Santiago Villaverde

Influence of pH over nitrifying biofilm activity in submerged biofilters

Abstract The influence of pH over nitrification in submerged biofilters has been studied through the observation of three pH effects over the nitrifying biofilm: activation-deactivation, substrate limitation, and free ammonia inhibition. Within a pH range of 5.0–9.0, a pH increase of one unit produce a 13% increase on the nitrification efficiency. A stoichiometry of 7.1 mg CaCO 3 /mg N was measured for the process, which became alkalinity limited below pH 5.0. The highest activity of ammonium oxidizers and the highest values of volatile attached solids (VAS) in the filter were obtained at pH 8.2. The concentration of volatile attached solids (VAS) was regulated by free ammonia inhibition, substrate limitation (NH 4 + concentration), and reactor hydrodynamics. Inhibition by free ammonia controlled bacteria activity at pH greater than 7.5, observing an increase of microorganisms concentration in the filter as a specific free ammonia concentration (mg NH 3free -N gVAS −1 ) decreased. A nitrite accumulation of up to 80–90% was obtained for specific inhibitory concentrations greater than 1.5 mg NH 3free -N gVAS −1 resulting from the selective inhibition of nitrite oxidizers.

Efficient nutrient removal from swine manure in a tubular biofilm photo-bioreactor using algae-bacteria consortia

Concentrated animals feeding operations (CAFOs) often pose a negative environmental impact due to the uncontrolled spreading of manure into soils that ends up in the release of organic matter and nutrients into water bodies. Conventional aerobic methods treating CAFOs wastewater require intensive oxygenation, which significantly increases the operational costs. The alternative proposed in this research is the application of micro-algae based systems by taking advantage of the cost-effective in situ oxygenation via photosynthesis. A 4.9 L enclosed tubular biofilm photo-bioreactor was inoculated with an algal-bacterial consortium formed by the micro-algae Chlorella sorokiniana and a mixed bacterial culture from an activated sludge process. C. sorokiniana delivers the O(2) necessary to accomplish both organic matter and ammonium oxidation. The reactor was fed with diluted swine wastewater containing 180, 15 and 2,000 mg/L of NH(4) (+)-N, soluble P and total COD, respectively. The photo-bioreactor exhibited good and sustained nutrient removal efficiencies (up to 99% and 86% for NH(4) (+) and PO(4) (3-), respectively) while total COD was removed up to 75% when the biofilm was properly established. Liquid superficial velocities up to 0.4 m/s (achieved by culture broth recirculation) hindered the formation of a stable biofilm, while operation at velocities lower than 0.1 m/s supported stable process performance. The high shear stress imposed by the centrifugal recirculation pump disintegrated the large aggregates detached from the biofilm, which resulted in a poor settling performance and therefore poor COD removal efficiencies. Enclosed biofilm photo-bioreactors therefore offer a potentially more economical alternative to conventional tertiary treatments process.

Two-phase partitioning bioreactors in environmental biotechnology

Two-phase partitioning bioreactors (TPPBs) in environmental biotechnology are based on the addition of a non-aqueous phase (NAP) into a biological process in order to overcome both mass-transfer limitations from the gas to aqueous phase and pollutant-mediated inhibitions. Despite constituting a robust and reliable technology in terms of pollutant biodegradation rates and process stability in wastewater, soil, and gas treatment applications, this superior performance only applies for a restricted number of pollutants or contamination events. Severe limitations such as high energy requirements, high costs of some NAPs, foaming, or pollutant sequestration challenge the full-scale application of this technology. The introduction of solid NAPs into this research field has opened a promising pathway for the future development of TPPBs. Finally, this work reviews fundamental aspects of NAP selection and mass transfer and identifies the niches for future research: low energy-demand bioreactor designs, experimental determination of partial mass transfers, and solid NAP tailoring.

New process for simultaneous removal of nitrogen and sulphur under anaerobic conditions

A granular activated carbon (GAC) anaerobic fluidised-bed reactor treating vinasse from an ethanol distillery of sugar beet molasses was operated for 90 days, the first 40 days of start-up followed by 50 days of operation at constant organic loading rate of 1.7g COD/Ld. The reactor showed good performance in terms of organic matter removal but an anomalous behaviour in terms of unusual high concentrations of molecular nitrogen in the biogas. The analysis of the different nitrogenous and sulphur compounds and the mass balances of these compounds in the liquid and gas phases clearly indicated an uncommon evolution of nitrogen and sulphur in the reactor. About 50% of the nitrogen entering the reactor as total Kjeldahl nitrogen (TKN) was removed from the liquid phase appearing as N2 in the gas phase. Simultaneously, only 20% of the S-SO4(2-) initially present in the influent appears as S-S2- in the effluent or S-H2S in the biogas, indicating that 80% of the sulphur is removed. This behaviour has not been reported previously in the literature. These observations may suggest a new anaerobic removal process of ammonia and sulphate according to an uncommon mechanism involving simultaneous anaerobic ammonium oxidation and sulphate reduction.

