Protozoan Bacterivory in the Ice and the Water Column of a Cold Temperate Lagoon

Abstract Bacterial abundance and bacterivorous protist abundance and activity were examined in ice-brine and water column communities of a cold temperate Japanese lagoon (Saroma-Ko Lagoon, Hokkaido, 44°N, 144°E), during the late winter phase of ice community development (February–March 1992). Bacterial abundance averaged 6 and 1 × 10⁵ cells ml⁻¹ in the ice-brine and plankton samples, respectively, and generally decreased during the sampling period. Bacterivorous protists, identified based on direct observation of short-term (<1 h) ingested fluorescently labeled bacteria (FLB) in their food vacuoles, were largely dominated by flagellates, mainly cryothecomonad-type and chrysomonad-like cells and small dinoflagellates of the genus Gymnodinium. Bacterivorous ciliates included mainly the prostomatid Urotricha sp., the scuticociliates Uronema and Cyclidium, the choreotrichs Lohmaniella oviformis and Strobilidium, and the hypotrich Euplotes sp. Protist abundance averaged 4 × 10³ and 8.1 cells ml⁻¹ in the ice-brine and 0.3 × 10³ and 1.2 cells ml⁻¹ in the plankton, for flagellates and ciliates, respectively. In contrast to bacteria, the abundance of protists generally increased throughout the sampling period, indicating predator–prey interactions. Protistan bacterivory, measured from the rate of FLB disappearance over 24 h, averaged 36% (ice) and 24% (plankton) of bacterial standing stock and exhibited the same seasonal pattern as for protist abundance. The calculated specific clearance (range, 2–67 nl protozoa⁻¹ h⁻¹) and ingestion (<1–26 particles protozoa⁻¹ h⁻¹) rates were likely to be minimal estimates and grazing impact may have been higher on occasion. Indications for the dependence of ``bacterivorous protists' on nonbacterial food items were also provided. Although alternative sources of bacterial loss are likely to be of importance, this study provides evidence for the potential of protozoan assemblages as bacterial grazers in both sea ice-brine biota and water column at the southern limit of sea ice in the northern hemisphere. Received: 30 July 1998; Accepted: 18 November 1998     

Trophic interactions among heterotrophic microplankton,nanoplankton, and bacteria in Lake Constance

A considerable portion of the pelagic energy flow in Lake Constance (FRG) is channelled through a highly dynamic microbial food web. In-situ experiments using the lake water dilution technique according to Landry & Hasset (1982) revealed that grazing by heterotrophic nanoflagellates (HNF) smaller than 10 µm is the major loss factor of bacterial production. An average flagellate ingests 10 to 100 bacteria per hour. Nano- and micro-ciliates have been identified as the main predators of HNF. If no other food is used between 3 and 40 HNF are consumed per ciliate and hour. Other protozoans and small metazoans such as rotifers are of minor importance in controlling HNF population dynamics.Clearance rates varied between 0.2 and 122.8 nl HNF^–1 h^–1 and between 0.2 and 53.6 µl ciliate^–1 h^–1, respectively.Ingestion and clearance rates measured for HNF and ciliates are in good agreement with results obtained by other investigators from different aquatic environments and from laboratory cultures. Both the abundance of all three major microheterotrophic categories — bacteria, HNF, and ciliates — and the grazing pressure within the microbial loop show pronounced seasonal variations.     

Grazing by the Antarctic sea-ice ciliate Pseudocohnilembus

Potential uptake and clearance rates of fluorescent microspheres (FM) from 0.25 to 4.05 μm diameter were determined for the non-loricate ciliate Pseudocohnilembus sp. from Antarctic sea ice. The percentage of ciliate cells that ingested FM after 20 min incubation decreased with increasing particle diameter. Pseudocohnilembus sp. ingested FM between 0.25 and 4.05 μm in diameter. We offered FM at concentrations less than natural concentrations for plankton plus detrital material and obtained clearance rates less than those previously reported for bactivorous ciliates. Clearance rates were 3.6–5.4 nl cell⁻¹ h⁻¹ for FM 0.5 and 1 μm diameter, respectively, but decreased to 1.1 nl cell⁻¹ h⁻¹ for 1.97 μm diameter and 1.4 nl cell⁻¹ h⁻¹ for 4.05-μm-diameter FM. Clearance and uptake rates of FM 0.5 and 1 μm diameter indicate that Pseudocohnilembus sp. principally grazes on bacteria-sized particles. However, it can also ingest organisms as large as nanoplankton and may graze particles as small as femtoplankton and colloids. This suggests a feeding strategy that may suit the temporal and spatial changes in food availability in the sea-ice habitat. Accepted: 13 August 2000     

