Unusual bloom of star-like prosthecate bacteria and filaments as a consequence of grazing pressure

In seawater used for shrimp aquaculture in French Polynesia, the grazing of small bacteria (rods and coccoids) allowed the growth ofAncalomicrobium cells (to more than 2×10⁶ cells ml^–1) and large filaments > 10m in length (5×10⁶ cells ml^–1). Their contribution to the increase in total bacterial number after grazing was 27.8 and 9.8%, respectively. These large bacteria are not grazed on by microflagellates, but are available for mesoplankton larvae.     

Tris not only controls the pH in microalgal cultures,but also feeds bacteria

Tris (Tris(hydroxymethyl)amino methane), a compound often used as a buffer in microalgal culture media, sustains active bacterial growth in non-axenic microalgal cultures when sodium phosphate is present. The low pH levels caused by bacterial growth and probably the depletion of phosphorus in the medium caused the collapse ofPhaeodactylum tricornutum cultures resulting in a reduction of microalgal growth from 32 x 10⁶ to 1.1 x 10⁶ cells ml^–1. This emphasizes the need for care when interpreting the results of non-axenic microalgae cultures in which Tris or other organic buffer is added.     

Recovery Patterns of Moina Macrocopa Exposed Previously to Different Concentrations of Cadmium and Methyl Parathion: Life-Table Demography and Population Growth Studies

In most toxicity studies using Cladocera, bioassays are routinely done to determine median lethal concentration (LC₅₀) or the responses to sublethal exposure. However, information on the patterns of recovery of cladocerans exposed to different concentrations of toxicants is scarce. This is important because cladocerans exposed to toxicants for a short duration may later recuperate under favourable conditions. Using the life table demographic and population growth, the present study was conducted to evaluate the recovery patterns of Monia macrocopa exposed to five different concentrations (0, 25, 50, 75 and 100% of 24 h LC₅₀ for CdCl₂ or methyl parathion) and then returned to toxicant-free medium containing alga (Chlorella vulgaris) at low (0.25 × 10⁶cells ml^–1), medium (0.5 × 10⁶cells ml^–1) or high (1 × 10⁶cells ml^–1) levels. We measured selected life history variables such as average lifespan, life expectancy at birth, gross and net reproductive rates, generation time and the rate of population increase. Results indicated that regardless of food concentration, surviving individuals of M. macrocopa exposed to a median lethal concentration did not recover. The effect of food level was significant at 25 and 50% of the median lethal concentration for cadmium or methyl parathion. Age-specific fecundity curves showed that exposure to either toxicant for a duration as short as 24 h at one-fourth of the LC₅₀ showed reduced output of offspring, especially at a lower food level. At and above exposures of 0.037 mgl^–1 of methyl parathion, no reproduction occurred. The highest gross and net reproductive rates (127 and 55 offspring female^–1) were obtained in controls at the high (1 × 10⁶ cells ml^–1) algal food level. The rate of population increase obtained from life table data was around 0.7 per day in controls but decreased when exposed to toxicant concentrations. The rates of population increase per day derived from population growth data varied from 0.22 to 0.33 per day for the controls, depending on the food levels.     

Life table demography and population growth of Brachionus variabilis Hempel, 1896 in relation to Chlorella vulgaris densities

We studied the life history variables and population growth characteristics of Brachionus variabilis, which was recorded for the first time from Mexico. The animals were fed Chlorella, using five concentrations (0.25, 0.5, 1, 2 and 4 × 10⁶ cells ml^–1) at 25 °C. Food density was observed to have significant effect on life expectancy, average lifespan, gross reproductive rate, net reproductive rate, generation time and population growth rate. The average lifespan ranged from 3 to 6 days depending on the food density. The net reproductive rate ranged from 2 to 7 neonates female^–1 d^–1. The rate of population increase per day varied from 0.14 to 0.35. The highest net reproductive rate and average lifespan and life expectancy were recorded at Chlorella concentrations of 1 × 10⁶ and 2 × 10⁶ cells ml^–1.     

Callus and suspension cultures for biomass production ofCynara cardunculus (Compositae)

Summary Friable calli, obtained from hypocotyl and leaf segments ofCynara cardunculus, were used for the production of cell suspension cultures in liquid Gamborg B₅ medium supplemented as for callus obtention. The low inoculum cell suspension cultures (6.9×10⁴ cells.ml^–1) presented a td=136.8 hours while those obtained with a high inoculum (15.6×10⁴ cells.ml^–1) reached a td=33.6 hours.     

