The functional responses of two benthic algivorous ciliated protozoa with differing feeding strategies

Surface-associated algivorous ciliated protozoa are common in the benthos of streams, but little is known about the feeding ecology of these organisms. We compared the functional responses of two algivorous ciliated protozoa, Oxytricha fallax (a filter feeder) and Trithigmostoma cucullulus (an encounter feeder). The ciliates were fed ¹⁴C-labeled Navicula cryptocephala in laboratory feeding experiments to determine their potential to consume significant amounts of algal prey. Logistic regression, and plots of the proportion of N. cryptocephala ingested vs. the total number offered, indicated functional responses of a typical rectangular hyperbolic (type II) form for both ciliates. Ingestion rates were estimated from regressions of the number of ¹⁴C-labeled N. cryptocephala cells ingested per ciliate vs. time. Maximum feeding rates and half-saturation concentrations were estimated by fitting the observed ingestion rates and experimental algal densities to a function of the Michaelis-Menten enzyme kinetics form using nonlinear regression. For O. fallax, the maximum feeding rate was estimated to be 1.07 N. cryptocephala cells per minute, and the half-saturation concentration was 3.9 × 102 N. cryptocephala per square centimeter. For T. cucullulus the maximum feeding rate was estimated to be 0.2 N. cryptocephala per minute, and the half-saturation concentration was 5.4 × 10³ N. cryptocephala per square centimeter. The data were also fitted using only the number of cells ingested at 60 and 120 min, by converting the endpoint consumption to rates. For O. fallax, the estimated maximum feeding rates were 1.3 and 1.0 N. cryptocephala per minute for 60 and 120 min, respectively, and estimated half-saturation concentrations were 5.1 × 10² and 3.5 × 10² N. cryptocephala per square centimeter. For T. cucullulus, estimated maximum feeding rates were 0.6 and 0.4 N. cryptocephala per minute for 60 and 120 min, respectively, and estimated half-saturation concentrations were 1.5 × 10⁴ and 1.1 × 104 N. cryptocephala per square centimeter. These results suggest that kinetic methods for estimating ingestion rates are more accurate than endpoint determinations. Based on field observations of periphyton densities, these ciliates potentially are consuming 4.8% of the total available standing crop of diatom biomass per day and this could represent up to 16% of total available daily primary production.     

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances

Grazing controls bacterial abundances and composition in many ecosystems. In marine systems, heterotrophic flagellates (HFs) are important predators. Assemblages of HFs are primarily formed by species still uncultured; therefore, many aspects of their trophic behaviour are poorly known. Here, we assessed the functional response of the whole assemblage and of four taxa grown in an unamended seawater incubation. We used fluorescently labelled bacteria to create a prey gradient of two orders of magnitude in abundance and estimated ingestion rates. Natural HFs had a half-saturation constant of 6.7 × 10⁵ prey ml⁻¹, a value lower than that of cultured flagellates and within the range of marine planktonic bacterial abundances. Minorisa minuta was well adapted to low prey abundances and very efficient in ingesting bacteria. MAST-4 and MAST-7 were also well adapted to the typical marine abundances but less voracious. In contrast, Paraphysomonas imperforata, a typical cultured species, did not achieve ingestion rate saturation even at the highest prey concentration assayed. Our study, beside to set the basis for the fundamental differences between cultured and uncultured bacterial grazers, indicate that the examined predator taxa have different functional responses, suggesting that they occupy distinct ecological niches according to their grazing strategies and prey preferences.     

Influence of bacterial density and water temperature on the grazing activity of two freshwater ciliates

1. The influences of bacterial density and water temperature on the grazing activity of the ciliates Uronema sp. and Colpoda inflata were studied. The conditions assayed were two prey densities (10⁶ and 4 × 10⁷ bacteria ml^?1) and three water temperatures (10, 15 and 22 °C). 2. The response of the ciliates was measured from changes in protistan biovolumes and specific clearance rates. At high prey density, both ciliates showed lower biovolumes as water temperature increased, while at low prey density this tendency was minimized. 3. At the intermediate temperature of 15 °C both ciliates filtered ten times more body volume when bacteria were scarce; however, the ingested bacteria were fewer than at high prey density. At low prey density, a decrease from 15 to 10 °C evidenced different strategies of the two ciliates, which led to a similar ingestion of bacteria: C. inflata reduced its specific clearance rates and increased its biovolume, while Uronema sp. did not show changes. At high prey density, an increase from 15 to 22 °C caused lower biovolumes and a noticeable increase in specific clearance rates in both ciliates, indicating opportunist behaviour.     

