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.     

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.     

Density and diversity of protozoa in some arid Australian soils

This is the first extensive study of soil protozoa of arid lands. Twenty-six samples from litters, soils, termitaria, and a cyanobacterial crust, collected from central and south Australian arid lands, were analyzed for numbers and species of gymnamoebae, ciliates, and testacea. Amoebae ranged from 1,000-5,000/g of material, and were two orders of magnitude more abundant than ciliates. Both groups increased in abundance and species richness from bare soils through spinifex to mulga to chenopod vegetations. Testacea ranged 900-5,000/g with similar species richness throughout vegetations, but reached 11,900/g with a doubling of species in a refugium in Kings Canyon. The most prevalent species of amoebae, ciliates, and testacea were taxa associated with ephemeral and disturbed habitats (r-selection). The cyanobacterial crust might be considered a micro-refugium because it contained a number of non-encysting protozoa, including Thecamoeba sp. and Nassula picta, feeding on cyanobacterial filaments. The numbers and species richness of protozoa under shrubs were greater than in bare soils, supporting the resource island hypothesis that desert plants create soil heterogeneity by localizing soil fertility under their canopies.     

Protozoa in relation to Rhizobium S-12 and Azotobacter chroococcum in soil

Summary The red sandy loam soils of Bangalore were found to contain Colpoda spp. and Uroleptus spp. as the predominant protozoa. The effect of these protozoa on Rhizobium S-12 and Azotobacter chroococcum which gain entry into soil as bacterial inoculants was studied. In the presence of protozoa a fall in the population of Rhizobium S-12 and A. chroococcum in soil suggested that these bacteria serve as prey for the protozoa. But complete devouring of the prey by the predators never occurred. In broth culture decline in the population of the two bacteria in the presence of protozoa was much quicker compared to soil. Several bacteria survived in broth too suggesting that soil does not prevent the predator from finding its prey. This upholds the recent view that the protozoa devour bacteria if they are sufficiently close to each other, that the energy gained by devouring the cell is greater than that required for hunting. Reduced nodulation and not absence of nodules, in soybean by Rhizobium S-12 in presence of protozoa further supports this view. re]19751105     

悉生培养微缩体系食细菌线虫提高土壤激素含量的机制

研究土壤食细菌线虫与细菌的相互作用及其生态功能是土壤生态学的核心内容之一。食细菌线虫取食细菌可以促进土壤中氮素的矿化,提高氮素养分的供给,改善土壤的营养条件,从而促进植物的生长发育。土壤食细菌线虫促进植物根系生长的"养分作用机制"已得到确认,而"激素作用机制"还存在争议。从供试土壤中筛选获得一株高效产IAA细菌和两种不同cp值的食细菌线虫,通过设置简化的悉生培养系统,对这两种土著食细菌线虫与土著产IAA细菌之间的相互作用,及其对土壤中IAA含量变化的影响进行研究。结果表明:两种食细菌线虫的取食均能促进细菌数量和活性的增强,食细菌线虫与产IAA细菌相互作用也能显著增加土壤中IAA的含量;这些促进作用受到接种食细菌线虫的种类以及培养时间的影响:在培养第10天和第20天时,接种cp值为1的中杆属食细菌线虫显著增加了产IAA细菌的数量;在培养第10天和第30天时,相比较接种cp值为2的头叶属食细菌线虫,接种中杆属食细菌线虫显著提高了土壤中IAA的含量。     

Isolation and assessment of gut bacteria from the Formosan subterranean termite,Coptotermes formosanus (Isoptera: Rhinotermitidae), for paratransgenesis research and application

