Abundance and distribution of anaerobic protozoa and their contribution to methane production in Lake Cadagno (Switzerland)

Abstract In the uupermost layers of the anoxic sediment in Lake Cadagno, 9 different species of anaerobic protozoa were identified. The total number of these organisms was about 580 cells·ml⁻¹ sediment. Most pf these protozoa contained endosymbiotic methanogenic bacteria which in total amounted to 10⁶ methanogens·ml⁻¹ sediment. In addition to the methanogenic endosymbionts, cells of Metopus setosus and Caenomorpha lata also contained a non-fluorescent bacterial rod inside the cytoplasm. In some individual cells of C. lata this second type of endosymbiotic bacterium was sometimes the only endosymbiont observed. Contrary to earlier suggestions, anaerobic protozoa do not seem to play a major role in methane production at least in Lake Cadagno. No significant methane production due to the anaerobic protozoa and their methanogenic endosymbionts was found in situ. Isolated ciliates and amoebae produced methane at 12°C, but not at 6°C, probably as a result of temperature limitation. In the sediment of Lake Cadagno sulfate reduction seemed to be the dominant terminal degradation process.     

Growth monitoring of <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> by pressure measurement

In life support systems, such as the MELiSSA (Micro-Ecological Life Support Alternative) project, developed by the European Space Agency, the aim is to understand and assemble artificial ecosystems for ensuring human subsistence in space. Fibrobacter succinogenes, an anaerobic bacterium, was used for the degradation of vegetable wastes produced in higher plants chambers, but the process does not allow the monitoring of biomass concentration and degradation rates. This study proposes a growth and a degradation monitoring technique using pressure measurements. First, volatile fatty acids (VFA) production was compared with biomass growth and with CO₂ production. The experiments were carried out in batch and fed-batch processes on glucose and on vegetables. The results have shown that a link could be established between VFA production, degradation rate and gas pressure measurements. Thus, the pressure could be used both as a relevant variable for online evaluation of biomass growth and of degradation of complex vegetable wastes.     

Anaerobic and aerobic biodegradation of chlorophenols using UASB and ASG bioreactors

Chlorophenol degradation was studied by combined anaerobic–aerobic treatments as a single or multi-substrate system. 2,4-Dichlorophenol (2,4-DCP) was degraded to the extent of 52 and 78% in up-flow anaerobic sludge blanket (UASB) and aerobic suspended growth (ASG) reactors respectively, at organic loading rates of 0.18kg/m³/day and hydraulic retention time of 26.4h in the presence of glucose. The UASB represents the dominating facultative anaerobic microbial population. When the effluent from the anaerobic reactor (UASB) was subjected to aerobic treatment on the ASG reactor, 2,4-DCP and COD removals of 86 and 95% respectively were achieved. Aerobic degradation of chlorophenol by acclimated mixed bacterial isolates was found to be sequential: 2-Chlorophenol (2-CP) and 4-CP were degraded first, followed by 2,4-DCP and 2,4,6-Trichlorophenol (2,4,6-TCP) while the contrary was obtained in anaerobic degradation. In anaerobic degradation by acclimated mixed bacterial cells, 2,4-DCP and 2,4,6-TCP were degraded first followed by mono-chlorophenols. The anaerobic/aerobic bioreactors were most efficient when operated in sequence (series) rather than in parallel.     

Response of Benthic Protozoa and Thraustochytrid Protists to Fish Farm Impact in Seagrass (Posidonia oceanica) and Soft-Bottom Sediments

We investigated the impact of fish farm biodeposition on benthic bacteria, thraustochytrid protists, and heterotrophic protozoa (nanoflagellates and ciliates) in an oligotrophic area of the Mediterranean Sea. The fish farm impact was investigated both on a seagrass (Posidonia oceanica) bed and on soft bottom sediments. In both systems, sediment samples were collected with a multicontrol sampling strategy (i.e., beneath the fish farm and at three control sites per system). The uneaten food pellets supplied to the fish determined the accumulation of sediment organic matter and the enhancement of protein content in impacted sediments (both seagrass bed and soft sediments). In both systems, the abundance and biomass of heterotrophic protists increased significantly beneath the fish farm, but the structure of the protist assemblages responded differently in vegetated and unvegetated sediments. Thraustochytrid abundance increased significantly in impacted seagrass. These results provide evidence that the structure of protist assemblages respond significantly to fish farm biodeposition and indicate that the monitoring of these benthic components provides complementary information for the assessment of the fish farm impact on the benthic systems.     