Growth of Nitrosomonas europaea in batch and continuous culture

Summary A clear medium has been used to grow pur cultures of Nitrosomonas europaea in flasks and in a continuous culture apparatus.Of several metallic ions examined in flask cultures of Nitrosomonas, Fe at 2 ppm and Co, Mn and Zn at 1 ppm were not toxic, Ni and Cr at concentrations greater than 0.25 ppm inhibited growth and Cu stopped growth completely at 0.5 ppm and inhibited at 0.1 ppm. Stainless steel of the specification EN58 B did not affect growth.In the continuous culture vessel, Nitrosomonas showed a growth response to Fe only when the population exceeded about 500×10⁶ organisms/ml. The minimum doubling time was about 8 hours in flasks and 11 hours in the culture vessel. With effective aeration and automatic P_H control, cultures of Nitrosomonas were grown successfully in continuous culture and gave a yield of 2.14 g dry weight of bacteria from 30 litres of culture in 5 days.     

Drosophila cells can be grown to high cell densities in a bioreactor

Summary Cell cultures of the fruitfly, Drosophila melanogaster, were grown in a bioreactor to high cell densities: 5.7×10⁷ cells/ml, in batch, semi-batch and continuous flow modes. Using a semi-batch culture mode 500g wet weight of cell mass was produced in 16 days, using 5 l of cheap, commercially available, serum-free medium.     

The production of bacteriophage μ2

In three series of experiments, 3-l., 20-l., and 150-l. bacterial cultures were grown in stirred, deep culture vessels to average bacterial cell densities of 71 × 10⁸, 63 × 10⁸, and 43 × 10⁸ viable organisms per milliliter, respectively, and then infected with phage. The average yield of progeny phage in each case was ca. 3000 mpfu (minimum plaque-forming units) per cell. Thus, the average mass of phage obtained in the 3-l. experiments was not less than 124 mg./l., calculated from the plaque counts, assuming a particle size of 3.6 × 10⁶ Daltons for the μ2 phage. This is about twentyfold higher than is obtainable by conventional methods in aerated, shaken culture flasks. The actual phage yields are probably much higher than the minimum values calculated from plaque counts. For example, in the case of one of our culture lysates which was purified at King's College, the efficiency of plating was shown to be only 19%. The carbon dioxide evolution rate of cultures was measured and used as a guide to the time at which phage should be added. In this way, greater control of cultural conditions was obtained than is possible in shaken flasks. For the best yield of phage per milliliter of culture, the optimum time for phage infection was such that bacterial lysis just prevented the carbon dioxide evolution rate from reaching its potential maximum. The major factor influencing the phage yield per milliliter of culture was the aeration capacity of the culture vessel used. All had maximum aeration capacities much higher than those obtainable in shaken culture flasks. Cultures grown and infected in 3-l. Vessel operated under conditions of low aeration gave poor yields of phage. The reason for this are discussed.     

In vitro Decline in Plant Cultures: Detection of a Legion of Covert Bacteria as the Cause for Degeneration of Long-term Micropropagated Triploid Watermelon Cultures

In vitro cultures of triploid seedless watermelon cv. Arka Manik displayed a decline in shoot and root growth after 4–5 years of active culturing. Visibly clean cultures upon indexing on enriched media showed covert bacteria, and a significant improvement in proliferation and rooting in response to surface sterilization. The bacteria however survived endophytically. Low pH and reduced clarity of agar gelled medium were found to mask the expression of bacteria in the tissue culture medium. Gentamycin, streptomycin or broad-spectrum bactericide cefazolin provided as a 2 ml overlay in the medium in factorial combination at 0 or 50 mg l^–1 resulted in selective suppression of some bacteria depending on the treatment and eight bacterial clones comprising of four Gram-positive (Bacillus spp.) and four-Gram negative (3 × Pseudomonas spp. and 1 × Aeromonas sp.) strains were isolated from the cultures. Provision of 50 mg l^–1 gentamycin in 2 ml overlay in the multiplication or rooting medium coupled with occasional decontamination of cultures helped in circumventing the decline problem. The plants established in the field after 6 years of active in vitro culturing appeared normal and fertile suggesting the feasibility of keeping cultures for long periods, thus saving time and other resources. Freeing the cultures from covert bacteria was complicated by the presence of different bacterial types and this will be addressed later.     

