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 = C(g)/C(NAP), where C(g) and C(NAP) represent the pollutant concentration in the gas phase and NAP, respectively). Despite the three NAPs exhibited a similar affinity for hexane (K approximately 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.

A step-forward in the characterization and potential applications of solid and liquid oxygen transfer vectors.

Silicone oil 20 and 200 cSt, a perfluorocarbon (FC40TM), heptamethylnonane, Kraton, Elvax, and Desmopan were evaluated for their ability to enhance oxygen transfer in stirred tank and airlift reactors (STR and ALR, respectively). None of the vectors tested was either toxic or biodegradable and they exhibited a moderate affinity for oxygen (gas/vector partitioning coefficients K(g)/(v) = C(g) times C(v)(-1) ranging from 3 to 5.1). FC40 was highly volatile, while KratonTM and ElvaxTM exhibited a low thermal stability, which constitutes a serious handicap for their implementation in fermentations. Silicone oil 20 cSt and Desmopan supported the highest oxygen transfer rates under abiotic conditions in both STR and ALR designs, with enhancement factors of up to 90% and 250%, respectively, compared to control tests (deprived of vector). The fact that these vectors showed the highest K (g/v) proved that, besides the classical selection criteria, the in situ hydrodynamic behavior (which affects K ( L ) a) must be considered for vector selection. The use of silicone oil 20 cSt and Desmopan in glucose-supplemented Saccharomyces cerevisiae fermentations resulted in a two- and threefold increase in biomass productions, respectively. The better performance of Desmopan in terms of biomass growth enhancement, together with the absence of the operational problems inherent to the use of liquid vectors (such as intensive foaming, high cost, and difficult solvent recovery), make solid vectors a promising and cost-effective alternative in the future developments of two-phase partitioning bioreactors.

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.

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.

Continuous cultures of Pseudomonas putida mt-2 overcome catabolic function loss under real case operating conditions.

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h(-1) under toluene loading rates of 259 +/- 23 and 801 +/- 78 g m(-3) h(-1) (inlet toluene concentrations of 3.5 and 10.9 g m(-3), respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol(-) population at day 4 to approx. 100% Tol(+) cells from day 13 onward, sustaining elimination capacities of 232 +/- 10 g m(-3) h(-1) at 3.5 g Tol m(-3) and 377 +/- 13 g m(-3) h(-1) at 10.9 g Tol m(-3), which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol(-) variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.

Microalgae-based processes for the biodegradation of pretreated piggery wastewaters.

The potential and limitations of photosynthetic oxygenation on carbon and nitrogen removal from swine slurry were investigated in batch experiments using Chlorella sorokiniana and an acclimated activated sludge as model microorganisms. While algal-bacterial systems exhibited similar performance than aerated activated sludge in tests supplied with four and eight times diluted slurry, a severe inhibition of the biodegradation process was recorded in undiluted and two times diluted wastewater. Daily pH adjustment to 7 in enclosed algal-bacterial tests at several swine slurry dilutions allowed the treatment of up to two times diluted slurries (containing up to 1,180 mg N-NH(4)(+) l(-1)). The combination of high pH levels and high NH(4)(+) concentrations was thus identified as the main inhibitory factor governing the efficiency of photosynthetically oxygenated processes treating swine slurry. Measurements of soluble total organic carbon (TOC) and volatile fatty acids (VFA) present in the slurry suggested that VFA degradation (mainly acetic and propionic acid) accounted for most of the soluble TOC removal, especially during the initial stages of the biodegradation process. On the other hand, assimilation into biomass and nitrification to NO(2)(-) constituted the main NH(4)(+) removal processes in enclosed algal-bacterial systems.

Response of Pseudomonas putida F1 cultures to fluctuating toluene loads and operational failures in suspended growth bioreactors.

The response of Pseudomonas putida F1 to process fluctuations and operational failures during toluene biodegradation was evaluated in a chemostat suspended growth bioreactor. The ability of P. putida F1 to rapidly increase its specific toluene degradation capacity resulted in no significant variation in process removal efficiency when toluene load was increased from 188 to 341 g m(-3) h(-1). Likewise, bacterial activity rapidly reached steady state performance (in less than 1.5 h after the restoration of steady state operational conditions) following an 8 h process shutdown, or after episodes of toluene or mineral nutrients deprivation. Process performance was however highly sensitive to pH, as pH levels below 4.5 dramatically inhibited bacterial activity, decreasing severely process robustness and inducing a cycle of periodic process collapses and recoveries. This pH mediated deterioration of bacterial activity was confirmed by further respirometric tests, which revealed a 50-60% reduction in the O(2) consumption rate during the degradation of both toluene and 3-methyl catechol when pH decreased from 5.05 to 4.55. Finally, process robustness was quantified according to methods previously described in literature.

