Andrew J. Daugulis

ENHANCED BIODEGRADATION OF PHENOL BY A MICROBIAL CONSORTIUM IN A SOLID-LIQUID TWO PHASE PARTITIONING BIOREACTOR

Two phase partitioning bioreactors (TPPBs) operate by partitioning toxic substrates to or from an aqueous, cell-containing phase by means of second immiscible phase. Uptake of toxic substrates by the second phase effectively reduces their concentration within the aqueous phase to sub-inhibitory levels, and transfer of molecules between the phases to maintain equilibrium results in the continual feeding of substrate based on the metabolic demand of the microorganisms. Conventionally, a single pure species of microorganism, and a pure organic solvent, have been used in TPPBs. The present work has demonstrated the benefits of using a mixed microbial population for the degradation of phenol in a TPPB that uses solid polymer beads (comprised of ethylene vinyl acetate, or EVA) as the second phase. Polymer modification via an increase in vinyl acetate concentration was also shown to increase phenol uptake. Microbial consortia were isolated from three biological sources and, based on an evaluation of their kinetic performance, a superior consortium was chosen that offered improved degradation when compared to a pure strain of Pseudomonas putida ATCC 11172. The new microbial consortium used within a TPPB was capable of degrading high concentrations of phenol (≈2000mgl−1), with decreased lag time (10h) and increased specific rate of phenol degradation (0.71g phenolg−1cellh). Investigation of the four-member consortium showed that it consisted of two Pseudomonas sp., and two Acinetobacter sp., and tests conducted upon the individual isolates, as well as paired organisms, confirmed the synergistic benefit of their existence within the consortium. The enhanced effects of the use of a microbial consortium now offer improved degradation of phenol, and open the possibility of the degradation of multiple toxic substrates via a polymer-mediated TPPB system.

A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.

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.

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

Abstract 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.

Bioproduction of the aroma compound 2-phenylethanol in a solid-liquid two-phase partitioning bioreactor system by Kluyveromyces marxianus.

The rose‐like aroma compound 2‐phenylethanol (2‐PE) is an important fragrance and flavor ingredient. Several yeast strains are able to convert l‐phenylalanine (l‐phe) to 2‐PE among which Kluyveromyces marxianus has shown promising results. The limitation of this process is the low product concentration and productivity primarily due to end product inhibition. This study explored the possibility and benefits of using a solid–liquid Two‐Phase Partition Bioreactor (TPPB) system as an in situ product removal technique. The system applies polymer beads as the sequestering immiscible phase to partition 2‐PE and reduce the aqueous 2‐PE concentration to non‐inhibitory levels. Among six polymers screened for extracting 2‐PE, Hytrel® 8206 performed best with a partition coefficient of 79. The desired product stored in the polymer was ultimately extracted using methanol. A 3 L working volume solid–liquid batch mode TPPB using 500 g Hytrel® as the sequestering phase generated a final overall 2‐PE concentration of 13.7 g/L, the highest reported in the current literature. This was based on a polymer phase concentration of 88.74 g/L and aqueous phase concentration of 1.2 g/L. Even better results were achieved via contact with more polymers (approximately 900 g) with the aqueous phase applying a semi‐continuous reactor configuration. In this system, a final 2‐PE concentration (overall) of 20.4 g/L was achieved with 1.4 g/L in the aqueous and 97 g/L in the polymer phase. The overall productivities of these two reactor systems were 0.38 and 0.43 g/L h, respectively. This is the first report in the literature of the use of a polymer sequestering phase to enhance the bioproduction of 2‐PE, and exceeds the performance of two‐liquid phase systems in terms of productivity as well as ease of operation (no emulsions) and ultimate product recovery. Biotechnol. Bioeng. 2009; 104: 332–339

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.

