Application of solid–liquid TPPBs to the production of L‐phenylacetylcarbinol from benzaldehyde using Candida utilis

The biotransformation of benzaldehyde and glucose to L ‐phenylacetylcarbinol (PAC) using Candida utilis was demonstrated in a solid–liquid two‐phase partitioning bioreactor (TPPB) with the aim of reducing substrate, product, and by‐product toxicity via sequestration. Previous work in the field had used octanol as the sequestering phase of liquid–liquid TPPBs but was limited by the toxic effects of octanol on C. utilis. To improve solvent selection in any future studies, the critical log P of C. utilis was determined in the current study to be 4.8 and can be used to predict biocompatible solvents. Bioavailability tests showed alkanes and alkenes to be non‐bioavailable. As polymers are biocompatible and non‐bioavailable, a wide range of commercially available polymers was screened and it was demonstrated that polymer softness plays a key role in absorptive capability. The polymer Hytrel G3548L was selected as the second phase to sequester benzaldehyde, PAC, and benzyl alcohol, with partition coefficients of 35, 7.5, and 10, respectively. With a 9% by volume partitioning phase, 13.6 g/L biomass of C. utilis achieved an overall PAC concentration of 11 g/L, a 1.9‐fold improvement over the single‐phase case. Benzyl alcohol concentration was 4.5 g/L, a 1.6‐fold reduction. The volumetric productivity was 0.85 g/L h, a 1.2‐fold improvement over the single‐phase system. These results demonstrate a promising starting point for solid–liquid TPPBs for PAC production. Biotechnol. Bioeng. 2010;107:633–641. © 2010 Wiley Periodicals, Inc.     

A novel solid-liquid two-phase partitioning bioreactor for the enhanced bioproduction of 3-methylcatechol

The bioproduction of 3-methylcatechol from toluene via Pseudomonas putida MC2 was performed in a solid-liquid two-phase partitioning bioreactor with the intent of increasing yield and productivity over a single-phase system. The solid phase consisted of HYTREL, a thermoplastic polymer that was shown to possess superior affinity for the inhibitory 3-methylcatechol compared to other candidate polymers as well as a number of immiscible organic solvents. Operation of a solid-liquid biotransformation utilizing a 10% (w/w) solid (polymer beads) to liquid phase ratio resulted in the bioproduction of 3-methylcatechol at a rate of 350 mg/L-h, which compares favorably to the single phase productivity of 128 mg/L-h. . HYTREL polymer beads were also reconstituted into polymer sheets, which were placed around the interior circumference of the bioreactor and successfully removed 3-methylcatechol from solution resulting in a rate of 3-methylcatechol production of 343 mg/L-h. Finally, a continuous biotransformation was performed in which culture medium was circulated upwards through an external extraction column containing HYTREL beads. The design maintained sub lethal concentrations of 3-methylcatechol within the bioreactor by absorbing produced 3-methylcatechol into the polymer beads. As 3-methylcatechol concentrations in the aqueous phase approached 500 mg/L the extraction column was replaced (twice) with a fresh column and the process was continued representing a simple and effective approach for the continuous bioproduction of 3-methylcatechol. Recovery of 3-methylcatechol from HYTREL was also achieved by bead desorption into methanol.     

Transformation of ferulic acid to vanillin using a fed‐batch solid–liquid two‐phase partitioning bioreactor

Amycolatopsis sp. ATCC 39116 (formerly Streptomyces setonii) has shown promising results in converting ferulic acid (trans‐4‐hydroxy‐3‐methoxycinnamic acid; substrate), which can be derived from natural plant wastes, to vanillin (4‐hydroxy‐3‐methoxybenzaldehyde). After exploring the influence of adding vanillin at different times during the growth cycle on cell growth and transformation performance of this strain and demonstrating the inhibitory effect of vanillin, a solid–liquid two‐phase partitioning bioreactor (TPPB) system was used as an in situ product removal technique to enhance transformation productivity by this strain. The thermoplastic polymer Hytrel^® G4078W was found to have superior partitioning capacity for vanillin with a partition coefficient of 12 and a low affinity for the substrate. A 3‐L working volume solid–liquid fed‐batch TPPB mode, using 300 g Hytrel G4078W as the sequestering phase, produced a final vanillin concentration of 19.5 g/L. The overall productivity of this reactor system was 450 mg/L. h, among the highest reported in literature. Vanillin was easily and quantitatively recovered from the polymers mostly by single stage extraction into methanol or other organic solvents used in food industry, simultaneously regenerating polymer beads for reuse. A polymer–liquid two phase bioreactor was again confirmed to easily outperform single phase systems that feature inhibitory or easily further degraded substrates/products. This enhancement strategy might reasonably be expected in the production of other flavor and fragrance compounds obtained by biotransformations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:207–214, 2014     

