Nitrification activity in the presence of 2-chlorophenol using whole nitrifying cells and cell-free extracts: batch and SBR assays.

Kinetic assays with a nitrifying consortium with whole nitrifying cells amended with 5 mg 2-CP-C/L and 100, 200, 300, or 500 mg NH4+-N/L were carried out in batch and nitrifying sequencing batch reactor (SBR) cultures. No nitrification activity was observed in batch assays with 100 mg NH4+-N/L and 5 mg 2-CP-C/L. Nevertheless, increasing the ammonium concentration from 200 to 500 mg NH4+-N/L allowed simultaneous ammonium and nitrite oxidation even in the presence of 5 mg 2-CP-C/L plus the halogenated compound consumption. Under these conditions, the ammonium monooxygenase enzyme participated in 2-CP consumption. Complete nitrification and simultaneous elimination of 5 mg 2-CP-C/L were achieved in the SBR amended with 200-500 mg NH4+-N/L. The inhibitory effect of 2-CP on the nitrite oxidation process completely disappeared under these conditions. Assays with nitrifying cell-free extracts, ammonium (100 mg NH4+-N/L), and 2-CP (5 mg 2-CP-C/L) were also conducted. In the absence of 2-CP, the nitrifying cell-free extracts maintained up to 60% of the nitrifying activity compared to whole-cells. Contrary to whole-cell assays, cell-free extracts were capable of simultaneously oxidizing ammonium and consuming 2-CP. However, the inhibitory effect of 2-CP on nitrification was still present as lower specific rates of ammonium consumption and nitrate production were obtained. Thus, these assays indicate that the presence of 2-CP affects both, the ammonium transport mechanism and the activity of nitrifying enzymes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03764-z.

Effect of sulfate and lactate loading rates on the respiration process and microbial population changes measured by ecological indices.

In a sulfate reducing process, increasing loading rates and sulfide accumulation may induce population changes resulting in decreasing effectiveness of the process. Thus, the relationship between microbial metabolism changes and population dynamics was studied. An upflow anaerobic sludge blanket reactor was operated at different sulfate loading rates (SLR), from 290 to 981 mg SO4-S/L d at a constant carbon/sulfur ratio of 0.75. When the SLR was increased, the total organic carbon and sulfate consumption efficiencies decreased to nearly 30% and 25%, respectively. The acetate and propionate yields increased with increasing SLR and 385±7 mg sulfide-S/L d was reached. The ecological indices, determined by random amplified polymorphic DNA and denaturing gradient gel electrophoresis techniques, diversity and evenness were found to be constant, and similarity coefficient values remained higher than 76%. The results suggest that the microbial population changes were negligible compared with metabolic changes when SLR was increased. The sulfide accumulation did not modify the microbial diversity. The sequencing of 16S rRNA genes showed strains related to sulfate reducing, fermentation, and methanogenesis processes. The results indicated that the decreasing of effectiveness, under the experimental conditions tested, was dependent more on operational parameters than microbial changes.

Kinetic and physiological evaluation of ammonium and nitrite oxidation processes in presence of 2-chlorophenol.

The effect of 2-chlorophenol (2-CP) on ammonium and nitrite-oxidizing processes was kinetically evaluated in batch cultures with nitrifying sludge at steady state. Assays with ammonium or nitrite as energy source and 2.5, 5.0, or 10.0 mg 2-CP-C/l were conducted. Control assays without 2-CP were also performed. Ammonium-oxidizing activity was completely inhibited at the different 2-CP concentrations, whereas nitrite-oxidizing activity was present as nitrite was completely consumed and converted to nitrate irrespectively of 2-CP concentration. In the presence of 2.5 and 5.0 mg 2-CP-C/l, no significant effect on specific rates of nitrite consumption and nitrate production was observed, but a significant decrease on these parameters was observed at 10.0 mg 2-CP-C/l. The nitrifying sludge previously exposed to 2-CP was unable to completely recover its ammonium and nitrite oxidation capacity. Nevertheless, complete 2-CP consumption was achieved in all assays. The effect of 2-CP on ammonium oxidation was observed at kinetic and metabolic pathway level, whereas the effect on nitrite oxidation was observed only at kinetic level. The results obtained in this work evidenced that in order to achieve a successful nitrification process the presence in wastewater of even 2.5 mg 2-CP-C/l should be avoided.

Simultaneous oxidation of ammonium and p-cresol linked to nitrite reduction by denitrifying sludge.

The metabolic capability of denitrifying sludge to oxidize ammonium and p-cresol was evaluated in batch cultures. Ammonium oxidation was studied in presence of nitrite and/or p-cresol by 55 h. At 50 mg/L NH4+-N and 76 mg/L NO2--N, the substrates were consumed at 100% and 95%, respectively, being N2 the product. At 50 mg/L NH4+-N and 133 mg/L NO2--N, the consumption efficiencies decreased to 96% and 70%, respectively. The increase in nitrite concentration affected the ammonium oxidation rate. Nonetheless, the N2 production rate did not change. In organotrophic denitrification, the p-cresol oxidation rate was slower than ammonium oxidation. In litho-organotrophic cultures, the p-cresol and ammonium oxidation rates were affected at 133 mg/L NO2--N. Nonetheless, at 76 mg/L NO2--N the denitrifying sludge oxidized ammonium and p-cresol, but at different rate. Finally, this is the first work reporting the simultaneous oxidation of ammonium and p-cresol with the production of N2 from denitrifying sludge.

p-Cresol biotransformation by a nitrifying consortium.

