D. Puyol

Comparison of UASB and EGSB performance on the anaerobic biodegradation of 2,4-dichlorophenol

The anaerobic degradation of 2,4-dichlorophenol (2,4-DCP) in upflow anaerobic sludge blanket (UASB) and expanded granular sludge bed (EGSB) reactors using glucose as main carbon source was studied. The performance of both systems was compared in terms of 2,4-DCP and COD removal efficiencies, methane production, stability, granular sludge adaptability as well as reversion of the bacterial inhibition. Both organic and 2,4-DCP loading rates were incrementally varied through the experiments. With loading rates of 1.9 gCODL(-1)d(-1) and 100mg 2,4-DCP L(-1)d(-1), 75% and 84% removal efficiencies of this compound, accompanied by COD consumption efficiencies of 61% and 80% were achieved in the UASB and EGSB reactors, respectively. In these conditions, methane production reached 0.088 L CH(4)g(-1) COD in the EGSB reactor whereas in the UASB reactor was almost negligible. Decreasing the 2,4-DCP loading rate to 30 mgL(-1)d(-1) an improvement in the methane production was observed in both reactors (methanogenic activity of 0.148 and 0.192 L CH(4)g(-1) COD in UASB and EGSB reactors, respectively). Efficiency of dechlorination was improved in both reactors from around 30% to 80% by reducing to one-half the COD due to a decreasing of the 4-chlorophenol concentration accumulated in the effluents of both reactors. The dechlorination efficiency of the UASB reactor was dramatically inhibited at a 2,4-DCP feed concentration above around 210 mgL(-1) because of 2,4-DCP accumulation in the effluent. SEM studies revealed no significant morphological changes in the sludge granules.

Pre-exposure to nitrite in the absence of ammonium strongly inhibits anammox.

Anaerobic ammonium oxidizing bacteria (Anammox) are known to be inhibited by their substrate, nitrite. However, the mechanism of inhibition and the physiological conditions under which nitrite impacts the performance of anammox bioreactors are still unknown. This study investigates the role of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added. Batch experiments were carried out with anammox granular biofilm pre-exposed to nitrite over a range of concentrations and durations in the absence of ammonium. The effect of pre-exposure to nitrite alone compared to nitrite simultaneously fed with ammonium was evaluated by measuring the anammox activity and the accumulation of the intermediate, nitric oxide. The results show that the inhibitory effect was more dramatic when bacteria were pre-exposed to nitrite in absence of ammonium, as revealed by the lower activity and the higher accumulation of nitric oxide. The nitrite concentration causing 50% inhibition was 53 and 384 mg N L(-1) in the absence or the presence of ammonium, respectively. The nitrite inhibition was thus 7.2-fold more severe in the absence of ammonium. Biomass exposure to nitrite (25 mg N L(-1)), in absence of ammonium, led to accumulation of nitric oxide. On the other hand when the biomass was exposed to nitrite in presence of ammonium, accumulation of nitric oxide was only observed at much higher nitrite concentrations (500 mg N L(-1)). The inhibitory effect of nitrite in the absence of ammonium was very rapid. The rate of decay of the anammox activity was equivalent to the diffusion rate of nitrite up to 46% of activity loss. The results taken as a whole suggest that nitrite inhibition is more acute when anammox cells are not actively metabolizing. Accumulation of nitric oxide in the headspace most likely indicates disruption of the anammox biochemistry by nitrite inhibition, caused by an interruption of the hydrazine synthesis step.

Kinetic characterization of Brocadia spp.-dominated anammox cultures.

In this study, kinetic analyses were conducted for two Brocadia-dominated enrichment cultures, granular and flocculent, retrieved from lab-scale anaerobic ammonium oxidation (anammox) reactors. Substrate KS ranged from 0.35 to 0.69 mMN and VSmax ranged from 0.67 to 0.88 mmol Ng(-1)VSSh(-1). The model respected the experimentally measured stoichiometry of N-compounds, serving as an independent validation. Growth kinetics of the flocculent sludge was also studied, which indicates a μmax of 0.0984 d(-1) and a YX/S of 0.105 mol C-biomass mol(-1)NH4(+). The flocculent enrichment culture was used to determine the stoichiometric equation. The kinetic parameters of the Brocadia spp. cultures measured here can be used for optimizing applications of the anammox process.

Role of biogenic sulfide in attenuating zinc oxide and copper nanoparticle toxicity to acetoclastic methanogenesis

Soluble ions released by zinc oxide (ZnO) and copper (Cu(0)) nanoparticles (NPs) have been associated with toxicity to methanogens. This study evaluated the role of biogenic sulfide in attenuating ZnO and Cu(0) NP toxicity to methanogens. Short- and long-term batch experiments were conducted to explore ZnO and Cu(0) NPs toxicity to acetoclastic methanogens in sulfate-containing (0.4mM) and sulfate-free conditions. ZnO and Cu(0) were respectively 14 and 7-fold less toxic in sulfate-containing than in sulfate-free assays as indicated by inhibitory constants (Ki). The Ki with respect to residual soluble metal indicated that soluble metal was well correlated with toxicity irrespective of the metal ion source or presence of biogenic sulfide. Long-term assays indicated that ZnO and Cu(0) NPs caused different effects on methanogens. ZnO NPs without protection of sulfide caused a chronic effect, whereas Cu(0) NPs caused an acute effect and recovered. This study confirms that biogenic sulfide effectively attenuates ZnO and Cu(0) NPs toxicity to methanogens by the formation of metal sulfides.

