An investigation of the pathogenic and non-pathogenic free-living amoebae in an activated-sludge plant

Pathogenic and non-pathogenic free-living amoebae from an activated-sludge plant were investigated. The greatest species diversity occurred in the raw, settled sewage and at the beginning of the aeration tank, whilst the lowest number of species occurred in the final sedimentation tanks. Thirteen species of free-living amoebae representing eleven genera were isolated; these included three species of Acanthamoeba (A. castellanii, A. culbertsoni and A. polyphaga), all of wich were pathogenic to mice with varying degrees of virulence. The implications for the public health risk of the presence of pathogenic free-living amoebae in activated-sludge plants are discussed.     

Isolation of amoebae of the genera Naegleria and Acanthamoeba from public fountains in Galicia (N.W. Spain)

Tests for free-living amoebae in water from 11 public fountains in Galicia were conducted at two seasons of the year, winter and summer. Only one fountain gave a negative result for the presence of amoebae in both samples. Twenty five strains were isolated, 14 belonging to the genus Acanthamoeba and 10 to the genus Naegleria. The Acanthamoeba strains were made up of the species A. polyphaga, A. quina, A. castellanii and A. paradivionensis. No relation was found between temperature, presence of free chlorine in the water and whether the water was drinkable and the presence or absence of amoebae, nor with the number of strains present in the water samples. The pathogenic capacity of the strains isolated from the genera Naegleria and Acanthamoeba was tested in vivo. It was found that of the 22 strains that would grow at 37 °C, 4 (3 strains of Acanthamoeba and 1 of Naegleria) caused the death of a statistically significant number of mice that had been inoculated intracerebrally, and the presence of amoebae was confirmed in the brains of all the animals inoculated. Intranasal inoculation caused less mice deaths than intracerebral inoculation, and less organs were found containing amoebae.     

Isolation of free-living pathogenic amoebae from thermal spas in N.W. Spain

We tested for the presence of free-living amoebae in water from 12 thermal spas in northwest Spain; 8 tested positive. No relationship was observed between temperature, pH or bacterial contamination and either the presence of amoebae or the, number of strains per sample. A total of 13 strains were found: 5 of Vahlkampfia, 2 of Naegleria, 3 of Acanthamoeba and 3 of other genera. Those Naegleria and Acanthamoeba strains which survived at 37 ? were tested for pathogenicity by intracerebral and intranasal inoculation of mice; only 2 strains (both of the species Acanthamoeba polyphaga) caused death.     

Amoebae in a waste stabilization pond system in Mexico

A protozoological survey was performed to analyze the organisms of the subphylum Sarcodina Schmarda, present in waste stabilization ponds located at Santo Tomás Atzingo, México, from March to December 1981. The amoebae isolated were identified and counted. Several physicochemical parameters were also determined and correlated with the biological data. Thirteen amoebic species were isolated and identified: one definite pathogen, Entamoeba histolytica Schaudinn; three species that have shown pathogenic strains for humans: Acanthamoeba polyphaga Page, Acanthamoeba castellanii Douglas, and Naegleria sp. (Alexeieff) Calkins; two opportunist species: Entamoeba coli (Grassi) Casagrandi and Barbagallo, and Iodamoeba bühschlii (Von Prowazek) Dobell; and seven free-living amoebae: Acanthamoeba astronyxis Ray and Hayes, Amoeba proteus (Pallas) Leidy, Hartmannella exundans Page, Hartmannella vermiformis Page, Pelomyxa palustris Leidy, Vahlkampfia avara Page, and Vahlkampfia russelli Singh. A clear correlation between the number of amoebae and the temperature and alkalinity of the system was found. The removal capacity of the ponds for E. histolytica varied from 30 to 100% during the survey. The amoebae showed a succession in space and time in the system studied.     

