Lacipirellula parvula gen. nov., sp. nov., representing a lineage of planctomycetes widespread in low-oxygen habitats, description of the family Lacipirellulaceae fam. nov. and proposal of the orders Pirellulales ord. nov., Gemmatales ord. nov. and Isosphaerales ord. nov.

Pirellula-like planctomycetes are ubiquitous aquatic bacteria, which are often detected in anoxic or micro-oxic habitats. By contrast, the taxonomically described representatives of these bacteria, with very few exceptions, are strict aerobes. Here, we report the isolation and characterization of the facultatively anaerobic planctomycete, strain PX69T, which was isolated from a boreal lake. Its 16S rRNA gene sequence is affiliated with the Pirellula-related Pir4 clade, which is dominated by environmental sequences retrieved from a variety of low-oxygen habitats. Strain PX69T was represented by ellipsoidal cells that multiplied by budding and grew on sugars, some polysaccharides and glycerol. Anaerobic growth occurred by means of fermentation. Strain PX69T grew at pH 5.5-7.5 and at temperatures between 10 and 30°C. The major fatty acids were C18:1ω9c, C16:0 and C16:1ω7c; the major intact polar lipid was dimethylphosphatidylethanolamine. The complete genome of strain PX69T was 6.92Mb in size; DNA G+C content was 61.7mol%. Among characterized planctomycetes, the highest 16S rRNA gene similarity (90.4%) was observed with 'Bythopirellula goksoyri' Pr1d, a planctomycete from deep-sea sediments. We propose to classify PX69T as a novel genus and species, Lacipirellula parvula gen. nov., sp. nov.; the type strain is strain PX69T (=KCTC 72398T=CECT 9826T=VKM B-3335T). This genus is placed in a novel family, Lacipirellulaceae fam. nov., which belongs to the order Pirellulales ord. nov. Based on the results of comparative genome analysis, we also suggest establishment of the orders Gemmatales ord. nov. and Isosphaerales ord. nov. as well as an emendation of the order Planctomycetales.

Biogas Production: Microbiology and Technology.

Biogas, containing energy-rich methane, is produced by microbial decomposition of organic material under anaerobic conditions. Under controlled conditions, this process can be used for the production of energy and a nutrient-rich residue suitable for use as a fertilising agent. The biogas can be used for production of heat, electricity or vehicle fuel. Different substrates can be used in the process and, depending on substrate character, various reactor technologies are available. The microbiological process leading to methane production is complex and involves many different types of microorganisms, often operating in close relationships because of the limited amount of energy available for growth. The microbial community structure is shaped by the incoming material, but also by operating parameters such as process temperature. Factors leading to an imbalance in the microbial community can result in process instability or even complete process failure. To ensure stable operation, different key parameters, such as levels of degradation intermediates and gas quality, are often monitored. Despite the fact that the anaerobic digestion process has long been used for industrial production of biogas, many questions need still to be resolved to achieve optimal management and gas yields and to exploit the great energy and nutrient potential available in waste material. This chapter discusses the different aspects that need to be taken into consideration to achieve optimal degradation and gas production, with particular focus on operation management and microbiology.

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

BACKGROUND: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. CONCLUSIONS: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

Isolation and Cultivation of Anaerobes.

Anaerobic microorganisms play important roles in different biotechnological processes. Their complex metabolism and special cultivation requirements have led to less isolated representatives in comparison to their aerobic counterparts. In view of that, the isolation and cultivation of anaerobic microorganisms is still a promising venture, and conventional methodologies as well as considerations and modifications are presented here. An insight into new methodologies and devices as well as a discussion on future perspectives for the cultivation of anaerobes may open the prospects of the exploitation of these microorganisms as a source for biotechnology.

A method to analyze, sort, and retain viability of obligate anaerobic microorganisms from complex microbial communities.

A high speed flow cytometric cell sorter was modified to maintain a controlled anaerobic environment. This technology enabled coupling of the precise high-throughput analytical and cell separation capabilities of flow cytometry to the assessment of cell viability of evolved lineages of obligate anaerobic organisms from cocultures.