Starved anammox cells are less resistant to NO2- inhibition

Anaerobic ammonium oxidizing (anammox) bacteria are be inhibited by their terminal electron acceptor, nitrite. Serious nitrite inhibition of the anammox bacteria occurs if the exposure coincides with the absence of the electron donating substrate, ammonium and pH < 7.2. Starvation of biomass occurs during underloading of bioreactors or biomass storage. This work investigated the effect of starvation on the sensitivity of anammox bacteria to nitrite exposure. Batch activity tests were carried out evaluating the response of anammox biomass subjected to different levels of starvation upon exposure to nitrite in the presence and absence of ammonium (active- and resting-cells, respectively). The response of the bacteria was evaluated by measuring the specific anammox activity and the evolution of the ATP content in the biomass over time. The 50% inhibitory concentrations of nitrite in starved- and fresh-resting-cells was 7 mg N L(-1) and 52 mg N L(-1), respectively. By contrast, only moderate nitrite inhibition occurred to starved anammox biomass when exposed to nitrite and ammonium simultaneously. Maximum ATP levels were observed in fresh cells. The ATP content in starved resting cells peaked 2-3 h after addition of NO2(-)(-). The response was hindered in cells starved for long periods. These findings agreed with a bioreactor study in which underloading of anammox biomass (0.10 g N L(-1) d(-1)) decreased its tolerance to a nitrite (only) exposure (101 mg NO2(-)-N L(-1)) and completely disrupted the N removal capacity of the biomass. A similar accumulation of 108 mg NO2(-)-N L(-1) after operation at 0.95 g N L(-1) d(-1) did not cause observable inhibition of the bacteria. The results taken as a whole demonstrate that starved anammox biomass is highly sensitive to nitrite toxicity. An explanation is proposed based on energy requirements to translocate nitrite in the cell.

Cosmetic wastewater treatment by upflow anaerobic sludge blanket reactor

Anaerobic treatment of pre-settled cosmetic wastewater in batch and continuous experiments has been investigated. Biodegradability tests showed high COD and solid removal efficiencies (about 70%), being the hydrolysis of solids the limiting step of the process. Continuous treatment was carried out in an upflow anaerobic sludge blanket reactor. High COD and TSS removal efficiencies (up to 95% and 85%, respectively) were achieved over a wide range of organic load rate (from 1.8 to 9.2g TCODL(-1)day(-1)). Methanogenesis inhibition was observed in batch assays, which can be predicted by means of a Haldane-based inhibition model. Both COD and solid removal were modelled by Monod and pseudo-first order models, respectively.

Vacuum promotes metabolic shifts and increases biogenic hydrogen production in dark fermentation systems

The successful operation of any type of hydrogen-producing bioreactor depends on the performance of the microorganisms present in the system. Both substrate and partial gas pressures are crucial factors affecting dark fermentation metabolic pathways. The main objective of this study was to evaluate the impact of both factors on hydrogen production using anaerobic granular sludge as inoculum and, secondly, to study the metabolic shifts of an anaerobic community subjected to low partial gas pressures. With this goal in mind, seven different wastewater (four synthetic media, two industrial wastewater, and one domestic effluent) and the effect of applying vacuum on the systems were analyzed. The application of vacuum promoted an increase in the diversity of hydrogenproducing bacteria, such as Clostridium, and promoted the dominance of acetoclastic- over hydrogenotrophic methanogens. The application of different media promoted a wide variety of metabolic pathways. Nevertheless, reduction of the hydrogen partial pressure by application of vacuum lead to further oxidation of reaction intermediates irrespective of the medium used, which resulted in higher hydrogen and methane production, and improved the COD removal. Interestingly, vacuum greatly promoted biogenic hydrogen production from a real wastewater, which opens possibilities for future application of dark fermentation systems to enhance biohydrogen yields.

Effect of 2,4,6-trichlorophenol on the microbial activity of adapted anaerobic granular sludge bioaugmented with Desulfitobacterium strains