Population growth of Lepadella patella (O. F. Müller, 1786) at different algal (Chlorella vulgaris) densities and in association with Philodina roseola Ehrenberg, 1832

Population growth of Lepadella patella was studied using Chlorella as the sole food at five concentrations ranging from 0.25 × 10⁶ to 4.0 × 10⁶ cells ml^–1 at 25 °C for 22 days. The population densities increased with increasing algal concentration up to 1.0 × 10⁶ cells ml^–1. The population growth of L. patella was lower at algal concentration of 2.0 × 10⁶ cells ml^–1 and above. In a separate experiment, we tested the influence of the bdelloid rotifer Philodina roseola on the population growth of L. patella at different ratios of initial inoculation densities using 1.0 × 10⁶ cells ml^–1 of Chlorella at 28 °C. Despite lower initial inoculation densities compared with those in the controls, both L. patella and P. roseola showed higher peak abundances when grown together. The maximum peak abundance values recorded for L. patella and P. roseola were 830 and 230 ind. ml^–1, respectively, at an inoculation ratio of 1:1.     

Improvement of the production of 5-methoxypodophyllotoxin using a new selected root culture of Linum flavum L.

Differences in transformation of the tomato cultivar (Ohio 7870, Roma, UCD82b) by wild-type Agrobacterium strains (A6, A66, A281) were identified in a leaf disk assay system. Transformation was expressed as the percentage of explants producing callus on hormone-free medium and was confirmed by opine production. Ohio 7870 and Roma were more readily transformed than UCD82b by all three strains of A. tumefaciens. Cotyledons and older true leaves of all three cultivars were more readily transformed than younger leaves. Transformation was biphasic over the bacterial concentrations tested (2×10³–7×10⁹ colony forming units ml^-1; cfu ml^-1) for all cultivars and leaf ages, and was greatest at 5×10⁸ cfu ml^-1. Transformation decreased significantly at levels less than 2×10⁷ cfu ml^-1 and slightly at concentrations higher than 5×10⁸ cfu ml^-1. UCD82b tissue was more necrotic than Ohio 7870 or Roma after incubation with bacteria, which may account for reduced transformation of this cultivar.     

Abundance and growth response of microalgae at Megalon Embolon solar saltworks in northern Greece: An aquaculture prospect

There is continuous interest in many countries in maintaining and manipulating the rich ecological value of hypersaline ecosystems for aquaculture. The Megalon Embolon solar saltworks (northern Greece) were studied in sites of increasing salinity of 60–144 ppt to evaluate Dunaliella salina abundance and microalgal composition, in relation to physical and chemical parameters. Cluster and ordination analyses were performed based on the biotic and abiotic data matrices. Using fresh aliquots from 60 and 140 ppt salinity waters, phytoplankton performance was appraised with flask cultures in the laboratory by varying the inorganic PO₄-P concentration at 23 °C and 30 °C. At the saltworks, among the most abundant microalgae identified were species of the genera Dunaliella, Chlamydomonas, Amphora, Navicula, and Nitzschia. Dunaliella salina populations were predominant comprising 5–22% of the total microalgal assemblages during spring, but only 0.3–1.0% during the summer, when grazing by Artemia parthenogenetica and Fabrea salina was intense. D. salina cell density in April–July was in the range of 0.4–12.5 × 10⁶ L⁻¹ with typical densities of 1.5–4.5 × 10⁶ L⁻¹. Overall, microalgal densities were high in salinities of ≥100 ppt when inorganic-P concentrations were ≥0.20 mg L⁻¹ within saltworks waters. Multivariate analysis of species abundance showed that algal growth responses were primarily related to variation in salinity and inorganic-P concentrations, but also to NO₃-N concentration. In the laboratory, experiments indicated effective fertilization and denser microalgal growth under high inorganic PO₄-P applications (4.0 and 8.0 mg L⁻¹) at 60 ppt salinity and 23 °C. The lower PO₄-P applications (0.6–2.0 mg L⁻¹) were more effective at 60 ppt salinity and 30 °C. At 140 ppt salinity, microalgal growth response was less obvious at any of the corresponding phosphorus concentrations or temperatures. In both salinity experiments, Dunaliella salina bloomed easily and was predominant among the microalgae. Our observations indicate that Dunaliella salina populations and the overall rich microalgal profile of the saltworks, along with their performance in laboratory mono–and mixed cultures hold promise for mass cultivation within the M. Embolon saltworks basins.     

