Phenotypic Properties and Microbial Diversity of Methanogenic Granules from a Full-Scale Upflow Anaerobic Sludge Bed Reactor Treating Brewery Wastewater

Methanogenic granules from an anaerobic bioreactor that treated wastewater of a beer brewery consisted of different morphological types of granules. In this study, the microbial compositions of the different granules were analyzed by molecular microbiological techniques: cloning, denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH), and scanning and transmission electron microscopy. We propose here that the different types of granules reflect the different stages in the life cycle of granules. Young granules were small, black, and compact and harbored active cells. Gray granules were the most abundant granules. These granules have a multilayer structure with channels and void areas. The core was composed of dead or starving cells with low activity. The brown granules, which were the largest granules, showed a loose and amorphous structure with big channels that resulted in fractured zones and corresponded to the older granules. Firmicutes (as determined by FISH) and Nitrospira and Deferribacteres (as determined by cloning and sequencing) were the predominant Bacteria. Remarkably, Firmicutes could not be detected in the brown granules. The methanogenic Archaea identified were Methanosaeta concilii (70 to 90% by FISH and cloning), Methanosarcina mazei, and Methanospirillum spp. The phenotypic appearance of the granules reflected the physiological condition of the granules. This may be valuable to easily select appropriate seed sludges to start up other reactors.     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.     

Development of brown fat cells in monolayer culture. II. Ultrastructural characterization of precursors, differentiating adipocytes and their mitochondria

The stroma of mature brown fat has been shown to contain cells which can proliferate and accumulate fat in monolayer cultures, and which have inherent characteristics distinct from those of white fat precursor cells. The purpose of the present investigation was to characterize by electron microscopic analysis these brown fat cells and their subsequent development when they were grown in vitro. By comparison with the existing ultrastructural data on brown fat in situ, it could thus be determined whether or not the precursor cells have the capacity to differentiate in culture. The stromal-vascular fraction isolated from the brown fat of weaned rats was identified as containing adipocyte stem cells, preadipocytes, endothelial cells and a few mature adipocytes. During the first week in culture (i.e., growth phase to confluence), when multilocular fat accumulation occurred, the mitochondria of the preadipocytes developed cristae and matrix granules, as they do in differentiating brown fat in situ. Such granules have been shown to be a sign of intense inner membrane synthetic activity. After confluence, the mitochondria regressed in internal structure and became morphologically more similar to white fat mitochondria. It was concluded that mature brown fat contains precursor cells which can differentiate in vitro. However, this differentiation was incomplete, and the necessity of specific factors for a full mitochondrial development in brown fat is discussed.     

Molecular biology techniques used in wastewater treatment: An overview

Identification of microorganisms by conventional methods requires the isolation of pure cultures followed by laborious characterization experiments. These procedures are therefore inadequate for study of the biodiversity of a natural or engineered ecosystem. A new set of molecular techniques developed during the 1990s revolutionized microbial ecology research. Among these techniques, cloning and the creation of a gene library, denaturant gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization with DNA probes (FISH) stand out. Cloning provides very precise taxonomical information, but is time consuming and requires specialized personnel and so its introduction in wastewater treatment has been slow. DGGE is a rapid and simple method that provides characteristic band patterns for different samples, allowing quick sample profiling, while retaining the possibility of a more thorough genetic analysis by sequencing of particular bands. FISH makes possible to identify microorganisms at any desired taxonomical level, depending on the specificity of the probe used. It is the only quantitative molecular biology technique, although quantification is either complex or tedious and subjective. Combination with a confocal laser-scanning microscope allows the visualization of three-dimensional microbial structures (granules, biofilms). The methods discussed have deepened our understanding of the microbiology of biological wastewater treatment. PCR-based methods (cloning and DGGE) have proved suitable for identifying the microorganisms that form the sludge. Both DGGE and FISH have been extensively employed. FISH is currently being used for elucidation of the composition, quantification and distribution of different bacterial groups in granules and biofilms, as well as their structure and architecture.     

Description of the phylogenetic structure of hydrolytic prokaryotic complex in the soils

With the help of the molecular-biological method of cell hybridization in situ (FISH), the abundance of a physiologically active hydrolytic prokaryotic complex in chernozem and gley-podzolic soils is determined. The total proportion of metabolically active cells, which were detected by hybridization with universal probes as representatives of the domains Bacteria and Archaea, in samples of the studied soil, was from 38% for chernozem up to 78% for gley-podzolic soil of the total number of cells. The differences in the structure of chitinolytic and pectinolytic prokaryotic soil complexes are detected. Along with the high abundance of Actinobacteria and Firmicutes in the soils with chitin, an increase in phylogenetic groups such as Alphaproteobacteria and Bacteroidetes is observed.     

Layered structure of bacterial and archaeal communities and their in situ activities in anaerobic granules

The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH(4), H(2), pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH(4), H(2), pH, and ORP revealed that acid and H(2) production occurred in the upper part of the granule, below which H(2) consumption and CH(4) production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H(2) was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH(4) in the inner layer. We determined the effective diffusion coefficient for H(2) in the anaerobic granules to be 2.66 x 10(-5) cm(2) s(-1), which was 57% in water.     