The anaerobic degradation of 2,4,6-trichlorophenol (246TCP) has been studied in batch experiments. Granular sludges previously acclimated to 2,4-dichlorophenol (24DCP) and then adapted to at a load of 330 μM 246TCPd(-1) in two expanded granular sludge bed (EGSB) reactors were used. One of the reactors had been bioaugmented with Desulfitobacterium strains whereas the other served as control. 246TCP was tested at concentrations between 250 and 760 μM. The study focused on the fate of both fermentation products and chlorophenols derived from dechlorination of 246TCP. This compound mainly affected the biodegradation of acetate and propionate, which were inhibited at 246TCP concentrations above 380 μM. Lactate and ethanol were also accumulated at 760 μM 246TCP. Methanogenesis was strongly inhibited at 246TCP concentrations higher than 380 μM. A diauxic production of methane was observed, which can be described by a kinetic model in which acetoclastic methanogenesis was inhibited, whereas hydrogenotrophic methanogenesis was hardly affected by 246TCP. The similarity of the kinetic parameters obtained for the control and the bioaugmented sludges (K(i)=175-200 μM 246TCP and n=7) suggests that methanogenesis is not affected by the bioaugmentation. Moreover, the 246TCP dechlorination occurred mainly at ortho position, successively generating 24DCP and 4-chlorophenol (4CP), which was identified as final product. The bioaugmentation does not significantly improve the anaerobic biodegradation of 246TCP. It has been shown that the active biomass is capable of bioaccumulating 246TCP and products from dechlorination, which are subsequently excreted to the bulk medium when the biomass becomes active again. A kinetic model is proposed which simultaneously explains 246TCP and 24DCP reductive dechlorinations and includes the 246TCP bioaccumulation. The values of the kinetic parameters for 246TCP dechlorination were not affected by bioaugmentation (V(max)=5.3 and 5.1 μM h(-1) and K(s)=5.8 and 13.1 μM for control and bioaugmented sludges, respectively).

Inhibition of methanogenesis by chlorophenols: a kinetic approach.

Chlorophenols exert a crucial effect on the methanogenesis, considerably reducing both maximum methane potential and methanogenic rates. However, there is not enough information about the kinetic mechanism of chlorophenols toxicity on the methanogenesis, which is a key aspect for the control of the anaerobic digesters because of the sensitivity and the potential for energy recovery derived from methane release. The International Water Association-Anaerobic Digestion Model No. 1 (IWA-ADM1) can be adapted to a wide range of situations by updating or changing the equations in the model. The present study proposes a general kinetic model for methanogenesis. This model has been applied to predict the inhibition of methanogenesis by chlorophenols, and it can be used for updating the IWA-ADM1 when treating inhibitory compounds. The model was calibrated and validated using a wide broad of experimental sets of data of methane production by granular sludge in the presence of 2,4-dichlorophenol (24 DCP), 2,4,6-trichlorophenol (246TCP) and pentachlorophenol (PCP) in batch assays. A lag-phase of the effect of chlorophenols on the methanogenesis by non-adapted sludge was detected and modeled by the kinetic model proposed. In addition, the inhibitory effect of PCP was more pronounced on the acetoclastic methanogenesis than on the hydrogenotrophic one. Non-competitive and uncompetitive inhibition types were detected using 24 DCP and 246 TCP, whereas a suicide or irreversible inhibition type was observed in the case of PCP. Values of inhibition constants considerably varied depending on the chlorophenol used, between 45 mg24DCPL(-1), 41-51 mg246TCPL(-1) and 0.9-7.8 mgPCPL(-1). The higher toxicity of PCP is related with its hydrophobicity, which was determined by adsorption tests and using partition coefficients n-octanol/water. Modeling was accompanied by high statistical support in all cases, which confirmed the validation of the model proposed.

The role of pH on the resistance of resting‐ and active anammox bacteria to NO2− inhibition

The anaerobic oxidation of ammonium (anammox) uses nitrite as terminal electron acceptor. The nitrite can cause inhibition to the bacteria that catalyze the anammox reaction. The literature shows a great divergence on the levels of NO2− causing inhibition. Moreover, the conditions influencing the resistance of anammox bacteria to NO2− inhibitory effect are not well understood. This work investigated the effect of the pH and the concentration of nitrite on the activity and metabolism of anammox granular sludge under different physiological conditions. Batch activity tests in a range of pH values were carried out in which either actively metabolizing cells or resting cells were exposed to nitrite in the presence or absence of the electron donating substrate ammonium, respectively. The response of the bacteria was evaluated by analyzing the specific anammox activity, the accumulation of nitric oxide, and the evolution of the ATP content in the biomass. Additionally, the effect of the pH on the tolerance of the biomass to single substrate feeding interruptions was evaluated in continuous anammox bioreactors. The results show that the influence of the pH on the NO2− inhibition of anammox bacteria is greater under non‐metabolizing conditions than during active metabolism. The exposure of resting cells to NO2− (100 mg N L−1) at pH values below 7.2 caused complete inhibition of the anammox activity. The inhibition was accompanied by accumulation of the intermediate, nitric oxide, in the gas phase. In contrast, just mild inhibition was observed for resting cells exposed to the same NO2− concentration at pH values higher than 7.5 or any of the pH values tested in assays with actively metabolizing cells. ATP initially increased and subsequently decreased in time after resting cells were exposed to NO2− suggesting an active response of the cells to nitrite stress. Furthermore, bioreactors operated at pH lower than 6.8 had greater sensitivity to NO2− during an ammonium feed interruption than a bioreactor operated at pH 7.1. The results suggest that the ability of resting cells to tolerate NO2− inhibition is seriously impeded at mildly acidic pH values; whereas actively metabolizing biomass is resistant to NO2− toxicity over a wide range of pH values. Biotechnol. Bioeng. 2014;111: 1949–1956.