Effect of salinity on competition between the rotifers Brachionus rotundiformis Tschugunoff and Hexarthra jenkinae (De Beauchamp) (Rotifera)

We studied the effect of different concentrations (0, 3, 6, 9 and 12 g l^–1) of sodium chloride at one food level of Chlorella (1×10⁶ cells ml^–1) on competition between the rotifers B. rotundiformis and H. jenkinae, both of which were isolated from a saline lake. The population growth experiments were conducted for 3 weeks. Both the rotifer species did not survive beyond one week at a salinity of 0 g l^–1. Regardless of salt concentration and the presence of a competitor, H. jenkinae reached higher densities than B. rotundiformis. When grown alone, both B. rotundiformis and H. jenkinae showed optimal peak population densities at the salinity of 6 and 9 g l^–1. Since biomass wise, B. rotundiformis was larger than H. jenkinae, it showed a lower numerical abundance. Thus, the maximum peak population densities of B. rotundiformis and H. jenkinae recorded in this study were 107±3 and 203±28 ind. ml^–1. The maximal rates of population increase for B. rotundiformis and H, jenkinae when grown alone were 0.264±0.003 and 0.274±0.004, respectively. Our results also indicated that B. rotundiformis and H. jenkinae coexisted better at a salinity of 6 and 9 g l^–1 of sodium chloride while a salinity of 3 g l^–1 favoured Hexarthra over B. rotundiformis. At 12 g l^–1, both the rotifer species grown alone or together showed lower growth rates compared to those at lower salinity levels. Except 0 g l^–1, in all other salinity treatments, H. jenkinae was a superior competitor to B. rotundiformis.     

Effect of the epizoic rotifer Brachionus rubens on the population growth of three cladoceran species

Using population densities and growth rates as criteria, we studied interactions between the epizoic rotifer Brachionus rubens and each of three cladoceran species differing in size and reproductive rates — Daphnia carinata, Moina macrocopa and Ceriodaphnia rigaudi. In all mixed — species experiments, B. rubens existed in both the epizoic mode, attached to the cladoceran host, and in the free-swimming mode. Rotifer population growth rates were significantly depressed in the presence of M. macrocopa, presumably as a consequence of exploitative and interference competition. The largest cladoceran, D. carinata probably did not suppress B. rubens, because the epizoic component of the rotifer population escaped from the deleterious effects of mechanical interference. Peak population numbers and initial population growth rates reached by all three cladocerans were lower in the presence of B. rubens, probably because of the adverse effects of the epizoic infestation, which was maximal on D. carinata and least on C. rigaudi. In mixed-species cultures of D. carinata and M. macrocopa, the presence of B. rubens helped D. carinata coexist with M. macrocopa, which otherwise would have suppressed the Daphnia.     

Monoxenic culture of the anaerobic ciliate Trimyema compressum Lackey

Sapropelic ciliates from anoxic mud samples were enriched and cultivated in monoculture together with natural food bacteria growing on cellulose. The ciliates lacked cytochrome oxidase and contained bluish fluorescent endosymbionts. One of the anaerobic ciliates, Trimyema compressum, contained methanogenic bacteria as was shown by methane formation. During continued cultivation, T. compressum gradually lost its endosymbionts. With SEM microscopy no episymbiotic bacteria could be detected.From enrichment cultures of T. compressum, anaerobic bacteria were isolated in pure culture. One of the strains, a Bacteroides spec., proved capable of serving as food bacteria, thus allowing establishment of monoxenic T. compressum cultures. These cultures exhibited a requirement for sterols as growth factors. The doubling time of this ciliate was 13 h at 28°C. The highest yield obtained was 2100 cells/ml.Dedicated to Holger W. Jannasch on the occasion of his 60th birthday     

Isolation and purification of Australian isolates of the toxic cyanobacteriumMicrocystis aeruginosa Kütz

