Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor.

A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-1 sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l-1 h-1. This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH.

Benzene/toluene/p-xylene degradation. Part II. Effect of substrate interactions and feeding strategies in toluene/benzene and toluene/p-xylene fermentations in a partitioning bioreactor.

A two-phase aqueous/organic partitioning bioreactor scheme was used to degrade mixtures of toluene and benzene, and toluene and p-xylene, using simultaneous and sequential feeding strategies. The aqueous phase of the partitioning bioreactor contained Pseudomonas sp. ATCC 55595, an organism able to degrade benzene, toluene and p-xylene simultaneously. An industrial grade of oleyl alcohol served as the organic phase. In each experiment, the organic phase of the bioreactor was loaded with 10.15 g toluene, and either 2.0 g benzene or 2.1 g p-xylene. The resulting aqueous phase concentrations were 50 mg/l, 25 mg/l and 8 mg/l toluene, benzene and p-xylene respectively. The simultaneous fermentation of benzene and toluene consumed these compounds at volumetric rates of 0.024 g l-1 h-1 and 0.067 g l-1 h-1, respectively. The simultaneous fermentation of toluene and p-xylene consumed these xenobiotics at volumetric rates of 0.066 g l-1 h-1 and 0.018 g l-1 h-1, respectively. A sequential feeding strategy was employed in which toluene was added initially, but the benzene or p-xylene aliquot was added only after the cells had consumed half of the initial toluene concentration. This strategy was shown to improve overall degradation rates, and to reduce the stress on the microorganisms. In the sequential fermentation of benzene and toluene, the volumetric degradation rates were 0.056 g l-1 h-1 and 0.079 g l-1 h-1, respectively. In the toluene/p-xylene sequential fermentation, the initial toluene load was consumed before the p-xylene aliquot was consumed. After 12 h in which no p-xylene degradation was observed, a 4.0-g toluene aliquot was added, and p-xylene degradation resumed. Excluding that 12-h period, the microbes consumed toluene and p-xylene at volumetric rates of 0.074 g l-1 h-1 and 0.025 g l-1 h-1, respectively. Oxygen limitation occurred in all fermentations during the rapid growth phase.

Simultaneous biodegradation of benzene, toluene, and p-xylene in a two-phase partitioning bioreactor: concept demonstration and practical application.

In this work, a mixture of benzene, toluene, and p-xylene was simultaneously biodegraded by Pseudomonas sp. ATCC 55595 in a two-phase partitioning bioreactor. This bioreactor consisted of a 1-L cell-containing aqueous medium phase and a 500-mL immiscible organic phase. The organic solvent systematically selected for use in the bioreactor was Adol 85 NF, an industrial-grade, biocompatible solvent. In the first of three experiments, the organic phase was loaded with 2.0 g of benzene, 10.15 g of toluene, and 2.1 g of p-xylene, which partitioned into the aqueous phase at concentrations of 25, 50, and 8 mg/L, respectively. The system ultimately biodegraded all of the substrates within 144 h. During the rapid growth phase of this fermentation, the cells were oxygen-limited. This fermentation was therefore repeated using an enriched air supply to remove the oxygen limitation. The use of enriched air ultimately reduced the length of the fermentation to 108 h, thereby improving the overall volumetric consumption rates. Finally, 500 mL of Adol were used to recover 2.0 g of benzene, 10.15 g of toluene, and 2.1 g of p-xylene from silica sand that was contaminated as part of a simulated soil "spill". The solvent washing procedure was able to recover greater than 99% of each compound from the contaminated soil. The Adol was then transferred to the two-phase bioreactor to permit biological treatment of the BTX contaminants. This process was repeated when the initial BTX load had been consumed almost to exhaustion, and the solvent was able to recover the contaminants at greater than 99% efficiency once again. The system was ultimately able to degrade 4.0 g of benzene, 20.2 g of toluene, and 4.2 g of p-xylene within 144 h. These results represent an unprecedented level of BTX degradation and illustrate a potential practical application for this novel biotechnology.

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol.

A two-phase partitioning bioreactor containing Pseudomonas putida ATCC 11172 was used to degrade high concentrations of phenol in batch and fed-batch mode. The 2-1 (nominal volume) partitioning bioreactor employs a 1-1 cell-containing aqueous phase, and a 500-ml immiscible and biocompatible second organic phase (2-undecanone), which partitions the toxic substrate into the aqueous phase at a rate based on the metabolic activity of the microorganisms. Using this reactor configuration, operated in batch mode, 10-g phenol was degraded to completion within 84-h. The system was, however, oxygen-limited during the rapid growth phase of the fermentation. A second experiment, using enriched air to prevent oxygen limitation, resulted in the complete degradation of 10-g phenol within 72-h. The use of a sequential feeding strategy, in which a 10-g phenol load was added in sequential 5-g aliquots, resulted in a significant reduction in the lag phase, from 36-h to 12-h, and the consumption of 10-g phenol in 60 h. Finally, fed-batch fermentation was used to attempt to determine the ultimate capacity of the system to degrade phenol. The organic phase was loaded with 10-g phenol, the microorganisms were allowed to consume this aliquot almost to completion, and a second 10-g aliquot was then added. The organic phase was spiked in this manner a total of four times, resulting in the degradation of 46.55-g phenol within 12 days. The system was also monitored for nutrient depletion, and a nutrient-feeding schedule was formulated, in response to the mass of phenol consumed.

Biodegradation of phenol at high initial concentrations in two-phase partitioning batch and fed-batch bioreactors.

A two-phase organic-aqueous system was used to degrade phenol in both batch and fed-batch culture. The solvent, which contained the phenol and partitioned it into the aqueous phase, was systematically selected based on volatility, solubility in the aqueous phase, partition coefficient for phenol, biocompatibility, and cost. The two-phase partitioning bioreactor used 500 mL of 2-undecanone loaded with high concentrations of phenol to deliver the xenobiotic to Pseudomonas putida ATCC 11172 in the 1-L aqueous phase, at subinhibitory levels. The initial concentrations of phenol selected for the aqueous phase were predicted using the experimentally determined partition coefficient for this ternary system of 47.6. This system was initially observed to degrade 4 g of phenol in just over 48 h in batch culture. Further loading of the organic phase in subsequent experiments demonstrated that the system was capable of degrading 10 g of phenol to completion in approximately 72 h. The higher levels of phenol in the system caused a modest increase in the duration of the lag phase, but did not lead to complete inhibition or cell death. The use of a fed-batch approach allowed the system to ultimately consume 28 g of phenol in approximately 165 h, without experiencing substrate toxicity. In this system, phenol delivery to the aqueous phase is demand based, and is directly related to the metabolic activity of the cells. This system permits high loading of phenol without the corresponding substrate inhibition commonly seen in conventional bioreactors.