Anaerobic ammonium-oxidizing bacteria gain antibiotic resistance during long-term acclimatization.

Three broad-spectrum antibiotics, amoxicillin (AMX), florfenicol (FF) and sulfamethazine (SMZ), that inhibit bacteria via different target sites, were selected to evaluate the acute toxicity and long-term effects on anaerobic ammonium oxidation (anammox) granules. The specific anammox activity (SAA) levels reduced by approximately half within the first 3 days in the presence of antibiotics but no nitrite accumulation was observed in continuous-flow experiments. However, the SAA levels and heme c content gradually recovered as the antibiotic concentrations increased. Extracellular polymeric substances (EPS) analysis suggested that anaerobic ammonium-oxidizing bacteria gradually developed a better survival strategy during long-term acclimatization, which reduced the antibiotic stress via increased EPS secretion that provided a protective 'cocoon.' In terms of nitrogen removal efficiency, anammox granules could resist 60 mg-AMX L(-1), 10 mg-FF L(-1) and 100 mg-SMZ L(-1). This study supported the feasibility of using anammox granules to treat antibiotic-containing wastewater.

Anaerobic ammonium oxidation (anammox) under realistic seasonal temperature variations: Characteristics of biogranules and process performance.

In this study, the effects of realistic seasonal temperatures on the nitrogen removal performance of anaerobic ammonium oxidation (anammox) and the properties of the anammox granules were comparatively investigated for 330 days. The results demonstrated that the nitrogen removal efficiency (NRE), nitrogen loading rate (NLR) and nitrogen removal rate (NRR) were decreased dramatically, as the temperature decreased from 31.2 to 2.5 °C. However, the nitrogen removal performance recovered andante as the temperature increased gradually. After low temperature exposure, the settleability tended to worsen, and granules appeared to be more irregular with a smaller average granule diameter, and the extracellular polymeric substances (EPS) content increased slightly, while the specific anammox activity (SAA) decreased obviously. This realistic seasonal temperatures based research was an illation of the actual operation, and could be potentially implemented to maintain stability for the application of anammox technology.

Reduced bacterial colony count of anaerobic bacteria is associated with a worsening in lung clearance index and inflammation in cystic fibrosis.

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

Aminocella lysinolytica gen. nov., sp. nov., a L-lysine-degrading, strictly anaerobic bacterium in the class Clostridia isolated from a methanogenic reactor of cattle farms.

A strictly anaerobic bacterial strain (WN037(T)) was isolated from a methanogenic reactor. Cells were Gram-positive rods. Strain WN037(T) was asaccharolytic. The strain fermented L-lysine in the presence of B-vitamin mixture or vitamin B12 and produced acetate and butyrate. L-arginine and casamino acids poorly supported the growth. Strain WN037(T) used neither other amino acids nor organic acids examined. The strain had C18:1 ω7c, C16:0 and C18:1 ω7c DMA as the predominant cellular fatty acids. The genomic DNA G + C content was 44.2 mol %. Phylogenetic analysis based on the 16S rRNA gene sequence placed strain WN037(T) in the family Eubacteriaceae in the class Clostridia. The closest relative was Eubacterium pyruvativorans (sequence similarity, 92.8 %). Based on the comprehensive analyses, the novel genus and species, Aminocella lysinolytica gen. nov., sp. nov. was proposed to accommodate the strain. The type strain is WN037(T) (= JCM 19863(T) = DSM 28287(T)).

Starved anammox cells are less resistant to NO2⁻ inhibition.