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

A pair of rosette glands in the embryo and zoeal larva of an estuarine crab Sesarma haematocheir, and classification of the tegumental glands in the embryos of other crabs

A pair of rosette glands (one of the tegumental glands in crustaceans) is present at the root of the dorsal spine of the thorax in mature embryos of the estuarine crab Sesarma haematocheir. Each rosette gland is spherical, 45-50 microm in diameter. This gland consists of three types of cells: 18-20 secretory cells, one central cell, and one canal cell. The secretory cells are further classified into two types on the basis of the morphology of secretory granules. There are 17-19 a cells, and only one b cell per rosette gland. An a cell contains spherical secretory granules of 2-3 microm in diameter. The granules are filled with highly electron-dense materials near the nucleus but have lower electron-density near the central cell. The secretory granules contained in the b cell have an irregular shape and are 1-1.5 microm in diameter. The density of the materials in the granules is uniform throughout the cytoplasm. The secretory granules contained in both the a and b cells are produced by the rough endoplasmic reticulum. Materials in the granules are exocytotically discharged into the secretory apparatus inside the secretory cell, sent to the extracellular channels in the central cell, and secreted through the canal cell. The rosette gland can be distinguished from the epidermal cells 2 weeks after egg-laying and the gland matures just before hatching. Materials produced by this gland are secreted after hatching and secretion continues through five stages of zoeal larvae. These rosette glands were never found in the megalopal larva. Rosette glands are found in the embryos of Sesarma spp. and Uca spp. In other crabs, tegumental glands are also found at the same position as in the embryo of S. haematocheir, but the fine structure of their glands is largely different from that of the rosette gland. On the basis of the morphology of secretory cells (a-g cell types), the tegumental glands of a variety of crab embryos can be classified into four types, including rosette glands (type I-IV). The function of these tegumental glands is not yet known, but different types of the gland seem to reflect the phylogeny of the crabs rather than differences of habitat.     

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

Characterization of microbial communities of biofilters by phospholipid fatty acid analysis and rRNA targeted oligonucleotide probes

The microbial community of a biofilter for waste gas treatment of an animal rendering plant was characterized by the analyses of the phospholipid fatty acids (PLFAs) of the filter material. For these analyses five samples of one filter were taken in intervals between one and two months. The main components of the PLFA profiles were straight chain saturated, monounsaturated and cyclopropyl fatty acids. Terminally branched and 10-methyl branched fatty acids were present in minor amounts. The structure and succession of the microbial community was interpreted by the presence and quantitative changes of diagnostic fatty acids. The stability of diagnostic fatty acids in relation to varying incubation parameters was tested for a number of bacterial isolates from biofilters representing different phylogenetic branches. For two samples, the data from the PLFA-analyses were compared with data obtained by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes specific for the alpha-, beta- and gamma-subclass of the Proteobacteria, the Actinobacteria (Firmicutes with high G+C content) and the Firmicutes with low G+C content. These data indicated a dominating number of Proteobacteria (54% and 35% of DAPI-stained cells), in which the gamma-Proteobacteria represented the main fraction. Actinobacteria were detected in minor amounts, the number of Firmicutes with low G+C content was near the detection limit of the method. About half of the cells detected with a probe specific for Bacteria did not hybridize with the probes specific for the alpha-, beta- and gamma subclass of the Proteobacteria and the two subgroups of the Firmicutes. The results of both methods, the fluorescence in situ hybridization (FISH) and the PLFA analyses corresponded well and were best suited to confirm and complement each other.     

大鼠背根神经节细胞—氧化氮合酶与乙酰胆碱酯酶共存的组织化学研究

用还原型尼克项胺腺嘌呤二核苷磷酸黄递酶反应,结合乙酸胆碱酯酶组织化学技术,观察了SD大鼠背根神经节,发现背根神经节内存在有四种不同显色的神经元。（1）AChE阳性神经元（8％）。（2）NADPH－d阳性神经元（2％）。（3）NADPH－d与AChE双显色神经元（90％）。（4）少数两种酶反应均为阴性神经元。其中双显色神经元胞质内含有蓝色和棕色两种颗粒。这证明背根节内存在许多NADPH－d与AChE共存的神经元。提示了背根节内NADPH－d与AChE双染神经元可能与痛感知的传递有关。     

Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria

We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.     

