Molecular assessment of complex microbial communities degrading long chain fatty acids in methanogenic bioreactors

Microbial diversity of anaerobic sludge after extended contact with long chain fatty acids (LCFA) was studied using molecular approaches. Samples containing high amounts of accumulated LCFA were obtained after continuous loading of two bioreactors with oleate or with palmitate. These sludge samples were then incubated in batch assays to allow degradation of the biomass-associated LCFA. In addition, sludge used as inoculum for the reactors was also characterized. Predominant phylotypes of the different samples were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Fingerprinting analysis showed changes in bacterial and archaeal communities during LCFA accumulation and degradation. Full-length 16S rRNA gene sequences of 22 clones, representing the predominant bacteria and archaea, were determined. Most bacterial clones (80%) clustered within the Clostridiaceae. Two major groups of methanogens were identified: hydrogen- and formate-utilizing organisms, closely related to Methanobacterium, and acetoclastic organisms closely related to Methanosaeta and Methanosarcina. Quantification by FISH and real-time PCR showed that the relative abundance of archaea increased during degradation of biomass-accumulated LCFA. These results provide insight into the importance and dynamics of balanced communities of bacteria and methanogens in LCFA-accumulation/degradation cycles.     

Biodegradation of methyl <Emphasis Type="Italic">t</Emphasis>-butyl ether by aerobic granules under a cosubstrate condition

Aerobic granules efficient at degrading methyl tert-butyl ether (MTBE) with ethanol as a cosubstrate were successfully developed in a well-mixed sequencing batch reactor (SBR). Aerobic granules were first observed about 100 days after reactor startup. Treatment efficiency of MTBE in the reactor during stable operation exceeded 99.9%, and effluent MTBE was in the range of 15–50 μg/L. The specific MTBE degradation rate was observed to increase with increasing MTBE initial concentration from 25 to 500 mg/L, which peaked at 22.7 mg MTBE/g (volatile suspended solids)·h and declined with further increases in MTBE concentration as substrate inhibition effects became significant. Microbial-community deoxyribonucleic acid profiling was carried out using denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S ribosomal ribonucleic acid. The reactor was found to be inhabited by several diverse bacterial species, most notably microorganisms related to the genera Sphingomonas, Methylobacterium, and Hyphomicrobium vulgare. These organisms were previously reported to be associated with MTBE biodegradation. A majority of the bands in the reactor represented a group of organisms belonging to the Flavobacteria–Proteobacteria–Actinobacteridae class of bacteria. This study demonstrates that MTBE can be effectively degraded by aerobic granules under a cosubstrate condition and gives insight into the microorganisms potentially involved in the process.     

口腔链球菌摄取生物素与口内白色念珠菌生长的关系

为了探讨口腔细菌生态平衡机制,本文体外检测了４株口腔链球菌（Ｓ．ｓａｌｉｖａｒｉｕｓ,Ｓ．ｓａｎｇｕｉｓ,Ｓ．ｍｕｔａｎｓ和Ｓ．ｍｉｔｉｏｒ）和白色念珠菌生长对生物素的依赖性。结果显示白色念珠菌和口腔链球菌的生长绝对依赖生物素,前者最大半数生长需要生物素约１０ｐＭｏｌ／Ｌ,其最大生长率所需生物素约为１００ｐＭｏｌ／Ｌ;而后者最大半数生长需生物素为５２５ｐＭｏｌ／Ｌ。正常人血清和唾液中生物素的含量约为２１２２９４ｐＭｏｌ／Ｌ。生物素饥饿状态下的白色念珠菌和口腔链球菌,以及唾液离心沉淀物（绝大多数为细菌）摄取生物素具温度依赖性,而代谢抑制剂（叠氮化合物、氰化物和醋酸碘）和某些抗生素（杆菌肽、短杆菌肽、万古霉素和二性霉素）具选择性抑制效应,提示生物素的摄取和跨膜转运是一依赖能量的主动过程。用唾液沉淀物和口腔链球菌吸收培养基中的生物素或与白色念珠菌共同培养,显著抑制白色念珠菌的生长。本文首次提供实验证据显示口腔优势菌可能通过竞争生物素等营养成份,抑制白色念珠菌生长,以维持口腔微生物之生态平衡。     

Bacterial community dynamics involved in Reactive Orange M2R dye degradation using a real time quantitative PCR and scale up studies using sequence batch reactor