Seasonal distribution of air-borne pathogenic and free-living amoebae in Mexico City and its suburbs

A survey was carried out over a one-year period to isolate amoebae suspended in the air of Mexico City and its suburbs. Sampling stations were placed at the four cardinal points of the metropolitan area. Selective media were used to culture the amoebae isolated. Specialized taxonomic keys and physical and physiological tests were used for identification, and a statistical analysis was performed to determine the correlations between physico-chemical and biological parameters. 108 strains were isolated, of which 19 were pathogenic via intracerebral inoculation and 9 via intranasal inoculation. Species of the generaAcanthamoeba, Vahlkampfia andHartmannella were most abundant.Acanthamoeba polyphaga showed the highest abundance. Several times during the period of the study SO₂ O₃, CO, NO and NO₂ exceeded the permissible levels established by the Mexican government. The ability of amoebae to form cysts and cyst size were important factors for their presence, survival, abundance and diversity in the atmosphere. The main source of air-borne amoebae was the soil. Factors that favored the incidence and diversity of the isolates were wind speed and direction, low relative humidity, generation of frequent dust-storms, resuspension of amoebae by vehicular traffic, proximity to garbage dumps and large extensions of bare soil. Soil cover was a factor associated with a reduction in the incidence and diversity of the aerial amoebae. This study demonstrates that there are viable cysts of amoebae in the atmosphere of Mexico City, that may have potential importance in the case of certain kinds of human allergies and diseases. Further research is needed to find out the aerial presence of viable cysts of obligatory, amphizoic or opportunistic amoebic parasites, and to clarify the qualitative and quantitative effects of the local meteorological and physico-chemical environment on the free-living amoebae present in the atmosphere.     

Distribution of and insights from soil protozoa of the Olympic coniferous rain forest

Decomposition occurs in the surface litter and soil to support temperate rainforests, but little is known about the protozoa that stimulate bacterial activity and turnover. I examined surface litter and top soils, fallen logs, and epiphytes within 2 m from the soil surface in Olympic National Park, USA, of the Pacific Northwest Temperate Coniferous Rain Forest. Ciliates in surface litter numbered 180-580 g⁻¹ dry weight, but were reduced by 20-60% in the underlying soils. Testate amoebae numbered 18,000-77,000 g⁻¹ dry weight in both litter and soil although they were often more abundant in underlying soils. Rotting logs, essential for tree regeneration, supported similar numbers of ciliates, but twice the numbers of testate amoebae. In three epiphytic soils, ciliates numbered 350-550, and testate amoebae 35,000-195,000 g⁻¹ dry weight of soil. In these soils, 26 species of gymnamoebae, 64 species of ciliates, and 113 species of testate amoebae were found. About 65% of the individuals in ciliate and 45% in testate amoebae populations were small, r-selected taxa. Rain forest soil protozoa have distinct testate amoebae populations, and are characterized by enormous biodiversity, the dominance of acrostome species, the proliferation of Euglypha and Nebela species, and the appearance of aquatic taxa. Ecological succession of ciliates and testate amoebae follows an additive (non-replacement) pattern according to a neutral model. The large numbers of persistent r-selected species respond to ecosystem disturbances by mobilizing quickly to resume the bacterivory necessary to help restore the recovering above-ground plant community.     

Amoebae from a take-all suppressive soil which feed on Gaeumannomyces graminis tritici and other soil fungi

Amoebae were isolated from soil of the Waite Institute permanent pasture plot which is suppressive to take-all of wheat. Nine species of amoebae belonging to eight genera were tested for their mycophagy against Gaeumannomyces graminis var. tritici, Cochliobolus sativus and Phytophthora cinnamomi. Members of the genera, Gephyramoeba, Mayorella, Saccamoeba, Thecamoeba and an unidentified species of the order Leptomyxida, were mycophagous. Feeding of mycophagous amoebae and their ability to perforate and lyse melanized propagules of fungi are discussed.     