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

With nearly three decades of development, Pichia pastoris (P. pastoris) has become a powerful eukaryotic protein expression system for the expression of thousands of proteins both on a laboratory and industrial scale. In addition, it has also been extensively used as a cell factory for the production of a variety of chemicals. This review summarizes the bottlenecks and solutions in achieving high‐level secretory protein expression with P. pastoris and then outlines its applications on chemical production with an emphasis on its role as whole‐cell biocatalyst. Furthermore, current challenges and future directions of this important system are also discussed.     

Solvent selection for enhanced bioproduction of 3-methylcatechol in a two-phase partitioning bioreactor

The biotransformation of toluene to 3-methycatechol (3MC) via Pseudomonas putida MC2 was used as a model system for the development of a biphasic process offering enhanced overall volumetric productivity. Three factors were investigated for the identification of an appropriate organic solvent and they included solvent toxicity, bioavailability of the solvent as well as solvent affinity for 3MC. The critical log P (log P(crit)) of the biocatalyst was found to be 3.1 and log P values were used to predict a solvent's toxicity. The presence of various functional groups of candidate solvents were used to predict the absorption of 3MC and it was found that solvents possessing polarity showed an affinity towards 3MC. Bis (2-ethylhexyl) sebecate was selected for use in the biphasic system as it fulfilled all selection criteria. A two-phase biotransformation with BES and a 50% phase volume ratio, achieved an overall volumetric productivity of 440 mg 3MC/L-h, which was an improvement by a factor of approximately 4 over previously operated systems. Additional work focused on reducing the toluene feed in order to minimize possible toxicity and decrease loss of substrate (toluene), a result of volatilization. Toluene losses were reduced by a factor of 4, compared to previously operated systems, without suffering an appreciable loss in overall volumetric productivity.     

Enhanced degradation of phenanthrene in a solid–liquid two‐phase partitioning bioreactor via sonication

The current article examined the feasibility of inducing improved delivery and degradation of phenanthrene in a solid–liquid partitioning bioreactor system at bench scale by means of ultrasonic energy input. Initial degradation rates of phenanthrene by a microbial consortium, delivered from Desmopan, were improved 2.7‐fold in the presence of sonication relative to unsonicated controls. Results demonstrated that an operating window involving on/off sonication cycling improved substrate delivery and rational selection of ultrasound cycling profiles could lead to even further enhancements. Additionally, all results were obtained in a conventional bioreactor with commercial ultrasonic equipment and a commercially available polymer. Subsequent DGGE analysis demonstrated that the sonication cycles selected maintained consortium compositions, relative to control cases, and suggest that exposure would not reduce degradative capabilities under the periods of irradiation examined. Finally, consortium members were identified as belonging to the Pandoraea, Sphingobium, and Pseudoxanthomonas genera. Comparison of genetic sequences in the Ribosomal Database Project revealed that some of the bacterial members, identified at the strain level, had been previously observed in PAH degradations, while others have been reported only in the degradation of other aromatics, such as pesticides. Biotechnol. Bioeng. 2010;105: 997–1001. © 2009 Wiley Periodicals, Inc.     

A two‐phase partitioning airlift bioreactor for the treatment of BTEX contaminated gases

This investigation characterizes a novel 11 L airlift two‐phase partitioning bioreactor (TPPB) for the treatment of gases contaminated with a mixture of benzene, toluene, ethylbenzene, and o‐xylene (BTEX). The application of the TPPB technology in an airlift bioreactor configuration provides a novel technology that reduces energy intensity relative to traditional stirred tank TPPB configurations. The addition of a solid second phase of silicone rubber beads (10%, v/v) or of a liquid second phase of silicone oil (10%, v/v) resulted in enhanced performance of the airlift bioreactor relative to the single phase case, with 20% more BTEX being removed from the gas phase during an imposed transient loading. During a 4 h loading step change of three times the nominal loading (60 g m^?3 h^?1), overall removal efficiencies for the airlift TPPBs containing a liquid or solid phase remained above 75%, whereas the single phase airlift had an overall removal efficiency of 47.1%. The airlift TPPB containing a silicone rubber second phase was further characterized by testing performance during steady‐state operation over a range of loadings and inlet gas flow rates in the form of a 3² factorial experimental design. Optimal operating conditions that avoid oxygen limitations and that still have a slow enough gas flow rate for sufficient BTEX transfer from the gas phase to the working volume are identified. The novel solid–liquid airlift TPPB reduces energy inputs relative to stirred tank designs while being able to eliminate large amounts of BTEX during both steady‐state and fluctuating loading conditions. Biotechnol. Bioeng. 2009;103: 1077–1086. © 2009 Wiley Periodicals, Inc.     

