Colony Structure of a Consortium of Nitrifying Bacteria

Colonies produced by a consortium of nitrifying bacteria were studied using light and electron microscopy. The colonies were obtained by direct plating of inoculum from a two-stage nonsterile chemostat fermentor and by repeatedly passing the microbial community of the fermentor through selective media containing ammonium or nitrite. The colonies studied can be characterized by a specific combination of six types of cells differing in their ultrastructure and spatial location within the colony. The types of cells occurring within a given colony were found to depend on the nitrogen compound present in the medium. As a result of our study, morphological features of colonial bacterial communities were identified. The proposed approach can be viewed as a method to describe microbial associations and communities.     

Characterization of the Microbial Community in a Partial Nitrifying Sequencing Batch Biofilm Reactor

A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon–Wiener index (H′) results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.     

Measuring Antimicrobial Activity Against Biofilm Bacteria

Standardization of methodology and interpretation has proved essential to scientific progress in studies of the activity of antimicrobial agents against planktonic bacteria. Current studies of antimicrobial activity against biofilm bacteria lack standardization of methodology. The principles applied to standardization of methods for planktonic bacteria can serve as a template in developing standards for studying biofilm bacteria. Such standards are essential to allow meaningful comparison between published studies.     

T_RFLP技术及其在硝化细菌群落分析中的应用

T_RFLP是建立在PCR基础上的,一种不依赖于传统培养方法的微生物生态学的研究方法.具有快速、灵敏的特点.自1997年首次被报道以来,T_RFLP技术已广泛应用于菌种鉴定、群落对比分析、群落中系统发育种群多样性的评估等领域,并成为环境微生物群落结构分析的强有力工具之一.目前T_RFLP在国内的应用较少,硝化细菌的群落分析上还未见报道.但作为一种研究微生物群落结构特征的理想方法,将会得到广泛地应用.本文主要介绍了T_RFLP的基本原理,概括了在微生物群落分析上的应用,阐述了硝化细菌传统研究的局限性及T_RFLP在硝化细菌群落结构分析上的应用前景.     

几种理化因子对海洋硝化细菌去除氨氮效果的影响

研究pH、温度、供氧状况、投菌量、氨氮初始浓度等理化因子对海洋硝化细菌去除氨氮效果的影响。结果表明,各种理化因子对硝化细菌去除氨氮效果均有明显影响,海洋硝化细菌的最佳作用条件是pH 8.5,25℃,好氧,对氨氮的去除效果随着投菌量的增加而增强。在适宜条件下,海洋硝化细菌对海水中氨氮具有较好的去除效果。研究海洋硝化细菌在去除氨氮过程中D IN之间相互转化关系发现,在处理系统中随着NH4 -N含量的降低,NO2--N、NO3--N含量持续增加,大约有66.57%的D IN在海洋硝化细菌作用过程中以其他形式脱离了处理系统。     

Effects of pH and Oxygen and Ammonium Concentrations on the Community Structure of Nitrifying Bacteria from Wastewater