Equine stomachs harbor an abundant and diverse mucosal microbiota

Little is known about the gastric mucosal microbiota in healthy horses, and its role in gastric disease has not been critically examined. The present study used a combination of 16S rRNA bacterial tag-encoded pyrosequencing (bTEFAP) and fluorescence in situ hybridization (FISH) to characterize the composition and spatial distribution of selected gastric mucosal microbiota of healthy horses. Biopsy specimens of the squamous, glandular, antral, and any ulcerated mucosa were obtained from 6 healthy horses by gastroscopy and from 3 horses immediately postmortem. Pyrosequencing was performed on biopsy specimens from 6 of the horses and yielded 53,920 reads in total, with 631 to 4,345 reads in each region per horse. The microbiome segregated into two distinct clusters comprised of horses that were stabled, fed hay, and sampled at postmortem (cluster 1) and horses that were pastured on grass, fed hay, and biopsied gastroscopically after a 12-h fast (cluster 2). The types of bacteria obtained from different anatomic regions clustered by horse rather than region. The dominant bacteria in cluster 1 were Firmicutes (>83% reads/sample), mainly Streptococcus spp., Lactobacillus spp. and, Sarcina spp. Cluster 2 was more diverse, with predominantly Proteobacteria, Bacteroidetes, and Firmicutes, consisting of Actinobacillus spp. Moraxella spp., Prevotella spp., and Porphyromonas spp. Helicobacter sp. sequences were not identified in any of 53,920 reads. FISH (n = 9) revealed bacteria throughout the stomach in close apposition to the mucosa, with significantly more Streptococcus spp. present in the glandular region of the stomach. The equine stomach harbors an abundant and diverse mucosal microbiota that varies by individual.     

Analysis of Microbial Community during Biofilm Development in an Anaerobic Wastewater Treatment Reactor

The formation, structure, and biodiversity of a multispecies anaerobic biofilm inside an Upflow Anaerobic Sludge Bed (UASB) reactor fed with brewery wastewater was examined using complementary microbial ecology methods such us fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and cloning. The biofilm development can be roughly divided into three stages: an initial attachment phase (0-36 h) characterized by random adhesion of the cells to the surface; a consolidation phase (from 36 h to 2 weeks) defined by the appearance of microcolonies; and maturation phase (from 2 weeks to 2 months). During the consolidation period, proteobacteria with broad metabolic capabilities, mainly represented by members of alpha-Proteobacteria class (Oleomonas, Azospirillum), predominated. Beta-, gamma-, delta- (both syntrophobacteria and sulfate-reducing bacteria) and epsilon- (Arcobacter sp.) Proteobacteria were also noticeable. Archaea first appeared during the consolidation period. A Methanospirillum-like methanogen was detected after 36 h, and this was followed by the detection of Methanosarcina, after 4 days of biofilm development. The mature biofilm displayed a hill and valley topography with cells embedded in a matrix of exopolymers where the spatial distribution of the microorganisms became well-established. Compared to the earlier phases, the biodiversity had greatly increased. Although alpha-Proteobacteria remained as predominant, members of the phyla Firmicutes, Bacteroidete, and Thermotogae were also detected. Within the domain Archaea, the acetoclastic methanogen Methanosaeta concilii become dominant. This study provides insights on the trophic web and the shifts in population during biofilm development in an UASB reactor.     

The ultrastructure of leucocytes in carp (Cyprinus carpio)

Leucocytes from anterior kidney, middle kidney, spleen, thymus and peripheral blood of Common carp were examined. Lymphocytes were found to have a similar fine structure to that of mammals. Thrombocytes had a similar appearance to lymphocytes, but the former were sometimes distinguishable by vesicles in series and/or microtubules below the plasma membrane. Blast cells, monocytes and clearly identifiable plasma cells were also seen. Neutrophils had varying proportions of at least two types of granules, which often presented inclusions. Owing to their different granules, eosinophils and basophils were morphologically distinguishable, but differentiated cells with equal proportions of the two types of granules were also seen. In addition, cells of uncertain nature, possessing rod-shaped granules, were observed. The different leucocyte types showed the same morphology in the different lymphoid organs and in peripheral blood, although they were present in different proportions.     

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).^1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.^3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.^4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.^18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),^8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),^9,10 and Bacteroidetes (Bac303).¹¹ In addition, specific probes binding to Streptococcus mutans (MUT590)^12,13 and Porphyromonas gingivalis (POGI)^13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.^5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations^3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.     

Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria

We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.     

Presence of anaerobic bacteroides in aerobically grown microbial granules

Microbial granules were grown in a column-type sequential aerobic sludge blanket reactor inoculated with activated sludge flocs taken from a wastewater treatment plant and containing a medium with glucose as the main carbon source. The reactor selected for granules that could settle rapidly by employing a short settling time of 2 min. Matured granules with diameters between 2 and 3 mm were examined for anaerobic bacteria as their presence can signal the onset of diffusion limitation problems that can potentially diminish granule stability due to the bacterial production of fermentation gases and organic acids under anaerobic conditions. To detect the anaerobes in the granules, clones were constructed from 16S rRNA PCR amplicons. Two sequence types associated with a strict anaerobe Bacteroides spp. were identified from these clones. Fluorescence in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM) demonstrated that cells of Bacteroides spp. were concentrated at a depth of approximately 800 mm below the surface of the granule. Cell enumeration using flow cytometry showed that the percentage of labeled cells of Bacteroides spp. compared to total bacterial cells in the granules was 0.56%. This is the first study to use a suite of culture-independent techniques to report the presence of a defined species of anaerobic bacteria in aerobically grown microbial granules.