The oxidizing ability of a nitrifying consortium exposed to p-cresol (25 mg CL(-1)) was evaluated in batch cultures. Biotransformation of the phenolic compound was investigated by identifying the different intermediates formed. p-Cresol inhibited the ammonia-oxidizing process with a decrease of 83% in the specific rate of ammonium consumption. After 48 h, ammonium consumption efficiency was 96+/-9% while nitrate yield reached 0.95+/-0.06 g NO(3)(-)-Ng(-1)NH(4)(+)-N consumed. High value for nitrate production yield showed that the nitrifying metabolic pathway was only affected at the specific rate level being nitrate the main end product. The consortium was able to totally oxidize p-cresol at a specific rate of 0.17+/-0.06 mg p-cresol-Cmg(-1) microbial protein h(-1). p-Cresol was first transformed to p-hydroxybenzaldehyde and p-hydroxybenzoate, which were later completely mineralized. In the presence of allylthiourea, a specific inhibitor of ammonia monooxygenase (AMO), p-cresol was oxidized to the same intermediates and in a similar pattern as obtained without the AMO inhibitor. AMO seemed not to be involved in the p-cresol oxidation process. When p-hydroxybenzaldehyde was added (25 mg CL(-1)), the nitrifying process was inhibited in the same way as observed with p-cresol, indicating that p-hydroxybenzaldehyde could be the main compound responsible for nitrification inhibition. p-Hydroxybenzaldehyde was accumulated during 15 h before complete consumption at a specific rate value eight times lower than the p-cresol consumption rate. Results showed that p-hydroxybenzaldehyde oxidation was the limiting step in p-cresol mineralization by the nitrifying consortium.

Toluene mineralization by denitrification in an up flow anaerobic sludge blanket (UASB) reactor.

In order to examine the effect of easily degradable substrate such as acetate on toluene mineralization by denitrification, an upflow anaerobic sludge blanket (UASB) reactor in steady state was set up. The experimentation was carried out in two stages. Initially, the reactor was fed with a carbon loading rate of 250 mg acetate-C L-1 d-1 as electron source. Nitrate loading rate (mg ) was adjusted to obtain a constant C/N ratio of 1.4. In the second stage, five toluene-C loading rates (TLR, mg toluene-C L-1 d-1), 25, 50, 75, 100 and 125, were assessed while total carbon loading rate and C/N were maintained constant at 250 mg C L-1 d-1 and 1.4, respectively. In so doing, acetate-C loading rate (mg acetate-C L-1 d-1) was gradually substituted by toluene-C. When acetate-C was the only electron source a dissimilative denitrifying process resulted as indicated by bicarbonate yield YHCO3, mg produced/mg carbon consumed) of 0.74 +/- 0.005 and denitrifying yield (YN2, mg N2 produced/mg consumed) of 0.89 +/- 0.042. The addition of different TLR did not affect the biological process as consumption carbon efficiency (CCE) values remained up to 95% +/- 3.5 and YHCO3 and YN2 values were higher than 0.71 +/- 0.03 and 0.88 +/- 0.01, respectively. Toluene mineralization by denitrification in continuous culture was successfully achieved. A simple UASB denitrifying reactor system has promising applications for complete conversion of nitrate, toluene and acetate into N2 and CO2 with a minimal sludge production.

Determination of denitrifying sludge settleability using contact angle measurements.

The adaptation of the technique for measuring the contact angle (CA) in order to follow the changes of sludge settleability of a denitrifying sludge surface is described. Denitrifying sludge was continuously fed into an upflow anaerobic sludge blanket (UASB) reactor with acetate and nitrate. The loss of settleability in the sludge was induced by decreasing the hydraulic residence time (HRT) from 1 d to 0.125 d. CA was measured with air and nitrogen bubbles. A comparison between sludge volume index (SVI) and the CA and their correlation with sludge settleability was carried out. Results showed that CA method had a high correlation with sludge settleability. Moreover the CA method showed to be simpler and less time consuming than the SVI method.

Denitrification in presence of benzene, toluene, and m-xylene: kinetics, mass balance, and yields.

Denitrification of the electron donors toluene-C (15-100 mg/L), m-xylene-C (15-70 mg/L), benzene-C (5-25 mg/L), and acetate-C as experimental reference (50-140 mg/L) was carried out in batch culture. An initial concentration of 1.1 +/- 0.15 g of volatile suspended solids/L of denitrifying sludge without previous exposure to aromatic compounds was used as inoculum. The results showed toluene and nitrate consumption efficiency (ET and EN, respectively) of 100%. Toluene was completely mineralized (oxidized) to CO2. In all cases, the N2 (YN2) and HCO3-yields (YHCO3) were 0.97 +/- 0.01 and 0.8 +/- 0.05, respectively. The consumption efficiency (EX) of m-xylene (53 +/- 5.7%) was partial. The YN2 and YHCO3 were 0.96 +/- 0.01 and 0.86 +/- 0.02, respectively. Benzene was not consumed under denitrifying conditions. The specific consumption rates of toluene (qT) and m-xylene (qX) were lower than that of acetate (qA). The differences in specific consumption rates were probably owing to the negative effect of benzene, toluene, and isomers of xylene on the cell membrane.