Bacterial consortium volatile suspended soil (VSS) (Vatva soil sample) with a capability of azo dye Reactive Orange M2R (ROM2R) decolorization and degradation (shown in our earlier studies using Fourier transform infrared spectroscopy (FTIR) and phytotoxicity studies) was isolated from industrial wastewaters by enrichment culture technique. The present study was carried out to study bacterial population dynamics in consortium Vatva soil sample (VSS) during azo dye ROM2R degradation and to identify the consortium members that were actively involved in the degradation process. To achieve this goal, a real-time Polymerase chain reaction (PCR) assay targeting species-specific region of 16S rDNA of each consortial bacteria was developed to provide quantitative information about the bacterial abundance during azo dye degradation. The real-time PCR assay indicated that Pseudomonas aeruginosa (VSS-6) dominated consortium bacterial community during the active continuous bioremediation process. Attempt has been made to scale up from 100 ml volume to 10 L operation volume with intermittent additions (batch fed loadings) in a Sequence batch reactor (SBR). The development of VSS consortium biomass (MLVSS), changes in COD and biochemical oxygen demand, and the dye degradation were studied under conditions simulating the operations of biological effluent treatment in an attempt to develop a commercially applicable dye effluent treatment process unit.     

Relative contribution of bacterial and photosynthetic production to metazooplankton as carbon sources

Percentages of label transfer (PLT) from bacteria and autotrophsto metazooplankton during 4 h were determined in parallel usingdissolved organic and inorganic¹³C tracers, respectively, inin situ batch incubations, to estimate the relative contributionof each production to the metazooplankton food requirement.The batch incubation for each pathway was done eight times,respectively, during 20 days in a mesocosm where continuousspecies succession of organisms was observed. The PLT in thetwo pathways, dependent on metazooplankton abundance and speciescomposition rather than changes in the activities of producers(bacteria or autotrophs), showed a similar tendency of changeand were of a similar magnitude: mean 1.0% (0.09–2.7%)in the photosynthetic pathway and 0.5% (0.22–1.5%) inthe bacterial pathway. This finding suggests that metazooplanktonare supported by entire microbial food webs including both thebacterial and photosynthetic pathways.     

Community dynamics of a mixed-bacterial culture growing on petroleum hydrocarbons in batch culture

The effects of various hydrocarbon substrates, and a chemical surfactant capable of enhancing crude-oil biodegradation, on the community structure of a mixed-bacterial inoculum were examined in batch culture. Of 1000 TSA-culturable isolates, 68.6% were identified at the genus level or better by phospholipid fatty acid analysis over 7-day time course experiments. Cultures were exposed to 20 g/L Bow River crude oil with and without 0.625 g/L Igepal CO-630 (a nonylphenol ethoxylate surfactant), 5 g/L saturates, 5 g/L aromatics, or 125 g/L refinery sludge. A group of six genera dominated the cultures: Acinetobacter, Alcaligenes, Ochrobactrum, Pseudomonas/Flavimonas, Stenotrophomonas, and Yersinia. Species from four of the genera were shown to be capable of hydrocarbon degradation, and counts of hydrocarbon degrading and total heterotrophic bacteria over time were nearly identical. Pseudomonas/Flavimonas and Stenotrophomonas normally dominated during the early portions of cultures, although the lag phase of Stenotrophomonas appears to have been increased by surfactant addition. Acinetobacter calcoaceticus was the most frequently isolated microorganism during exposure to the saturate fraction of crude oil. Regardless of substrate, the culture medium supported a greater variety of organisms during the latter portions of cultures. Understanding the community structure and dynamics of mixed bacterial cultures involved in treatment of heterogeneous waste substrates may assist in process development and optimization studies.     

Enhanced hyaluronic acid production by a two-stage culture strategy based on the modeling of batch and fed-batch cultivation of Streptococcus zooepidemicus

This study aimed to enhance hyaluronic acid (HA) production by a two-stage culture strategy based on the modeling of batch and fed-batch culture of Streptococcus zooepidemicus. Batch culture had higher specific HA synthesis rate while fed-batch culture had higher specific cell growth rate. The lower specific HA synthesis rate in fed-batch culture resulted from the competition of cell growth for the common precursors at a low substrate concentration. Based on the modeling of batch and fed-batch culture of S. zooepidemicus, a two-stage culture strategy was proposed to enhance HA production. S. zooepidemicus were cultured in a fed-batch mode with sucrose concentration maintained at 1.0+/-0.2g/L during 0-8h and then batch culture was performed during 8-20h with an initial sucrose concentration of 15g/L. With the proposed two-stage culture strategy, HA production was increased to 6.6g/L compared with 5.0g/L in batch culture with the same total sucrose. The enhanced HA production by the proposed two-stage culture strategy resulted from the decreased inhibition of cell growth and the increased transformation rate of sucrose to HA.     

