Impact of protozoan grazing on bacterial community structure in soil microcosms

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.     

Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a prey–predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.     

Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Phagotrophic Protist Diversity in the Groundwater of a Karstified Aquifer – Morphological and Molecular Analysis

To clarify the structure of microbial food webs in groundwater, knowledge about the protist diversity and feeding strategies is essential. We applied cultivation‐dependent approaches and molecular methods for further understanding of protist diversity in groundwater. Groundwater was sampled from a karstified aquifer located in the Thuringian Basin (Thuringia, Germany). Cultivable protist abundance estimated up to 8,000 cells/L. Eleven flagellates, 10 naked amoebae, and one ciliate morpho‐species were detected in groundwater enrichment cultures. Most of the flagellates morpho‐species, typically < 10 μm, were sessile or free swimming suspension feeders, e.g., Spumella spp., Monosiga spp., and mobile, surface‐associated forms that grasp biofilms, e.g., Bodo spp. Naked amoebae, typically < 35 μm, that grasp biofilms were represented by, e.g., Vahlkampfia spp., Vannella spp., and Hartmanella spp. The largest fraction of the 18S rRNA gene sequences was affiliated with Spumella‐like Stramenopiles. Besides, also sequences affiliated with fungi and metazoan grazers were detected in clone libraries of the groundwater. We hypothesize that small sized protist species take refuge in the structured surface of the fractures and fissures of the karstified aquifer and mainly feed on biofilm‐associated or suspended bacteria.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Soil Columns

The enhanced mineralization of immobilized nitrogen by bacteriophagous protozoa has been thought to favor the nitrification process in soils in which nitrifying bacteria must compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the flagellate Adriamonas peritocrescens on the competition for ammonium between the chemolithotrophic species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi was studied in soil columns, which were continuously percolated with media containing 5 mM ammonium and different amounts of glucose at a dilution rate of 0.007 h^-1 (liquid volumes). A. globiformis won the competition for ammonium. The grazing activities of the flagellates had two prominent effects on the competition between N. europaea and A. globiformis. First, the distribution of ammonium over the profile of the soil columns was more uniform in the presence of flagellates than in their absence. In the absence of flagellates, relatively high amounts of ammonium accumulated in the upper layer (0 to 3 cm), whereas in the underlying layers the ammonium concentrations were low. In the presence of flagellates, however, considerable amounts of ammonium were found in the lower layers, whereas less ammonium accumulated in the upper layer. Second, the potential ammonium-oxidizing activity of N. europaea was stimulated in the presence of flagellates. The numbers of N. europaea at different glucose concentrations in the presence of flagellates were comparable to those in the absence of protozoa. However, in the presence of flagellates, the potential ammonium-oxidizing activities were four to five times greater than those in the absence of protozoa.     

Distribution and diversity of soil protozoa in the McMurdo Dry Valleys of Antarctica

The polar desert soils of the McMurdo Dry Valley region support a limited water film community dominated by flagellates, amoebae, and nematodes. This study describes the protozoa and compares their distribution to nematodes. In 50 samples collected from 12 locations, rotifers and tardigrades were infrequent, and ciliates and testacea were rare. Soil protozoa occurred at all sites but the dominant nematode, Scottnema lindsayae (Timm 1971), did not, indicating soil habitat factors limiting nematode distribution are not limiting to protozoa. In contrast to the nematode species, which are all endemic to Antarctica, there were no endemic protozoan morphospecies found in our samples. The protozoan abundance was several orders of magnitude greater than that of the nematodes, and the species diversity was much greater. Most of the protozoa grew better at lower incubation temperatures. The ubiquitous distribution of protozoa suggests their importance in soil food webs and nutrient cycling in the dry valleys.     

