Physiological adaptation and population dynamics of a nitrifying sludge exposed to ampicillin.

Antibiotics in wastewater treatment plants can alter the physiological activity and the structure of microbial communities through toxic and inhibitory effects. Physiological adaptation, kinetic, and population dynamics behavior of a nitrifying sludge was evaluated in a sequential batch reactor (SBR) fed with 14.4 mg/L of ampicillin (AMP). The addition of AMP did not affect ammonium consumption (100 mg NH4+-N/L) but provoked nitrite accumulation (0.90 mg NO2--N formed/mg NH4+-N consumed) and an inhibition of up to 67% on the nitrite oxidizing process. After 30 cycles under AMP feeding, the sludge recovered its nitrite oxidizing activity with a high nitrate yield (YNO3-) of 0.87 ± 0.10 mg NO3--N formed/mg NH4+-N consumed, carrying out again a stable and complete nitrifying process. Increases in specific rate of nitrate production (qNO3-) showed the physiological adaptation of the nitrite oxidizing bacteria to AMP inhibition. Ampicillin was totally removed since the first cycle of addition. Exposure to AMP had effects on the abundance of bacterial populations, promoting adaptation of the nitrifying sludge to the presence of the antibiotic and its consumption. Nitrosomonas and Nitrosospira always remained within the dominant genera, keeping the ammonium oxidizing process stable while an increase in Nitrospira abundance was observed, recovering the stability of the nitrite oxidizing process. Burkholderia, Pseudomonas, and Thauera might be some of the heterotrophic bacteria involved in AMP consumption.

Long-term post-storage reactivation of a nitrifying sludge in a sequential batch reactor: physiological and kinetic evaluation.

Production, preservation and recovery of sludge with stabilized nitrifying activity over long time can be difficult. Information on the ability of nitrifying sludge to regain its nitrifying activity after long-term storage is still scarce. In this work, the physiological and kinetic changes during the reactivation and stabilization of a nitrifying sludge previously exposed to ampicillin (AMP) were evaluated in a sequential batch reactor (SBR) after its long-term storage (1 year) at 4 °C. After storage, both ammonium and nitrite oxidizing processes were slow, being nitrite oxidation the most affected step. During the reactivation stage (cycles 1-6), physiological and kinetic activity of the nitrifying sludge improved through the operating cycles, in both its ammonium oxidizing and nitrite oxidizing processes. At the end of the reactivation stage, complete nitrifying activity was achieved in 10 h, reaching ammonium consumption efficiencies (ENH4 +) close to 100% and nitrate yields (YNO3 -) of 0.98 mg NO3 --N/mg NH4 +-N consumed without nitrite accumulation. During the stabilization stage (cycles 7-17), results indicated that the sludge could maintain a steady-state respiratory process with restoration percentages of 100% for nitrifying specific rates (qNH4 + and qNO3 -) with respect to their values obtained before storage. Furthermore, during the addition of 15 mg AMP/L (cycles 18-21), the sludge preserved its metabolic capacity to biodegrade 90% of AMP in 2 h. Therefore, long-term storage of nitrifying sludge could be used to preserve nitrifying inocula as bioseeds for bioremediation and bioaugmentation strategies.

Biological ammonium and sulfide oxidation in a nitrifying sequencing batch reactor: Kinetic and microbial population dynamics assessments.

A kinetic study was carried out in a sequencing batch reactor (SBR) (125 mg NH4+-N/L) inoculated with a physiologically stable nitrifying sludge not previously acclimated to sulfur compounds and fed at different initial sulfide concentrations (2.5-20.0 mg HS--S/L). Up to 10.0 mg HS--S/L, the nitrifying process kept stable and complete, reaching an ammonium consumption efficiency (ENH4+) of 100% and a nitrate yield (YNO3-) of 0.95 ± 0.03 mg NO3--N/mg NH4+-N consumed. At 15.0 and 20.0 mg HS--S/L, after an initial alteration in the nitrite oxidizing process, the YNO2- was decreasing throughout the cycles and the YNO3- increasing, obtaining in the last cycle at 20.0 mg HS--S/L, an ENH4+ of 100%, a YNO2- of zero, and a YNO3- of 0.80 mg NO3--N/mg NH4+-N consumed. At the end of the period at 20.0 mg HS--S/L, the specific rates of ammonium consumption and nitrate formation were 15 and 55% lower than their respective values in the control period without sulfide addition, showing that the sludge had a better metabolic adaptation for ammonium oxidizing activity than for nitrite oxidizing activity. The sludge acquired a higher sulfide oxidation capacity along the cycles. Bacterial population dynamics assessment indicated that the ammonium oxidizing bacteria (AOB) community was more diverse and stable than the nitrite oxidizing bacteria (NOB) community. The use of consortia with a previously stabilized nitrifying activity in SBR may constitute an alternative for eliminating simultaneously ammonium by nitrification and sulfide by sulfide oxidation and be implemented for the treatment of wastewater with ammonium and sulfide.