Paratransgenesis targeting the gut protozoa is being developed as an alternative method for the control of the Formosan subterranean termite (FST). This method involves killing the cellulose‐digesting gut protozoa using a previously developed antiprotozoal peptide consisting of a target specific ligand coupled to an antimicrobial peptide (Hecate). In the future, we intend to genetically engineer termite gut bacteria as “Trojan Horses” to express and spread ligand‐Hecate in the termite colony. The aim of this study was to assess the usefulness of bacteria strains isolated from the gut of FST as “Trojan Horses.” We isolated 135 bacteria from the guts of workers from 3 termite colonies. Sequencing of the 16S rRNA gene identified 20 species. We tested 5 bacteria species that were previously described as part of the termite gut community for their tolerance against Hecate and ligand‐Hecate. Results showed that the minimum concentration required to inhibit bacteria growth was always higher than the concentration required to kill the gut protozoa. Out of the 5 bacteria tested, we engineered Trabulsiella odontotermitis, a termite specific bacterium, to express green fluorescent protein as a proof of concept that the bacteria can be engineered to express foreign proteins. Engineered T. odontotermitis was fed to FST to study if the bacteria are ingested. This feeding experiment confirmed that engineered T. odontotermitis is ingested by termites and can survive in the gut for at least 48 h. Here we report that T. odontotermitis is a suitable delivery and expression system for paratransgenesis in a termite species.     

三种纤毛虫对土壤微生物量和有效氮磷含量的影响

采用土壤培养方法研究了 3种纤毛虫对土壤微生物量及氮磷转化的影响 ,结果表明 ,向土壤接种肾形虫 ( Colopodia sp.)、尖毛虫 ( Oxytricna sp.)和澳毛虫 ( Australothrix sp.) ,特别是澳毛虫 ,显著地降低了土壤微生物碳。说明供试的原生动物与微生物之间存在消长关系。接种澳毛虫显著地降低了土壤有效磷含量 ,而肾形虫和尖毛虫对土壤有效磷含量影响很小 ,仅在培养后期显著地降低了土壤铵态氮含量 ,3种原生动物特别是澳毛虫 ,显著地降低了土壤氮矿化量和硝态氮含量 ,但提高了土壤铵态氮含量 ,说明 3种原生动物抑制了硝化作用 ,而增强了氨化作用。     

Identification of protozoa in dairy lagoon wastewater that consume Escherichia coli O157:H7 preferentially

Escherichia coli O157:H7 (EcO157), an agent of life threatening hemolytic-uremic syndrome, resides in ruminants and is released in feces at numbers as high as 10 million cells/gram. EcO157 could survive in manure for as long as 21 months, but we observed a 90% decrease in cells of an outbreak strain of EcO157 within half a day in wastewater from dairy lagoons. Although chemical, environmental and biological factors may be responsible for this decrease, we observed an 11-fold increase in native protozoa when wastewater was re-inoculated with 2×10(7) cells of EcO157/mL. These protozoa engulfed the green fluorescent protein labeled EcO157 within 2 hours after inoculation, but expelled vacuoles filled with live EcO157 cells within 3 days into surrounding wastewater, whereas other protozoa retained the EcO157-filled vacuoles for 7 days. EcO157 was not detected by confocal microscopy either inside or outside protozoa after 7 days. Mixed cultures of protozoa enriched from wastewater consumed EcO157 preferentially as compared to native aerobic bacteria, but failed to eliminate them when EcO157 cells declined to 10(4)/mL. We isolated three protozoa from mixed cultures and typed them by 18S sequencing as Vorticella microstoma, Platyophyra sp. and Colpoda aspera. While all three protozoa internalized EcO157, only Platyophyra and Colpoda acted as predators. Similar to mixed cultures, these protozoa failed to eliminate EcO157 from PBS containing no other supplemental nutrients or prey. However, spiking PBS with cereal grass medium as nutrients induced predation of EcO157 by Platyophyra sp. after 3 days or enhanced predation by Colpoda after 5 days. Therefore, attempts to enrich protozoa to decrease EcO157 from dairy lagoons, may correspond to an increase in protozoa similar to Vorticella and possibly facilitate transport of bacterial pathogens to food crops grown in proximity.     