Shifts in nitrifying community structure and function in response to different ammonium concentrations (50, 500, 1,000, and 3,000 mg of N liter⁻¹), pH values (pH 6.0, 7.0, and 8.2), and oxygen concentrations (1, 7, and 21%) were studied in experimental reactors inoculated with nitrifying bacteria from a wastewater treatment plant. The abilities of the communities selected for these conditions to regain their original structures after conditions were returned to the original conditions were also determined. Changes in nitrifying community structure were determined by performing an amplified ribosomal DNA (rDNA) restriction analysis of PCR products obtained with ammonia oxidizer-specific rDNA primers, by phylogenetic probing, by small-subunit (SSU) rDNA sequencing, and by performing a cellular fatty acid analysis. Digestion of ammonia-oxidizer SSU rDNA with five restriction enzymes showed that a high ammonium level resulted in a great community structure change that was reversible once the ammonium concentration was returned to its original level. The smaller changes in community structure brought about by the two pH extremes, however, were irreversible. Sequence analysis revealed that the highest ammonium environment stimulated growth of a nitrifier strain that exhibited 92.6% similarity in a partial SSU rRNA sequence to its nearest relative, Nitrosomonas eutropha C-91, although the PCR product did not hybridize with a general phylogenetic probe for ammonia oxidizers belonging to the β subgroup of the class Proteobacteria. A principal-component analysis of fatty acid methyl ester data detected changes from the starter culture in all communities under the new selective conditions, but after the standard conditions were restored, all communities produced the original fatty acid profiles.Autotrophic nitrifying bacteria that oxidize ammonium to nitrite and nitrate are found in soils, sediments, wastewaters, freshwater, and marine water and on building facades. They are essential components of the nitrogen (N) cycle, linking the most reduced and most oxidized forms of inorganic N. Nitrification occurs as a two-step process carried out by two distinct groups of bacteria; ammonia-oxidizing bacteria convert ammonia to nitrite, and then nitrite oxidizers convert nitrite to nitrate (22, 30). Environmental factors control the rate of nitrification. The most significant environmental factors are substrate concentration, pH, temperature, and oxygen availability (12, 23). Nitrifying bacteria exhibit different substrate concentration sensitivities (26). Media containing low substrate concentrations (10 mg of NH₄⁺ liter⁻¹) can give larger most-probable-number counts of ammonia oxidizers than media containing higher NH₄⁺ concentrations (6, 26). Also, ammonia oxidation is inhibited at high substrate concentrations. The growth rates of Nitrosomonas spp. cultures were reduced in the presence of 1,050 to 2,800 mg of NH₄⁺-N liter⁻¹ (16). Substrate inhibition of ammonia oxidation has also been observed in studies of wastewater systems (23). Natural environments, such as soil and water, usually contain 1 to 10 mg of NH₄⁺-N liter⁻¹ (22), yet liquid wastes from animal farms give rise to concentrations up to 1,600 or 5,600 mg of NH₄⁺-N liter⁻¹ (5, 17). Free ammonia (NH₃) rather than the total ammonium concentration inhibits ammonia oxidizers (1). As the ratio between the ionized form and the nonionized form depends on pH, the toxicity of ammonium also depends on the environmental pH.The pH range for growth of pure cultures of ammonia oxidizers is 5.8 to 8.5, and the pH range for growth of nitrite oxidizers is 6.5 to 8.5 (30). Nitrification was inhibited at pH values below 5.8 in our preliminary experiments performed with an enriched culture of nitrifiers obtained from wastewater. Yet in natural environments, such as soil, nitrification has been reported to occur at pH values below 4.0 (7, 29).Limiting amounts of dissolved oxygen (concentrations below 2 mg liter⁻¹) inhibit nitrification and cause nitrite accumulation or nitrous and nitric oxide production (9, 21). Ammonia-oxidizing bacteria are the key functional group in removing ammonium from wastewaters. Knowledge of the effect of oxygen on nitrification and nitrifying populations has economic importance since aeration of activated sludge is one of the most costly items in the operation of a wastewater treatment plant (21).In environments with high inputs of ammonium, such as wastewaters, biooxidation of this substrate increases the oxygen uptake and lowers the pH. Such modifications of the environment not only affect the production of nitrite and nitrate but can also select a different nitrifying community that is perhaps specialized for these new conditions. Nitrification does occur in extreme environments that pure cultures of nitrifiers cannot tolerate (4). In this study we examined extreme environments in which nitrifying bacteria may be viable but have not been cultured thus far.Because of the difficulty of obtaining nitrifier isolates, nucleic acid-based methods have greatly aided studies of the diversity of nitrifiers (11, 20, 27, 28). Recent molecular investigations have provided valuable information concerning the diversity of ammonia oxidizers in natural environments (5, 15, 20, 25). However, no previous study has focused on the structural or compositional responses of nitrifying communities to perturbations in the environment. In the present laboratory study we examined the effects of high ammonium concentrations, different pH values, and different oxygen concentrations on nitrification and on the community structure of nitrifying bacteria from wastewater. To test the abilities of the communities to regain their original structures, growth of nitrifying communities under the new conditions was followed by incubation under the original conditions.     

