzzy tracks, whereas mutants capable of axenic growth produced clear, sharp tracks. The sharp track morphology was found to be a recessive phenotype that segregates with axenicity and probably requires a previously unidentified axenic mutation. Axenic and nonaxenic strains also differed in their ability to pinocytose. When the two types of cells were shifted from bacterial growth plates to nutrient media, within 24 h the axenic strain established a rapid rate of pinocytosis, approximately 100-fold higher than the low rate detectable for the nonaxenic strain. However, track formation did not appear to be directly related to endocytosis. Electron microscopic examination of cells during track formation showed that both axenic and nonaxenic strains accumulated gold particles on their surfaces, but neither strain internalized the gold to any significant degree. Observation of living cells revealed that axenic strains collected all particles that they contacted, whereas wild-type strains left many particles undisturbed. The size of the gold particle clusters discarded by the cells also contributed to track morphology.     

Successful nodulation of Casuarina by Frankia in axenic conditions

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: This axenic plant nodulation system is of major interest in analysing the roles of Frankia genes in nodulation pathways.     

The effect of bacteria on interspecific relationships between the euryhaline rotifer Brachionus rotundiformis and the harpacticoid copepod Tigriopus japonicus

Inconsistent results have been obtained on the population growth of Brachionus rotundiformis and Tigriopus japonicus, when results from single-species and two-species mixed cultures are compared. Bacteria growth was not regulated in these experiments, which could be the cause for this. In order to test this possibility, we conducted similar experiments under axenic and synxenic (with presence of one species of bacteria) conditions. The population growth of B. rotundiformis was suppressed by the presence of T. japonicus in axenic cultures. T. japonicus could not persist in axenic cultures, but its population increased when grown in synxenic cultures. T. japonicus used RT bacteria strain as a food source, while these bacteria were toxic to B. rotundiformis. These results suggest that bacteria can modify the interspecific relationship between B. rotundiformis and T. japonicus.     

A New Method For Axenic Mass Cultivation of Paramecium Tetraurelia*

SYNOPSIS A method is described for the axenic mass cultivation of Paramecium tetraurelia strains 51s and 299s. the ciliate is grown in an enriched axenic medium developed by Soldo, Godoy & van Wagtendonk. Under continuous shaking on a rotary shaker, cultures were grown in one-liter Erlenmeyer flasks with 330 ml medium yield cell densities of 32,000 cell/ml and 20,000 cells/ml for strains 299s and 51s respectively. Doubling time is considerably shorter under these conditions than in the conventional static cultures. A 20-liter airlift bioreactor is described in detail which can be used successfully to otain up to 100 g wet weight of Paramecium in a single run; in this reactor the cell density reaches 38,000 cells/ml for strain 299s. and 23,000 cells/ml for 51s. This technic should facilitate the study of minor protein components of the ciliate.     

Novel approach for the development of axenic microalgal cultures from environmental samples

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence‐activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

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

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

Iron Inefficiency in Maize Mutant ys1 (Zea mays L. cv Yellow-Stripe) Is Caused by a Defect in Uptake of Iron Phytosiderophores

To determine the Fe inefficiency factors in the maize mutant ys1 (Zea mays L. cv Yellow Stripe), root exudates of Fe-inefficient ys1 and of two Fe-efficient maize cultivars (Alice, WF9) were collected in axenic nutrient solution cultures. Analysis by thin-layer chromatography and high-performance liquid chromatography revealed that under Fe deficiency ys1 released the phytosiderophore 2[prime]-deoxymugineic acid (DMA) in quantities similar to those of Alice and WF9. Under nonaxenic conditions, DMA released by plants of all three cultivars was rapidly decomposed by microorganisms in the nutrient solution. Uptake experiments with 59Fe-labeled DMA, purified from root exudates of either Fe-deficient Alice or ys1 plants, showed up to 20 times lower uptake and translocation of 59Fe in ys1 than in Alice or WF9 plants. The presence of microorganisms during preculture and short-term uptake experiments had no significant effect on uptake and translocation rates of 59Fe in Alice and ys1 plants. We conclude that Fe inefficiency in the maize mutant ys1 is the result of a defect in the uptake system for Fe-phytosiderophores.     

Primordia initiation of mushroom (Agaricus bisporus) strains on axenic casing materials

The mushroom (Agaricus bisporus) has a requirement for a "casing layer" that has specific physical, chemical and microbiological properties which stimulate and promote the initiation of primordia. Some of these primordia then may develop further into sporophores, involving differentiation of tissue. Wild and commercial strains of A. bisporus were cultured in axenic and nonaxenic microcosms, using a rye grain substrate covered by a range of organic and inorganic casing materials. In axenic culture, A. bisporus (commercial strain A15) was capable of producing primordia and mature sporophores on charcoal (wood and activated), anthracite coal, lignite and zeolite, but not on bark, coir, peat, rockwool, silica or vermiculite. Of six strains tested, only the developmental variant mutant, B430, produced rudimentary primordia on axenic peat-based casing material. However, none of these rudimentary primordia developed differentiated tissues or beyond 4 mm diameter, either on axenic casing material in the microcosms or in larger-scale culture. In larger-scale, nonaxenic culture, strain B430 produced severely malformed but mature sporophores in similar numbers to those of other strains. Typically, 3-6% of primordia developed into mature sporophores, but significant differences in this proportion, as well as in the numbers of primordia produced, were recorded between 12 A. bisporus strains.     

Glycollate production and excretion byAlcaligenes eutrophus

Autotrophic cultures of the facultative chemolithotrophAlcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO₂ to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic conditions.Extracts from autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction ofd0ribulose 1,5-diphosphate carboxylase under the same conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - HPMS 2-pyridyl-hydroxymethane sulphonie acid - RuDP d-ribulose 1,5-diphosphate