Optimal cell density and multiplicity of infection for the propagation of human rotavirus in monkey kidney cells

The human rotavirus titer was optimal at an infection cell density of about 4–8 × 10⁴ cells cm^–2 in monkey kidney cell cultures. The highest viral titers (3.8 × 10⁷ TCID₅₀ ml^–1 and 3.7 × 10⁷ TCID₅₀ ml^–1) were obtained at an multiplicity of infection of 0.05 in well plate and T-flask, respectively.     

Dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells

Production of a TGF receptor with high density baculovirus infected Sf-9 cells (7×10⁶cells ml^-1) served as a test run for a retrofitted 150 L microbial fermentor. The entire 110 L batch run was performed in serum free medium, with an addition of a concentrated amino acid and yeastolate mixture at the time of infection. This addition strategy has been proven effective at a small scale by enabling cultures to maintain maximum product yield. In the bioreactor however, while cellular growth was comparable to that of the smaller scale control, TGF receptor production was three fold below the control. To minimize the mechanical stress, low flow rate of pure oxygen was used to control the dissolved oxygen at 40%. As a consequence, it seems that this aeration strategy involved an accumulation of dissolved carbon dioxide that in turn inhibited the protein production. A model has been developed that estimated the CO₂ partial pressure in the culture to be in the vicinity of 0.15 atm. The effect of dissolved CO₂ at this concentration has been assessed at smaller scale for TGF receptor and -gal expression, in controlled atmosphere incubators.Abbreviations CST cumulative sparging time (s) - dpi days post-infection (d) - CO₂/O₂ diffusivities ratio (=0.784 at 27 °C) - DO % saturation of dissolved oxygen (%) - hpi hours post-infection (h) - He Henry coefficient (He_{CO 2}=32.1×10^-3M atm^-1, He_{O 2}=1.23×10^-3 M atm^-1 at 27 °C) - k_La volumetric liquid-side mass transfer coefficient (h^-1) - LDH Lactate dehydrogenase - MOI multiplicity of infection (pfu cell^-1) - N agitation speed (rpm) - OTR oxygen transfer rate (mole O₂ ml^-1 h^-1) - OUR oxygen uptake rate (mole O₂ ml^-1 h^-1) - p partial pressure (atm) - P total pressure (atm) - pfu plaque forming units - q specific consumption or production rate (mole cell^-1 h^-1) - Q_{H, OUT} headspace outlet gas flow rate - Q_S,NOM nominal volumetric sparged gas flow rate (92 ml s^-1 at bioreactor conditions) - R ideal gas constant (82.05 ml atm mole^-1 K^-1) - SR_{V L} molar sparging rate per unit liquid volume (mole ml^-1 h^-1) - SSR specific sparging rate (mole cell^-1 h^-1) - T temperature (C or K) - V_L culture volume (ml) - VVD volume of feed per volume of culture per day (d^-1) - X cell concentration (cell ml^-1) - Y yield coefficient Indexes CO₂, O₂ related to CO₂ or O₂, respectively - glc glucose - H headspace gas phase or gas/liquid interface - L liquid phase - lact lactate - S sparged phase or gas/liquid interface - V viable     

Induction of diapausing amictic eggs in Synchaeta pectinata

Amictic females of a clone of S. pectinata from Star Lake (Norwich, Vermont) may produce diapausing as well as non-diapausing (subitaneous) eggs. The proportion of diapausing eggs produced in cultures was unaffected by temperature (12 vs 19 °C) or rotifer population density (minima of 0.33 vs 3 ind. ml^–1) at 19 °C. However, at 19 °C this proportion was higher in cultures maintained at a low food level suppressing reproduction (5 × 10³ cells ml^–1 Cryptomonas erosa) than in those maintained at a high food level (2 × 10⁴ cells ml^–1); the treatment effect was marginally significant (p=0.067). Consistent with the effect of low food availability, a period of starvation was very effective in inducing the development of diapausing eggs. None of 19 females cultured individually from hatching at 19 °C on C. erosa (2 × 10⁴ cells ml^–1) in 1-ml volumes produced any diapausing eggs in 4 days (0 out of 349 eggs), while 13 out of 16 females subjected to a 15-hour starvation period 6 hours after birth produced one or more diapausing eggs during that time (34% of the 158 eggs produced by the 16 females were diapausing). Diapausing eggs produced and left at 19 °C hatched after 4 to 13 days. Those produced in cultures with a low food level took significantly longer to hatch (9.7 days) than those produced in cultures with a high food level (8.1 days) (p=0.022). In natural communities, S. pectinata should be able to respond directly and rapidly to poor food conditions by producing eggs that undergo an obligatory dormant period before resuming development.     

