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-l 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. Received: 16 November 1998 / Received revision: 28 March 1999 / Accepted: 9 April 1999     

Dynamic modeling and optimal fed-batch feeding strategies for a two-phase partitioning bioreactor

A dynamic model for the degradation of phenol in a two-phase partitioning bioreactor has been developed based on mechanistic balances around the bioreactor. The key process characteristics including substrate transfer between the organic and aqueous phases, substrate inhibition, oxygen limitation, and cell entrainment were incorporated into the model. The model predictions were validated against existing experimental data obtained for a 2-L bioreactor, and good correlation was observed for the time frames of the simulations, as well as for trends in cell and substrate concentrations. Optimal fed-batch, phenol feeding strategies were then developed based on two approaches: (1) maximization of phenol consumption in a fixed time interval and (2) consumption of a fixed amount of phenol in minimal time. The optimal feeding policies, determined using the Iterative Dynamic Programming algorithm, provided substantial improvements in the amount of phenol consumed when compared to a typical experimental heuristic approach. For example, 45.73 g of phenol was predicted to be consumed in 50 h (not including lag phase) using the optimal feeding profile compared to 10.26 g of phenol consumed in the simulated experimental approach. Oxygen limitation was predicted to be a recurring operational challenge in the partitioning bioreactor, and had a strong impact on the optimization results.     

Expanded application of a two-phase partitioning bioreactor through strain development and new feeding strategies

This research demonstrated the microbial treatment of concentrated phenol wastes using a two-phase partitioning bioreactor (TPPB). TPPBs are characterized by a cell-containing aqueous phase and an immiscible and biocompatible organic phase that partitions toxic substrates to the cells on the basis of their metabolic demand and the thermodynamic equilibrium of the system. Process limitations imposed by the capability of wild-type Pseudomonas putida ATCC 11172 to utilize long chain alcohols were addressed by strain modification (transposon mutagenesis) to eliminate this undesirable biochemical characteristic, enabling use of a range of previously bioavailable organics as delivery solvents. Degradation of phenol in a system with the modified strain as catalyst and industrial grade Adol 85 NF (primarily oleyl alcohol) as the solvent was demonstrated, with the system ultimately degrading 36 g of phenol within 38 h. Volumetric phenol consumption rates by wild type P. putida ATCC 11172 and the genetically modified derivative revealed equivalent phenol degrading capabilities (0.49 g/L x h vs 0.47 g/L x h respectively, in paired fermentations), with the latter presenting a more efficient remediation option due to decreased solvent losses arising from the modified strain's forced inability to consume the delivery solvent as a substrate. Two feeding strategies and system configurations were evaluated to expand practical applications of TPPB technology. The ability to operate with a lower solvent ratio over extended periods revealed potential for long-term application of TPPB to the treatment of large masses of phenol while minimizing solvent costs. Repeated recovery of 99% of phenol from concentrated phenol solutions and subsequent treatment within a TPPB scheme demonstrated applicability of the approach to the remediation of highly contaminated "effluents" as well as large masses of bulk phenol. Operation of the TPPB system in a dispersed manner, rather than as two distinct phases, resulted in volumetric consumption rates similar to those previously achieved only in systems operated with enriched air.     

Effect of directional switching frequency on toluene degradation in a vapor-phase bioreactor