Control of N-acetylglucosaminidase specific activity in myxamoebae of Dictyostelium discoideum

Myxamoebae of Dictyostelium discoideum were grown with Aerobacter aerogenes as substrate, on nutrient agar plates or in shaken culture, with mean doubling times that varied between 3 and 6 hr. Growth in axenic culture was with mean doubling times of 5–6 or 8 hr. The specific activity of N-acetylglucosaminidase was three to four times higher in myxamoebae grown axenically than in myxamoebae grown with A. aerogenes and there was no correlation between enzyme specific activity and myxamoebal growth rate. High specific activities of N-acetylglucosaminidase were also found in myxamoebae grown with gram-positive bacteria (Bacillus megaterium, Staphylococcus lactis) as substrate while low specific activities were found in myxamoebae grown with the gram-negative bacteria Acinetobacter lwoffi and Pseudomonas aeruginosa as well as with A. aerogenes. A preparation of the cell envelope of A. aerogenes was nearly as effective as the intact bacteria at depressing myxamoebal N-acetylglucosaminidase specific activity. Lipopolysaccharide extracted from the cell envelope of gram-negative bacteria, or the lipid A component of the lipopolysaccharide, also depressed N-acetylglucosaminidase specific activity when added to axenic cultures of myxamoebae.     

Enhanced Production of Human Recombinant Proteins from CHO cells Grown to High Densities in Macroporous Microcarriers

Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q _P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q _P with increasing cell densities up to a threshold of >1 × 10⁸ cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q _P any further. A positive linear correlation (r ² = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 10⁶ to 3 × 10⁸ cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.     

Characteristics of mixotrophic growth of Synechocystis sp. in an enclosed photobioreactor

Synechocystis sp. PCC 6803 was grown in a 2.5 l enclosed photobioreactor on medium with or without glucose. The incident light intensities ranged from 1.5 klux to 7 klux. The highest average specific growth rates of mixotrophic culture and photoautotrophic culture were, respectively, 1.3 h^–1 at a light intensity of 7 klux on 3.2 g l^–1 glucose and 0.3 h^–1 at both light intensities of 5 klux and 7 klux. The highest cell density 2.5 g l ^–1 was obtained at both of light intensities 5 klux and 7 klux on 3.2 g glucose l^–1. Glucose consumption decreased with decreasing light intensity. The energy yields of mixotrophic cultures were 4 to 6 times higher than that of photoautotrophic cultures. Light favored mixotrophic growth of Synechocystis sp. PCC 6803, especially at higher light intensities (5–7 klux).     

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10² to 4.6×10²/ml for seawater, 4.0×10⁴ to 4.3×10⁵/g wet wt for eelgrass-bed sediment, and 2.1 × 10⁵ to 1.2 × 10⁷/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.     

The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis

Summary Suspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10^-6M 2,4-D. Transfer of cells to media with 10^-5M kinetin or benzyladenine and no auxin or 10^-7M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10^-5M kinetin and with 10^-7 M NAA or 5×10^-5M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10^-5M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10^-7M but not at 10^-6M. At 10^-7M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4–8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.Issued as NRCC No. 11388.Abbreviations used: 2,4-D=2,4-dichlorophenoxyacetic acid, NAA + -naphthaleneacetic acid.     

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

Summary We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10⁷ protoplasts.ml^–1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10⁶ protoplasts.ml^–1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.     

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.     

Effects of temperature and food level on growth and development of a planktonic water mite

We analysed the relative effects of food availability and temperature on rates of growth and development of a predatory planktonic water mite, Piona exigua. Growth in length of mites fed Daphnia, Ceriodaphnia and Chydorus was analysed by Gompertz or von Bertalanffy curves; these curves were compared by parallel curve analysis. Growth rates of nymphs and adult female mites increased with temperature; the duration of the imagochrysalis stage decreased. Females grown at 10 °C were smaller at final size than females grown at 15 °C, 18 °C or 22 °C. Females reared at food levels of 15 or 30 prey l⁻¹ grew more slowly and were smaller than those provided with 60 or 120 prey l⁻¹. Nymphs grew more slowly when Daphnia were the only prey, than when smaller prey were available. Food level did not affect nymph growth at 10 °C or 15 °C, but growth at 18 °C or 22 °C may have been slowed at the lowest food levels. Synergistic effects of temperature and food level on nymph growth were apparent only from analysis of growth curves and not from stage duration data.     