Anaerobic ammonium oxidizing (anammox) bacteria are be inhibited by their terminal electron acceptor, nitrite. Serious nitrite inhibition of the anammox bacteria occurs if the exposure coincides with the absence of the electron donating substrate, ammonium and pH < 7.2. Starvation of biomass occurs during underloading of bioreactors or biomass storage. This work investigated the effect of starvation on the sensitivity of anammox bacteria to nitrite exposure. Batch activity tests were carried out evaluating the response of anammox biomass subjected to different levels of starvation upon exposure to nitrite in the presence and absence of ammonium (active- and resting-cells, respectively). The response of the bacteria was evaluated by measuring the specific anammox activity and the evolution of the ATP content in the biomass over time. The 50% inhibitory concentrations of nitrite in starved- and fresh-resting-cells was 7 mg N L(-1) and 52 mg N L(-1), respectively. By contrast, only moderate nitrite inhibition occurred to starved anammox biomass when exposed to nitrite and ammonium simultaneously. Maximum ATP levels were observed in fresh cells. The ATP content in starved resting cells peaked 2-3 h after addition of NO2(-)(-). The response was hindered in cells starved for long periods. These findings agreed with a bioreactor study in which underloading of anammox biomass (0.10 g N L(-1) d(-1)) decreased its tolerance to a nitrite (only) exposure (101 mg NO2(-)-N L(-1)) and completely disrupted the N removal capacity of the biomass. A similar accumulation of 108 mg NO2(-)-N L(-1) after operation at 0.95 g N L(-1) d(-1) did not cause observable inhibition of the bacteria. The results taken as a whole demonstrate that starved anammox biomass is highly sensitive to nitrite toxicity. An explanation is proposed based on energy requirements to translocate nitrite in the cell.

Identification of Synergistetes in endodontic infections.

The bacterial phylum Synergistetes consists of Gram-negative anaerobes. Oral Synergistetes are divided in two main clusters, namely A and B. Increasing evidence demonstrates their involvement in etiology of oral infections, including apical periodontitis. This condition causes bone loss around the apex of the tooth, subsequent to pulp inflammation (pulpitis). Although the presence of Synergistetes has been confirmed in endodontic infections by molecular methods, these have not been morphologically identified in the affected apical region, and their prevalence among different endodontic infections has not been determined. Therefore, the aim of this study was to evaluate the prevalence, levels and morphology of oral Synergistetes clusters A and B, in apical root canal samples obtained of teeth with irreversible pulpitis, pulp necrosis and apical periodontitis, or previously root-filled teeth with apical periodontitis. For their detection, fluorescence in situ hybridization and epifluorescence microscopy were used. Synergistetes cluster A was not detected in pulpitis, but was found in both apical periodontitis groups, more frequently and at higher ranges in teeth which were previously root-filled. Microscopically, they appeared as straight or slightly curved long rods. Synergistetes cluster B was not detected in any of the cases. Fusobacteria and Actinomyces, which are well-established taxa in endodontic infections, were detected more frequently and at higher ranges than Synergistetes. In conclusion, Synergistetes cluster A constitutes part of the mixed apical microbiota in apical periodontitis, and may be involved in its pathogenesis.

Rapid isolation of a facultative anaerobic electrochemically active bacterium capable of oxidizing acetate for electrogenesis and azo dyes reduction.

In this study, 27 strains of electrochemically active bacteria (EAB) were rapidly isolated and their capabilities of extracellular electron transfer were identified using a photometric method based on WO3 nanoclusters. These strains caused color change of WO3 from white to blue in a 24-well agar plate within 40 h. Most of the isolated EAB strains belonged to the genera of Aeromonas and Shewanella. One isolate, Pantoea agglomerans S5-44, was identified as an EAB that can utilize acetate as the carbon source to produce electricity and reduce azo dyes under anaerobic conditions. The results confirmed the capability of P. agglomerans S5-44 for extracellular electron transfer. The isolation of this acetate-utilizing, facultative EBA reveals the metabolic diversity of environmental bacteria. Such strains have great potential for environmental applications, especially at interfaces of aerobic and anaerobic environments, where acetate is the main available carbon source.

Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum.

BACKGROUND: Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO(2) in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life. RESULTS: The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO(2) reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome. CONCLUSIONS: The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation.

Anaerobic ammonium-oxidizing bacteria: unique microorganisms with exceptional properties.

