Isolation and Maintenance.- Isolation and Differentiation of Medaka Embryonic Stem Cells.- Maintenance of Chicken Embryonic Stem Cells In Vitro.- Derivation and Culture of Mouse Trophoblast Stem Cells In Vitro.- Derivation, Maintenance, and Characterization of Rat Embryonic Stem Cells In Vitro.- Derivation, Maintenance, and Induction of the Differentiation In Vitro of Equine Embryonic Stem Cells.- Generation and Characterization of Monkey Embryonic Stem Cells.- Derivation and Propagation of Embryonic Stem Cells in Serum- and Feeder-Free Culture.- Signaling in Embryonic Stem Cell Differentiation.- Internal Standards in Differentiating Embryonic Stem Cells In Vitro.- Matrix Assembly, Cell Polarization, and Cell Survival.- Phosphoinositides, Inositol Phosphates, and Phospholipase C in Embryonic Stem Cells.- Cripto Signaling in Differentiating Embryonic Stem Cells.- The Use of Embryonic Stem Cells to Study Hedgehog Signaling.- Transfection and Promoter Analysis in Embryonic Stem Cells.- SAGE Analysis to Identify Embryonic Stem Cell-Predominant Transcripts.- Utilization of Digital Differential Display to Identify Novel Targets of Oct3/4.- Gene Silencing Using RNA Interference in Embryonic Stem Cells.- Genetic Manipulation of Embryonic Stem Cells.- Efficient Transfer of HSV-1 Amplicon Vectors Into Embryonic Stem Cells and Their Derivatives.- Lentiviral Vector-Mediated Gene Transfer in Embryonic Stem Cells.- Use of the Cytomegalovirus Promoter for Transient and Stable Transgene Expression in Mouse Embryonic Stem Cells.- Use of Simian Immunodeficiency Virus Vectors for Simian Embryonic Stem Cells.- Generation of Green Fluorescent Protein-Expressing Monkey Embryonic Stem Cells.- DNA Damage Response and Mutagenesis in Mouse Embryonic Stem Cells.- Ultraviolet-Induced Apoptosis in Embryonic Stem Cells In Vitro.- Use of Embryonic Stem Cells in Pharmacological and Toxicological Screens.- Use of Differentiating Embryonic Stem Cells in Pharmacological Studies.- Embryonic Stem Cells as a Source of Differentiated Neural Cells for Pharmacological Screens.- Use of Murine Embryonic Stem Cells in Embryotoxicity Assays.- Use of Chemical Mutagenesis in Mouse Embryonic Stem Cells.- Epigenetic Analysis of Embryonic Stem Cells.- Nuclear Reprogramming of Somatic Nucleus Hybridized With Embryonic Stem Cells by Electrofusion.- Methylation in Embryonic Stem Cells In Vitro.- Tumor-Like Properties.- Identification of Genes Involved in Tumor-Like Properties of Embryonic Stem Cells.- In Vivo Tumor Formation From Primate Embryonic Stem Cells.- Animal Models and Therapy.- Directed Differentiation and Characterization of Genetically Modified Embryonic Stem Cells for Therapy.- Use of Differentiating Embryonic Stem Cells in the Parkinsonian Mouse Model.

Isolation and characterization

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

/pdf/cell-biology-and-molecular-basis-of-denitrification-red4l5iffz.pdf

Cell biology and molecular basis of denitrification.

Phosphate solubilizing bacteria and their role in plant growth promotion

Dissimilatory Fe(III) and Mn(IV) reduction.

Removal of nutrients in various types of constructed wetlands.

Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h−1, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research.

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

An autotrophic, synthetic medium for the enrichment of anaerobic ammonium-oxidizing (Anammox) micro-organisms was developed This medium contained ammonium and nitrite, as the only electron donor and electron acceptor, respectively, while carbonate was the only carbon source provided Preliminary studies showed that the presence of nitrite and the absence of organic electron donors were essential for Anammox activity The conversion rate of the enrichment culture in a fluidized bed reactor was 3 kg NH4 + m-3 d-1 when fed with 30 mM NH4 + This is equivalent to a specific anaerobic ammonium oxidation rate of 1000-1100 nmol NH4 +h-1 (mg volatile solids)-1 The maximum specific oxidation rate obtained was 1500 nmol NH4 +h-1 (mg volatile solids)-1 Per mol NH4 + oxidized, 0041mol CO2 were incorporated, resulting in a estimated growth rate of 0001 h-1 The main product of the Anammox reaction is N2, but about 10% of the N-feed is converted to NO3 - The overall nitrogen balance gave a ratio of NH4 --conversion to NO2 --conversion and NO3 --production of 1:1-31++006:022+002 During the conversion of NH4 + with NO2 -, no other intermediates or end-products such as hydroxylamine, NO and N2O could be detected Acetylene, phosphate and oxygen were shown to be strong inhibitors of the Anammox activity The dominant type of micro-organism in the enrichment culture was an irregularly shaped cell with an unusual morphology During the enrichment for Anammox micro-organisms on synthetic medium, an increase in ether lipids was observed The colour of the biomass changed from brownish to red, which was accompanied by an increase in the cytochrome content Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment

https://repository.tudelft.nl/islandora/object/uuid%3Afd4a0685-4292-4498-911f-9f85302e3291/datastream/OBJ/download

Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43°C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1.4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.

/pdf/key-physiology-of-anaerobic-ammonium-oxidation-3sy39zw8eb.pdf

Key Physiology of Anaerobic Ammonium Oxidation

Organic matter must be removed from sewage to protect the quality of the water bodies that it is discharged to. Most current sewage treatment plants are aimed at removing organic matter only. They are energy-inefficient, whereas potentially the organic matter could be regarded as a source of energy. However, organic carbon is not the only pollutant in sewage: Fixed nitrogen such as ammonium (NH4+) and nitrate (NO3−) must be removed to avoid toxic algal blooms in the environment. Conventional wastewater treatment systems for nitrogen removal require a lot of energy to create aerobic conditions for bacterial nitrification, and also use organic carbon to help remove nitrate by bacterial denitrification (see the figure). An alternative approach is the use of anoxic ammonium-oxidizing (anammox) bacteria, which require less energy ( 1 ) but grow relatively slowly. We explore process innovations that can speed up the anammox process and use all organic matter as much as possible for energy generation.

http://science.sciencemag.org/content/sci/328/5979/702.full.pdf

J.G. Kuenen

Papers

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor

Key Physiology of Anaerobic Ammonium Oxidation

Sewage Treatment with Anammox

Completely autotrophic nitrogen removal over nitrite in one single reactor