Recovery of Amoebae Community in the Root Soil of <Emphasis Type="Italic">M. sativa</Emphasis> after a Strong Contamination Pulse with n-Hexane

Microbial food webs tolerate toxic compounds depending on individualistic species resistance and their ability of using alternate food sources. Soil polluted with low-molecular weight volatile organics, such as hexane, diminishes bacterial and fungal communities despite its short residence time. Survival of microbial species depends on perturbation intensity, which in turn restricts resources for amoebae survival in polluted soil. Soil functional recovery from anthropogenic perturbations depends on microbial organic matter (OM) metabolization of pollutants. However, reconfiguration of amoebae community after soil exposure remains largely unknown. A microcosms study was carried out to determine the effects of hexane on the community structure of soil amoebae as well as the importance of Medicago sativa on amoebae community recovering. Hexane had a negative impact on species richness and structure of the amoebae community 24 h after pollution. There was a significant increase in species richness and number of amoebae 30 days after contamination. These two parameters further increased after 60 days from contamination. After 30 days of the initial trophozoites extinction caused by Hexane, M. sativa’s. Root zone showed a significant increase of both species richness and number of individuals. This recovery trend was kept after 60 days when the highest values in species richness and abundance of individuals were shown in both polluted and non-polluted microcosms. In conclusion, M. sativa’s root zone speeds up recovery of the amoebae community structure after pollution exposure.     

Influence of some agroforestry practices on the temporal structures of nematodes in western Kenya

The influence of agroforestry practices on the temporal fluctuation of nematodes was studied in western Kenya. The experiment comprised a fallow phase, which had the following treatments: (1) maize/beans intercropping; (2) maize/beans intercropping with rock phosphate; (3) Crotalaria fallow; (4) Crotalaria fallow with rock phosphate; (5) Crotalaria fallow with rock phosphate (+ Calliandra and napier hedges); and a cultivation phase when all plots were planted to maize and beans. There were strong seasonal fluctuations in the abundance of both free-living and plant-parasitic nematodes. The abundance of plant-parasitic nematodes did not vary among cropping systems during the fallow phase, but varied significantly during the cultivation phase of the experiment. Pratylenchus spp. appeared to be stimulated by the application of phosphorus while Scutellonema spp. had higher populations in the maize crops, which were planted after a Crotalaria fallow. In both the fallow and the cultivation phases, the sampling date had a significant impact on nematode abundance. There were more plant-parasitic nematode species during the fallow phase of the study, but the evenness of the different nematode communities was significantly greater in the cultivation phase of the experiment.     

富营养化水体中芦苇和菖蒲浮床氮净化能力比较研究

以小兴凯湖水为原水配制不同富营养化程度的污水,比较了芦苇和菖蒲浮床系统对TN的去除效果,分析了植物根系特征和植物吸收氮总量与氮去除效率和速率之间的关系,研究氮去除的主要途径和主要影响因素。结果表明,芦苇和菖蒲浮床对TN均有较强的净化能力,在TN浓度分别为9．63、4．58mg·L^-1的污水中,芦苇浮床对TN的平均去除率分别为91．5％和84．2％,菖蒲浮床的去除效率分别为89．9％和82．8％,植物吸收对氮去除的贡献率为36．4％～77．1％。两种污水中,芦苇浮床在生长末期前2d对TN的平均去除速率分别为4．20、1．77mg·L^-1·d^-1,显著高于菖蒲浮床的1．75、1．04mg·L^-1·d^-1。两种植物的根系总长度、表面积和体积3个指标均与TN去除速率呈正相关关系（R≥0．826,P〈0．01）,根系发达程度是影响TN去除速率的主要因素。     