Anaerobic ammonium-oxidizing (anammox) bacteria defy many microbiological concepts and share numerous properties with both eukaryotes and archaea. Among their most intriguing characteristics are their compartmentalized cell plan and archaeon-like cell wall. Here we review our current knowledge about anammox cell biology. The anammox cell is divided into three separate compartments by bilayer membranes. The anammox cell consists of (from outside to inside) the cell wall, paryphoplasm, riboplasm, and anammoxosome. Not much is known about the composition or function of both the anammox cell wall and the paryphoplasm compartment. The cell wall is proposed to be proteinaceous and to lack both peptidoglycan and an outer membrane typical of Gram-negative bacteria. The function of the paryphoplasm is unknown, but it contains the cell division ring. The riboplasm resembles the standard cytoplasmic compartment of other bacteria; it contains ribosomes and the nucleoid. The anammoxosome occupies most of the cell volume and is a so-called "prokaryotic organelle" analogous to the eukaryotic mitochondrion. This is the site where the anammox reaction takes place, coupled over the curved anammoxosome membrane, possibly giving rise to a proton motive force and subsequent ATP synthesis. With these unique properties, anammox bacteria are food for thought concerning the early evolution of the domains Bacteria, Archaea, and Eukarya.

Cellulosomes: highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates.

Cellulosomes can be described as one of nature's most elaborate and highly efficient nanomachines. These cell bound multienzyme complexes orchestrate the deconstruction of cellulose and hemicellulose, two of the most abundant polymers on Earth, and thus play a major role in carbon turnover. Integration of cellulosomal components occurs via highly ordered protein:protein interactions between cohesins and dockerins, whose specificity allows the incorporation of cellulases and hemicellulases onto a molecular scaffold. Cellulosome assembly promotes the exploitation of enzyme synergism because of spatial proximity and enzyme-substrate targeting. Recent structural and functional studies have revealed how cohesin-dockerin interactions mediate both cellulosome assembly and cell-surface attachment, while retaining the spatial flexibility required to optimize the catalytic synergy within the enzyme complex. These emerging advances in our knowledge of cellulosome function are reviewed here.

Culturing aerobic and anaerobic bacteria and mammalian cells with a microfluidic differential oxygenator.

In this manuscript, we report on the culture of anaerobic and aerobic species within a disposable multilayer polydimethylsiloxane (PDMS) microfluidic device with an integrated differential oxygenator. A gas-filled microchannel network functioning as an oxygen-nitrogen mixer generates differential oxygen concentration. By controlling the relative flow rate of the oxygen and nitrogen input gases, the dissolved oxygen (DO) concentration in proximal microchannels filled with culture media are precisely regulated by molecular diffusion. Sensors consisting of an oxygen-sensitive dye embedded in the fluid channels permit dynamic fluorescence-based monitoring of the DO concentration using low-cost light-emitting diodes. To demonstrate the general utility of the platform for both aerobic and anaerobic culture, three bacteria with differential oxygen requirements (E. coli, A. viscosus, and F. nucleatum), as well as a model mammalian cell line (murine embryonic fibroblast cells (3T3)), were cultured. Growth characteristics of the selected species were analyzed as a function of eight discrete DO concentrations, ranging from 0 ppm (anaerobic) to 42 ppm (fully saturated).

Principal component analysis and quantitative image analysis to predict effects of toxics in anaerobic granular sludge.

Principal component analysis (PCA) was applied to datasets gathering morphological, physiological and reactor performance information, from three toxic shock loads (SL1 - 1.6 mg(detergent)/L; SL2 - 3.1mg(detergent)/L; SL3 - 40 mg(solvent)/L) applied in an expanded granular sludge bed (EGSB) reactor. The PCA allowed the visualization of the main effects caused by the toxics, by clustering the samples according to its operational phase, exposure or recovery. The aim was to investigate the variables or group of variables that mostly contribute for the early detection of operational problems. The morphological parameters showed to be sensitive enough to detect the operational problems even before the COD removal efficiency decreased. As observed by the high loadings in the plane defined by the first and second principal components. PCA defined a new latent variable t[1], gathering the most relevant variability in dataset, that showed an immediate variation after the toxics were fed to the reactors. t[1] varied 262%, 254% and 80%, respectively, in SL1, SL2 and SL3. The high loadings/weights of the morphological parameters associated with this new variable express its influence in shock load monitoring and control, and consequently in operational problems recognition.

