Showing papers by "J.G. Kuenen published in 1996"

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

Abstract: 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

Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions

Abstract: Nitrous oxide can be a harmful by-product in nitrogen removal from wastewater. Since wastewater treatment systems operate under different aeration regimens, the influence of different oxygen concentrations and oxygen fluctuations on denitrification was studied. Continuous cultures of Alcaligenes faecalis TUD produced N2O under anaerobic as well as aerobic conditions. Below a dissolved oxygen concentration of 5% air saturation, the relatively highest N2O production was observed. Under these conditions, significant activities of nitrite reductase could be measured. After transition from aerobic to anaerobic conditions, there was insufficient nitrite reductase present to sustain growth and the culture began to wash out. After 20 h, nitrite reductase became detectable and the culture started to recover. Nitrous oxide reductase became measurable only after 27 h, suggesting sequential induction of the denitrification reductases, causing the transient accumulation of N2O. After transition from anaerobic conditions to aerobic conditions, nitrite reduction continued (at a lower rate) for several hours. N2O reduction appeared to stop immediately after the switch, indicating inhibition of nitrous oxide reductase, resulting in high N2O emissions (maximum, 1.4 mmol liter-1 h-1). The nitrite reductase was not inactivated by oxygen, but its synthesis was repressed. A half-life of 16 to 22 h for nitrite reductase under these conditions was calculated. In a dynamic aerobic-anaerobic culture of A. faecalis, a semisteady state in which most of the N2O production took place after the transition from anaerobic to aerobic conditions was obtained. The nitrite consumption rate in this culture was equal to that in an anaerobic culture (0.95 and 0.92 mmol liter-1 h-1, respectively), but the production of N2O was higher in the dynamic culture (28 and 26% of nitrite consumption, respectively).

Heat flux measurements for the fast monitoring of dynamic responses to glucose additions by yeasts that were subjected to different feeding regimes in continuous culture.

Abstract: Heat measurements have been successfully as an analytical tool for the study of the dynamics of energy metabolism of Saccharomyces cerevisiae and Candida utilis grown in continuous culture under fluctuating substrate supply. A low average dilution rate (D = 0.05 h-1) was maintained either by adding the medium as continuously (dropwise) as possible or (blockwise) by adding the medium at high speed during a short period (D = 0.5 h-1 for 40 s) and not at all during the following period (D = 0.00 h-1 for 360 s). The resulting biological activity was monitored on-line with conventional (O2 and CO2) off-gas analyses, DOT measurements, and heat flux measurements. In C. utilis cultures, the biomass-specific maximum oxygen consumption rate (qO2,max), the biomass yield (Ys,x), and the dynamic responses to a glucose pulse and to a change in feeding regime were not significantly affected by different preceding feeding regimes. In contrast, S. cerevisiae grown in continuous culture with blockwise feed showed a 50% increase in qO2,max and a 25% drop in Ys,x compared to the culture grown with dropwise feed. The dynamic response to a glucose pulse (0.6 g L-1) was slower for the continuous (dropwise) than for the blockwise grown S. cerevisiae. With a second testing method for the dynamic response of the yeasts, the feeding regime was changed. The blockwise fed S. cerevisiae proved to be better "trained" to cope with sudden changes in glucose supply and, therefore, was more "shockproof" toward a change in feeding regime. This clearly points to major differences in the intracellular metabolic flux control between the yeasts. These findings are of relevance for industrial baker's yeast production, where reactor mixing times of one to several minutes are not uncommon. The observed, heat production, together with the dissolved oxygen concentration, appeared to give the fastest response to actual changes in the culture. It is suggested that heat measurements can be a very useful tool to monitor and control the growth of S. cerevisiae in laboratory and industrial fermenter operations.

Purification and characterization of a periplasmic Thiosulfate dehydrogenase from the obligately autotrophic Thiobacillus sp. W5.

Abstract: A periplasmic thiosulfate dehydrogenase (EC 1.8.2.2) was purified to homogeneity from the neutrophilic, obligately chemolithoautotrophicThiobacillus sp. W5. A five-step procedure resulted in an approximately 2,300-fold purification. The purified protein had a molecular mass of 120±3 kDa, as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 33±1 kDa and 27±0.5 kDa, as determined by SDS-PAGE. UV/visible spectroscopy revealed that the enzyme contained haemc; haem staining showed that both subunits contained haemc. A haemc content of 4 mol per mol of enzyme was calculated using the pyridine haemochrome test. The pH optimum of the enzyme was 5.5 At pH 7.5, the Km and Vmax were 120±10 μM and 1,160±30 U mg-1, respectively. The absence of 2-heptyl-4-hydroquinoline-N-oxide (HQNO) inhibition for the oxidation of thiosulfate by whole cells suggested that the electrons enter the respiratory chain at the level of cytochromec. Comparison with thiosulfate dehydrogenases from otherThiobacillus species showed that the enzyme was structurally similar to the thiosulfate dehydrogenase of the acidophilic, facultatively chemolithoautotrophicThiobacillus acidophilus, but not to the thiosulfate dehydrogenases published for the obligately chemolithoautotrophicThiobacillus tepidarius andThiobacillus thioparus.