2种植物浮床对含抗生素养殖废水的净化效果

[目的]探讨湿地植物种类、浮床覆盖面积和处理时间3个因素对含抗生素养殖废水净化效果的影响,为高效利用湿地植物浮床技术去除养殖废水中的传统污染物和抗生素提供依据。[方法]分别利用巴拉草(Brachiaria mutica)和短叶茳芏(Cyperus malaccensis Lam. var.brevifolius)构建了植物浮床,每种植物浮床设置了20%,40%,60%这3个水面覆盖面积,监测了植物浮床在不同处理时间对养殖废水中的化学需氧量(COD)、氮磷和磺胺嘧啶的净化效果。[结果]各处理水质各指标随着运行时间持续变好:pH值变中性;溶解氧(DO)含量增加;氧化还原电位(ORP)上升;COD,氨氮(NH_3-N),总氮(TN),总磷(TP)和抗生素磺胺嘧啶(SDZ)浓度持续降低;不同植物种类对水体pH值和ORP值,DO和COD的含量无显著影响,但显著影响水体营养盐的去除;巴拉草浮床对水体中NH_3-N,TN和TP的去除效果优于短叶茳芏浮床;两种植物对SDZ的去除效率相当;总体上,60%的覆盖面积更加有利于水体中污染物的去除,但不利于DO扩散。[结论] 60%覆盖面积的巴拉草浮床,在运行47 d时,去除养殖废水中COD(88%),NH_3-N(97%),TN(89%),TP(94%)和SDZ(43%)的效果最好。     

Mycophagous amoebae in Australian forest soils

Twenty-seven soils, mostly from forested locations in southeastern Australia and Western Australia, were surveyed for the presence of mycophagous vampyrellid amoebae. Characteristic perforations and lysis of conidia of Cochliobolus sativus incubated in 20 of these soils indicated the presence of these amoebae. Isolations of Arachnula impatiens and an amoeba which may be a species of Theratromyxa were obtained from 15 soils. A. impatiens perforated and lysed hyphae and chlamydospores of Phytophthora cinnamomi in culture.     

Infection of Mayorella penardi (Gymnamoebia) in oak-hornbeam forest soil by the ectoparasitic fungus Amoebophilus simplex (Zygomycota)

During research on naked amoebae in terrestrial habitats in the oak-hornbeam forests of Malé Karpaty Mts. (Western Slovakia), a mass fungal infection in Mayorella penardi Page, 1972 (Euamoebida: Paramoebidae) was observed. In one of the 243 soil and moss samples examined a remarkably high percentage (80%) of amoebae were infected by the ectoparasitic soil fungus Amoebophilus simplex Barron, 1983 (Zygomycota: Zoopagales), as revealed by detailed light microscopical observations of infected specimens. The parasitic fungus displayed characteristic cylindrical conidia (length 10 μm, breadth 3 μm) projecting out from the posterior part of the amoeba cell. Infecting conidium frequently produced a chain of two to three conidia and, rarely, secondary chains from the distal end. Two to four infections in a single amoeba were frequently observed. From the present study, and from earlier records, it can be deduced that Amoebophilus simplex is specialized in parasitizing mayorellid amoebae, maybe even Mayorella penardi exclusively. Two other species (Korotnevella diskophora and Korotnevella stella) from the family Paramoebidae were recorded in the same sample, but neither of them showed signs of infection.     

Testate amoebae (protista) of an elevational gradient in the tropical mountain rain forest of Ecuador

We investigated the species composition of testate amoebae at three altitudes (1000, 2000 and 3000 m) and two horizons (L and F/H/Ah) of a tropical mountain rain forest in southern Ecuador. A total of 135 species and intraspecific taxa of testate amoebae were found (36 samples). Rarefaction plots suggest that only few more species are to be expected. Species number in the L horizon was at a maximum at 2000 m (109 species) and similar at 1000 and 3000 m (75 and 71 species, respectively). Species numbers in the F/H/Ah horizon were 29, 67 and 48 at 1000, 2000 and 3000 m, respectively. The density of testate amoebae in the L horizon increased significantly in the order 1000<3000<2000 m with 3147±129, 9198±144 and 12,497±1317 ind. g⁻¹ dry matter and in the F/H/Ah horizon with 176±25, 3118±97 and 4986±102 ind. g⁻¹ dry matter, respectively. The significant Horizon×Elevation interaction reflects the exceptionally low abundance of testate amoebae in the Ah horizon at 1000 m. The results suggest that species richness of testate amoebae does not decrease continuously with elevation; rather, it peaks at an intermediate elevation. Further, the data suggest that diversity, but not density of testate amoebae in tropical forests exceeds that in temperate forests. Morphological features of testate amoebae reflected semiaquatic habitat conditions. The great majority of testate amoebae species of the studied tropical mountain rain forests are geographically widespread, including temperate regions; however 9 of the species (i.e. 6.7%) are considered tropical, some of these species likely represent Gondwana relicts.     