对虾养殖池塘底质修复技术对原生动物群落结构的影响

在对虾养殖池塘水体中共检测到原生动物86种,其中纤毛虫48种;鞭毛虫25种,肉足虫13种.原生动物优势种主要由一些运动能力相对较弱或固着生长的种类组成,如褐砂壳虫(Difflugia avellana)、瓜形膜袋虫(Cyclidium citrullus)、透明鞘居虫(Vaginicola crystalline)、多态喇叭虫(Stentor polymorphrus)和钟虫(Vorticella sp.)等,这些原生动物有以腐质和藻类为食、耐污性较强的特点.结果表明:在养殖初期,使用底质修复技术的试验池塘原生动物群落结构恢复比对照塘快,PFU群集的原生动物种类数和密度比对照塘多,水质比对照塘好,嗜污性相对较强的一些纤毛虫如尾草履虫(Paramecium caudaium)、拟瘦尾虫(Paruroleptus spp.)等比对照塘出现晚.在随后的养殖过程中,使用底质修复技术的试验塘原生动物群落结构比对照塘稳定,原生动物种类数和密度起伏小,pH值、透明度变化幅度也小于对照池塘,没有发生病害,水质较对照塘好.     

Rumen Ciliate Fauna of Alaskan Moose (Alces americana), Musk-Ox (Ovibos moschatus) and Dall Mountain Sheep (Ovis dalli)*

Total numbers, generic distribution and percentage species distribution were determined for the ciliate protozoa in rumen contents obtained from Alaskan moose (Alces americana), musk-ox (Ovibos moschatus) and Dall mountain sheep (Ovis dalli). The musk-ox has a fauna somewhat similar to that previously observed in reindeer and caribou. In contrast, only protozoa in the genus Entodinium were observed in moose, while Dall mountain sheep have a fauna unique among Alaskan ruminants studied to date. Other than Entodinium exiguum which was common to all animals, only 2 additional species of Entodinium, observed in the moose and musk-ox, occurred in more than one animal species. Four new species of protozoa are described, Entodinium dalli sp.n., Entodinium constrictum sp.n. and Polyplastron alaskum sp.n. from the Dall mountain sheep and Entodinium alces sp.n. from moose.     

冬季上海市城区河道中原生动物群落的结构特征

于2007年11月至2008年1月,对上海市城区经过初步修复的四条河道(午潮港、横港、朝阳河和曹杨环浜)中原生动物的群落结构和多样性进行了调查.共鉴定出原生动物88种,其中鞭毛虫31种,肉足虫13种,纤毛虫44种.除曹杨环浜无优势种外,其他三条河道的种类均以耐污种类为优势种,如波豆虫 Bodo sp.、滴虫 Monas sp.、隐藻 Cryptomonas sp.等;四条河道均呈现纤毛虫和鞭毛虫种类较为丰富,肉足虫相对较少的现象.四条河道中原生动物多样性指数的平均值变动在1.14～2.87之间;密度变动在900.35～2837.89 ind/ml间.分析发现,前期生态治理较为成功的曹杨环浜具有多样性指数高、密度大、无明显优势种等特征,而其他三条河道中原生动物种类相对较单一,且分布不均匀,主要优势种为一些耐污种类.提示经过人工合理的修复,可以有效改善受污染河道的状况.因此对已经初步修复河道的生态系统进行评估,探索和制定出有针对性的修复方案,并对其加以合理的维护,对建立健康、稳态的河道生态系统是十分必要的.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Concentrations of Pathogens and Indicators in Animal Feces in the Sydney Watershed

A fecal analysis survey was undertaken to quantify animal inputs of pathogenic and indicator microorganisms in the temperate watersheds of Sydney, Australia. The feces from a range of domestic animals and wildlife were analyzed for the indicator bacteria fecal coliforms and Clostridium perfringens spores, the pathogenic protozoa Cryptosporidium and Giardia, and the enteric viruses adenovirus, enterovirus, and reovirus. Pathogen and fecal indicator concentrations were generally higher in domestic animal feces than in wildlife feces. Future studies to quantify potential pathogen risks in drinking-water watersheds should thus focus on quantifying pathogen loads from domestic animals and livestock rather than wildlife.     

Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.     