Changes in the Spoilage-Related Microbiota of Beef during Refrigerated Storage under Different Packaging Conditions

The microbial spoilage of beef was monitored during storage at 5°C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O₂ and 40% CO₂ (MAP2), and (iii) 20% O₂ and 40% CO₂ (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

Impairment of T Cell Function in Parasitic Infections

In mammals subverted as hosts by protozoan parasites, the latter and/or the agonists they release are detected and processed by sensors displayed by many distinct immune cell lineages, in a tissue(s)-dependent context. Focusing on the T lymphocyte lineage, we review our present understanding on its transient or durable functional impairment over the course of the developmental program of the intracellular parasites Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi in their mammalian hosts. Strategies employed by protozoa to down-regulate T lymphocyte function may act at the initial moment of naïve T cell priming, rendering T cells anergic or unresponsive throughout infection, or later, exhausting T cells due to antigen persistence. Furthermore, by exploiting host feedback mechanisms aimed at maintaining immune homeostasis, parasites can enhance T cell apoptosis. We will discuss how infections with prominent intracellular protozoan parasites lead to a general down-regulation of T cell function through T cell anergy and exhaustion, accompanied by apoptosis, and ultimately allowing pathogen persistence.     

Identification and Analysis of Putative Homologues of Mechanosensitive Channels in Pathogenic Protozoa

Mechanosensitive channels play important roles in the physiology of many organisms, and their dysfunction can affect cell survival. This suggests that they might be therapeutic targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, dysentery, leishmaniasis and trypanosomiasis that are responsible for millions of deaths each year worldwide. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of mechanosensitive channels. Entamoeba histolytica, Leishmania spp., Trypanosoma cruzi and Trichomonas vaginalis have genes encoding homologues of Piezo channels, while most pathogenic protozoa have genes encoding homologues of mechanosensitive small-conductance (MscS) and K⁺-dependent (MscK) channels. In contrast, all parasites examined lack genes encoding mechanosensitive large-conductance (MscL), mini-conductance (MscM) and degenerin/epithelial Na⁺ (DEG/ENaC) channels. Multiple sequence alignments of evolutionarily distant protozoan, amoeban, plant, insect and vertebrate Piezo channel subunits define an absolutely conserved motif that may be involved in channel conductance or gating. MscS channels are not present in humans, and the sequences of protozoan and human homologues of Piezo channels differ substantially. This suggests the possibility for specific targeting of mechanosensitive channels of pathogens by therapeutic drugs.     

Persistence of free-living protozoan communities across rearing cycles in commercial poultry houses

The introduction and survival of zoonotic bacterial pathogens in poultry farming have been linked to bacterial association with free-living protozoa. To date, however, no information is available on the persistence of protozoan communities in these environments across consecutive rearing cycles and how it is affected by farm- and habitat-specific characteristics and management strategies. We therefore investigated the spatial and temporal dynamics of free-living protozoa in three habitats (pipeline, water, and miscellaneous samples) in three commercial poultry houses across three rearing cycles by using the molecular fingerprinting technique denaturing gradient gel electrophoresis (DGGE). Our study provides strong evidence for the long-term (ca. 6-month) persistence of protozoa in broiler houses across consecutive rearing cycles. Various free-living protozoa (flagellates, ciliates, and amoebae), including known vectors of bacterial pathogens, were observed during the down periods in between rearing cycles. In addition, multivariate analysis and variation partitioning showed that the protozoan community structure in the broiler houses showed almost no change across rearing cycles and remained highly habitat and farm specific. Unlike in natural environments, protozoan communities inside broiler houses are therefore not seasonal. Our results imply that currently used biosecurity measures (cleaning and disinfection) applied during the down periods are not effective against many protozoans and therefore cannot prevent potential cross-contamination of bacterial pathogens via free-living protozoa between rearing cycles.     

Rhizosphere bacterial community composition responds to arbuscular mycorrhiza, but not to reductions in microbial activity induced by foliar cutting

Differences in bacterial community composition (BCC) between bulk and rhizosphere soil and between rhizospheres of different plant species are assumed to be strongly governed by quantitative and qualitative rhizodeposit differences. However, data on the relationship between rhizodeposit amounts and BCC are lacking. Other soil microorganisms, e.g. arbuscular mycorrhizal fungi (AMF), may also influence BCC. We simulated foliar herbivory (cutting) to reduce belowground carbon allocation and rhizodeposition of pea plants grown either with or without AMF. This reduced soil respiration, rhizosphere microbial biomass and bacteriovorous protozoan abundance, whereas none of these were affected by AMF. After labelling plants with (13)CO(2), root and rhizosphere soil (13)C enrichment of cut plants were reduced to a higher extent (24-46%) than shoot (13)C enrichment (10-24%). AMF did not affect (13)C enrichment. Despite these clear indications of reduced rhizosphere carbon-input, denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes PCR-amplified targeting DNA and RNA from rhizosphere soil did not reveal any effects of cutting on banding patterns. In contrast, AMF induced consistent differences in both DNA- and RNA-based DGGE profiles. These results show that a reduction in rhizosphere microbial activity is not necessarily accompanied by changes in BCC, whereas AMF presence inhibits proliferation of some bacterial taxa while stimulating others.     