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

PCP degradation is mediated by closely related strains of the genus Sphingomonas

There have been numerous reports in the literature of diverse bacteria capable of degrading pentachlorophenol (PCP). In order to gain further insight into the phylogenetic relationships of PCP-degrading bacteria, we examined four strains: Arthrobacter sp. strain ATCC 33790, Flavobacterium sp. strain ATCC 39723, Pseudomonas sp. strain SR3, and Sphingomonas sp. strain RA2. These organisms were isolated from different geographical locations and all of them degrade high concentrations (100–200 mg/L) of PCP. Southern blot analyses determined that these bacteria all harbour DNA that encodes similar, if not identical, genes involved in PCP degradation. Comparison of the 16S rRNA nucleotide sequences revealed that these organisms were very closely related and, in fact, represent a monophyletic group. The 16S rRNA analyses together with fatty acid and sphingolipid analyses strongly suggest that the four strains are members of the genus Sphingomonas . The close relationship of the four organisms is supported by nucleotide sequence analysis data of the pcpB locus encoding PCP-4-monooxygenase, the first enzyme in the PCP degradative pathway.     

复合碳源培养对酒糟厌氧消化液中丙酸互营氧化菌及群落结构的影响

丙酸累积是影响厌氧消化系统稳定性的主要因素,为了考察酒糟厌氧消化过程中间代谢产物的累积情况,以总固体含量(TS)（质量分数） 5%和7%的白酒糟为发酵原料进行了批次试验。结果表明,乙酸(最高浓度33~129 mmol/L)、丙酸(39~61 mmol/L)、丁酸(5~44 mmol/L)和15种氨基酸(0.01~0.3 mmol/L)为主要中间代谢产物。为了探究其中关键的丙酸降解菌群,以酒糟原始沼液JO为植种源,10 mmol/L丙酸和0.1 mmol/L混合氨基酸为复合碳源进行富集培养,获得中温厌氧丙酸-氨基酸培养系JO-AP。高通量测序分析表明,互营丙酸降解菌与厚壁菌门（Firmicutes）的丙酸厌氧降解菌（Pelotomaculum schinkii）近缘,16S rRNA基因相似性100%,占细菌总丰度的16.7%。对比酒糟原始沼液JO、丙酸培养系JO-P及丙酸-氨基酸培养系JO-AP中的主要功能菌群,发现采用单一碳源和复合碳源获得的优势互营丙酸降解菌不同;传统培养与分子生物学技术相结合可以更全面地掌握系统中的微生物群落组成。     

Uptake and incorporation of the products of proteolysis by the rumen bacteriumBacteroides ruminicola R8/4

The polypeptides of the proteolytic rumen bacteriumBacteroides ruminicola R8/4 grown in the presence of either leaf Fraction 1 protein, bovine serum albumin, or Bactocasitone as sole nitrogen source were separated by SDS-polyacrylamide gel electrophoresis. Over 40 polypeptides were resolved; the pattern for organisms grown on Fraction 1 protein was similar but not identical to that of the serum albumin and Bactocasitone-grown bacteria. All the bacterial polypeptides were distinguishable from the polypeptides of Fraction 1 protein (and serum albumin). The stained pattern was the same for organisms sampled at intervals during the growth of a batch culture. After incubation of the growing organisms with [¹⁴C]-Fraction 1 protein, all the bacterial polypeptides were labeled. Bacteria grown in the presence of nonlabeled Fraction 1 protein and a mixture of [^14C]-labeled amino acids incorporated label into all the polypeptides; the bacteria did not grow in the absence of intact protein, and then virtually no label was incorporated from the amino acid mixture.     

Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility

The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.     

Nitrification and anammox with urea as the energy source

Urea is present in many ecosystems and can be used as an energy source by chemolithotrophic aerobic ammonia oxidizing bacteria (AOB). Thus the utilization of urea in comparison to ammonia, by AOB as well as anaerobic ammonia oxidizing (Anammox) bacteria was investigated, using enrichments cultures, inoculated with activated sludge, and molecular ecological methods. In batch enrichment cultures grown with ammonia a population established in 2 weeks, which was dominated by halophilic and halotolerant AOB as determined by fluorescence in situ hybridization (FISH) experiments, with the 16S rRNA targeting oligonucleotide probe NEU. In other batch enrichment cultures using urea, the AOB population was assessed by PCR amplification, cloning and phylogenetic analysis of amoA and ribosomal 16S rRNA genes. While only one of the 48 16S rRNA gene clones could be identified as AOB (Nitrosomonas oligotropha), the amoA approach revealed two more AOB, Nitrosomonas europaea and Nitrosomonas nitrosa to be present in the enrichment. FISH analysis of the enrichment with probe NEU and newly designed probes for a specific detection of N. oligotropha and N. nitrosa related organisms, respectively, showed that N. oligotropha-like AOB formed about 50% of the total bacterial population. Also N. nitrosa (about 15% of the total population) and N. europaea (about 5% of the total population) were relatively abundant. Additionally, continuous enrichments were performed under oxygen limitation. When ammonia was the energy source, the community in this reactor consisted of Anammox bacteria and AOB hybridizing with probe NEU. As the substrate was changed to urea, AOB related to N. oligotropha became the dominant AOB in this oxygen limited consortium. This resulted in a direct conversion of urea to dinitrogen gas, without the addition of organic carbon.     

