J.G. Kuenen

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

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, 0.041mol CO2 were incorporated, resulting in a estimated growth rate of 0.001 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++0.06:0.22+0.02. 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.

Sewage Treatment with Anammox

Wastewater treatment including high rate anammox processes have the potential to become energy-neutral or even energy-producing. 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.

Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor

Summary: A novel metabolic pathway for anaerobic ammonium oxidation with nitrite as the electron acceptor has been elucidated using 15N-Iabelled nitrogen compounds. These experiments showed that ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor. The hydroxylamine itself is most likely derived from nitrite. Batch experiments in which ammonium was oxidized with hydroxylamine transiently accumulated hydrazine. The conversion of hydrazine to dinitrogen gas is postulated as the reaction generating electron equivalents for the reduction of nitrite to hydroxylamine. During the conversion of ammonium, a small amount of nitrate was formed from some of the nitrite. The addition of NH2OH to an operating fluidized bed system caused a stoichiometric increase in the ammonium conversion rate (1 mmol I-1 h-1) and a decrease in the nitrate production rate (0.5 mmol I-1 h-1). Addition of hydrazine also caused a decrease in nitrate production. On the basis of these findings, it is postulated that the oxidation of nitrite to nitrate could provide the anaerobic ammonium-oxidizing bacteria with the reducing equivalents necessary for CO2 fixation.

Thiosphaera pantotropha gen. nov. sp. nov., a facultatively anaerobic, facultatively autotrophic sulfur bacterium

Summary: During studies on a desulphurizing, denitrifying effluent-treatment system, an organism which is able to grow aerobically and anaerobically on reduced sulphur compounds and hydrogen, while fixing carbon dioxide, was isolated. The new isolate is also capable of mixotrophic and heterotrophic growth on a wide range of substrates, and is therefore a facultatively aerobic, facultative autotroph. Comparisons with two similar species, Thiobacillus A2 and Paracoccus denitrificans, showed that the new isolate is significantly different from the other two, and merits separate classification. In view of its ability to oxidize reduced sulphur compounds, and because it is a chain-forming coccus rather than a rod, the new isolate has been given the generic name of Thiosphaera, and the species name pantotropha in recognition of its wide range of possible substrates.

Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria

Reports of the simultaneous use of oxygen and denitrification by different species of bacteria have become more common over the past few years. Research with some strains (e.g. Thiosphaera pantotropha) has indicated that there might be a link between this ‘aerobic denitrification’ and a form of nitrification which requires rather than generates energy and is therefore known as heterotrophic nitrification. This paper reviews recent research into heterotrophic nitrification and aerobic denitrification, and presents a preliminary model which, if verified, will provide at least a partial explanation for the simultaneous occurrence of nitrification and denitrification in some bacteria.

Aerobic Denitrification in Various Heterotrophic Nitrifiers

Various heterotrophic nitrifiers have been tested and found to also be aerobic denitrifiers. The simultaneous use of two electron acceptors (oxygen and nitrate) permits these organisms to grow more rapidly than on either single electron acceptor, but generally results in a lower yield than is obtained on oxygen, alone. One strain, formerly known as “Pseudomonas denitrificans”, was grown in the chemostat and shown to achieve nitrification rates of up to 44 nmol NH3 min−1 mg protein−1 and denitrification rates up to 69 nmol NOinf3sup−1min−1 mg protein−1.Unlike Thiosphaera pantotropha, this strain needed to induce its nitrate reductase. However, the remainder of the denitrifying pathway was constitutive and, like T. pantotropha, “Ps. denitrificans” probably possesses the copper nitrite reductase.

Novel principles in the microbial conversion of nitrogen compounds

Some aspects of inorganic nitrogen conversion by microorganisms like N2O emission and hydroxylamine metabolism studied by Beijerinck and Kluyver, founders of the Delft School of Microbiology, are still actual today. In the Kluyver Laboratory for Biotechnology, microbial conversion of nitrogen compounds is still a central research theme. In recent years a range of new microbial processes and process technological applications have been studied. This paper gives a review of these developments including, aerobic denitrification, anaerobic ammonium oxidation, heterotrophic nitrification, and formation of intermediates (NO2-, NO, N2O), as well as the way these processes are controlled at the genetic and enzyme level.

Diversity, Activity, and Abundance of Sulfate-Reducing Bacteria in Saline and Hypersaline Soda Lakes

ABSTRACT Soda lakes are naturally occurring highly alkaline and saline environments. Although the sulfur cycle is one of the most active element cycles in these lakes, little is known about the sulfate-reducing bacteria (SRB). In this study we investigated the diversity, activity, and abundance of SRB in sediment samples and enrichment cultures from a range of (hyper)saline soda lakes of the Kulunda Steppe in southeastern Siberia in Russia. For this purpose, a polyphasic approach was used, including denaturing gradient gel electrophoresis of dsr gene fragments, sulfate reduction rate measurements, serial dilutions, and quantitative real-time PCR (qPCR). Comparative sequence analysis revealed the presence of several novel clusters of SRB, mostly affiliated with members of the order Desulfovibrionales and family Desulfobacteraceae. We detected sulfate reducers and observed substantial sulfate reducing rates (between 12 and 423 μmol/dm3 day−1) for most lakes, even at a salinity of 475 g/liter. Enrichments were obtained at salt saturating conditions (4 M Na+), using H2 or volatile fatty acids as electron donors, and an extremely halophilic SRB, strain ASO3-1, was isolated. Furthermore, a high dsr gene copy number of 108 cells per ml was detected in a hypersaline lake by qPCR. Our results indicate the presence of diverse and active SRB communities in these extreme ecosystems.

Nested PCR-Denaturing Gradient Gel Electrophoresis Approach To Determine the Diversity of Sulfate-Reducing Bacteria in Complex Microbial Communities

ABSTRACT Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.

Aerobic Denitrification - Old Wine in New Bottles

The evidence concerning aerobic denitrification over the past 100 years has been reviewed and the conclusion reached that the denitrification systems of some bacteria are inhibited by oxygen, other species are capable of aerobic denitrification, or co-respiration of nitrate and oxygen. Possible mechanisms and ecological implications are discussed.