A potential method to improve biomass distribution and the stability of vapor-phase bioreactors is to operate them in a directionally switching mode such that the contaminant air stream direction is periodically reversed through the reactor. In this study, the effect of switching frequency (SF) on bioreactor performance and biodegradation activity was investigated at 1-, 3- and 7-day SFs using toluene as a model compound. Rapid losses of biodegradation capacity and serious bioreactor instability were observed in the bioreactor operated at a 1-day SF. It is hypothesized that the frequent dynamic loading conditions at the 1-day SF hindered biofilm development and ultimately bioreactor stability. In contrast, bioreactors operated at the 3- and 7-day SFs achieved overall removal efficiencies of greater than 99% for 72 and 59 days of operation, respectively. Following each air-stream reversal, the bioreactor operated at the 7-day SF required 48 h to fully restore biodegradation capacity in the inlet bioreactor section. The 1-day SF bioreactor required no such reacclimation period. The toluene-degrading activity in the inlet section of the 7-day SF bioreactor dropped by 71% during the 7-day cycle, whereas it decreased by only 11% in the inlet of the 3-day SF bioreactor. These declines suggest that continuous or near-continuous exposure to toluene can inhibit microbial activity. Of the three SFs examined, the 3-day SF yielded the most efficient bioreactor performance by balancing reacclimation requirements with biodegradation activity losses.     

Addressing biofilter limitations: A two-phase partitioning bioreactor process for the treatment of benzene and toluene contaminated gas streams

A two-phase partitioning bioreactor (TPPB) achievedsimultaneous and continuous removal and degradation of benzene and toluene froman air stream. The aqueous-organic system utilized n-hexadecane as the organicphase, and the organism Alcaligenes xylosoxidans Y234 in the aqueous phaseto achieve the degradation of benzene and toluene. The system, which operates asa well-mixed dispersion and is therefore resistant to substrate surges, was firstshown to be capable of utilizing toluene while operating at a loading capacity of 235 g m^-3 h^-1with an elimination capacity of 233 g m^-3 h^-1. It was also determined that to characterize TPPB performance in terms of elimination capacity thedefinition of elimination capacity must be extended to include the cell mass present, a readilycontrollable variable given the nature of the system. Based on this criterion, it wasestimated that for a cell concentration of 1 g l^-1 present in the TPPB, thepotential maximum toluene elimination capacity is 1290 g m^-3 h^-1 whichis substantially higher than any toluene elimination capacity achieved by biofiltersat a high removal efficiency. If no other factor were to limit the system, eliminationcapacities could be many times higher still, and are dependent on maintaining desiredcell concentrations above 1 g l^-1. The TPPB was then operated at nominalloading capacities of 63 g m^-3h^-1 (benzene) and 51 g m^-3 h^-1 (toluene) at a removal efficiency greater than 99% to demonstratedthe applicability of this system in dealing with two chemical species simultaneously. TPPBsystems therefore have been shown to be effective at removing gaseous organiccontaminants at high removal efficiencies while also possessing desirable operatingfeatures, such as providing and maintaining high cell concentrations throughout thereactor, and a capacity to effectively deal with high contaminant loadings.     

Amplification of toluene dioxygenase genes in a hybrid Pseudomonas strain to enhance the biodegradation of benzene, toluene, and p-xylene mixture

A hybrid metabolic pathway through which benzene, toluene, and p-xylene (BTX) mixture could be simultaneously mineralized was previously constructed in Pseudomonas putida TB101 (Lee, Roh, Kim, Biotechnol. Bioeng 43: 1146-1152, 1994). In this work, we improved the performance of the hybrid pathway by cloning the todC1C2BA genes in the broad-host-range multicopy vector RSF1010 and by introducing the resulting plasmid pTOL037 into P. putida mt-2 which harbors the archetypal TOL plasmid. As a result, a new hybrid strain, P. putida TB103, possessing the enhanced activity of toluene dioxygenase in the hybrid pathway was constructed. The degradation rates of benzene, toluene, and p-xylene by P. putida TB103 were increased by about 9.3-, 3.7-, and 1.4-fold, respectively, compared with those by previously constructed P. putida TB101. Apparently, this improved capability of P. putida TB103 for the degradation of BTX mixture resulted from the amplification of the todC1C2BA genes. Furthermore, a relatively long lag period for benzene degradation observed when P. putida TB101 was used for the degradation of BTX mixture at low dissolved oxygen (DO) tension disappeared when P. putida TB103 was employed. (c) 1995 John Wiley & Sons, Inc.     