Pelagic Ciliates in a Large Mesotrophic Lake: Seasonal Succession and Taxon‐Specific Bacterivory in Lake Constance

The taxonomic composition of the ciliate assemblage and their taxon‐specific bacterial grazing rates in Lake Constance were investigated over the course of one year. Bacterial grazing rates were measured using natural fluorescently labelled bacteria (FLB) and compared to bacterial production. Small species such as Balanion planctonicum/Urotricha furcata and Rimostrombidium spp./Halteria sp. were the most numerous ciliates on the annual average. Larger ciliates such as Rimostrombidium lacustris and Limnostrombidium spp. contributed significantly to total ciliate biomass, but were relatively unimportant as bacterial grazers. Per capita ingestion rates ranged from 0–194 bacteria ciliate⁻¹ h⁻¹ and changed seasonally up to a hundredfold within a given taxon. Approximately 1% of the bacterial production were removed by the ciliate community on the annual average. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Benthic Bacterial Production and Protozoan Predation in a Silty Freshwater Environment

The interrelation of heterotrophic bacteria with bacterivorous protists has been widely studied in pelagic environments, but data on benthic habitats, especially in freshwater systems, are still scarce. We present a seasonal study focusing on bacterivory by heterotrophic nanoflagellates (HNF) and ciliates in the silty sediment of a temperate macrophyte-dominated oxbow lake. From January 2001 to February 2002 we monitored the standing stock of bacteria and protozoa, bacterial secondary production (BSP, ³H-thymidine, and ¹⁴C-leucine incorporation), and grazing rates of HNF and ciliates on bacteria (FLB uptake) in the oxic sediment of the investigated system. BSP ranged from 470 to 4050 µg C L^–1 wet sediment h^–1. The bacterial compartment turned out to be highly dynamic, indicated by population doubling times (0.6–10.0 d), which were comparable to those in the water column of the investigated system. Yet, the control mechanisms acting upon the bacterial population led to a relative constancy of bacterial standing stock during a year. Ingestion rates of protozoan grazers were 0–20.0 bacteria HNF^–1 h^–1 and 0–97.6 bacteria ciliate^–1 h^–1. HNF and ciliates together cropped 0–14 (mean 4)% of BSP, indicating that they did not significantly contribute to benthic bacterial mortality during any period of the year. The low impact of protozoan grazing was due to the low numbers of HNF and ciliates in relation to bacteria (1.8–3.5 × 10⁴ bacteria HNF^–1, 0.9–3.1 × 10⁶ bacteria ciliate^–1). Thus, grazing by HNF and ciliates could be ruled out as a parameter regulating bacterial standing stock or production in the sediment of the investigated system, but the factors responsible for the limitation of benthic protistan densities and the fate of benthic BSP remained unclear.     

The importance of phytoflagellate, heterotrophic flagellate and ciliate grazing on bacteria and picophytoplankton sized prey in a coastal marine environment

The uptake of bacteria and picoplankton sized fluorescentlylabelled beads was measured off the west coast of the SouthIsland of New Zealand in winter. Phytoflagellates and heterotrophicflagellates showed similar grazing rates on 0.49 µm beads,with mean clearance rates of 1.1 and 1.8 nl ind.^–1 h^–1,respectively. Clearance rates for 1.09 µm beads were 0.9nl ind.^–1 h^–1 for heterotrophic flagellates and0.5 nl ind.^–1 h^–1 for phytoflagellates. Non-loricateciliates had clearance rates of 1.5 µl ind.^–1 h^–1for the picoplankton sized particles. The heterotrophic flagellatesshowed no significant difference between clearance rates of0.49 and 1.09 µm particles. Phytoflagellates, however,showed an apparent preference for the smaller particles. Themeasurement of significant grazing by phytoflagellate populationsin the marine environment is important and indicates that weneed to reassess our concepts of food web structure.     