T_RFLP技术及其在硝化细菌群落分析中的应用

T_RFLP是建立在PCR基础上的, 一种不依赖于传统培养方法的微生物生态学的研究方法。具有快速、灵敏的特点。自1997年首次被报道以来, T_RFLP技术已广泛应用于菌种鉴定、群落对比分析、群落中系统发育种群多样性的评估等领域, 并成为环境微生物群落结构分析的强有力工具之一。目前T_RFLP在国内的应用较少, 硝化细菌的群落分析上还未见报道。但作为一种研究微生物群落结构特征的理想方法, 将会得到广泛地应用。本文主要介绍了T_RFLP的基本原理, 概括了在微生物群落分析上的应用, 阐述了硝化细菌传统研究的局限性及T_RFLP在硝化细菌群落结构分析上的应用前景。     

Mechanisms for Soil Moisture Effects on Activity of Nitrifying Bacteria

Moisture may limit microbial activity in a wide range of environments including salt water, food, wood, biofilms, and soils. Low water availability can inhibit microbial activity by lowering intracellular water potential and thus reducing hydration and activity of enzymes. In solid matrices, low water content may also reduce microbial activity by restricting substrate supply. As pores within solid matrices drain and water films coating surfaces become thinner, diffusion path lengths become more tortuous, and the rate of substrate diffusion to microbial cells declines. We used two independent techniques to evaluate the relative importance of cytoplasmic dehydration versus diffusional limitations in controlling rates of nitrification in soil. Nitrification rates in shaken soil slurries, in which NH(inf4)(sup+) was maintained at high concentrations and osmotic potential was controlled by the addition of K(inf2)SO(inf4), were compared with rates in moist soil incubations, in which substrate supply was controlled by the addition of NH(inf3) gas. Comparison of results from these techniques demonstrated that diffusional limitation of substrate supply and adverse physiologic effects associated with cell dehydration can explain all of the decline in activity of nitrifying bacteria at low soil water content. However, the relative importance of substrate limitation and dehydration changes at different water potentials. For the soil-microbial system we worked with, substrate limitation was the major inhibiting factor when soil water potentials were greater than -0.6 MPa, whereas adverse physiological effects associated with cell dehydration were more inhibiting at water potentials of less than -0.6 MPa.     

Nitrifying Bacteria in Wastewater Reservoirs

Deep wastewater reservoirs are used throughout Israel to store domestic wastewater effluents for summer irrigation. These effluents contain high concentrations of ammonia (≤5 mM) that are frequently toxic to photosynthetic microorganisms and that lead to development of anoxic conditions. Population dynamics of nitrifying bacteria and rates of nitrification were studied in two wastewater reservoirs that differed in organic load and degree of oxygenation and in the laboratory under controlled conditions, both by serial dilutions in mineral medium and microscopically with fluorescein isothiocyanate-conjugated antibodies prepared against local isolates. The difference in counts by the two methods was within 1 order of magnitude. In the laboratory, an O₂ concentration of 0.2 mg liter⁻¹ was close to optimal with respect to growth of NH₃ oxidizers on domestic wastewater, while O₂ concentrations of 0.05 mg liter⁻¹ supported significant rates of nitrification. It was found that even hypertrophic anaerobic environments such as the anaerobic hypolimnion of the wastewater reservoir or the anaerobic settling ponds are capable of sustaining a viable, although not actively nitrifying, population of Nitrosomonas spp. and Nitrobacter spp., in contrast to their rapid decline when maintained anaerobically in mineral medium in the laboratory. Nitrification rates of NH₃ in effluents during storage in the reservoirs were slower by 1 to 2 orders of magnitude compared with corresponding rates in water samples brought to the laboratory. The factors causing this inhibition were not identified.     

Nitrification and Autotrophic Nitrifying Bacteria in a Hydrocarbon-Polluted Soil

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.     