Tertiary treatment of the liquid fraction of pig manure with Phragmites australis

Since 2003, the pig industry in Flanders (Belgium) is obliged to process a portion of the nutrient overproduction. In general, pig manure processing occurs as follows: i) separation into liquid and solid fractions, ii) conversion of the solid fraction to an exportable product (e.g. composting) and iii) reduction of nutrient contents in the liquid fraction before discharge into surface water or spreading on arable land. The aim of this study was to evaluate the potential of constructed wetlands (CWs) planted with Phragmites australis to reduce nitrogen (N), phosphorus (P) and chemical oxygen demand (COD) in the liquid fraction to levels below discharge criteria. In addition, the removal efficiency of heavy metals (Cu, Zn) present at elevated levels in the liquid fraction was evaluated. A greenhouse experiment was conducted with subsurface flow (SSF) reed beds (2 × 0.125 × 0.11 m) filled with sand, loam, clayey sand or expanded clay (argex). The liquid manure load was set at 1 mm per day. Removal efficiencies varied between 64–75% for COD, 73–83% for N and 71–92% for P, depending on the matrix material used. However, effluent levels still remained significantly above the Flemish legal discharge criteria of 2 mgl^?1, 15 mgl^?1 and 125 mgl^?1 for P, N and COD respectively.     

Ability of different plant species to promote microbiological processes in semiarid soil

In semiarid climate soils, the establishment of a plant cover is fundamental to avoid degradation and desertification processes. A better understanding of the ability of plants to promote soil microbial processes in these conditions is necessary for successful soil reclamation. Six different plant species were planted in a semiarid soil, in order to know which species are the most effective for the reclamation of semiarid areas. Six years after planting, the rhizosphere soils were studied by measuring chemical (pH, electrical conductivity, total organic carbon and other carbon fractions), physical (% of saggregates), microbiological (microbial biomass carbon and soil respiration), and biochemical (dehydrogenase, phosphatase, β-glucosidase and urease activities) parameters. In general, in all the soil–plant systems plant nutrients, organic matter and microbial activity increased compared to the control soil. For some species, such as Rhamnus lycioides, the increase in the total organic carbon content (TOC) in the rhizosphere zone was almost 200%. A positive correlation was found between TOC and water-soluble carbon (p<0.001); both parameters were negatively correlated with electrical conductivity. Microbial biomass carbon and soil respiration were highest in the rhizosphere of Stipa tenacissima (98% and 60%, respectively, of increase on soil control values) and Rosmarinus officinalis (94% and 51%, respectively, of increase on soil control values). These microbiological parameters were correlated with the percentage of stable aggregates (p<0.01). Enzyme activities were affected by the rhizosphere, their values depending on the shrub species.     

Ammonia Removal from Oil Refinery Effluent in Vertical Upflow Macrophyte Column Systems

Constructed vertical macrophyte systems, for nitrogen removal from oil refinery wastewater, were investigated. Detailed studies were carried out in laboratory columns (diameter, 0.06 m; depth, 0.5 m; operating volume, 0.6 L) planted with common reed, Phragmites australis. Through a vertical flow format, collected oil refinery wastewater was supplied directly to the columns. Wastewater quality varied through the experimental period with initial ammonia concentrations ranging from 3 to 20 mg N L^-1. Effective ammonia removal was obtained for the planted columns with a hydraulic detention time of 5 hr. Removal efficiencies above 90% was obtained for high (above 6 mg N L^-1) ammonia inflow concentrations. A satisfactory ammonia removal was obtained at shorter detention times for the low initial concentrations. Longer detention times also provided organic nitrogen removal. Recirculation of the flow, which provides the same total detention time but a higher hydraulic loading, provides the possibility to adjust the flow rate and the inflow ammonia concentration with detention time to achieve a target outflow concentration.     