A study of feeding in predacious ciliates using prey ciliates labeled with fluorescent microspheres

Feeding in predacious estuarine ciliates was investigated ina series of laboratory experiments using a new method of preylabeling which facilitates microscopic indentification of ingestedprey items. Ingestion rates of Mesodinium pulex, Euplotes vannusand E.woodruffi were estimated using the appearance, insidethe predator, of bacteriovorous ciliates (Metanophrys sp., Cyclidiumsp.and Pleuronema sp ) labeled with fluorescent microspheres. Preyremain motile and have presumably unaltered surface characteristics.Ingestion rates of log-growth phase predators increased withprey density. Mesodinium pulex ingested 0 15–0.32 cellsh^–1 over a prey concentration of 60–2300 ml^–1.Maximum ingestion rates of E. woodruffi and E. vannus were 4.5and 3.4 cells h^–1 respectively, estimated at prey abundancesof 75 and 172 cells ml^–1 respectively. Comparisons offeeding rates on prey of different sizes, and the effects ofstarvation, indicated that ingestion is likely limited by differentfactors in ‘raptorial’ (M pulex) and ‘filterfeeding’ (Euplotes spp.) predators.     

Protozoan growth rates in Antarctic lakes

The growth rates of heterotrophic nanoflagellates (HNAN), mixotrophic cryptophytes, dinoflagellates and ciliates in field assemblages from Ace Lake in the Vestfold Hills (eastern Antarctica) and Lakes Fryxell and Hoare (McMurdo Dry Valleys, western Antarctica), were determined during the austral summers of 1996/1997 and 1997/1998. The response of the nanoflagellates to temperature differed between lakes in eastern and western Antarctica. In Ace Lake the available bacterial food resources had little impact on growth rate, while temperature imposed an impact, whereas in Lake Hoare increased bacterial food resources elicited an increase in growth rate. However, the incorporation of published data from across Antarctica showed that temperature had the greater effect, but that growth is probably controlled by a suite of factors not solely related to bacterial food resources and temperature. Dinoflagellates had relatively high specific growth rates (0.0057–0.384 h⁻¹), which were comparable to Antarctic lake ciliates and to dinoflagellates from warmer, lower latitude locations. Temperature did not appear to impose any significant impact on growth rates. Mixotrophic cryptophytes in Lake Hoare had lower specific growth rates than HNAN (0.0029–0.0059 h⁻¹ and 0.0056–0.0127 h⁻¹, respectively). They showed a marked seasonal variation in growth rate, which was probably related to photosynthetically active radiation under the ice at different depths in the water column. Ciliates' growth rates showed no relationship between food supply and mean cell volume, but did show a response to temperature. Specific growth rates ranged between 0.0033 and 0.150 h⁻¹ for heterotrophic ciliates, 0.0143 h⁻¹ for a mixotrophic Plagiocampa species and 0.0075 h⁻¹ for the entirely autotrophic ciliate, Mesodinium rubrum. The data indicated that the scope for growth among planktonic Protozoa living in oligotrophic, cold extreme lake ecosystems is limited. These organisms are likely to suffer prolonged physiological stress, which may account for the highly variable growth rates seen within and between Antarctic lakes. Accepted: 7 December 1999     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Epizooic ciliates (Vorticella sp.) compete for food with their host Daphnia longispina in a small polyhumic lake

Summary Clearance rates of epizooic ciliates (Vorticella sp.) were measured together with their host, a planktonic cladoceran Daphnia longispina by using fluorescent latex beads as tracers of food. Vorticellans and their host graze on food of same size range (nanoplanktonic algae and bacteria). Individual clearance rates of Vorticella averaged 6.9 and 7.0 l ind^-1 h^-1 and those of Daphnia 463 and 708 l ind^-1 h^-1 for beads with diameter 2.00 and 3.92 m. On the average, epizooic vorticellans together on the carapace of Daphnia cleared particles with rates representing 25–33% of that the host cleared, the maximum rates being 50–80%. In a steeply stratified polyhumic lake vorticellans take advantage of following Daphnia to food patches and they can severely compete for food with their host.     

Production of levanase byRhodotorula sp.

Rhodotorula sp. produced a high yield of levanase (12.5 nkat/mL) in shake flasks in basal medium containing 1% maltose as the sole carbon source. Among the different carbon sources used, maltose was found to be the best for levanase production. The optimum temperature and pH for levanase production were 30°C and 6, respectively. In a batch reactor the enzyme productivity was higher (500 nkat L⁻¹ h⁻¹) than in shaken flasks (347 nkat L⁻¹ h⁻¹).     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Biodesulfurization of dibenzothiophene by growing cells of Gordonia sp. in batch cultures

A new isolate, identified as Gordonia sp. ZD-7 by 16S rDNA sequence analysis, grew in n-hexadecane containing dibenzothiophene (DBT) which was degraded from 2.8 mM to 0.2 mM within 48 h. Biodesulfurization could be repeatedly performed for more than 190 h, with average desulfurization rates of 5 mmol DBT kg cells (dry wt)⁻¹ h⁻¹.     