A systematic selection of the non-aqueous phase in a bacterial two liquid phase bioreactor treating alpha-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 alpha-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 alpha-pinene mass transport characteristics. FC 40, HMS, HMN, and silicone oil at 10% (v/v) enhanced alpha-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 alpha-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 alpha-pinene directly from the non aqueous phase.

Toluene biodegradation by Pseudomonas putida F1: targeting culture stability in long-term operation.

The stability of Pseudomonas putida F1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h(-1)). Each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the TOL plasmid. At all tested conditions, P. putida F1 exhibited stable performance as shown by the constant values of the specific toluene degradation yield, CO2 produced versus toluene degraded yield, and biomass concentration within each steady state. Benzyl alcohol, a curing agent causing TOL plasmid deletion in Pseudomonas strains, was present in the cultivation medium as a result of the monooxygenation of toluene by the diooxygenase system of P. putida F1. However, no mutant population growing at the expense of the extracellular excreted carbon or lysis products was established in the chemostat as confirmed by the constant dissolved total organic carbon (TOC) concentration and fraction of toluene degrading cells (approx. 100%). In addition, batch experiments conducted with both lysis substrate and toluene simultaneously confirmed that P. putida F1 preferentially consumed toluene rather than extracellular excreted carbon.

Two-phase partitioning bioreactors for treatment of volatile organic compounds.

Two-phase partitioning bioreactors (TPPBs) allow the biological removal of volatile organic compounds (VOCs) from contaminated gas streams at unprecedented rates and concentrations. TPPBs are constructed by adding a non-aqueous phase (e.g. hexadecane, silicone oil) to an aqueous phase that contains the microorganisms responsible for degrading the VOCs. Presence of a water-immiscible phase improves the transfer of hydrophobic substrates (e.g. hexane, oxygen) or reduces the toxicity of inhibitory substances (e.g. benzene, toluene) to the microorganisms present in the aqueous phase. The non-aqueous phase is selected based on cost, safety, good partitioning properties towards the target pollutants, biocompatibility, and non-biodegradability. TPPBs have hitherto been designed as laboratory-scale well-mixed stirred-tank reactors or as biofilters that contain a non-aqueous phase. Scale-up and industrial use of TPPBs require elucidation and modeling of the mechanisms of substrate transfer and uptake; understanding of the mechanisms of microbial selection; identification or synthesis of new inexpensive and robust non-aqueous phases; and generation of suitable guidelines for process design and control.

New insights on toluene biodegradation by Pseudomonas putida F1: influence of pollutant concentration and excreted metabolites.

The influence of toluene concentration on the specific growth rate, cellular yield, specific CO(2), and metabolite production by Pseudomonas putida F1 (PpF1) was investigated. Both cellular yield and specific CO(2) production remained constant at 1.0 +/- 0.1 g biomass dry weight (DW) g(-1) toluene and 1.91 +/- 0.31 g CO(2) g(-1) biomass, respectively, under the tested range of concentrations (2-250 mg toluene l(-1)). The specific growth rate increased up to 70 mg toluene l(-1). Further increases in toluene concentration inhibited PpF1 growth, although inhibitory concentrations were far from the application range of biological treatment processes. The specific ATP content increased with toluene concentration up to toluene concentrations of 170 mg l(-1). 3-Methyl catechol (3-MC) was never detected in the cultivation medium despite being an intermediary in the TOD pathway. This suggested that the transformation from toluene to 3-MC was the limiting step in the biodegradation process. On the other hand, benzyl alcohol (BA) was produced from toluene in a side chain reaction. This is, to the best of our knowledge, the first reported case of methyl monoxygenation of toluene by PpF1 not harboring the pWW0 TOL plasmid. In addition, the influence of 3-MC, BA, and o-cresol on toluene degradation was investigated respirometrically, showing that toluene-associated respiration was not significantly inhibited in the presence of 10-100 mg l(-1) of the above-mentioned compounds.

Combined anaerobic-aerobic treatment of azo dyes--a short review of bioreactor studies.

The most logical concept for the removal of azo dyes in biological wastewater treatment systems is based on anaerobic treatment, for the reductive cleavage of the dyes' azo linkages, in combination with aerobic treatment, for the degradation of the products from azo dye cleavage, aromatic amines. Since the 1990s, several research papers have been published on combined, sequential or integrated, anaerobic-aerobic bioreactor treatment of azo dye-containing wastewater. The extent of azo dye reduction in the anaerobic phase of those bioreactor systems was generally high, albeit the process often required long reaction times, a limitation that can easily be remedied by making use of the property of redox mediators to speed up the process. The consequent removal of aromatic amines under aerobic conditions was less unequivocal. Although analytical data indicate that many of the aromatic amines were removed from the wastewater, and although the limited amount of available toxicity data all show far-reaching detoxification during aerobic treatment, it is clear that not all aromatic amines can be completely mineralized.