Protistan bacterivory in an oligomesotrophic lake: Importance of attached ciliates and flagellates

Seasonal and depth variations of the abundance, biomass, and bacterivory of protozoa (heterotrophic and mixotrophic flagellates and ciliates) were determined during thermal stratification in an oligomesotrophic lake (Lake Pavin, France). Maximal densities of heterotrophic flagellates (1.9 × 10³ cells ml^–1) and ciliates (6.1 cells ml^–1) were found in the metalimnion. Pigmented flagellates dominated the flagellate biomass in the euphotic zone. Community composition of ciliated protists varied greatly with depth, and both the abundance and biomass of ciliates was dominated by oligotrichs. Heterotrophic flagellates dominated grazing, accounting for 84% of total protistan bacterivory. Maximal grazing impact of heterotrophic flagellates was 18.9 × 10⁶ bacteria 1^–1h^–1. On average, 62% of nonpigmented flagellates were found to ingest particles. Ciliates and mixotrophic flagellates averaged 13% and 3% of protistan bacterivory, respectively. Attached protozoa (ciliates and flagellates) were found to colonize the diatom Asterionella formosa. Attached bacterivores had higher ingestion rates than free bacterivorous protozoa and may account for 66% of total protozoa bacterivory. Our results indicated that even in low numbers, epibiotic protozoa may have a major grazing impact on free bacteria. Correspondence: C. Amblard.     

A Mesocosm Study of the Changes in Marine Flagellate and Ciliate Communities in a Crude Oil Bioremediation Trial

Protozoan grazers play an important role in controlling the density of crude-oil degrading marine communities as has been evidenced in a number of microcosm experiments. However, small bioreactors contain a low initial titre of protozoa and the growth of hydrocarbon-depleting bacteria is accompanied by the fast depletion of mineral nutrients and oxygen, which makes microcosms rather unsuitable for simulating the sequence of events after the oil spill in natural seawater environment. In the present study, the population dynamics of marine protozoan community have been analysed in a 500 l mesocosm experiment involving bioaugmented oil booms that contained oil sorbents and slow-release fertilisers. A significant increase in numbers of marine flagellates and ciliates on biofilms of oil-degrading microbes was microscopically observed as early as 8 days after the start of the experiment, when protozoa exhibited a population density peak making up to 3,000 cells ml⁻¹. Further, the protozoan density varied throughout the experiment, but never dropped below 80 cells ml⁻¹. An 18S rRNA gene-based fingerprinting analysis revealed several changes within the eukaryotic community over the whole course of the experiment. Initial growth of flagellates and small ciliates was followed by a predominance of larger protozoa. According to microscopic observations and SSU rRNA molecular analyses, most predominant were the ciliates belonging to Euplotidae and Scuticociliatia. This is the first study to characterise the eukaryotic communities specifically in a large-scale oil bioremediation trial using both microscopy-based and several molecular techniques.     

Functional integration of Methanobacterium formicicum into the anaerobic ciliate Trimyema compressum

Monoxenic cultures of the anaerobic, endosymbiont-free ciliate Trimyema compressum were incubated with low numbers of Bacteroides sp. strain WoCb15 as food bacteria and two strains (DSM 3636 and 3637) of Methanobacterium formicicum, which originally had been isolated from the anaerobic protozoa Metopus striatus and Pelomyxapalustris. The ciliate which had lost its original endosymbiotic methanogens ingested both strains of M. formicicum. The methanogenic bacteria were found intact in large vacuoles in contrast to the food bacteria which were digested. Single methanogens were separated from the vacuoles and appeared surrounded by a membrane in the cytoplasm of the ciliate. After 2 months of incubation, the methanogenic bacteria still exhibited the typical bluish fluorescence and the new symbiotic association of M. formicicum and T. compressum excreted methane. Increasing the growth rate of the ciliates by large numbers of food bacteria resulted in a loss of the methanogenic bacteria, due to statistical outgrowth.     

Impairment of nucleoid segregation and cell division at high osmolarity in a strain of Escherichia coli overproducing the chaperone DnaK

Abstract Fourteen species of ciliates, seven of which are new, were found living in a sample of anoxic water collected from a small lake in Spain. The species belong to all six orders in which anaerobic ciliates have been described and they include the first anaerobic representatives of the order Prostomatida. This surprising diversity is probably sustained because it embraces all ciliate feeding types, and because protozoa are the only important consumers of the diversity of microbes in anoxic habitats. Six of the anaerobic ciliate species have aerobic congeners; this strengthens the contention that anaerobic ciliates evolved independently from aerobes belonging to several taxonomic groups.     