不同富集和分离培养条件下的含酚废水处理生物膜微生物群落结构与活性比较分析

采用Biolog和变性梯度凝胶电泳(DGGE)技术研究了不同苯酚浓度培养对焦化废水处理厂反硝化池生物膜样品中微生物群落结构和代谢类型的影响。DGGE结果表明, 不同浓度苯酚和不同培养方式富集培养后, 细菌16S rDNA的部分条带分布谱形发生改变, 还有部分条带只受到了苯酚浓度变化的影响; 富集培养过程中由于碳源组成相对焦化废水简单, DGGE条带所代表的优势微生物多样性有所降低。Biolog试验结果表明, 生物膜样本的细菌群落代谢能力最强; 低浓度苯酚富集后的样品能利用的底物碳源类型最丰富。对Biolog试验结果的主成分分析显示, 相同浓度苯酚富集培养后的细菌群落代谢功能多样性相似, 但从DGGE结果看出其结构组成产生了变化。富集培养使样品微生物群落的代谢功能发生改变, 低浓度的苯酚富集增加了群落中微生物的代谢类型。而不同条件获得的分离物其苯酚降解能力的初步分析也表明, 富集与分离条件对苯酚降解菌的分离能力和得到的菌株特性具有差别。     

Abundance, Activity, and Community Structure of Pelagic Methane-Oxidizing Bacteria in Temperate Lakes

The abundance and activity of methane-oxidizing bacteria (MOB) in the water column were investigated in three lakes with different contents of nutrients and humic substances. The abundance of MOB was determined by analysis of group-specific phospholipid fatty acids from type I and type II MOB, and in situ activity was measured with a ¹⁴CH₄ transformation method. The fatty acid analyses indicated that type I MOB most similar to species of Methylomonas, Methylomicrobium, and Methylosarcina made a substantial contribution (up to 41%) to the total bacterial biomass, whereas fatty acids from type II MOB generally had very low concentrations. The MOB biomass and oxidation activity were positively correlated and were highest in the hypo- and metalimnion during summer stratification, whereas under ice during winter, maxima occurred close to the sediments. The methanotroph biomass-specific oxidation rate (V) ranged from 0.001 to 2.77 mg CH₄-C mg⁻¹ C day⁻¹ and was positively correlated with methane concentration, suggesting that methane supply largely determined the activity and biomass distribution of MOB. Our results demonstrate that type I MOB often are a large component of pelagic bacterial communities in temperate lakes. They represent a potentially important pathway for reentry of carbon and energy into pelagic food webs that would otherwise be lost as evasion of CH₄.     

Changes in Activity and Community Structure of Methane-Oxidizing Bacteria over the Growth Period of Rice

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.     

Structure and Dynamics of Exopolymers in an Intertidal Diatom Biofilm

Diatom biofilms growing at the surface of the intertidal mudflat of Marennes Oléron, France, were incubated for 48 h in the laboratory under simulated conditions of high- and low tide (immersed and emersed in seawater) and day and night (illuminated or dark conditions). The biofilms were subsequently sampled using the cryolander technique, without disturbing the structure. The samples were kept in liquid nitrogen until they were transferred to the cooled stage of a field-emission cryo-scanning electron microscope, which was used to study the structural relationships between the sediment particles, the diatoms and the different types of extracellular polymeric substances (EPS) produced by these organisms. The diatoms were most abundant at the sediment surface when incubated in the light under emersed conditions. In the dark or when immersed, the diatoms migrated into the sediment. In the light, the diatoms were coated with EPS, while this was not the case when incubated in the dark. When immersed, the sediment surface appeared smooth as the result of the deposition of mud. Under emersed conditions, the coarser silt grains were prominently present. These grains were wrapped with organic matter and bound together through threads of EPS. This was the case both in light and in dark incubated sediment. It is proposed that this latter type of EPS contributes to the increased erosion threshold of intertidal mudflats colonized by biofilms of diatoms.     

Influences of Infaunal Burrows on the Community Structure and Activity of Ammonia-Oxidizing Bacteria in Intertidal Sediments

Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O₂ concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O₂ transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH₄⁺ consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH₄⁺ and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O₂ transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH₄⁺ consumption capacity. Consequently, the infaunal burrow became an important site for NH₄⁺ consumption in the intertidal sediment.     

Community Structure, Cellular rRNA Content, and Activity of Sulfate-Reducing Bacteria in Marine Arctic Sediments

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell⁻¹ day⁻¹), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.     