Dynamics of cyanophage-like particles and algicidal bacteria causing Microcystis aeruginosa mortality

The dynamics of cyanophage-like particles and algicidal bacteria that infect the bloom-forming cyanobacterium Microcystis aeruginosa was followed in a hyper-eutrophic pond from September 1998 to August 1999. The densities of M. aeruginosa ranged between 4.0 × 10⁵ and 1.9 × 10⁷ cells ml⁻¹, whereas those of algicidal bacteria were between 4.0 and 5.1 × 10² plaque-forming units (PFU) ml⁻¹ and those of cyanophage-like particles were between <5.0 × 10² and 7.1 × 10³ PFU ml⁻¹. A significant relationship was found between the densities of algicidal bacteria and M. aeruginosa (r = 0.81, n = 69, P < 0.001), suggesting that the dynamics of the algicidal bacteria may regulate the abundance of M. aeruginosa. Occasional peaks of density of cyanophage-like particles were detected in October, June, and August, when sharp declines in M. aeruginosa cell densities were also observed. The densities of cyanophage-like particles became undetectable when the abundance of M. aeruginosa was low, suggesting the density-dependent infection of M. aeruginosa by cyanophage-like particles. Thus, we suggest that infections of both algicidal bacteria and cyanophage-like particles are important biological agents that decompose blooms of M. aeruginosa in freshwater environments. Received: August 31, 2000 / Accepted: December 6, 2000     

Improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection

The extracellular domain of the epidermal growth factor receptor (EGFR) was expressed using the baculovirus expression vector system. The maximum level of the EGFR extracellular domain secreted into the medium in Sf-9 (Spodoptera frugiperda or fall armyworm) cell batch culture was approximately 2.5 g ml^–1. In order to increase this yield, a process was developed that included the following sequence of steps: batch growth to maximum cell density, infection of the cells with recombinant virus, and replacement of spent medium. By using this process, the specific yield of recombinant protein, which in batch culture drops when infection is carried out at densities greater than 3 × 10⁶ cells ml^–1, can be maintained at a maximum in cultures infected at densities of 10⁷ cells ml^–1 or greater. The process, when applied to 3-1 and 11-1 bioreactor cultures, allowed a maximum volumetric yield of triple the maximum value attainable in batch culture. Spent-medium analysis indicates that medium replacement provides certain nutrients that could otherwise be limiting for recombinant protein production.     

Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters

Somatic embryo cultures of Picea mariana and the species complex P. glauca-engelmannii were each grown in 7.5-l-capacity mechanically-stirred bioreactors containing 61 medium (LP, von Arnold and Eriksson) with 30 mm sucrose. Growth of both species occurred with no observable signs of shear stress due to mechanical agitation. Growth kinetics were analysed using an array of parameters (settled culture volume, packed culture volume, osmolarity, conductivity, pH). These were compared with fresh weight, dry weight, and somatic embryo number in order to determine what parameters were highly correlated with growth and embryo number. Increasing the sucrose concentration from 30 mm to 60 mm resulted in an increase in biomass and total number of somatic embryos. For P. mariana a maximum dry weight of 6.3 gl^–1 and 3076 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 10–12 days of culture. For P. glauca-engelmannii a maximum dry weight of 4.3 gl^–1 and 2278 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 6–8 days culture. For all sucrose concentrations, fresh weight, dry weight and embryo number were closely correlated with packed culture volume and conductivity for P. mariana, and settled culture volume, packed culture volume and conductivity for P. glauca-engelmannii.Correspondence to: D. I. Dunstan     

A microcarrier culture method for the production of large quantities of viable Chlamydia pneumoniae

 We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on microcarrier beads. Infection of the cells in microcarrier culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption. The yield in microcarrier culture was similar to that of microtitre-plate culture using centrifugation-assisted infection (120×10⁶ and 225×10⁶ bacteria/10⁶ HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively). However, up to 64% savings in labour time and 27% savings in culture medium were achieved if the microcarrier culture method was used instead of the microtitre-plate culture method. The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24–1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection or PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35°C or 37°C). The yield of microcarrier cultures was the same at an incubation temperature of 35°C or 37°C in contrast to an increased production at 35°C in the microtitre-plate culture method using centrifugation-assisted infection. In conclusion, the microcarrier culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically. Received: 27 December 1995/Received revision: 4 April 1996/Accepted: 15 April 1996     

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.