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.     

Metabolic activity of pathogenic bacteria during semicontinuous anaerobic digestion.

In natural environments such as anaerobic digesters, bacteria are frequently subjected to the stress of nutrient fluxes because of the continual changes in the flow of nutrients, and to survive, they must be capable of adapting readily to nutrient changes. In this study, the metabolic activities of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes, and Campylobacter jejuni were studied within culture bags (Versapor-200 filters, 0.22-microns pore size) in laboratory anaerobic digesters. The metabolic activity of these bacteria was indicated by their adenylate energy charge (EC) ratios and their ability to incorporate [3H]thymidine, which was related to the respective changes in viable numbers within the culture bags during anaerobic digestion. Fluctuations in the adenylate EC ratios, the uptake of [3H]thymidine, and the viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes cells were probably due to constant changes in the amount of available nutrients within the anaerobic digesters. The viability of S. typhimurium increased quickly after a fresh supply of nutrients was added to the system as indicated by the uptake of [3H]thymidine and an increase in the adenylate EC ratios. The viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes organisms declined rapidly from 10(7) to 10(8) CFU/ml to 10(3) to 10(4) CFU/ml and remained at this level for an indefinite period. The decimal reduction time calculated during the period of exponential decline ranged from 0.8 to 1.2 days for these bacteria. C. jejuni had the greatest mean decimal reduction time value (3.6 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge.

Activated sludge communities which performed enhanced biological phosphate removal (EBPR) were phylogenetically analyzed by 16S rRNA-targeted molecular methods. Two anaerobic-aerobic sequencing batch reactors were operated with two different carbon sources (acetate vs. a complex mixture) for three years and showed anaerobic-aerobic cycles of polyhydroxybutyrate- (PHB) and phosphate-accumulation characteristic for EBPR-systems. In situ hybridization showed that the reactor fed with the acetate medium was dominated by bacteria phylogenetically related to the Rhodocyclus-group within the beta-Proteobacteria (81% of DAPI-stained cells). The reactor with the complex medium was also predominated by this phylogenetic group albeit at a lesser extent (23% of DAPI-stained cells). More detailed taxonomic information on the dominant bacteria in the acetate-reactor was obtained by constructing clone libraries of 16S rDNA fragments. Two different types of Rhodocyclus-like clones (R1 and R6) were retrieved. Type-specific in situ hybridization and direct rRNA-sequencing revealed that R6 was the type of the dominant bacteria. Staining of intracellular polyphosphate- and PHB-granules confirmed that the R6-type bacterium accumulates PHB and polyphosphate just as predicted by the metabolic models for EBPR. High similarities to 16S rDNA fragments from other EBPR-sludges suggest that R6-type organisms were present and may play an important role in EBPR in general. Although the R6-type bacterium is closely related to the genus Rhodocyclus, it did not grow phototrophically. Therefore, we propose a provisional new genus and species Candidatus Accumulibacter phosphatis.     

Two unusual budding bacteria isolated from a swimming pool

S ly , L.I. & H argreaves , M.H. 1984. Two unusual budding bacteria isolated from a swimming pool. Journal of Applied Bacteriology 56 , 479–486.
Two unusual strains of budding bacteria were isolated on a Millipore Pseudomonas Count Water Tester during routine monitoring of Pseudomonas aeruginosa counts in a swimming pool. The first isolate has been identified as Blastobacter sp. It was a yellow-pigmented, Gram negative rod-shaped organism with a polar holdfast by which it attached to solid surfaces or other cells to form rosettes. The cells reproduced by asymmetric division or budding at the free pole of the cell, producing motile daughter cells with a single polar flagellum. The second isolate, which has not yet been identified, was a red-pigmented, Gram negative rod-shaped organism which produced one or more buds at each pole of the cell. Cell division appears to occur by both binary fission and by budding. Both organisms were strict aerobes, catalase and oxidase positive and did not produce acid from glucose in Hugh and Leifson medium.