Growth pattern and cellular nitrogen and phosphorus contents of the dinoflagellate Peridinium penardii (Lemm.) Lemm. causing a freshwater red tide in a reservoir

The population growth pattern and related changes in both the nitrogen and phosphorus contents in the cell of the dinoflagellate Peridinium penardii (Lemm.) Lemm., which formed a freshwater red tide in a reservoir, were studied in situ. An exponential increase with time in population density was found. A specific growth rate of 0.25 d^–1 was observed. The cellular content of phosphorus per cell decreased from 6.0 × 10^–5 µg to 9.2 × 10^–6 µg along with an increase in population density from 8.0 × 10² cells ml^–1 to 2.5 × 10⁴ cells ml^–1. A prominent change in the cellular nitrogen did not occur. Decreasing cell content and continuous uptake of phosphorus were advantageous for P. penardii to form a freshwater red tide under P-limited conditions.     

A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta, Ulvales)

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl₂ was developed for isolating rapidlyprotoplasts from different species of Monostroma,Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×10⁶ to 10.2 × 10⁶ cells g^–1f. wt thallus, and forEnteromorpha from 3.48 × 10⁶ to 11.7× 10⁶ cells g^–1 f. wt and forUlva from 4.58 × 10⁶ to 26.8 ×10⁶ cells g^–1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates.     

Development of in vitro procedures for regeneration of petiole and leaf expiants and production of protoplast-derived callus in Tanacetum vulgare L. (Tansy)

Tanacetum vulgare (Tansy) was established in vitro on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) using shoot tips and embryos. From petiole expiants 93% formed callus, and 27% produced shoots on MS medium containing 4.5 mg l^-1 NAA and BAP. NAA alone induced root formation from leaf expiants. Up to 7 ×10⁶ viable protoplasts were obtained by macerating 1 g of leaves in 0.5 % Macerozyme R-10, 1.0% Cellulase R10, and 1.0% Cellulysin. Cell division was observed 3–4 days after protoplast isolation at the optimum plating density of 0.2-0.4×10⁶ cells ml^-1. A total of 350 protoplast-derived calluses were produced on which nodules with meristematic zones developed. Roots regenerated on MS medium supplemented with BAP 3.0 mg 1^-1, NAA 2.0 mg l^-1, and 250 mg l^-1 casein hydrolysate, however no shoots have been obtained yet.Abbreviations BAP 6-benzylaminopurine - CH casein enzymatic hydrolysate - 2.4 D dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellic acid - IBA indole butyric acid - IPA 6-dimethylallylamino purine - KIN Kinetin - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

The maintenance ofBdellovibrio at low prey density

A mathematical model for the interaction ofBdellovibrio and its prey predicted that a relatively high prey density (7×10⁵ cells ml^–1) would be required for the establishment of an equilibrium in a mixed population [8]. The present report shows thatBdellovibrio can be maintained in a continuous culture when the prey cell density is much lower (2–5×10⁴ cells ml^–1), and closer to that of naturally occurring bacterial populations in sea waters.     