Isolation and laboratory culture ofMicrocystis aeruginosa Kütz. using a growth medium (MLA medium) suitable for both non-axenic and axenic cultures is described. Seventeen established strains ofM. aeruginosa were subjected to one or more of three purification methods: centrifugation cleaning, sulphide gradient selection, and antibiotic treatment (Imipenem^®). While each method purified only about half of the strains attempted, the selective application of each method, based on the morphological characteristics of the strains, succeeded in purifying 12 of the 17 strains. Three of the 5 strains not purified were contaminated with a sulphide-tolerant, Imipenem-resistant spirochaete,Spirochaeta cf.aurantia, which could not be detected on normal, broad spectrum bacterial test media. The presence of this bacterial species was detected only by phase contrast and DAPI (4,6-diamidino-2-phenylindole) stained fluorescence microscopy.Author for correspondence     

Induction of axenic culture of Arthrospira (Spirulina) platensis based on antibiotic sensitivity of contaminating bacteria

Arthrospira platensis SAG 21.99 and the isolated bacteria (Halomonas spp., Staphylococcus sp., etc.) from the culture of A. platensis SAG 21.99 were treated with five antibiotics to determine the minimal lethal concentrations. The combination of a washing step and a consecutive treatment with antibiotics, imipenem (100 μg ml⁻¹), neomycin (100 μg ml⁻¹) and cycloheximide (20 μg ml⁻¹), treatment step was highly effective in eliminating bacteria. An axenic culture of A. platensis SAG 21.99 could be induced within 3 days using this method. This technique is a simple and rapid method for obtaining axenic cultures of filamentous cyanobacteria.     

Reproductive isolation among geographically and temporally isolated marine Brachionus strains

Using a polyclonal antibody against the mate recognition pheromone (MRP) of Brachionus rotundiformis Koshiki strain, we investigated the behavioral reproductive isolation and the similarity of MRP among geographically and temporally isolated B. rotundiformis strains. Males of the Koshiki strain did not discriminate in mating attempts among females of the Koshiki strain and those of conspecific allopatric strains from Hamana, Fiji, Thailand and Spain. Likewise, Koshiki males attempted mating with statistically indistinguishable frequency with Koshiki females and B. plicatilis strains. However, copulation was not consummated between Koshiki males and B. plicatilis females. The amount of anti-MRP binding to three allopatric B. rotundiformis strains was similar to that of the Koshiki strain, but binding to Hamana and the B. plicatilis strain was significantly lower. Four temporally separated B. rotundiformis populations were hatched from resting eggs collected from 0, 5, 10 and 15 cm depth in the sediment of Kai-ike pond in Koshiki island, Japan. Sediment age was determined using the ²¹⁰Pb method, allowing us to estimate that resting eggs from 15 cm depth were produced 65 years ago. Results of mating assays and anti-MRP binding showed that no behavioral reproductive isolation exists among the four temporally isolated Koshiki strains. B. rotundiformis appears to be reproductively isolated from B. plicatilis, but heterospecific matings are still attempted between B. plicatilis and B. rotundiformis, suggesting that the MRP remains sufficiently similar to elicit circling behavior.     

Establishment of transformed root cultures of Perezia cuernavacana producing the sesquiterpene quinone perezone

Summary The sesquiterpene quinone currently known as perezone is abundantly produced by the roots of Perezia cuernavacana. This compound is of biotechnological interest since it may be used as a pigment and has several pharmacological properties. In this work we demonstrate that perezone is also produced in transformed root cultures of P. cuernavacana. Hairy roots were induced by inoculation of internodal segments of sterile plants of P. cuernavacana with Agrobacterium rhizogenes AR12 strain. The axenic liquid MS medium cultures of the hairy roots isolated from the internodes showed active growth in the absence of growth regulators. The transformed nature of the tissue was confirmed by genomic integration (PCR and slot blot hybridization) and expression (enzyme activity) of the marker gus-gene. The production of perezone by a transformed root culture was evidenced by IR spectroscopy. Our results offer an alternative for enhanced production of perezone and represent an advantage over its extraction from natural plant populations which present problems in their agronomic culture.     