A systematic selection of the non-aqueous phase in a bacterial two liquid phase bioreactor treating α-pinene

A systematic evaluation of the selection criteria of non-aqueous phases in two liquid phase bioreactors (TLPBs), also named two-phase partitioning bioreactors (TPPBs), was carried out using the biodegradation of α-pinene by Pseudomonas fluorescens NCIMB 11671 as a model process. A preliminary solvent screening was thus carried out among the most common non-aqueous phases reported in literature for volatile organic contaminants biodegradation in TLPBs: silicon oil, paraffin oil, hexadecane, diethyl sebacate, dibutyl-phtalate, FC 40, 1,1,1,3,5,5,5-heptamethyltrisiloxane (HMS), and 2,2,4,4,6,8,8-heptamethylnonane (HMN). FC 40, silicone oil, HMS, and HMN were first selected based on its biocompatibility, resistance to microbial attack, and α-pinene mass transport characteristics. FC 40, HMS, HMN, and silicone oil at 10% (v/v) enhanced α-pinene mass transport from the gas to the liquid phase by a factor of 3.8, 14.8, 11.4, and 8.6, respectively, compared to a single-phase aqueous system. FC 40 and HMN were finally compared for their ability to enhance α-pinene biodegradation in a mechanically agitated bioreactor. The use of FC 40 or HMN (both at 10% v/v) sustained non-steady state removal efficiencies (RE) and elimination capacities (EC) approximately 7 and 12 times higher than those achieved in the system without an organic phase, respectively. In addition, preliminary results showed that P fluorescens could uptake and mineralize α-pinene directly from the non aqueous phase.

A Comparative Study of Solid and Liquid Non-Aqueous Phases for the Biodegradation of Hexane in Two-Phase Partitioning Bioreactors

A comparative study of the performance of solid and liquid non‐aqueous phases (NAPs) to enhance the mass transfer and biodegradation of hexane by Pseudomonas aeruginosa in two‐phase partitioning bioreactors (TPPBs) was undertaken. A preliminary NAP screening was thus carried out among the most common solid and liquid NAPs used in pollutant biodegradation. The polymer Kraton G1657 (solid) and the liquid silicone oils SO20 and SO200 were selected from this screening based on their biocompatibility, resistance to microbial attack, non‐volatility and high affinity for hexane (low partition coefficient: K = Cg/CNAP, where Cg and CNAP represent the pollutant concentration in the gas phase and NAP, respectively). Despite the three NAPs exhibited a similar affinity for hexane (K ≈ 0.0058), SO200 and SO20 showed a superior performance to Kraton G1657 in terms of hexane mass transfer and biodegradation enhancement. The enhanced performance of SO200 and SO20 could be explained by both the low interfacial area of this solid polymer (as a result of the large size of commercial beads) and by the interference of water on hexane transfer (observed in this work). When Kraton G1657 (20%) was tested in a TPPB inoculated with P. aeruginosa, steady state elimination capacities (ECs) of 5.6 ± 0.6 g m−3 h−1 were achieved. These values were similar to those obtained in the absence of a NAP but lower compared to the ECs recorded in the presence of 20% of SO200 (10.6 ± 0.9 g m−3 h−1). Finally, this study showed that the enhancement in the transfer of hexane supported by SO200 was attenuated by limitations in microbial activity, as shown by the fact that the ECs in biotic systems were far lower than the maximum hexane transfer capacity recorded under abiotic conditions. Biotechnol. Bioeng. 2010;106: 731–740.