Bacterial community involved in traditional fermented soybean paste dajiang made in northeast China

Dajiang is a traditional fermented food prepared from soybeans that is still popular in northeast China. Although recent studies have revealed that a variety of bacterial species contribute to the production of fermented soybean products, little is known about bacterial communities involved in the fermentation of dajiang made in northeast China. In this study, 14 samples of naturally fermented dajiang were analyzed by denaturing gradient gel electrophoresis (DGGE) to determine the diversity of the bacteria involved in fermentation. Our results indicate that lactic acid bacteria, including Lactobacillus plantarum, uncultured Leuconostoc mesenteroides, Leuconostoc gasicomitatum, Enterococcus faecium, and Tetragenococcus halophilus, were the predominant species. This is the first report of Enterococcus spp. and Leuconostoc spp. in the Chinese fermented soybean paste dajiang using DGGE. The presence of Bacillus spp. (including Bacillus firmus), Oceanobacillus spp., and Paenibacillus glycanilyticus in the dajiang samples may be due to their salt tolerance. Potentially pathogenic Alphaproteobacteria and Staphylococcus epidermidis strains were also detected in this study. Moreover, three uncultured bacterial clones were found in some samples and require further study. The results reveal a high level of bacterial diversity in dajiang.     

The Truman Show for protozoan parasites: A review of in vitro cultivation platforms

Protozoan parasites are responsible for severe disease and suffering in humans worldwide. Apart from disease transmission via insect vectors and contaminated soil, food, or water, transmission may occur congenitally or by way of blood transfusion and organ transplantation. Several recent outbreaks associated with fresh produce and potable water emphasize the need for vigilance and monitoring of protozoan parasites that cause severe disease in humans globally. Apart from the tropical parasite Plasmodium spp., other protozoa causing debilitating and fatal diseases such as Trypanosoma spp. and Naegleria fowleri need to be studied in more detail. Climate change and socioeconomic issues such as migration continue to be major drivers for the spread of these neglected tropical diseases beyond endemic zones. Due to the complex life cycles of protozoa involving multiple hosts, vectors, and stringent growth conditions, studying these parasites has been challenging. While in vivo models may provide insights into host–parasite interaction, the ethical aspects of laboratory animal use and the challenge of ready availability of parasite life stages underline the need for in vitro models as valid alternatives for culturing and maintaining protozoan parasites. To our knowledge, this review is the first of its kind to highlight available in vitro models for protozoa causing highly infectious diseases. In recent years, several research efforts using new technologies such as 3D organoid and spheroid systems for protozoan parasites have been introduced that provide valuable tools to advance complex culturing models and offer new opportunities toward the advancement of parasite in vitro studies. In vitro models aid scientists and healthcare providers in gaining insights into parasite infection biology, ultimately enabling the use of novel strategies for preventing and treating these diseases.     

Seasonal and successional influences on bacterial community composition exceed that of protozoan grazing in river biofilms

The effects of protozoa (heterotrophic flagellates and ciliates) on the morphology and community composition of bacterial biofilms were tested under natural background conditions by applying size fractionation in a river bypass system. Confocal laser scanning microscopy (CLSM) was used to monitor the morphological structure of the biofilm, and fingerprinting methods (single-stranded conformation polymorphism [SSCP] and denaturing gradient gel electrophoresis [DGGE]) were utilized to assess changes in bacterial community composition. Season and internal population dynamics had a greater influence on the bacterial biofilm than the presence of protozoa. Within this general framework, bacterial area coverage and microcolony abundance were nevertheless enhanced by the presence of ciliates (but not by the presence of flagellates). We also found that the richness of bacterial operational taxonomic units was much higher in planktonic founder communities than in the ones establishing the biofilm. Within the first 2 h of colonization of an empty substrate by bacteria, the presence of flagellates additionally altered their biofilm community composition. As the biofilms matured, the number of bacterial operational taxonomic units increased when flagellates were present in high abundances. The additional presence of ciliates tended to at first reduce (days 2 to 7) and later increase (days 14 to 29) bacterial operational taxonomic unit richness. Altogether, the response of the bacterial community to protozoan grazing pressure was small compared to that reported in planktonic studies, but our findings contradict the assumption of a general grazing resistance of bacterial biofilms toward protozoa.     

