H.J.M. op den Camp

Molecular mechanism of anaerobic ammonium oxidation.

Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N2) to the atmosphere. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N2H4). Here we show that N2H4 is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N2H4. We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N2H4 synthesis and its oxidation to N2. These results present a new biochemical reaction forging an N–N bond and fill a lacuna in our understanding of the biochemical synthesis of the N2 in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.

Biochemistry and molecular biology of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N2 gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.

1994–2004: 10 years of research on the anaerobic oxidation of ammonium

The obligately anaerobic ammonium oxidation (anammox) reaction with nitrite as primary electron acceptor is catalysed by the planctomycete-like bacteria Brocadia anammoxidans, Kuenenia stuttgartiensis and Scalindua sorokinii. The anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. They have a unique prokaryotic organelle, the anammoxosome, surrounded by ladderane lipids, which exclusively contains the hydrazine oxidoreductase as the major protein to combine nitrite and ammonia in a one-to-one fashion. In addition to the peculiar microbiology, anammox was shown to be very important in the oceanic nitrogen cycle, and proved to be a very good alternative for treatment of high-strength nitrogenous waste streams. With the assembly of the K. stuttgartiensis genome at Genoscope, Evry, France, the anammox reaction has entered the genomic and proteomic era, enabling the elucidation of many intriguing aspects of this fascinating microbial process.

Microbial cycling of volatile organic sulfur compounds

Abstract. Microbial cycling of volatile organic sulfur compounds (VOSCs), especially dimethyl sulfide (DMS) and methanethiol (MT), is intensively studied because these compounds play an important role in the processes of global warming, acid precipitation, and the global sulfur cycle. VOSC concentrations in freshwater sediments are low due to the balance between the formation and degradation of these compounds. These reactions occur for the greater part at the oxic/anoxic interphase of sediment and water column. In contrast to marine ecosystems, where dimethylsulfoniopropionate is the main precursor of MT and DMS, in freshwater ecosystems, VOSCs are formed mainly by methylation of sulfide and to a lesser extent from the degradation of S-containing amino acids. One of the major routes for DMS and MT formation through sulfide methylation is anaerobic O-demethylation of methoxylated aromatic compounds. Inhibition studies have revealed that the major part of the endogenously produced MT and DMS is degraded anaerobically by methanogens. The major bacterial groups involved in formation and consumption of VOSCs are described.

Bacteria in the Intestinal Tract of Different Species of Arthropods

A bstractThe number of bacteria in the intestine of 12 species of arthropods, belonging to 7 different orders, was determined to obtain information about the significance of intestinal bacteria for the digestion of food. Therefore, a simple and effective method for direct counts of 4′, 6-diamidino-2-phenylindole (DAPI) stained bacteria from the gastrointestinal tract of arthropods was developed. The intestinal bacteria could be released from the gut wall by ultrasonic treatment in the presence of sodium tetrapyrophosphate (PPi). The bacterial counts ranged between 0.2 and 3.6 × 109 (ml gut)−1 in the foregut, 0.2 and 28 × 109 (ml gut)−1 in the midgut, and 0.1 and 190 × 109 (ml gut)−1 in the hindgut. The foregut and hindgut of Hylotrupes bajules larvae were devoid of bacteria; the whole intestinal tract of Eurycanta calcarata and Schistocerca gragaria contained only low numbers of bacteria. The population of bacteria in some parts of the intestinal tract of several arthropods were high enough to suggest a potential contribution to digestive processes.

Global impact and application of the anaerobic ammonium-oxidizing (anammox) bacteria

In the anaerobic ammonium oxidation (anammox) process, ammonia is oxidized with nitrite as primary electron acceptor under strictly anoxic conditions. The reaction is catalysed by a specialized group of planctomycete-like bacteria. These anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. The reactions are assumed to be carried out in a unique prokaryotic organelle, the anammoxosome. This organelle is surrounded by ladderane lipids, which make the organelle nearly impermeable to hydrazine and protons. The localization of the major anammox protein, hydrazine oxidoreductase, was determined via immunogold labelling to be inside the anammoxosome. The anammox bacteria have been detected in many marine and freshwater ecosystems and were estimated to contribute up to 50% of oceanic nitrogen loss. Furthermore, the anammox process is currently implemented in water treatment for the low-cost removal of ammonia from high-strength waste streams. Recent findings suggested that the anammox bacteria may also use organic acids to convert nitrate and nitrite into dinitrogen gas when ammonia is in short supply.