Combined effects of food resources and exposure to ammonium nitrogen on population growth performance in the bacterivorous ciliate Paramecium caudatum

Ciliated protozoa (ciliates) play vital roles in biological wastewater-treatment processes, however, combined effects of abiotic and biotic factors as well as the importance of species-specificity of bacterial food organisms on population growth dynamics remain poorly understood, which are hampering the management and optimization of biological wastewater treatment processes. This study investigated the effects of food resources and ammonium nitrogen (NH₄⁺) exposure, both independently and in combination, on the population growth of the bacterivorous ciliate Paramecium caudatum. Results showed that, when fed with two different bacterial food organisms, population growth performance of P. caudatum differed significantly and increased with the addition of protozoa pellet medium. When exposed to NH₄⁺ population growth declined and metabolic enzyme activities were altered. The negative effects of NH₄⁺ on population growth could be weakened by supplementing the food resource with protozoa pellet media. In brief, it was confirmed that the existence of interactive effect of food resources and ammonium nitrogen, as well as the importance of species-specificity of bacterial food organisms on the population growth performance of ciliates. These findings might lead to the development of a valuable strategy for improving the performance of biological wastewater-treatment processes.     

Ammonia inhibition in anaerobic digestion: A review

Even though ammonia is an essential nutrient for bacterial growth, it may inhibit methanogenesis during anaerobic digestion process if it is available at high concentrations. Therefore, ammonia is regarded as a potential inhibitor during anaerobic digestion, particularly when dealing with complex type of substrates such as manure or the organic fraction of municipal solid waste (OFMSW). Ammonia is produced through biological degradation of nitrogenous matter. Ammonium ion (NH₄⁺) and free ammonia (NH₃) are the two principal forms of inorganic ammonia nitrogen. Both forms can directly and indirectly cause inhibition in an anaerobic digestion system. Particularly, free ammonia (FAN) is a powerful inhibitor in an anaerobic digester above threshold concentrations. Process inhibition is related to the particular characteristics of the substrate to be anaerobically digested, pH, process temperature (mesophilic or thermophilic), type of the seed sludge (inoculum), the reactor configuration and to the concentrations of ammonium and ammonia. In this paper, ammonia inhibition in anaerobic digestion systems and the recovery efforts after inhibition are discussed. Furthermore, the impacts of ammonia inhibition on the microbial population available in anaerobic digesters, namely bacteria and Archaea, are also evaluated in detail.     

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

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

A comparison of two strains of the anaerobic ciliate Trimyema compressum

Two strains of the anaerobic ciliate Trimyema compressum, isolated from different habitats, were compared. The cytoplasm of the ciliates contained hydrogenosome-like microbodies and methanogenic bacteria; the latter were lost during continued cultivation. In addition both strains harbored a non-methanogenic endosymbiont, which was lost in strain K. The ciliates lacked cytochromes, cytochrome oxidase and catalase but contained superoxide dismutase. Hydrogenase activity could be demonstrated only in strain N. In monoxenic culture strain K needed sterols as growth factors. The cells of both strains reacted similarly with respect to oxygen tolerance (up to 0.5 mg O₂/l), inhibition of growth by cyanide and azide, and resistance to antimycin A. Only cells of strain N showed growth inhibition by chloramphenicol. It is concluded that Trimyema compressum is an anaerobic, microaerotolerant organism, its microbodies show more resemblance to hydrogenosomes than to mitochondria.     

Pentachlorophenol (PCP) dechlorination in horizontal-flow anaerobic immobilized biomass (HAIB) reactors

This study verifies the potential applicability of horizontal-flow anaerobic immobilized biomass (HAIB) reactors to pentachlorophenol (PCP) dechlorination. Two bench-scale HAIB reactors (R1 and R2) were filled with cubic polyurethane foam matrices containing immobilized anaerobic sludge. The reactors were then continuously fed with synthetic wastewater consisting of PCP, glucose, acetic acid, and formic acid as co-substrates for PCP anaerobic degradation. Before being immobilized in polyurethane foam matrices, the biomass was exposed to wastewater containing PCP in reactors fed at a semi-continuous rate of 2.0 μg PCP g⁻¹ VS. The applied PCP loading rate was increased from 0.05 to 2.59 mg PCP l⁻¹ day⁻¹ for R1, and from 0.06 to 4.15 mg PCP l⁻¹ day⁻¹ for R2. The organic loading rates (OLR) were 1.1 and 1.7 kg COD m⁻³ day⁻¹ at hydraulic retention times (HRT) of 24 h for R1 and 18 h for R2. Under such conditions, chemical oxygen demand (COD) removal efficiencies of up to 98% were achieved in the HAIB reactors. Both reactors exhibited the ability to remove 97% of the loaded PCP. Dichlorophenol (DCP) was the primary chlorophenol detected in the effluent. The adsorption of PCP and metabolites formed during PCP degradation in the packed bed was negligible for PCP removal efficiency.     

Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium

A novel denitrifying bacterium, strain 72Chol, was enriched and isolated under strictly anoxic conditions on cholesterol as sole electron donor and carbon source. Strain 72Chol grew on cholesterol with oxygen or nitrate as electron acceptor. Strictly anaerobic growth in the absence of oxygen was demonstrated using chemically reduced culture media. During anaerobic growth, nitrate was initially reduced to nitrite. At low nitrate concentrations, nitrite was further reduced to nitrogen gas. Ammonia was assimilated. The degradation balance measured in cholesterol-limited cultures and the amounts of carbon dioxide, nitrite, and nitrogen gas formed during the microbial process indicated a complete oxidation of cholesterol to carbon dioxide. A phylogenetic comparison based on total 16S rDNA sequence analysis indicated that the isolated micro-organism, strain 72Chol, belongs to the β2-subgroup in the Proteobacteria and is related to Rhodocyclus, Thauera, and Azoarcus species. Received: 16 July 1996 / Accepted: 5 December 1996     

Ability of rumen protozoa <Emphasis Type="Italic">Diploplastron affine</Emphasis> to utilize β-glucans

The ability of the rumen ciliates to utilize β-glucans other than cellulose and xylan is currently being recognized. The objective of the present study was to characterize the ability of the ciliate Diploplastron affine to digest some pachyman, laminarin, pustulan, curdlan and lichean. The protozoa were isolated from the rumen of sheep and either grown in vitro or inoculated into the rumen of ciliate-free sheep and maintained in natural conditions. In vitro culture studies showed that the enrichment of culture medium with the examined saccharides results in an increase in the number of ciliates in comparison to the control cultures. The increase was over 36 and 15 % when the growth medium was supplemented with pachyman (1,3-β-glucan) and pustulan (1,6-β-glucan), respectively. A positive correlation was also found between the population density of ciliates and the dose of saccharide supplemented to the growth medium. Enzyme studies were performed using the crude enzyme preparation obtained from ciliates treated with antibiotics. The ability of ciliates to digest the examined β-glucans was tested by the quantification of reducing sugars released from the mentioned substrates during the incubation with crude enzyme preparation. The results showed that D. affine ciliates were able to digest both of them. The mean degradation rate varied between 6.7 and 28.2 μmol/L glucose per mg protein per h for pustulan and lichean, respectively, whereas the digestion velocity was the highest at 5.0–5.5 pH and 45–50°C.     

Distributions and Biomass of Benthic Ciliates,Foraminifera and Amoeboid Protists in Marine,Brackish, and Freshwater Sediments

The quantitative importance of ciliates, foraminifers, and amoebae was investigated in marine, brackish, and freshwater sediments from 15 littoral stations. Total protozoan communities were usually dominated by ciliates in term of abundance, while amoebae often dominated in terms of biomass. Applying the biomass‐metabolic rate equation, ciliates, amoebae, and foraminifera were estimated to contribute 66% of the total abundance and 33% of the biomass, but up to 55% of the combined metabolic rate to the micro‐ and meiobenthos in the 15 sediments. Statistical analyses using ciliate data demonstrated: (1) species composition and community structures represented significant differences between freshwater and marine/brackish sediments, and subsequently between temperate and arctic sampling sites; (2) the occurrence of dominant ciliates and their allocation to feeding types indicated that herbivory was the most common feeding strategy in these sediments; (3) multivariate analyses showed all of the tested environmental factors (temperature, salinity, silt/clay, carbon, nitrogen, and chlorophyll a) to be important to varying degrees, but especially the combination of salinity, temperature, and silt/clay. Multiple factor effects or comprehensive influences might be important in regulating the distribution of protozoa in sediments. The importance of protozoa in sediment systems and the potential ecological significance of cysts are discussed.     