A single-cell view on the ecophysiology of anaerobic phototrophic bacteria.

Quantitative information on the ecophysiology of individual microorganisms is generally limited because it is difficult to assign specific metabolic activities to identified single cells. Here, we develop and apply a method, Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), and show that it allows simultaneous phylogenetic identification and quantitation of metabolic activities of single microbial cells in the environment. Using HISH-SIMS, individual cells of the anaerobic, phototropic bacteria Chromatium okenii, Lamprocystis purpurea, and Chlorobium clathratiforme inhabiting the oligotrophic, meromictic Lake Cadagno were analyzed with respect to H(13)CO(3)(-) and (15)NH(4)(+) assimilation. Metabolic rates were found to vary greatly between individual cells of the same species, showing that microbial populations in the environment are heterogeneous, being comprised of physiologically distinct individuals. Furthermore, C. okenii, the least abundant species representing approximately 0.3% of the total cell number, contributed more than 40% of the total uptake of ammonium and 70% of the total uptake of carbon in the system, thereby emphasizing that numerically inconspicuous microbes can play a significant role in the nitrogen and carbon cycles in the environment. By introducing this quantification method for the ecophysiological roles of individual cells, our study opens a variety of possibilities of research in environmental microbiology, especially by increasing the ability to examine the ecophysiological roles of individual cells, including those of less abundant and less active microbes, and by the capacity to track not only nitrogen and carbon but also phosphorus, sulfur, and other biological element flows within microbial communities.

[Effect of NO3(-)-N on CH4 emission during denitrification in subtropical soils].

Methane production and emission were investigated in 45 subtropical soil samples, collected from different land use and derived from different soil parent materials in Jiangxi province, by incubating flooded soil slurries in a closed system under N2 gas in the headspace after treatment with or without NO3(-)-N (200 mg x kg(-1)) for 28 days at 30 degrees C. The results indicated that the content and availability of soil organic C were the dominant factors influencing CH4 production and emission whether NO3(-)-N was added or not under this anaerobic incubation condition. Methane emission was higher in the soils derived from granite in the unamended soils and used for rice cultivation in the amended soils. During the anaerobic incubation, the NO3(-)-N added significantly inhibited the production and emission of CH4. The inhibitory effect of NO3(-)-N on CH4 emission might be stronger than that of N2O. The amount and rate of NO3(-)-N denitrified in the first 7 days of incubation determined the CH4 emission amount in the soils with NO3(-)-N. The Fe2+ content increased exponentially with the CH4 emission in 73% of the control soils without NO3(-)-N, which indicated that Fe3+ reduction processed simultaneously with CO2 reduction. Nitrate nitrogen inhibits not only the production and emission of CH4 but also the reduction of Fe3+.

Fervidobacterium changbaicum sp. nov., a novel thermophilic anaerobic bacterium isolated from a hot spring of the Changbai Mountains, China.

A thermophilic, obligately anaerobic, rod-shaped bacterium (strain CBS-1(T)) was isolated from a hot spring mixture of water and mud of the Changbai Mountains, China. Strain CBS-1(T) was found to be non-sporulating, Gram-negative, with optimal growth at 75-80 degrees C. It grew on a wide range of carbon sources, including glucose, lactose, maltose, starch, sorbitol and pyruvate amongst others. The DNA G+C content of strain CBS-1(T) was 31.9 mol%. The 16S rRNA gene sequence analysis indicated that the strain was a member of the genus Fervidobacterium. The high concentration of C(16 : 0) (52.2 %) in the fatty acid profile of the cell envelope supported its inclusion as a member of the genus Fervidobacterium. On the basis of the low values of DNA-DNA hybridization (25.8 and 20.5 %) and phenotypic features, strain CBS-1(T) represents a novel species of the genus Fervidobacterium, for which the name Fervidobacterium changbaicum sp. nov. is proposed. The type strain is CBS-1(T) (=DSM 17883(T)=JCM 13353(T)).