Determining the effect of solid and liquid vectors on the gaseous interfacial area and oxygen transfer rates in two-phase partitioning bioreactors

The effect of liquid and solid transfer vectors (silicone oil and Desmopan, respectively) on the gaseous interfacial area (a(g)) was evaluated in a two-phase partitioning bioreactor (TPPB) using fresh mineral salt medium and the cultivation broth of a toluene degradation culture (Pseudomonas putida DOT-T1E cultures continuously cultivated with and without silicone oil at low toluene loading rates). Higher values of a(g) were recorded in the presence of both silicone oil and Desmopan compared to the values obtained in the absence of a vector, regardless of the aqueous medium tested (1.6 and 3 times higher, respectively, using fresh mineral salt medium). These improvements in a(g) were well correlated to the oxygen mass transfer enhancements supported by the vectors (1.3 and 2.5 for liquid and solid vectors, respectively, using fresh medium). In this context, oxygen transfer rates of 2.5 g O(2)L(-1)h(-1) and 1.3 g O(2)L(-1)h(-1) were recorded in the presence of Desmopan and silicone oil, respectively, which are in agreement with previously reported values in literature. These results suggest that mass transfer enhancements in TPPBs might correspond to an increase in a(g) rather than to the establishment of a high-performance gas/vector/water transfer pathway.

Spatial distribution of respiratory activity in Pseudomonas putida 54G biofilms degrading volatile organic compounds (VOC).

Abstract All over the world, Microbial systems are used to clean soils, waters and air streams that have been contaminated with volatile organic compounds (VOC). Information about the structure and function of the microbes that metabolize these contaminants can be gained by studying these microbial systems. Here we describe the spatial patterns of respiratory activity in Pseudomonas putida 54G aerobic biofilms degrading two VOC, toluene and ethanol. Oxygen concentration profiles within the biofilm were measured using microsensors. These profiles are thought to be most accurate reflection of the structure and function of aerobic microbial biofilms. The degrading process certainly imposed a structural and functional patterns on the microbial biofilm community growing at the expense of the VOC substrate. Cryosectioning coupled with the staining of biofilm samples confirmed a high respiratory activity near the substratum, that decreased towards the biofilm/fluid interface. The accumulation of inactive cells in the outer biofilm layer protects the inner biofilm from high concentrations of toxic compounds and also limits the degradation rate. This stratification phenomenon appeared to be a general pattern for P. putida 54G biofilms degrading VOC.

Modeling photosynthetically oxygenated biodegradation processes using artificial neural networks.

The complexity of the mechanisms underlying organic matter mineralization and nutrient removal in algal-bacterial photobioreactors during the treatment of residual wastewaters has severely hindered the development of mechanistic models able to accurately describe these processes. Artificial neural networks (ANNs) are capable of inferring the complex relationships existing between input and output process variables without a detailed description of the mechanisms governing the process, and should therefore be more suitable for the modeling of photosynthetically oxygenated systems. Thus, a neural network consisting of a single hidden layer with four neurons accurately predicted the steady-state operation of a continuous stirred tank photobioreactor during salicylate biodegradation by an algal-bacterial consortium. Despite its simplicity and the low number of data sets for ANN training (23), this network topology exhibited a satisfactory fit for both training and testing data with correlation coefficients of 99%. Although the use of ANNs for modeling conventional wastewater treatment systems is not novel, this work constitutes, to the best of our knowledge, the first reported application of ANNs to photosynthetically oxygenated systems and one of the few models for microalgae-based treatment processes. This modeling approach is therefore expected to contribute to improve the understanding of the complex relationships between light, temperature, hydraulic retention time, pollutant concentration and process removal efficiency, which would eventually promote the development of algal-bacterial processes as a cost effective alternative for the treatment of industrial wastewaters.

Addressing the role of the extrusion pump-bearing pGRT1 plasmid in toluene biodegradation by Pseudomonas putida DOT-T1E under real case scenarios.

The role of both the plasmid pGRT1 and the solvent extrusion pump ttgGHI during toluene biodegradation in Pseudomonas putida DOT-T1E cultures was investigated in a sterile suspended growth bioreactor operated as chemostat and inoculated independently with a wild type strain, a mutant lacking the pGRT1 plasmid (P. putida DOT-TIE-100), and a mutant with a modified pGRT1 plasmid lacking the genes encoding the ttgGHI solvent efflux pump (P. putida DOT-TIE-28). A similar process performance was recorded in all tested strains at 4 g tol m(-3) and dilution rates (D) of 0.1 h(-1). However, operation at 10 g tol m(-3) and D of 0.2 h(-1) revealed a much lower toluene EC (285 g m(-3) h(-1)) in P. putida DOT-T1E-100 cultures when compared to wild type and P. putida DOT-T1E-28 cultures (483-498 g m(-3) h(-1)), which suggests that other mechanisms rather than solvent extrusion by the ttgGHI efflux pump supported this superior process performance. Finally, the plasmid pGRT1 analysed exhibited a remarkable stability towards toluene harmful mediated effects, regardless the strain or toluene loading tested.