Presence of Cryptosporidium spp. and Giardia duodenalis in Drinking Water Samples in the North of Portugal

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and β,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.     

Protozoan Response to the Addition of Bacterial Predators and Other Bacteria to Soil

Representatives of several categories of bacteria were added to soil to determine which of them might elicit responses from the soil protozoa. The various categories were nonobligate bacterial predators of bacteria, prey bacteria for these predators, indigenous bacteria that are normally present in high numbers in soil, and non-native bacteria that often find their way in large numbers into soil. The soil was incubated and the responses of the indigenous protozoa were determined by most-probable-number estimations of total numbers of protozoa. Although each soil was incubated with only one species of added bacteria, the protozoan response for the soil was evaluated by using most-probable-number estimations of several species of bacteria. The protozoa did not respond to incubation of the soil with either Cupriavidus necator, a potent bacterial predator, or one of its prey species, Micrococcus luteus. C. necator also had no effect on the protozoa. Therefore, in this case, bacterial and protozoan predators did not interact, except for possible competition for bacterial prey cells. The soil protozoa did not respond to the addition of Arthrobacter globiformis or Bacillus thuringiensis. Therefore, the autochthonous state of Arthrobacter species in soil and the survival of B. thuringiensis were possibly enhanced by the resistance of these species to protozoa. The addition of Bacillus mycoides and Escherichia coli cells caused specific responses by soil protozoa. The protozoa that responded to E. coli did not respond to B. mycoides or any other bacteria, and vice versa. Therefore, addition to soil of a nonsoil bacterium, such as E. coli, did not cause a general increase in numbers of protozoa or in protozoan control of the activities of other bacteria in the soil.     

The prevalence and diversity of intestinal parasitic infections in humans and domestic animals in a rural Cambodian village

In Cambodia, intestinal parasitic infections are prevalent in humans and particularly in children. Yet, information on potentially zoonotic parasites in animal reservoir hosts is lacking. In May 2012, faecal samples from 218 humans, 94 dogs and 76 pigs were collected from 67 households in Dong village, Preah Vihear province, Cambodia. Faecal samples were examined microscopically using sodium nitrate and zinc sulphate flotation methods, the Baermann method, Koga Agar plate culture, formalin-ether concentration technique and Kato Katz technique. PCR was used to confirm hookworm, Ascaris spp., Giardia spp. and Blastocystis spp. Major gastrointestinal parasitic infections found in humans included hookworms (63.3%), Entamoeba spp. (27.1%) and Strongyloides stercoralis (24.3%). In dogs, hookworm (80.8%), Spirometra spp. (21.3%) and Strongyloides spp. (14.9%) were most commonly detected and in pigs Isospora suis (75.0%), Oesophagostomum spp. (73.7%) and Entamoeba spp. (31.6%) were found. Eleven parasite species were detected in dogs (eight helminths and three protozoa), seven of which have zoonotic potential, including hookworm, Strongyloides spp., Trichuris spp., Toxocara canis, Echinostoma spp., Giardia duodenalis and Entamoeba spp. Five of the parasite species detected in pigs also have zoonotic potential, including Ascaris spp., Trichuris spp., Capillaria spp., Balantidium coli and Entamoeba spp. Further molecular epidemiological studies will aid characterisation of parasite species and genotypes and allow further insight into the potential for zoonotic cross transmission of parasites in this community.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate Adriamonas peritocrescens on the competition for limiting amounts of ammonium between the ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h^-1. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM. A. globiformis won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h^-1, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 × 10⁵ and 12 × 10⁵ flagellates ml^-1 were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h^-1, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.