Selective grazing of methanotrophs by protozoa in a rice field soil

Biological methane oxidation is a key process in the methane cycle of wetland ecosystems. The methanotrophic biomass may be grazed by protozoa, thus linking the methane cycle to the soil microbial food web. In the present study, the edibility of different methanotrophs for soil protozoa was compared. The number of methanotroph-feeding protozoa in a rice field soil was estimated by determining the most-probable number (MPN) using methanotrophs as food bacteria; naked amoebae and flagellates were the dominant protozoa. Among ten methanotrophic strains examined as a food source, seven yielded a number of protozoa comparable with the yield with Escherichia coli [10⁴ MPN (g soil dry weight)⁻¹], and three out of four Methylocystis spp. yielded significantly fewer numbers [10²–10³ MPN (g soil dry weight)⁻¹]. The lower edibility of the Methylocystis spp. was not explained either by their growth phase or by harmful effects on protozoa. Incubation of the soil under methane resulted in a higher number of protozoa actively grazing on methanotrophs, especially on the less-edible group. Protozoa isolated from the soil demonstrated a grazing preference on the different methanotrophs consistent with the results of MPN counts. The results indicate that selective grazing by protozoa may be a biological factor affecting the methanotrophic community in a wetland soil.     

Phylogeny of protozoa deduced from 5S rRNA sequences

Summary The nucleotide sequences of 5S rRNAs from three protozoa,Bresslaua vorax, Euplotes woodruffi andChlamydomonas sp. have been determined and aligned together with the sequences of 12 protozoa species including unicellular green algae already reported by the authors and others. Using this alignment, a phylogenic tree of the 15 species of protozoa has been constructed. The tree suggests that the ancestor for protozoa evolved at an early time of eukaryotic evolution giving two major groups of organisms. One group, which shares a common ancestor with vascular plants, contains a unicellular green flagellate (Chlamydomonas) and unicellular green algae. The other group, which shares a common ancestor with the multicellular animals, includes various flagellated protozoa (includingEuglena), ciliated protozoa and slime molds. Most of these protozoa appear to have separated from one another at a fairly early period of eukaryotic evolution.     

Contrasting Soil Ciliate Species Richness and Abundance between Two Tropical Plant Species: A Test of the Plant Effect

We still have a rudimentary understanding about the mechanism by which plant roots may stimulate soil microbial interactions. A biochemical model involving plant-derived biochemical fractions, such as exudates, has been used to explain this “rhizosphere effect” on bacteria. However, the variable response of other soil microbial groups, such as protozoa, to the rhizosphere suggests that other factors could be involved in shaping their communities. Thus, two experiments were designed to: (1) determine whether stimulatory and/or inhibiting factors associated with particular plant species regulate ciliate diversity and abundance and (2) obtain a better understanding about the mechanism by which these plant factors operate in the rhizosphere. Bacterial and chemical slurries were reciprocally exchanged between two plant species known to differ in terms of ciliate species richness and abundance (i.e., Canella winterana and plantation Tectona grandis). Analysis of variance showed that the bacteria plus nutrients and the nutrients only treatment had no significant effect on overall ciliate species richness and abundance when compared to the control treatment. However, the use of only colpodean species increased the taxonomic resolution of treatment effects revealing that bacterial slurries had a significant effect on colpodean ciliate species richness. Thus, for particular rhizosphere ciliates, biological properties, such as bacterial diversity or abundance, may have a strong influence on their diversity and possibly abundance. These results are consistent with a model of soil bacteria-mediated mutualisms between plants and protozoa.     

Restoration of the ability to settle bulking sludge by bacterial seeding in wastewater treatment

An attempt to restore the settling ability of denatured bulking sludge was under-taken by changing the microflora in waste treatment tanks. A cell suspension of mixed cultures of ten strains of bacteria, which were isolated from normal activated sludge from night soil plants, and a type culture Zoogloea ramigera IAM1236 was seeded into a laboratory-scale aeration tank containing bulking sludge collected from municipal night soil or a food processing (bean curd production) waste treatment plant. The tank was fed with synthetic wastewater or industrial waste and aerated for 22 days. After 5 days, the microflora in the sludge changed remarkably with the seeding of the bacterial culture; filamentous organisms disappeared and active protozoa (Vorticella sp., Epistylis sp., and Lecane sp.) appeared. The sludge became compact and settled rapidly. The SV₃₀ of the sludge temporarily increased, but in the end decreased from 97 to 20%. The CODcr value decreased from 300 to 20 ppm. In the tank without seeding, the sludge contained almost filamentous organisms only which floated and finally decomposed. The effects were confirmed by applied tests in 700-ton scale and 100-ton scale aeration tanks of municipal night soil and a food processing waste treatment plant, respectively.