Ampicillin biotransformation by a nitrifying consortium.

Ampicillin is a widely used ß-lactam antibiotic that has been detected in various effluents and can alter biological processes used in wastewater treatment such as nitrification. Physiological and kinetic behaviour of a nitrifying consortium in the presence of ampicillin (AMP) (10, 25, and 50 mg/L) was evaluated in batch cultures. Under the experimental conditions (320 ± 8 mg bacterial protein/L, C/N = 2.4, 24 h), the nitrifying behaviour was very similar among the controls without AMP and the assays with antibiotic, as there was no AMP effect on efficiency (ENH4+ = 99.7 ± 4.2%), yields (YNO2- = 0, YNO3- = 1.0 ± 0.1 mg N/mg NH4+-N consumed), neither specific rates of NH4+ oxidation and NO3- formation. Therefore, nitrifying bacteria were insensitive to AMP presence. At all assayed concentrations, after 24 h, 70.5 ± 3.7% of AMP was removed from the cultures through abiotic (16.0-16.5%), biosorption (23.2-47.0%), and biotransformation (10.0-29.8%) processes. With the increase in the initial AMP concentration, a greater participation of the biotransformation process, associated to an increase in the specific AMP consumption rate was attained. The sludge was able to completely oxidize NH4+ to NO3- by nitrification and eliminate AMP biologically, but without reaching its full mineralization.

Physiologic impact of 2-chlorophenol on denitrification process in mixture with different electron sources.

The aim of this study was to evaluate the physiologic behavior of sludge in the absence and presence of 2-chlorophenol (2-CP) with different electron donors (phenol, glucose, and acetate) during denitrification process. In batch assays with phenol in the presence of 2-CP, a significant decrease of phenol consumption efficiencies (E phenol) up to 99% was observed regarding the cultures without 2-CP. However, in most of the cases, nitrate consumption efficiencies ( E NO 3 - ), and yields of nitrogen gas ( Y N 2 ) and bicarbonate ( Y HCO 3 - ) were high, showing that the denitrifying respiratory process successfully occurred with phenol and 2-CP. The specific consumption rates of nitrate ( q NO 3 - ) and phenol (q phenol) decreased up to 6.0 and 32.3 times, respectively. In assays with glucose in the presence of 2-CP, the denitrifying performance was not significantly altered in terms of efficiencies and product yields; however, q NO 3 - was up to 1.6 times smaller than that obtained without 2-CP whereas q glucose was increased up to 1.17 times. In assays with acetate plus 2-CP, the E NO 3 - , E acetate, and Y N 2 values remained high but 2-CP caused a decrease in Y HCO 3 - . Moreover, q NO 3 - and q acetate increased up to 1.4 and 2.0 times, respectively. These results show that the negative or positive effects of 2-CP on denitrification process depend on the type and concentration of electron source. The obtained physiologic and kinetic information might be useful to define strategies to maintain successful denitrification processes in wastewater treatment bioreactors fed with 2-CP.

Simultaneous oxidation of ammonium and cresol isomers in a sequencing batch reactor: physiological and kinetic study.

The aim of this study was to evaluate the physiological and kinetic capacities of a nitrifying consortium to simultaneously oxidize ammonium (138 mg N/L day), m-cresol, o-cresol, and p-cresol (180 mg C/L day in mixture) in a sequencing batch reactor (SBR). A 1-L SBR was firstly operated without cresol addition (phase I) for stabilizing the nitrification respiratory process with ammonium consumption efficiencies close to 100 % and obtaining nitrate as the main end product. When cresols were added (phase II m-cresol (10, 20, and 30 mg C/L); phase III m-cresol (30 mg C/L) and o-cresol (10, 20, and 30 mg C/L); phase IV a mixture of three isomers (30 mg C/L each one)), inhibitory effects were evidenced by decreased values of the specific rates of nitrification compared with values from phase I. However, the inhibition diminished throughout the operation cycles, and the overall nitrifying physiological activity of the sludge was not altered in terms of efficiency and nitrate yield. The different cresols were totally consumed, being o-cresol the most recalcitrant. The use of SBR allowed a metabolic adaptation of the consortium to oxidize the cresols as the specific rates of consumption increased throughout the cycles, showing that this type of reactor can be a good alternative for treating industrial effluents in a unique reactor.