Insights on the role of vegetation on nitrogen cycling in effluent irrigated lands

This work investigated the effect of plant species (Eucalyptus camaldulensis vs Arundo donax) on N-turnover during land application of pre-treated municipal wastewater. The study was carried out in 40-L pots under field conditions and revealed strong effects of plant species on N uptake, potential nitrification rate (PNR), and gaseous losses of N. E. camaldulensis accumulated 38% more N in its biomass than A. donax. With regard to the soil N, there was no effect of plant species on total Kjeldahl N content. The lower PNR measured in pots planted with A. donax could not be explained by differences in archaeal or bacterial amoA gene copies; suggesting that plant species affected the activity of nitrifiers.The loads of NH₄⁺-N applied were not found to have delayed the oxidation of NH₄⁺-N, despite the differences in PNR, as indicated by soil solution and soil NH₄⁺-N concentrations in the rhizosphere of the two plant species throughout the period of the study. However, decreased concentrations of NO₃⁻-N were measured in the pots planted with A. donax from the end of June onwards. This finding implies increased losses of N through denitrification and reveals a direct effect of plant species on the activity of denitrifiers since differences in copy numbers of denitrification genes (nirK, nirS, nosZ) were observed only early in the season. Our findings reveal a critical role of plant species on N cycling in terrestrial environments with important implications for the management/restoration of N-polluted areas, such as riparian zones, and for land treatment systems and constructed wetlands.     

In vitro studies of the impact of the naked soil amoeba Thecamoeba similis Greef,feeding on phototrophic soil biofilms

The effect of grazing by the naked soil amoeba Thecamoeba similis feeding on algae of in vitro phototrophic soil biofilms was investigated in terms of carbon biomass of algae, bacteria and amoebae and compared to shifts in the microbial community determined by 16S rDNA t-RFLP patterns. In a first approach, grazing of T. similis on unialgal Xanthonema hormidioides biofilms growing on regosol was investigated. The growth rate of T. similis was 0.5 divisions per day and the production efficiency reached 25% (allocation of prey-carbon to predator-carbon). The algal biomass decreased from the original 100% down to 7% at the end of the experiment. The increase of egested residues enhanced bacterial growth; the biomass carbon ratio bacteria vs. algae increased from 0.25 to 4.5 indicating the degradation of the algal biofilm, while the 16S rDNA t-RFLP patterns indicated changes in the composition of the microbial community. A subsequent experiment was conducted by inoculating T. similis on a natural biofilm community consisting of trichomes of cyanobacteria, several species of Chlorophyceae and Xanthophyceae. The total algal biomass (including cyanobacteria) remained constant during the experiment. However, 73% of the algal biomass without cyanobacteria was grazed, while the amount of mucous Chlamydocapsa spec. doubled from 32% to 68%. The growth rate of T. similis was 0.4 divisions per day and the production efficiency was only 13%. The 16S rDNA t-RFLP patterns showed a shift of the microbial community. Our results give a first insight into the obviously high potential of naked soil amoebae feeding on algae to change the structures of both the algal and bacterial communities of phototrophic soil biofilms.     

Population dynamics of amoebae in soils suppressive and non-suppressive to wheat take-all

Populations of soil amoebae were estimated throughout two wheat crops and a fallow from wheat rotations suppressive and non-suppressive to take-all. The suppressive pasture-pasture-wheat plot showed higher rhizosphere populations of mycophagous amoebae and higher associations of mycophagous and other amoebae with wheat roots throughout the crop period than the non-suppressive continuous-wheat plot. Populations of mycophagous amoebae were positively and significantly (P = 0.05) correlated with populations of the take-all fungus in both soils, and with soil moisture in the non-suppressive soil. The possible role of mycophagous amoebae in the wheat rhizosphere in suppressive soil is discussed.