Bacterioplankton Community Structure and Dynamics after Large-Scale Release of Nonindigenous Bacteria as Revealed by Low-Molecular-Weight-RNA Analysis

[This corrects the article on p. 3388 in vol. 58.].     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Seasonal Community and Population Dynamics of Pelagic Bacteria and Archaea in a High Mountain Lake

The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenköllesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy. Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters. Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%). Bacteria of the Cytophaga-Flavobacterium cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively. A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences. Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH. About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes. The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn. Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.Until recently, microbial ecologist studying aquatic bacteria faced a basic dilemma: they could either measure the abundance, biomass, growth rates, activity, etc. of the “average” bacterium under in situ conditions (e.g., see reference 13), ignoring the phylogenetic and physiological diversity of microbial communities, or they could isolate and ecophysiologically characterize individual bacterial strains (e.g., see reference 36) but were then not able to tell if these microorganisms were also common in the environment. Consequently, little knowledge has been gathered about the spatial and temporal abundance fluctuations of defined phylogenetic groups and of individual bacterial species in natural habitats. Molecular biological techniques used to identify microbes in environmental samples have recently provided new tools to study bacterioplankton biodiversity (e.g., see references 1, 9, 14, 15, and 19) and the in situ abundances of bacteria and archaea that could not be adequately distinguished before (2, 4, 5, 25). Microbiologists are now in a position to potentially elucidate the biogeography (24), population dynamics, and successions (28) not only of a few morphologically conspicuous microbes but of a large number of species, most of which might still be uncharacterized.Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes selectively visualizes bacterial cells with defined phylogenetic affiliations (3, 5). Based on a rapidly growing set of 16S (and, to a lesser extend, 23S) rRNA sequence data, it is probably the phylogenetically most sophisticated (22) approach for whole-cell in situ identification. On the other hand, FISH of plankton samples can be performed with minimal laboratory requirements (16), and evaluation relies on epifluorescence microscopy, which is a standard technique of aquatic microbial ecologists, e.g., for counting (30) and sizing (33) of picoplankton. In contrast to other identification approaches, FISH largely conserves the gestalt of the targeted microorganisms, i.e., their morphologies, cell sizes (26, 34), and cellular rRNA content (7, 32). So, despite the limitations of the method (as discussed in reference 5), its potential for the identification and cytometric analysis of planktonic microbes is just about to be recognized.Recent investigations have reported that various freshwater microbial communities are dominated by bacteria which are phylogenetically affiliated with the alpha and beta subclasses of the class Proteobacteria (alpha- and beta-proteobacteria, respectively) and with members of the Cytophaga-Flavobacterium cluster (2, 6, 16, 19). These observations were based on single or short-term sampling schemes. The instantaneous community composition of the bacterioplankton, however, may not be representative for different seasons, and the typical ranges of annual community variability remain to be established.The size distribution of planktonic bacteria, and particularly the appearance of filamentous cells, has come into the focus of aquatic microbial ecology in the context of studies of predator-prey interactions. It has been shown both in the laboratory (18, 37) and in field experiments (20) that the filamentous morphotype is a phenotypic adaptation of some microbes to protistan grazing, but there are probably numerous other causes for bacteria to elongate far beyond their typical sizes (e.g., see reference 23). Threadlike bacteria have been observed throughout the year in the plankton of a hypertrophic lake (41) but were also found in midwinter in an oligotropic alpine lake (31).In earlier studies, we demonstrated FISH to be an appropriate tool for the monitoring of spatial (2) and short-term temporal (26) dynamics of different phylogenetic groups of the planktonic microbial community in a high mountain lake. Here we report on the seasonal and vertical abundance distributions of pelagic members of Bacteria and Archaea in Gossenköllesee and analysis of the community structure at different levels of taxonomic resolution. We applied published domain- and group-specific oligonucleotide probes (5) but also used the sequence information from a 16S rRNA-encoding gene (rDNA) library obtained from Gossenköllesee bacterioplankton 1 year earlier to construct specific probes targeted at individual bacterial populations. Particular attention was paid to the changes in abundance and taxonomic composition of the filamentous bacterial morphotypes which were recognized as a permanently important fraction of the planktonic procaryotes in Gossenköllesee. Additionally, we monitored the seasonal changes in the biomass size distributions of the nonfilamentous fraction of the pelagic microbial community.