Start-up of sequencing batch reactor with Thiosphaera pantotropha for treatment of high-strength nitrogenous wastewater and sludge characterization.

Biological treatment of high-strength nitrogenous wastewater is challenging due to low growth rate of autotrophic nitrifiers. This study reports bioaugmentation of Thiosphaera pantotropha capable of simultaneously performing heterotrophic nitrification and aerobic denitrification (SND) in sequencing batch reactors (SBRs). SBRs fed with 1:1 organic-nitrogen (N) and NH4+-N were started up with activated sludge and T. pantotropha by gradual increase in N concentration. Sludge bulking problems initially observed could be overcome through improved aeration and mixing and change in carbon source. N removal decreased with increase in initial nitrogen concentration, and only 50-60 % removal could be achieved at the highest N concentration of 1000 mg L-1 at 12-h cycle time. SND accounted for 28 % nitrogen loss. Reducing the settling time to 5-10 min and addition of divalent metal ions gradually improved the settling characteristics of sludge. Sludge aggregates of 0.05-0.2 mm diameter, much smaller than typical aerobic granules, were formed and progressive increase in settling velocity, specific gravity, Ca2+, Mg2+, protein, and polysaccharides was observed over time. Granulation facilitated total nitrogen (TN) removal at a constant rate over the entire 12-h cycle and thus increased TN removal up to 70 %. Concentrations of NO2--N and NO3--N were consistently low indicating effective denitrification. Nitrogen removal was possibly limited by urea hydrolysis/nitrification. Presence of T. pantotropha in the SBRs was confirmed through biochemical tests and 16S rDNA analysis.

d(1) haem biogenesis - assessing the roles of three nir gene products.

The synthesis of the modified tetrapyrrole known as d(1) haem requires several dedicated proteins which are coded for by a set of genes that are often found adjacent to the structural gene, nirS, for cytochrome cd(1) nitrite reductase. NirE, the product of the first gene in the nir biogenesis operon, was anticipated to catalyse the conversion of uroporphyrinogen III into precorrin-2; this was confirmed, but it was shown that this enzyme is less sensitive to product inhibition than similar enzymes that function in other biosynthetic pathways. Sequence analysis suggesting that one of these proteins, NirN, is a c-type cytochrome, and has similarity to the part of cytochrome cd(1) that binds d(1), was validated by recombinant production and characterization of NirN. A NirN-d(1) haem complex was demonstrated to release the cofactor to a semi-apo form of cytochrome cd(1) from which d(1) was extracted, suggesting a role for NirN in the assembly of cytochrome cd(1) (NirS). However, inactivation of nirN surprisingly led to only a marginal attenuation of growth of Paracoccus pantotrophus under anaerobic denitrifying conditions. As predicted, NirC is a c-type cytochrome; it was shown in vitro to be an electron donor to the NirN-d(1) complex.

Effect of periplasmic nitrate reductase on diauxic lag of Paracoccus pantotrophus.

Paracoccus pantotrophus expresses two nitrate reductases-membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To analyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with nap(-) mutant resulted in increased diauxic lag when switched from aerobic to anoxic respiration, suggesting Nap is responsible for shorter lags and helps in adaptation to anoxic metabolism after transition from aerobic conditions.

Effect of pH, temperature and substrate on N2O, NO and CO2 production by Alcaligenes faecalis p.

AIMS: To study the effect of pH, temperature and substrate on the magnitude of N(2)O and NO production by heterotrophic nitrifiers. METHODS AND RESULTS: The change in N(2)O and NO production by the heterotrophic nitrifiers Alcaligenes faecalis subsp. parafaecalis and Paracoccus pantotrophus because of variations in pH, temperature and substrate was studied in chemostat cultures under steady-state conditions. N(2)O, NO and CO(2) production increased with temperature between 4 and 32 degrees C. For N(2)O an optimum temperature of 28 degrees C was observed. No optimum temperature was found for NO. Highest N(2)O and CO(2) productions were observed at a pH of 7.0. However, besides having an optimum at pH 7.0, especially NO production but also N(2)O production increased significantly at pH

Regulation of the nap operon encoding the periplasmic nitrate reductase of Paracoccus pantotrophus: delineation of DNA sequences required for redox control.

Expression of the nap operon, encoding the periplasmic nitrate reductase in Paracoccus pantotrophus, is maximal when cells are grown aerobically, but not anaerobically, with butyrate. Two promoters, termed P1 and P2, control operon expression and the operon-proximal P2 promoter is primarily responsible for increased nap expression in the presence of butyrate. A near-perfect palindromic sequence is centred at +7, relative to the P2 transcription start site. Mutation of this palindrome demonstrated that it is important for regulation of nap operon expression in response to both the redox and the oxidation state of the carbon substrate. A 5' deletion analysis of the nap promoter fused to lacZ revealed that full redox control of expression was retained when the DNA sequence up to position -49 bp, relative to the operon-distal P1 transcription start site, was removed. Encroaching beyond this position resulted in an approximately 4-fold reduction in expression when cells were grown aerobically with butyrate. Additionally, point mutations at position -38 and -45 relative to P1 also resulted in a reduction in expression during aerobic growth with butyrate. A GC-rich region of nap promoter DNA, centred on position -41 relative to the P1 transcription start site is thus proposed as a second DNA motif that is important for efficient expression of the nap operon.

Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA.

Paracoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat (kg protein)(-1). The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase [3.3 mkat (kg protein)(-1)]. The inducible desulfonation reaction was not detected initially in cell extracts. However, a strongly induced protein with subunits of 8 kDa (alpha) and 42 kDa (beta) was found and purified. The corresponding genes had similarities to those encoding altronate dehydratases, which often require iron for activity. The purified enzyme could then be shown to convert 3-sulfolactate to sulfite and pyruvate and it was termed sulfolactate sulfo-lyase (Suy). A high level of sulfite dehydrogenase was also induced during growth with cysteate, and the organism excreted sulfate. A putative regulator, OrfR, was encoded upstream of suyAB on the reverse strand. Downstream of suyAB was suyZ, which was cotranscribed with suyB. The gene, an allele of tauZ, encoded a putative membrane protein with transmembrane helices (COG2855), and is a candidate to encode the sulfate exporter needed to maintain homeostasis during desulfonation. suyAB-like genes are widespread in sequenced genomes and environmental samples where, in contrast to the current annotation, several presumably encode the desulfonation of 3-sulfolactate, a component of bacterial spores.