Kinetic Constants for Biological Ammonium and Nitrite Oxidation Processes Under Sulfide Inhibition.

Inhibition of nitrification by sulfide was assessed using sludge obtained from a steady-state nitrifying reactor. Independent batch activity assays were performed with ammonium and nitrite as substrate, in order to discriminate the effect of sulfide on ammonium and nitrite oxidation. In the absence of sulfide, substrate affinity constants (K S,NH4 = 2.41 ± 0.11 mg N/L; K s, NO2 = 0.74 ± 0.03 mg N/L) and maximum specific rates (q max,NH4 = 0.086 ± 0.008 mg N/mg microbial protein h; q max,NO2 = 0.124 ± 0.001 mg N/mg microbial protein h) were determined. Inhibition of ammonium oxidation was no-competitive (inhibition constant (K i , NH4 ) of 2.54 ± 0.12 mg HS(-)-S/L) while inhibition of nitrite oxidation was mixed (competitive inhibition constant (K' i , NO2 ) of 0.22 ± 0.03 mg HS(-)-S/L and no-competitive inhibition constant (K i , NO2 ) of 1.03 ± 0.06 mg HS(-)-S/L). Sulfide has greater inhibitory effect on nitrite oxidation than ammonium oxidation, and its presence in nitrification systems should be avoided to prevent accumulation of nitrite. By simulating the effect of sulfide addition in a continuous nitrifying reactor under steady-state operation, it was shown that the maximum sulfide concentration that the sludge can tolerate without affecting the ammonium consumption efficiency and nitrate yield is 1 mg HS(-)-S/L.

Effect of phenol and acetate addition on 2-chlorophenol consumption by a denitrifying sludge.

Chlorophenols are widely distributed in the environment. Various strategies have been used to improve their biological elimination under anaerobic conditions; however, such information is still scarce. The aim of this study was to evaluate in batch assays the consumption of 2-chlorophenol (2-CP) by a denitrifying sludge and the influence of acetate or phenol as co-substrates in the 2-CP consumption. It was observed that phenol (69 and 92 mg phenol-C L(-1)) and acetate (60 and 108 mg acetate-C L(-1)) enhanced 2-CP consumption by the denitrifying sludge, increasing both the efficiency (up to 100%) and specific rate of 2-CP consumption. When phenol was added at 92 mg C L(-1), the specific consumption rate of 2-CP increased 2.6 times with respect to the control lacking co-substrates, whereas with acetate (108 mgC L(-1)) the increase was 9.0 times. Acetate appeared to be a better co-substrate for 2-CP consumption, obtaining a specific consumption rate of 2.48 +/- 0.14 mg 2-CP-C g(-1) VSS d(-1) at 108 mg acetate-C L(-1). The mass balance analysis indicated that the denitrifying sludge was able to simultaneously mineralize 2-CP, phenol or acetate (E2-CP, E(Phenol), and E(Acetate) close to 100% [E = consumption efficiency], Y(HCO3-) of 0.90 +/- 0.10 [Y = yield]) and reduce nitrate to nitrogen gas (E(NO3-) of 100% and Y(N2) of 0.96 +/- 0.02). It was shown that the addition of co-substrates as phenol or acetate could be a good alternative for improving the elimination of chlorophenols from wastewaters by denitrifying sludges.

Biodegradation of 2-chlorophenol (2CP) in an anaerobic sequencing batch reactor (ASBR).