Enhanced bioproduction of carvone in a two-liquid-phase partitioning bioreactor with a highly hydrophobic biocatalyst

The microbial biotransformation of (-)-trans-carveol to the flavor and fragrance compound (R)-(-)-carvone by Rhodococcus erythropolis DCL14 was carried out in a 3 L two phase partitioning bioreactor with an immiscible liquid second phase in an effort to improve upon the reactor performance achieved in a single aqueous phase system. The purpose of employing the liquid second phase is to minimize biotransformation rate inhibition due to the accumulation of the toxic substrate (cis-carveol) and product (carvone) in the aqueous phase. 1-Dodecene was chosen as the solvent for this application because it is biocompatible, non-biodegradable and has a superior affinity for the target product (carvone) relative to the other solvents tested. However, when 1-dodecene was used in the biotransformation, the extremely hydrophobic R. erythropolis DCL14 created an emulsion with the organic solvent with significant sequestering of the cells into the organic phase and negligible substrate conversion. To overcome these operational difficulties, silicone oil, which is considered a liquid polymer, was used with the aim of preventing emulsification and sequestration of cells in the non-aqueous phase. Although some emulsification of the water-silicone oil was again created by the cells, operability was improved and, in fed-batch mode, the system was able to convert approximately 2(1/2) times more carveol than a benchmark single aqueous phase system before substrate/product toxicity caused the biotransformation to stop. This study has demonstrated enhancement of a microbial biotransformation for the production of a high value nutraceutical compound via the use of a second partitioning phase, along with operational challenges arising from the use of a highly hydrophobic organism in such systems.     

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 © 2009 Wiley Periodicals, Inc.     

Degradation of xenobiotics in a partitioning bioreactor in which the partitioning phase is a polymer

Two-phase partitioning bioreactors (TPPBs) are characterized by a cell-containing aqueous phase and a second immiscible phase that contains toxic and/or hydrophobic substrates that partition to the cells at subinhibitory levels in response to the metabolic demand of the organisms. To date, the delivery phase in TPPBs has been a hydrophobic solvent that traditionally needed to possess a variety of important properties including biocompatibility, nonbioavailability, low volatility, and low cost, among others. In the present work we have shown that the organic solvent phase can be replaced by inexpensive polymer beads that function in a similar fashion as organic solvents, delivering a toxic substrate to cells based on equilibrium considerations. Specifically, 3.4 mm diameter beads of poly(ethylene-co-vinyl acetate) (EVA) were used to reduce the aqueous concentration of phenol in a bioreactor from toxic levels ( approximately 2,000 mg/L) to subinhibitory levels ( approximately 750 mg/L), after which Pseudomonas putida ATCC 11172 was added to the system and allowed to consume the total phenol loading. Thus, the beads absorbed the toxic substrate and released it to the cells on demand. The EVA beads, which could be reused, were able to absorb 14 mg phenol/g EVA. This work has opened the possibility of using widely mixed cultures in TPPB systems without concern for degradation of the delivery material and without concern of contamination.     

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

Transketolase is a proven biocatalytic tool for asymmetric carbon‐carbon bond formation, both as a purified enzyme and within bacterial whole‐cell biocatalysts. The performance of Pichia pastoris as a host for transketolase whole‐cell biocatalysis was investigated using a transketolase‐overexpressing strain to catalyze formation of l ‐erythrulose from β‐hydroxypyruvic acid and glycolaldehyde substrates. Pichia pastoris transketolase coding sequence from the locus PAS_chr1‐4_0150 was subcloned downstream of the methanol‐inducible AOX1 promoter in a plasmid for transformation of strain GS115, generating strain TK150. Whole and disrupted TK150 cells from shake flasks achieved 62% and 65% conversion, respectively, under optimal pH and methanol induction conditions. In a 300 μL reaction, TK150 samples from a 1L fed‐batch fermentation achieved a maximum l ‐erythrulose space time yield (STY) of 46.58 g L^?1 h^?1, specific activity of 155 U , product yield on substrate (Y_p/s) of 0.52 mol mol^?1 and product yield on catalyst (Y_p/x) of 2.23g . We have successfully exploited the rapid growth and high biomass characteristics of Pichia pastoris in whole cell biocatalysis. At high cell density, the engineered TK150 Pichia pastoris strain tolerated high concentrations of substrate and product to achieve high STY of the chiral sugar l ‐erythrulose. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:99–106, 2018     