Phosphonium ionic liquids for degradation of phenol in a two-phase partitioning bioreactor

Six ionic liquids (ILs), which are organic salts that are liquid at room temperature, were tested for their biocompatibility with three xenobiotic-degrading bacteria, Pseudomonas putida, Achromobacter xylosoxidans, and Sphingomonas aromaticivorans. Of the 18 pairings, seven were found to demonstrate biocompatibility, with one IL (trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl) amide) being biocompatible with all three organisms. This IL was then used in a two-phase partitioning bioreactor (TPPB), consisting of 1 l aqueous phase loaded with 1,580 mg phenol and 0.25 l IL, inoculated with the phenol degrader P. putida. This initially toxic aqueous level of phenol was substantially reduced by phenol partitioning into the IL phase, allowing the cells to utilize the reduced phenol concentration. The partitioning of phenol from the IL to the aqueous phase was driven by cellular demand and thermodynamic equilibrium. All of the phenol was consumed at a rate comparable to that of previously used organic-aqueous TPPB systems, demonstrating the first successful use of an IL with a cell-based system. A quantitative ³¹P NMR spectroscopic assay for estimating the log P values of ILs is under development.     

Delivery of benzene to Alcaligenes xylosoxidans by solid polymers in a two-phase partitioning bioreactor

Toxic levels of benzene were decreased to sub-inhibitory levels in a bioreactor via absorption by polymer beads or cylinders (poly(ethylene-co-vinyl acetate) or poly(styrene-co-butadiene)). After inoculation with Alcaligenes xylosoxidans, the remaining benzene in the aqueous phase as well as the benzene taken up by the polymers was degraded to completion. The capacity of these polymers for benzene, and benzene diffusivity within the polymers were also determined.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Removal and destruction of high concentrations of gaseous toluene in a two-phase partitioning bioreactor by Alcaligenes xylosoxidans

A two-phase bioreactor consisting of hexadecane dispersed in an aqueous, cell-containing medium (organic fraction = 0.33) was used to trap toluene vapours from an air stream. The affinity for toluene by the solvent resulted in high efficiency of removal and transfer to the aqueous phase based on equilibrium transfer. The system was readily able to handle a loading capacity of 748 mg l^–1 h^–1 at a toluene degradation efficiency of greater than 98%.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Degradation of toluene/heptene mixtures in a trickling-bed bioreactor

Laws and regulations for the reduction of solvent emissions tend to force increasingly lower limits on gaseous emissions. Biological waste-gas treatment is an ecological and rather cheap method for solvent reduction, which is often used for waste-gas streams containing two or more components. The microbiological degradation of toluene and heptane as well as of several mixtures of these substances were investigated in a laboratory plant trickling-bed reactor. It was found that toluene was degraded at up to three times the rate for the heptane. Heptane elimination in the mixtures was low and the toluene elimination rate at low organic loads approached 100%. *** DIRECT SUPPORT *** AG903066 00010     

The roles of intermediates in biodegradation of benzene,toluene, and p-xylene by Pseudomonas putida F1

Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1. Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively. Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis. When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.     

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.     

Combination of the tod and the tol pathways in redesigning a metabolic route of Pseudomonas putida for the mineralization of a benzene, toluene, and p-xylene mixture.

Construction of a hybrid strain which is capable of mineralizing components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture. Consequently, a hybrid strain was constructed by cloning todC1C2BA genes encoding toluene dioxygenase on RSF1010 and introducing the resulting plasmid into P. putida mt-2. The hybrid strain of P. putida TB105 was found to mineralize a benzene, toluene, and p-xylene mixture without accumulation of any metabolic intermediate.     