The aim of this study is to contribute to the knowledge about anaerobic digestion of 2-chlorophenol (2CP) in an anaerobic sequencing batch reactor (ASBR). Two reactors were set up (ASBR(A) and ASBR(B)). The ASBR(A) was fed with 2-chlorophenol (28-196 mg 2CP-C/L) and no other exogenous electron donor. The ASBR(B) was fed with a mixture of 2CP (28-196 mg 2CP-C/L) and phenol (28-196 mg phenol-C/L) as an electron donor. The process evaluation was conducted by three means: first by substrate consumption efficiency (E(2CP)), second, by biogas yield (Y(biogas-C/2CP-C)) and third, by the specific consumption rates (q(2CP)) as response variables. The 2CP consumption efficiency (90 ± 0.4%) was not influenced by the increase in the concentrations tested. In both reactors ASBR(A) and ASBR(B), both concentration as well as speed increased. Concentration increased from 28 to 114 mg 2CP-C/L. The specific consumption rate (q(2CP)) values were fivefold higher. However, a decrease of 37% was observed at 140 mg 2CP-C/L and one of 72% at 196 mg 2CP-C/L. The biogas yields (0.80 ± 0.06) remained stable in both reactors. In both reactors the biogas yield decreased to 78 ± 3% at 196 mg 2CP-C/L. We might assume this decrease was due to the accumulation of VFA. Finally, poor sludge settleability was determined only in the SBR(B) reactor at 140 and 196 mg 2CP-C/L. An increase was observed in both SVI ≤ 140 ± 5 mL/g and over exopolymeric protein ≤120 mg EP/L.

Methanization and mineralization of 2-chlorophenol by anaerobic digestion.

The aim of this study is to contribute to the knowledge about 2-Chlorophenol (2CP) mineralization and methanization in batch culture. This work was focused on evaluating the effect of: (i) the use of sludge with different periods of previous contact to 2CP, (ii) the electron donor addition in stoichiometric relation with 2CP and (iii) the presence of different initial oxygen concentrations. When compared with the control, 50 and 80 days of previous contact to 2CP resulted in a lag phase reduction of 57% and an increase in 2CP specific consumption rate (q(2CP)) of 114%. These results were obtained with no addition of an external electron donor. When acetate was used as an electron donor its consumption resulted independently of 2CP consumption. No lag phase and increase of 46% in q(2CP) was observed when phenol was used as an electron donor. In the third part when sludge without previous contact to 2CP was used, it was found that consumption efficiency (E(2CP)) and q(2CP) values did not increase in the presence of different oxygen concentrations. However, at the highest oxygen concentration, CH(4) yield (Y(CH(4))(-C/2CP-C)) and phenol yield (Y(phenol-C/2CP-C)) values decreased, while CO(2) yield value (Y(CO(2))(-C/2CP-C)) increased with regard to the methanogenic control. The use of sludge previously exposed to both 2CP and O(2) resulted in an increase in q(2CP) of 73%. However, among the different oxygen concentrations, no significant difference in E(2CP) or q(2CP) values was observed when compared to the control without oxygen. Therefore, previous contact to 2CP resulted in being a key factor for improving 2CP mineralization and methanization in batch culture.

Acetate enhances the specific consumption rate of toluene under denitrifying conditions.

Toluene is usually present in the environment as a contaminant along with other carbon sources which may influence its removal. In this work we studied the effect of a readily consumable carbon source such as acetate on toluene mineralization under denitrifying conditions. Continuous and batch cultures with stabilized denitrifying sludge were carried out. An upflow anaerobic sludge blanket reactor (UASB) was fed with several ratios of acetate-C/toluene-C loading rates (mg C/L-day: 100/0, 75/25, 50/50, and 0/100). Batch assays with different acetate-C/toluene-C ratios (10/70, 30/50, 50/30, and 65/20 mg C/L) were also done. As the acetate loading rate decreased in the culture, the carbon and nitrate consumption efficiency decreased by 40% and 34%, respectively. HCO(3) (-) and N(2) yields also decreased by 43%. Analysis of the denitrifying community using the denaturing gradient gel electrophoresis technique indicated that there was no clear relationship between its population profile and the metabolic pattern. In batch assays, when the acetate concentration was higher than that of toluene (65 mg acetate-C/L vs 20 mg toluene-C/L), the specific consumption rate of toluene (q(T)) was two times higher than in assays with 20 mg toluene-C/L as the sole electron source (0.006 mg C/mg volatile suspended solids-day). It is proposed that acetate can act by enhancing the growth of microbial populations and as a biochemical enhancer. The results show that acetate addition can be useful to improve the consumption rate of toluene in contaminated water.