Fungicide Enhancement of Nitrogen Fixation and Colonization of Phaseolus vulgaris by Rhizobium phaseoli

The number and weight of pods and the weight and nitrogen content of the tops of beans (Phaseolus vulgaris) derived from seeds inoculated with a thiram-resistant strain of Rhizobium phaseoli were increased if the seeds were treated with thiram before sowing in soil. A greater percentage of the nodules on 21-day-old plants were derived from the resistant strain, more nodules were formed, and these nodules were more effective in the presence of the fungicide than in its absence. These differences in nodule numbers were no longer present in 56-day-old plants, and only a small percentage of the nodules contained the resistant strain. The abundance of the fungicide-tolerant R. phaseoli increased rapidly soon after planting the seed and subsequently fell markedly, but the rate of decline was less if the seeds had been treated with the chemical. Protozoa also proliferated if thiram had not been applied to the seed, but their numbers were deleteriously influenced by thiram. Bdellovibrio, bacteriophages, and lytic micro-organisms acting on R. phaseoli were rare under these conditions. Ciliates and flagellated protozoa were initially suppressed by planting thiram-coated bean seeds in nonsterile soil, but the former were inhibited longer than the latter and the ciliate numbers never fully recovered if the seeds were treated with the fungicide. The resistant strain grew well in sterile soil also inoculated with a protozoa-free mixture of soil microorganisms whether thiram was added or not, but after an initial rise in numbers, its abundance fell if the mixture contained protozoa; the rate of this fall was delayed by the fungicide. The numbers of R. phaseoli were consistently less in sterile soil inoculated with the rhizobium plus a mixture of soil microorganisms containing ciliates and other protozoa than if the inoculum contained other protozoa but no ciliates. These results suggest that a suppression of protozoa, and possibly especially the ciliates, accounts for the enhanced growth of beans and the greater initial frequency of nodules formed by the thiram-resistant R. phaseoli in the presence of this fungicide. Thiram applied to uninoculated seed enhanced bean growth if thiram-resistant R. phaseoli were present in soil.     

Observations on changes in abundance of questing Ixodes ricinus,castor bean tick,over a 35‐year period in the eastern part of its range (Russia,Tula region)

Ixodes ricinus (Acari: Ixodidae) L. transmit a wide variety of pathogens to vertebrates including viruses, bacteria and protozoa. Understanding of the epidemiology of tick‐borne infections requires basic knowledge of the regional and local factors influencing tick population dynamics. The present study describes the results of monitoring of a questing I. ricinus population, conducted over 35 years (1977–2011) in the eastern, poorly studied part of its range (Russia, Tula region). We have found that the multiannual average abundance of ticks is small and varies depending on the biotope and degree of urban transformation. Tick abundance for the first 14 years of observations (1977–1990) was at the lower limit of the sensitivity of our methods throughout the study area (0.1–0.9 specimens per 1‐km transect). In the following 21 years (1991–2011), a manifold increase in abundance was observed, which reached 18.1 ± 1.8 individuals per 1‐km transect in moist floodplain terraces, and 4.8 ± 0.9 in xerophylic hill woods. Long‐term growth of tick abundance occurred in spite of a relatively constant abundance of small mammals and only minor fluctuations in the abundance of large wild animals. Climate and anthropogenic changes appear to be the main contributors to increased abundance of the tick.     

Bioaugmentation of Syntrophic Acetate-Oxidizing Culture in Biogas Reactors Exposed to Increasing Levels of Ammonia

The importance of syntrophic acetate oxidation for process stability in methanogenic systems operating at high ammonia concentrations has previously been emphasized. In this study we investigated bioaugmentation of syntrophic acetate-oxidizing (SAO) cultures as a possible method for decreasing the adaptation period of biogas reactors operating at gradually increased ammonia concentrations (1.5 to 11 g NH₄⁺-N/liter). Whole stillage and cattle manure were codigested semicontinuously for about 460 days in four mesophilic anaerobic laboratory-scale reactors, and a fixed volume of SAO culture was added daily to two of the reactors. Reactor performance was evaluated in terms of biogas productivity, methane content, pH, alkalinity, and volatile fatty acid (VFA) content. The decomposition pathway of acetate was analyzed by isotopic tracer experiments, and population dynamics were monitored by quantitative PCR analyses. A shift in dominance from aceticlastic methanogenesis to SAO occurred simultaneously in all reactors, indicating no influence by bioaugmentation on the prevailing pathway. Higher abundances of Clostridium ultunense and Tepidanaerobacter acetatoxydans were associated with bioaugmentation, but no influence on Syntrophaceticus schinkii or the methanogenic population was distinguished. Overloading or accumulation of VFA did not cause notable dynamic effects on the population. Instead, the ammonia concentration had a substantial impact on the abundance level of the microorganisms surveyed. The addition of SAO culture did not affect process performance or stability against ammonia inhibition, and all four reactors deteriorated at high ammonia concentrations. Consequently, these findings further demonstrate the strong influence of ammonia on the methane-producing consortia and on the representative methanization pathway in mesophilic biogas reactors.