Dynamic simulation of benzene vapor treatment by a two-phase partitioning bioscrubber: Part II: Model calibration,validation, and predictions

The two-phase partitioning bioscrubber (TPPB) model presented in Part I has been validated using experimental data under constant and transient operating conditions for the treatment of benzene waste gases by Achromobacter xylosoxidans Y234 with n-hexadecane as an immiscible, organic phase. Model calibration was performed to account for observed enhancements of benzene biodegradation rates in biphasic media, postulating that direct benzene uptake from dispersed organic droplets increased substrate bioavailability. This led to the use of an ‘effective dissolved substrate concentration’ to model cell specific growth rates. Model predictions were greatly improved using this empirical modification. The characteristics of the organic phase, both in terms of the volume fraction selected and its high equilibrium solubility, are found to be of critical importance for minimizing effluent gas VOC concentrations and stabilizing performance during transient operation. The biokinetic parameters μ_max and K_S are also particularly important, greatly influencing the response of the TPPB both during and while recovering from transient periods. K_S was found to be important for influencing steady-state benzene removal efficiencies under even invariant operation, leading to the conclusion that microorganisms which can maintain high rates of biological activity under very dilute substrate concentrations will make ideal biocatalysts in the TPPB.     

Enhancement of mass transfer characteristics and phenanthrene degradation in a two-phase partitioning bioreactor equipped with internal static mixers

Oxygen and substrate supply have always been considered physical constraints for the performance and operation of two-phase partitioning bioreactors (TPPB), widely used for the degradation of hydrophobic substrates. In this regard, the potential advantages of static mixers in upgrading the oxygen transfer and liquid-liquid dispersions in TPPB have been highlighted. In the present paper, the concomitant influence of static mixers on the gas-liquid mass transfer coefficient k _L a and on substrate bioavailability was examined in TPPB. The static method based on conventional forms was developed to estimate the oxygen volumetric mass transfer coefficient. Over a broad range of liquid and air flow rates, the presence of static mixers was found to significantly enhance k _L a relative to a mixer-free mode of operation. For identical conditions, static mixers improved the k _L a threefold. In the presence of external aeration supply, the boost in the k _L a was associated with an increase of 16% in the phenanthrene biodegradation rate due to bubble break up accomplished by the static mixers. On the other hand, static mixers were efficient in enhancing substrate bioavailability by improving the liquid-liquid interfacial area. This effect was reflected by a threefold increase in the degradation rate in the bioreactors with no external supply of air when equipped with static mixers.     

Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites

Benzene, toluene, and o-xylene (BTX) degradation by immobilized Pseudomonas putida F1 of postconsumer agave-fiber/polymer foamed-composites (AFPFC) and suspended cultures was studied under controlled conditions. Analyses using FTIR-ATR and SEM showed that P. putida F1 adhered onto the composite surface and developed a biofilm. In this sense, the AFPFC were successfully used as a support for bacterial immobilization. Both systems, immobilized and suspended cells of P. putida F1, were able to completely degrade benzene and toluene from initial concentrations of 15, 30, 60, and 90 mg l⁻¹. An inhibitory effect of the intermediary catechol from benzene degradation was observed in suspended cultures but it was not presented in the immobilized system. The degradation of o-xylene was partially accomplished in both systems. The Monod equation was used to model the experimental data obtained from the biodegradation kinetics, and they were adequately described with this model.     

Microbial degradation of high and low molecular weight polyaromatic hydrocarbons in a two-phase partitioning bioreactor by two strains of Sphingomonas sp.

A mixture of six polyaromatic hydrocarbons (naphthalene, phenanthrene, fluoranthene, pyrene, chyrysene and benzo[a]pyrene), varying in size from 2 to 5 rings, was dissolved in dodecane, and used as the delivery phase of a partitioning bioreactor. Two species of Sphingomonas were then used individually, and as a consortium, to determine which of the PAHs were degraded. Only low molecular weight PAHs (naphthalene, phenanthrene and fluoranthene) were degraded by the individual strains, but the consortium degraded all substrates either to completion or near completion.     

Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.