Microbial diversity of Minnesota peatlands

Microbial diversity, numbers, and metabolic activities in Minnesota peatlands were investigated using a variety of microbial enrichment and enumeration procedures together with radioisotopic measurements of microbial degradative processes. Minnesota peatlands were shown to contain large microbial populations of wide metabolic diversity. Direct counts of bacteria using epifluorescence microscopy indicated bacterial populations of about 10⁸ ml^–1 of peatland water, irrespective of depth. Radioisotopic most-probable-number (MPN) counts of heterotrophs able to mineralize¹⁴C-labeled substrates to¹⁴CO₂ showed significant populations of glucose degraders (10⁴–10⁶ ml^–1) as well as degraders of benzoate (10²–10³ ml^–1), 2,4-dichlorophenoxyacetate (10²–10⁵ ml^–1), and sphagnum (10³–10⁷ ml^–1) in the various peatlands examined. The MPNs of NO₃ ^– reducers varied from 10³–10⁶ ml^–1, SO₄ ^– reducers from 10²–10³ ml^–1, methanogenic bacteria from 10³–10⁶ ml^–1, and methane oxidizers from 10³–10⁴ ml^–1, depending on sampling site and depth. Eighty pure cultures of aerobic bacteria and fungi were isolated from Minnesota peats. Most of those cultures tested were able to grow on at least 20 organic compounds (carbohydrates, aromatic molecules, hydrocarbons, etc.) as sole sources of carbon and energy. One isolate, aBacillus, was able to fix atmospheric N₂. Several of the isolates were able to mineralize¹⁴C-labeled lignin.     

Food dependence and energetics of freeliving nematodes

Summary The food dependence of larval duration, fecundity and the intrinsic rate of natural increase follow a hyperbolic form, which can for the former be described by the Michaelis-Menten function.Maximal larval duration at 20° C is 62 h, maximal fecundity is 153 eggs per female and r _max is 1.136 per day. The lower food threshold is 10⁸ E. coli cells·ml^-1 (=0.06 mg dry weight·ml^-1) for larval growth and 2·10⁸ cells·ml^-1 for reproduction and r. 50% of maximal performances (K _s ) are attained at 5·10⁸ and 7.5·10⁸ cells·ml^-1 respectively.Reproductive effort at dense food is highest immediately after maturation (e.g. 50% of the total eggs produced by a female are laid within 2 days after onset of egg production). At lower food densities the reproductive effort is delayed.Larval mortality increases strongly below 10⁹ cells·ml^-1.The results reported sofar were obtained with E. coli cells which were harvested at the phase of decreasing population growth in batch cultures. With cells from the exponential and the stationary phase, performances are increased and decreased respectively. This is partly due to differences in bacterial biomass per unit cell, partly an expression of the change of nutritive value of bacterial cells with growth phases.     

Brachionus calyciflorus Pallas as agent for the removal of E. coli in sewage ponds

Brachionus calyciflorus (Pallas) is a common brachionid in sewage oxidation ponds. The uptake and assimilation of E. coli was optimal at concentrations of 2.7–6.9 × 10⁸ cells ml^–1 while assimilation coefficient per body weight of B. calyciflorus was found to be 10% · Ind.^–1 d^–1. More than two eggs per individual were produced during 24 hours when brachionids were fed with a mixutre of E. coli (10⁹ cells · ml^–1) and Chlorella spp. (10⁶ cells · ml^–1). The nutritional value of the mixture of E. coli and Chlorella spp. was found to be higher than that of bacteria alone.     

Acute and chronic toxicity of the pesticide methyl parathion to the rotifer Brachionus angularis (Rotifera) at different algal (Chlorella vulgaris) food densities

Methyl parathion is a commonly used insecticide in Mexico to eradicate insect pests. We evaluated the effects of this insecticide on rotifer B. angularis using both acute and chronic toxicity tests. Median lethal concentration (LC₅₀) of methyl parathion for B. angularis for a 24-h bioassay in the presence and absence of an algal diet was derived. Elevated LC₅₀ due to the survival of a greater number of test individuals in the presence of food was observed. Regardless of the toxicant concentration, population growth curves of the animals maintained at the low food level (0.75×10⁶ cells ml^-1) had a longer lag phase than those at the high food level (1.5×10⁶ cells ml^-1). Regardless of food level, an increase in the toxicant concentration in the medium resulted in decreased population growth. The lowest peak population density (50 ind. ml^-1) was observed at the highest toxicant concentration and the lower food level. The highest population density (200 ind. ml^-1) was observed in the controls at high food level. The rates of population increase per day (r) in the controls were higher (from 0.14 to 0.37 depending on the food level). Irrespective of food level, there was a decrease in the r values with increasing pesticide concentration in the medium. In order to detect the effect of population density on the growth rates in relation to the toxicant stress, we plotted the daily growth rate against initial density for the entire duration of the experiment. We observed the existence of a significantly inverse relation at all treatments except at the low food level and high toxicant concentrations (0.625 and 1.25 mg l^-1). We discuss the role of algae in the toxicity of methyl parathion to zooplankton.