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.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

An approach to measure ciliate grazing on living heterotrophic nanoflagellates

The complicated routes by which organic material is channelled up to higher trophic levels via bacteria and protozoans is a major issue in aquatic microbial ecology. Because of the fragile nature of protists it is not straightforward to perform experimental studies of prey–predator interactions. Here we present an approach for the assessment of ciliate grazing on living heterotrophic nanoflagellates. Stationary phase cultures of a heterotrophic nanoflagellate (Cafeteria sp.) were live-stained by allowing them to take up fluorescently labelled macromolecules. Controls revealed that this label persisted for several hours. Fluorescently labelled living flagellates (FLLF) were added into enriched natural assemblages of marine oligotrich ciliates and uptake of FLLF was monitored over time. Oligotrich ciliates did not incorporate fluorescent-labelled macromolecules but a linear FLLF uptake over time was observed for 20–30 min at 20°C. Ingestion rates were 21–46 FLLF h^–1 at a concentration of about 2×10⁴ FLLF ml^–1, which corresponded to clearance rates of 0.7–0.8 l ciliate^–1 h^–1. These results are in the same order of reported ciliate grazing on phytoplankton of similar size. This method represents a direct approach to measure ciliate grazing specifically on living heterotrophic nanoflagellates.     

Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil

A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 °C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l⁻¹ h⁻¹ at a dilution rate of 0.4 h⁻¹. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant K _S of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, μ_max, of 1.0 h⁻¹. Oxygen uptake rates of up to 2.9 g l⁻¹h⁻¹ were calculated and the yield coefficient, Y _X/O, was determined to be 0.29 g dry cell weight/g O₂. From an overall material balance the yield coefficient, Y _X/S, was estimated to be 0.60 g dry cell weight/g olive oil. Received: 8 January 1997 / Received revision: 30 April 1997 / Accepted: 4 May 1997     

Effects of marine ciliates on survivability of the first-feeding larval surgeonfish,Paracanthurus hepatus: laboratory rearing experiments

The contribution of ciliates as a food source to survival of first-feeding surgeonfish larvae, Paracanthurus hepatus, was examined in rearing experiments. The larvae were exposed to eight treatments; i.e. a tintinnid, Amphorellopsis acuta (1.0 × 10⁴, 5.1 × 10³ and 2.2 × 10³ cells l^–1) and a naked ciliate, Euplotes sp. (1.3 × 10⁴, 8.0 × 10³ and 5.0 × 10³ cells l^–1), plus two controls without ciliates. Highest survival of the larvae over the first 4–8 days was observed in the highest density of A. acuta. Rearing experiments also showed that the survivals of larvae fed with A. acuta were higher than those fed with Euplotes sp. Gut content analyses revealed loricae of A. acuta in the larvae. Although Euplotes sp. (lacking loricae) was never recognized in those larval guts, feeding on Euplotes sp. by larvae was confirmed using the ciliate labeled with fluorescent microspheres, implying that the feeding on naked ciliates by fish larvae has been overlooked. The results strongly suggested that both tintinnid and naked ciliates play important roles as alternative food sources to copepod nauplii by enhancing the survivability of fish larvae, especially those with a smaller mouth.     

Identification of novel denitrifying bacteria Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13 and application for removal of nitrate from industrial wastewater

Two novel denitrifying bacteria were successfully isolated from industrial wastewater and soil samples. Using morphological, biochemical/biophysical and 16S rRNA gene analyses, these two bacteria were identified as Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13, respectively. Both of these two bacteria showed efficient NO₃ ⁻-N removing abilities under a semi-anaerobic condition without obvious accumulation of NO₂ ⁻-N, N₂O-N and NH₄ ⁺-N. NO₃ ⁻-N removal from paper mill wastewater was also successful by treatments with either a denitrifier or an immobilization method. Therefore, this study provides valuable denitrifying bacteria in biotreatment of industrial wastewater and other environmental pollution caused by NO₃ ⁻/NO₂ ⁻.