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two‐phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt, and ionic liquid (IL)–salt). The systems composed of PEG 3350‐potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1‐fold purification) and t‐butanol‐20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8‐fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG‐salt ATPS for the potential recovery of SOD. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1326–1334, 2014     

A strategic approach for the design and operation of two‐phase partitioning bioscrubbers for the treatment of volatile organic compounds

A strategic approach for the design of two‐phase partitioning bioscrubbers (TPPBs) has been formulated using, as a basis, a re‐evaluation of extensive literature data available for the degradation of benzene by Achromobacter xylosoxidans Y234 in TPPBs with n‐hexadecane as the partitioning phase. Using a previously determined maintenance coefficient for benzene, we determined that an inlet benzene loading rate of 100 mg/h requires 5,928 mg cell mass at biological steady state and 243.0 mg O₂/h. The total oxygen‐transfer rates (TOTRs) into the TPPB increased by 83.5% with 33.3% of organic phase compared with a single aqueous phase and were significantly influenced by gas flow rate, whereas agitation has a minor affect. The fraction of organic phase used was suggested to be the primary parameter with which the TOTR into the TPPB may be altered. Although the presence of an organic solvent in the TPPB remarkably increased the TOTR, the total benzene transfer rate into the TPPB remained largely insensitive due to the intrinsic low Henry's law constant (or relatively high solubility) of benzene in water. Finally, we have integrated the elements of this analysis into a set of heuristic criteria that can serve as a guideline for the design of TPPB systems for future volatile organic compound treatment applications. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of bubble–liquid two‐phase turbulent hydrodynamics on cell damage in sparged bioreactor

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas–liquid bioreactors have not been performed to date. In this work, a second‐order moment (SOM) bubble–liquid two‐phase turbulent model based on the two‐fluid continuum approach is used to investigate the gas–liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble–liquid two‐phase fluctuation velocities correlation transport equations, the anisotropy of two‐phase stresses and the bubble–liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress‐induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:48–58, 2014     

Enhancement of a two-phase partitioning bioreactor system by modification of the microbial catalyst: demonstration of concept

Application of two-phase partitioning bioreactors (TPPB) to the degradation of phenol and xenobiotics has been limited by the fact that many organic compounds that would otherwise be desirable delivery solvents can be utilized by the microorganisms employed. The ability to metabolize the solvent itself could interfere with xenobiotic degradation, limiting remediation efficiency, and hence represents a microbial characteristic incompatible with process goals. To avoid the issue of bioavailability, previous TPPB applications have relied on complex and often expensive delivery solvents or suboptimal catalyst-solvent pairings. In an effort to enhance TPPB activity and applicability, a genetically engineered derivative of Pseudomonas putida ATCC 11172 mutated in its ability to utilize medium-chain-length alcohols was generated (AVP2) and applied as the catalyst within a TPPB system with decanol as the delivery solvent. Kinetic analysis verified that the genetic alteration had not negatively affected phenol degradation. The volumetric productivity of AVP2 (0.48 g/L x h(-1)) was equivalent to that seen for wild-type ATCC 11172 (0.51 g/L x h(-1)), but a comparison of initial cell concentrations and yields revealed an improved phenol-degrading efficiency for the mutant under process conditions. Yield coefficients, cell dry weight, and viable count determinations all confirmed the stability of the modified phenotype. This work illustrates the possibilities for TPPB process enhancement through a careful combination of genetic modification and solvent selection.     

Characterization and high‐yield production of non‐N‐glycosylated recombinant human BCMA‐Fc in Pichia pastoris

B‐cell maturation antigen (BCMA) fused at the C‐terminus to the Fc portion of human IgG1 (BCMA‐Fc) blocks B‐cell activating factor (BAFF) and proliferation‐inducing ligand (APRIL)‐mediated B‐cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA‐Fc (rhBCMA‐Fc) in Pichia pastoris under the control of the AOX1 methanol‐inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N‐linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed‐batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA‐Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA‐Fc possessed high‐binding affinity to APRIL and dose‐dependent inhibition of APRIL‐induced proliferative activity in vitro through three‐step purification. Thus, this yeast‐derived expression method could be a low‐cost and effective alternative to the production of rhBCMA‐Fc in mammalian cell lines.