Autotrophic methanotrophy in Verrucomicrobia: Methylacidiphilum fumariolicum SolV uses the Calvin Benson Bassham cycle for carbon dioxide fixation

Genome data of the extreme acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicumstrain SolV indicated the ability of autotrophic growth. This was further validated by transcriptome analysis, which showed that all genes required for a functional Calvin-Benson-Bassham (CBB) cycle were transcribed. Experiments with (13)CH(4) or (13)CO(2) in batch and chemostat cultures demonstrated that CO(2) is the sole carbon source for growth of strain SolV. In the presence of CH(4), CO(2) concentrations in the headspace below 1% (vol/vol) were growth limiting, and no growth was observed when CO(2)concentrations were below 0.3% (vol/vol). The activity of the key enzyme of the CBB cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), measured with a (13)C stable-isotope method was about 70 nmol CO(2) fixed · min(-1)· mg of protein(-1). An immune reaction with antibody against the large subunit of RuBisCO on Western blots was found only in the supernatant fractions of cell extracts. The apparent native mass of the RuBisCO complex in strain SolV was about 482 kDa, probably consisting of 8 large (53-kDa) and 8 small (16-kDa) subunits. Based on phylogenetic analysis of the corresponding RuBisCO gene, we postulate that RuBisCO of the verrucomicrobial methanotrophs represents a new type of form I RuBisCO.

Comparison of growth characteristics of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a defined medium.

Anaerobic fungi were isolated from rumen fluid of a domestic sheep (Ovis aries; a ruminant) and from faeces of five non-ruminants: African elephant (Loxodonta africana), black rhinoceros (Diceros bicornis), Indian rhinoceros (Rhinoceros unicornis), Indian elephant (Elephas maximus) and mara (Dolichotis patagonum). The anaerobic fungus isolated from the sheep was a Neocallimastix species and the isolates from non-ruminants were all species similar to Piromyces spp. A defined medium is described which supported growth of all the isolates, and was used to examine growth characteristics of the different strains. For each fungus the lipid phosphate content was determined after growth on cellobiose and the resulting values were used to estimate fungal biomass after growth on solid substrates. The ability of isolates from ruminants and non-ruminants to digest both wheat straw and cellulose was comparable. More than 90% and 60%, respectively, of filter paper cellulose and wheat straw were digested by most strains within 60-78 h. Growth of two fungi, isolated from rumen fluid of a sheep (Neocallimastix strain N1) and from faeces of an Indian rhinoceros (Piromyces strain R1), on cellobiose was studied in detail. Fungal growth yields on cellobiose were 64.1 g (mol substrate)-1 for N1 and 34.2 g mol-1 for R1. The major fermentation products of both strains were formate, lactate, acetate, ethanol and hydrogen.

Population Dynamics of Scytalidium thermophilum in Mushroom Compost and Stimulatory Effects on Growth Rate and Yield of Agaricus bisporus

SUMMARY: Mycelial growth of Agaricus bisporus on sterilized compost is strongly stimulated by pre-incubating the compost with the thermophilic fungus Scytalidium thermophilum. This stimulatory effect is not species specific, for either A. bisporus or S. thermophilum. Normal mushroom compost is almost completely colonized with S. thermophilum. In experimental composts a positive relation was found between the logarithm of mushroom yield of A. bisporusand the density of S. thermophilum. S. thermophilum provides for compost selectivity: it protects against negative effects of compost bacteria on mycelial growth of A. bisporus. S. thermophilum is inactivated by the growth of A. bisporusmycelium.

Bacteria associated with iron seeps in a sulfur-rich, neutral pH, freshwater ecosystem

The freshwater nature reserve De Bruuk is an iron- and sulfur-rich minerotrophic peatland containing many iron seeps and forms a suitable habitat for iron and sulfur cycle bacteria. Analysis of 16S rRNA gene-based clone libraries showed a striking correlation of the bacterial population of samples from this freshwater ecosystem with the processes of iron reduction (genus Geobacter), iron oxidation (genera Leptothrix and Gallionella) and sulfur oxidation (genus Sulfuricurvum). Results from fluorescence in situ hybridization analyses with a probe specific for the beta-1 subgroup of Proteobacteria, to which the genera Leptothrix and Gallionella belong, and newly developed probes specific for the genera Geobacter and Sulfuricurvum, supported the clone library data. Molecular data suggested members of the epsilonproteobacterial genus Sulfuricurvum as contributors to the oxidation of reduced sulfur compounds in the iron seeps of De Bruuk. In an evaluation of anaerobic dimethyl sulfide (DMS)-degrading activity of sediment, incubations with the electron acceptors sulfate, ferric iron and nitrate were performed. The fastest conversion of DMS was observed with nitrate. Further, a DMS-oxidizing, nitrate-reducing enrichment culture was established with sediment material from De Bruuk. This culture was dominated by dimorphic, prosthecate bacteria, and the 16S rRNA gene sequence obtained from this enrichment was closely affiliated with Hyphomicrobium facile, which indicates that the Hyphomicrobium species are capable of both aerobic and nitrate-driven DMS degradation.