Axenic Cultivation of the Cellulolytic Flagellate Trichomitopsis termopsidis (Cleveland) from the Termite Zootermopsis*

SYNOPSIS. Trichomitopsis termopsidis (Cleveland), a cellulolytic hindgut symbiote of the termite Zootermopsis, has been cultivated axenically under anaerobic conditions. The medium consists of cellulose, reduced glutathione, fetal calf serum, yeast extract, and autoclaved rumen fluid or autoclaved rumen bacteria, in a buffered salt solution the composition of which is based on an analysis of Zootermopsis hindgut fluid. The hindgut contents of surface-sterilized termites were inoculated into anaerobic buffer-containing cellulose and serum. Repeated passages yielded mixed cultures of T. termopsidis and termite hindgut bacteria. Flagellates were then inoculated into complete medium containing antibiotics, and after 2 passages, axenic cultures of T. termopsidis were obtained. Various nutritional supplements, including clarified rumen fluid or heat-killed bacteria of several known species failed to support the growth of T. termopsidis when substituted for autoclaved rumen fluid. The flagellates did not grow when any of several carbohydrates were substituted for cellulose. Electron microscopy of flagellates from axenic cultures revealed that cellulose particles and partially digested bacteria were present in food vacuoles. No endosymbiotic bacteria were present in the cytoplasm indicating that T. termopsidis does not depend on living prokaryotes for cellulose digestion. The results suggest that T. termopsidis possesses the enzyme cellulase.     

Bacterial response to extracellular dissolved organic carbon released from healthy and senescent Fragilaria crotonensis (Bacillariophyceae) in experimental systems

Responses of bacteria to dissolved organic carbon (DOC) released from healthy and senescent Fragilaria crotonensis (Bacillariophyceae) were examined in experimental systems. The alga released DOC actively, although the concentration fluctuated greatly in both the axenic (the alga alone) and the mixed (the alga plus the enriched bacteria) cultures. In the control (the bacteria alone) cultures, both DOC concentration and bacterial density were low and almost constant throughout the experiment: 5.0 mg C 1^–1 and 1.1 × 10⁵ cells ml^–1, respectively. In the mixed cultures, bacterial growth was negligible during the exponential growth phase of the alga, but rapid proliferation of the bacteria occurred after the onset of the stationary growth phase. As the bacterial population grew, the density of senescent algal cells also increased. When the bacteria were fed on the DOC from healthy algae, their growth rate was relatively low (0.44 d^–1), but the maximum cell density was high (6.4 × 10⁵ cells ml^–1). Conversely, when the bacteria fed on the DOC of senescent algae, they grew at a relatively high rate (0.51 d^–1), but the maximum cell density was low (2.8 × 10⁵ cells ml^–1). These results suggest that DOCs released from dominant phytoplankton species in different physiological states affect the biomass and activity of bacteria.     

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.     

Isolation and characterization of aerobic bacteria capable of the degradation of synthetic and natural melanoidins from distillery effluent

Melanoidins, complex biopolymer of amino-carbonyl compounds are the major coloring and polluting constituents of distillery wastewaters. In this study, three aerobic melanoidin-degrading bacteria (RNBS1, RNBS3 and RNBS4) were isolated from soil contaminated with distillery effluent and characterized as Bacillus licheniformis (RNBS1), Bacillus sp. (RNBS3) and Alcaligenes sp. (RNBS4) by biochemical tests and 16S rRNA gene sequence analysis. The degradation of synthetic and natural melanoidins was studied by using the axenic and mixed bacterial consortium. Results have revealed that the mixed consortium was more effective compared to axenic culture decolorizing 73.79 and 69.83% synthetic and natural melanoidins whereas axenic cultures RNBS1, RNBS3 and RNBS4 decolorized 65.88, 62.56 and 66.10% synthetic and 52.69, 48.92 and 59.64% natural melanoidins, respectively. The HPLC analysis of degraded samples has shown reduction in peak areas compared to controls, suggesting that decrease in color intensity might be largely attributed to the degradation of melanoidins by isolated bacteria.     