Evaluation of solvents for extractive butanol fermentation with Clostridium acetobutylicum and the use of poly(propylene glycol) 1200

SummaryIn an effort to improve the viability of acetone-butanol-ethanol fermentation by extractive fermentation, 63 organic solvents, including alkanes, alcohols, aldehydes, acids, and esters, were experimentally evaluated for biocompatibility with Clostridium acetobutylicum by observing gas evolution from cultures in contact with candidate solvents. Thirty-one of these solvents were further tested to determine their partition coefficient for butanol in fermentation medium. The biocompatible solvent with the highest partition coefficient for butanol (4.8), was poly(propylene glycol) 1200, which was selected for fermentation experiments. This is the highest partition coefficient reported to date for a biocompatible solvent. Extractive fermentations using concentrated feeds were observed to produce up to 58.6 g·l−1 acetone and butanol in 202 h, the equivalent of three control fermentations in a single run. Product yields (based on total solvent products and glucose consumed) of 0.234 g·g−1 to 0.311 g·g−1 and within run solvent productivities of 0.174 g·l−1·h−1 to 0.290 g·l−1·h−1 were consistentwith conventional fermentations reported in the literature. The extended run-time of the fermentation resulted in an overall improvement in productivity by reducing the fraction of between-run down-time for fermentor cleaning and sterilization.

Ethanol production using Zymomonas mobilis immobilized on an ion exchange resin

SummaryTen ion exchange resins as well as activated carbon and ceramic chips were examined for their ability to adsorb cells of Zymomonas mobilis. A cationic macroreticular resin was shown to be the most efficient adsorbant and was used to immobilize cells of Z. mobilis in a column bioreactor. The bioreactor was operated with a feed glucose concentration of 100 g/L at a dilution rate of 11.2 h-1 and a productivity based on void volume of 377 g ethanol/L-h was obtained with 80% substrate utilization. It was observed that the cell concentration in the bioreactor increased during continuous operation and that the form of Z. mobilis changed from single cells to filamentous forms of the bacterium. Plugging problems occurred after 200 h of operation as a result of excessive filamentous cell growth.

Development of a novel bioreactor system for treatment of gaseous benzene

A novel, continuous bioreactor system combining a bubble column (absorption section) and a two-phase bioreactor (degradation section) has been designed to treat a gas stream containing benzene. The bubble column contained hexadecane as an absorbent for benzene, and was systemically chosen considering physical, biological, environmental, operational, and economic factors. This solvent has infinite solubility for benzene and very low volatility. After absorbing benzene in the bubble column, the hexadecane served as the organic phase of the two-phase partitioning bioreactor, transferring benzene into the aqueous phase where it was degraded by Alcaligenes xylosoxidans Y234. The hexadecane was then continuously recirculated back to the absorber section for the removal of additional benzene. All mass transfer and biodegradation characteristics in this system were investigated prior to operation of the integrated unit, and these included: the mass transfer rate of benzene in the absorption column; the mass transfer rate of benzene from the organic phase into the aqueous phase in the two-phase bioreactor; the stripping rate of benzene out of the two-phase bioreactor, etc. All of these parameters were incorporated into model equations, which were used to investigate the effects of operating conditions on the performance of the system. Finally, two experiments were conducted to show the feasibility of this system. Based on an aqueous bioreactor volume of 1 L, when the inlet gas flow and gaseous benzene concentration were 120 L/h and 4.2 mg/L, respectively, the benzene removal efficiency was 75% at steady state. This process is believed to be very practical for the treatment of high concentrations of gaseous pollutants, and represents an alternative to the use of biofilters.

Recent advances in two-phase partitioning bioreactors for the treatment of volatile organic compounds.

Biological processes are considered to be the most cost-effective technology for the off-gas treatment of volatile organic compounds (VOC) at low concentrations. Two-phase partitioning bioreactors (TPPBs) emerged in the early 1990s as innovative multiphase systems capable of overcoming some of the key limitations of traditional biological technologies such as the low mass transfer rates of hydrophobic VOCs and microbial inhibition at high VOC loading rates. Intensive research carried out in the last 5 years has helped to provide a better understanding of the mass transfer phenomena and VOC uptake mechanisms in TPPBs, which has significantly improved the VOC biodegradation processes utilizing this technology platform. This work presents an updated state-of-the-art review on the advances of TPPB technology for air pollution control. The most recent insights regarding non-aqueous phase (NAP) selection, microbiology, reactor design, mathematical modeling and case studies are critically reviewed and discussed. Finally, the key research issues required to move towards the development of efficient and stable full-scale VOC biodegradation processes in TPPBs are identified.