Attachment,colonization and proliferation ofAzospirillum brasilense andEnterobacter spp. on root surface of grasses

Root colonization studies, employing immunofluorescence and using locally isolated strains, showed thatEnterbacter sp. QH7 andEnterobacter agglomerans AX12 attached more readily to the roots of most plants compared withAzospirillum brasilense JM82. Heat treatment of either root or inoculum significantly decreased the adsorption of bacteria to the root surface. Kallar grass and rice root exudates sustained the growth ofA. brasilense JM82,Enterobacter sp. QH7 andE. agglomerans AX12 in Hoagland and Fahraeus medium. All the strains colonized kallar grass and rice roots in an axenic culture system. However, in studies involving mixed cultures,A. brasilense JM82 was inhibited byEnterobacter sp. QH7 in kallar grass rhizosphere and the simultaneous presence ofEnterobacter sp. QH7 andE. agglomerans AX12 suppressed the growth ofA. brasilense JM82 in rice rhizosphere. The bacterial colonization pattern changed from dispersed to aggregated within 3 days of inoculation. The colonization sites corresponded mainly to the areas where root mucigel was present. The area around the point of emergence of lateral roots usually showed maximum colonization.     

The biology ofAzospirillum-sugarcane association II. Ultrastructure

Summary Tissue cultures of sugarcane support abundant growth ofAzospirillum brasilense (SP 7). Visible after 1–2 weeks as a white or pink slime, this growth reaches 2×10⁸ bacteria/mm² on the surface of callus. Growth of the bacterium is strictly extracellular in viable callus, and instances of intracellular growth result from rupture of the cell wall during senescence of callus tissue. A significant proportion of the bacterial population on callus is pleomorphic. Varying the nitrogen source in the nutrient medium caused no obvious effect on callus cell structure. The presence of the bacterium caused structural alterations in callus cells which did not inhibit overall growth of the bacterium. Growth of callus as tight groups of cells lacking intercellular spaces may be important for the establishment of a long-term association withAzospirillum. The interface of bacteria and live callus tissue is at the surface of tight cell groups. Browning of the surface cell layers of these groups in the presence ofAzospirillum is not of the rapid nature known for hypersensitivity reactions. Rather, this production of phenolics appears to be due to the accumulation of extracellular bacterial metabolites. The ultrastructure of this and other callus reactions is described. As evidenced by organogenesis, the associated cultures have remained viable for at least 18–20 months.Florida Agricultural Experiment Station Journal Series No. 1695.     

Effects of salinity, food level, and the presence of microcrustacean zooplankters on the population dynamics of rotifer Brachionus rotundiformis

We investigated the population dynamics of the rotifer Brachionus rotundiformis fed with the alga Isochrysis galbana at two food concentrations (3 × 10⁴ and 40 × 10⁴ cells ml⁻¹) and four salinity levels (5, 10, 20, and 30) in the presence and absence of two copepod species, Pseudodiaptomus annandalei and Apocyclops royi and one cladoceran, Diaphanosoma aspinosum. Both the density and population growth rate of B. rotundiformis increased at higher food concentration and at salinity levels of 10 and 20. Among the microcrustaceans, only P. annandalei had a significant negative effect on the growth rate of the rotifer population because of its efficient predation. In contrast, the presence of both A. royi and D. aspinosum did not affect the growth rates at any of the salinity and food levels. Brachionus rotundiformis had significantly larger size during the log-phase, particularly if P. annandalei was present. Thus, B. rotundiformis grows better at higher food level and medium salinity levels. Unlike the larger calanoid, P. annandalei, B. rotundiformis can definitely coexist with relatively small cyclopoid copepods (A. royi) and cladocerans (D. aspinosum), because of the absence of interference.     

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.     

Bacteria–algae association in batch cultures of phytoplankton from a tropical reservoir: the significance of algal carbohydrates

Summary 1. The dissolved organic matter, especially carbohydrates, released by phytoplanktonic organisms may be ecologically important, through its influence on carbon cycling and microbial diversity. Here axenic cultures of three phytoplanktonic species, Cryptomonas tetrapyrenoidosa (Cryptophyceae), Staurastrum orbiculare (Zygnematophyceae) and Thalassiosira duostra (Bacillariophyceae), were inoculated with a microbial community from the same habitat in which the algae had been isolated (a tropical reservoir). Replicate cultures were not inoculated.
2. In both axenic and co-inoculated cultures, phytoplanktonic density and extracellular carbohydrate production were monitored microscopically and by high performance liquid chromatography with a pulse amperometric detector, respectively. Bacterial population density was also monitored by epifluorescence microscope in the microbial co-inoculated cultures.
3. Both bacterial and phytoplanktonic densities increased for 11 days in all cases. The use of extracellular carbohydrates by bacteria was also showed for all phytoplanktonic species. Of the three species of phytoplankton, only T. duostra had a faster population growth in the presence of bacteria, and reached a higher biomass than in axenic culture.     

Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.     

BACTERIAL INFLUENCE UPON GROWTH AND HYDROCARBON PRODUCTION OF THE GREEN ALGA BOTRYOCOCCUS BRAUNII1

Batch cultures of the hydrocarbon-rich alga Botryococcus braunii, Kütz. (axenic strains, non-axenic strains, associations with selected microorganisms) were examined with regard to total biomass and hydrocarbons at the onset of the stationary phase. Pronounced variations, related to the origin of the strains and to growth conditions, were observed with axenic cultures. It also appeared that the presence of microorganisms is not essential for high hydrocarbon production. Nevertheless, numerous bacteria were shown to exert considerable influence, antagonistic or beneficial, on B. braunii growth yield and hydrocarbon production. Such effects were strongly dependent on the species involved and on culture conditions. The presence of various microorganisms can influence not only the quantity of hydrocarbons produced, but also their level in the algal biomass and their relative abundance. However, their chemical structure is not affected. Intricate relationships were observed in B. braunii-bacteria systems and numerous factors (including, in some cultures, large positive effects due to bacterially produced CO₂) were implicated. Accordingly, specific associations should provide appropriate conditions for renewable hydrocarbon production via B. braunii large scale cultures.     

Selectivity of food bacteria for the growth of anaerobic ciliate Trimyema compressum

The anaerobic ciliate Trimyema compressum was cultivated on various food bacteria. Significant growth was observed when Lactobacillus sp., Escherichia coli, Enterobacter aerogenes, Desulfovibrio vulgaris, Methanoculleus bourgense, or Pelobacter propionicus cells were fed to the ciliates. The highest cell yield which we obtained was ca. 9,000 cells/ml when feeding D. vulgaris. However, no growth of the ciliates was observed on the culture with Clostridium novyi, Propionibacterium sp., Desulfobulbus propionicus, Methanobrevibacter arboriphilicus, Methanobacterium sp., Methanosarcina barkeri, or Methanothrix soehngenii cells. The ciliates produced acetate and methane as major end products in any cultures and small amounts of propionate, butyrate and hydrogen were also detected in some cultures. Physiological studies on the food bacteria which we tested indicated that the growth of T. compressum depended on the bacterial species, but there was no apparent correlation between the digestibility and the basic properties of those bacteria (i.e. size of the bacteria, gram-staining properties, susceptibility to the known lytic enzymes, Archaea or Bacteria).     

Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal sea of Japan

Noxious red tides of the dinoflagellate Cochlodinium polykrikoides tend to be long lasting and cause mass mortalities of cultured and natural fish and invertebrates along the western coast of Japan and the southern coast of Korea. In order to assess the tolerance of C. polykrikoides to attack by algicidal bacteria, the effects of algicidal bacteria strains on the growth of three C. polykrikoides strains were examined in laboratory culture experiments. Algicidal bacteria used were two strains of Cytophaga (J18/M01 and AA8-2, direct attack type and wide prey range), three strains of Alteromonas (S, K, D) and one strain of Pseudoalteromonas (R, indirect attack type), which were all isolated by using Chattonella antiqua as a prey organism. Neither Cytophaga strain showed any algicidal activity. In the cases of Alteromonas and Pseudoalteromonas, some cultures of C. polykrikoides were killed, but at least 10 days or more were required for the death of this dinoflagellate. C. polykrikoides survived in the presence of algicidal bacteria in concentrations up to 10⁶–10⁷ cells ml⁻¹, which is enough for other red tide microalgae to be killed. On the contrary, the algicidal effects of bacteria on C. antiqua were detected clearly within a few days. These results imply that C. polykrikoides is resistant to the six algicidal bacteria examined, which may reflect the capacity for mixotrophy. This resistance of C. polykrikoides to algicidal bacteria could provide a selective advantage for survival compared to other microalgae susceptible to attack by algicidal bacteria and hence prolong red tides caused by this harmful dinoflagellate.