Phase Bioreactor Research Articles

Nano-injection molding with resin mold inserts for prototyping of nanofluidic devices for single molecular detection.

While injection molding is becoming the fabrication modality of choice for high-scale production of microfluidic devices, especially those used for in vitro diagnostics, its translation into the growing area of nanofluidics (structures with at least one dimension <100 nm) has not been well established. Another prevailing issue with injection molding is the high startup costs and the relatively long time between device iterations making it in many cases impractical for device prototyping. We report, for the first time, functional nanofluidic devices with dimensions of critical structures below 30 nm fabricated by injection molding for the manipulation, identification, and detection of single molecules. UV-resin molds replicated from Si masters served as mold inserts, negating the need for generating Ni-mold inserts via electroplating. Using assembled devices with a cover plate via hybrid thermal fusion bonding, we demonstrated two functional thermoplastic nanofluidic devices. The first device consisted of dual in-plane nanopores placed at either end of a nanochannel and was used to detect and identify single ribonucleotide monophosphate molecules via resistive pulse sensing and obtain the effective mobility of the molecule through nanoscale electrophoresis to allow its identification. The second device demonstrated selective binding of a single RNA molecule to a solid phase bioreactor decorated with a processive exoribonuclease, XRN1. Our results provide a simple path towards the use of injection molding for device prototyping in the development stage of any nanofluidic or even microfluidic application, through which rapid scale-up is made possible by transitioning from prototyping to high throughput production using conventional Ni mold inserts.

Continuous two phase partitioning bioreactor (C-TPPB) for removal of xenobiotics: Design criteria

This paper proposes a new continuous two phase bioreactor (C-TPPB) configuration for the removal and degradation of xenobiotic compounds from wastewaters. The reactor is made of three principal parts: a tube side in which the wastewater flows; a polymeric membrane which constitutes the tube walls; a shell side, set as a continuous stirred tank reactor (CSTR), in which the xenobiotics are degraded by means of a bacterial population. A mathematical model for the system description was developed and validated by means of fitting using literature experimental data obtained from a similar configuration. Since the nature of the fitting parameters did not depend upon the particular set-up, such validation procedure was possible. A dimensionless analysis was also carried out by defining a modified Stenton (St’) and Damköhler (Da’) number in order to detect the C-TTPB design criteria. The removal of 4-NtiroPhenol was reported as a case study and an algorithm for a preliminary design procedure was developed.

Slurry-Phase Bioremediation of Ogoni Land Crude Oil Contaminated Soil

The contamination of soil originated from evolutional activities such as transportation and spillage, as in the case of Ogoni land in the Niger Delta area of Nigeria, and it is a well-known environmental problem in Nigeria. This study examined the capacity of two indigenous bacteria, Pseudomonas aeruginosa, and Bacillus subtilis to remediate the hydrocarbon contaminated soil. The focus of the work was to isolate, identify, and determine the extent of bioremediation achieved in the soil contaminated with crude oil in Ogoni land, which was used as a case study for the slurry-phase bioremediation process. These organisms earlier mentioned were identified based on their morphological and biochemical characteristics. The bacteria species were considered separately and in combination in a Laboratory-scale slurry-phase in a 14-day treatment period using 30 g of soil for each case. The performance of each batch bioreactor was compared to the sterile control. The slurry phase bio-reactor, RA, treated with Pseudomonas sp., had an actual degradation of 31%, the slurry phase bio-reactor, RB, treated with Bacillus sp., had a real degradation of 35%. The slurry phase bio-reactor Rc, which contain both microorganisms had an actual degradation of 36%, the slurry phase bio-reactor RD had a real degradation of 0%. The results obtained confirm that the isolated bacterial from the soil can effectively utilize contaminating crude oil as a source of carbon and energy. The decrease of hydrocarbon in the sterile control was used as a basis for comparison of the petroleum hydrocarbons lost through the extraction process as microbiological activity was not possible. Results from the study have shown that P. aeruginosa and B. subtilis strains were effective for the slurry-phase bioremediation of hydrocarbon contaminated soil.

Simultaneous amylase production, raw cassava starch hydrolysis and ethanol production by immobilized Aspergillus awamori and Saccharomyces cerevisiae in a novel alternating liquid phase–air phase system

As a solution to the problems of mass transfer limitation in submerged cultures and scale up of solid-state/liquid-surface cultures, an alternating liquid phase–air phase bioreactor was developed. It consisted of a bioreactor equipped with a siphon system and a reservoir. Aspergillus awamori was immobilized in loofa sponge inside the bioreactor and culture broth was pumped from the reservoir into the bioreactor. Each time the culture broth level reached a critical level, the broth automatically siphoned back into the reservoir. Thus the immobilized cells were alternatingly submerged and exposed to air. The duration of each phase was controlled by the pumping rate and with an on-off timer. During amylase production from soluble starch and raw cassava starch, the optima ratios of the liquid to air phases were 12 h : 12 h and 3 h : 6 h respectively. Saccharomyces cerevisiae IR2 was immobilized in the reservoir and the system was used for simultaneous amylase production, hydrolysis and ethanol production from raw cassava starch. The process was very stable for more than 7 batches with high ethanol yield of 0.46 g-ethanol/g-starch and productivity of 1.73 g-ethanol/L/h. These values are high, the system can be scaled up, and thus it has many potential applications.

Constitutively solvent-tolerant Pseudomonas taiwanensis VLB120∆ C∆ ttgV supports particularly high-styrene epoxidation activities when grown under glucose excess conditions.

Solvent-tolerant bacteria represent an interesting option to deal with the substrate and product toxicity in bioprocesses. Recently, constitutive solvent tolerance was achieved for Pseudomonas taiwanensis VLB120 via knockout of the regulator TtgV, making tedious adaptation unnecessary. Remarkably, ttgV knockout increased styrene epoxidation activities of P. taiwanensis VLB120Δ C. With the aim to characterize and exploit the biocatalytic potential of P. taiwanensis VLB120Δ C and VLB120Δ CΔ ttgV, we investigated and correlated growth physiology, native styrene monooxygenase (StyAB) gene expression, whole-cell bioconversion kinetics, and epoxidation performance. Substrate inhibition kinetics was identified but was attenuated in two-liquid phase bioreactor setups. StyA fusion to the enhanced green fluorescent protein enabled precise enzyme level monitoring without affecting epoxidation activity. Glucose limitation compromised styAB expression and specific activities (30-40 U/g CDW for both strains), whereas unlimited batch cultivation enabled specific activities up to 180 U/g CDW for VLB120Δ CΔ ttgV strains, which is unrivaled for bioreactor-based whole-cell oxygenase biocatalysis. These extraordinarily high specific activities of constitutively solvent-tolerant P. taiwanensis VLB120∆ C∆ ttgV could be attributed to its high metabolic capacity, which also enabled high expression levels. This, together with the high product yields on glucose and biomass obtained qualifies the VLB120∆ ttgV strain as a highly attractive tool for the development of ecoefficient oxyfunctionalization processes and redox biocatalysis in general.

Bioremediation of chlorpyrifos contaminated soil by two phase bioslurry reactor: Processes evaluation and optimization by Taguchi's design of experimental (DOE) methodology

Two phase bioreactor was constructed, designed and developed to evaluate the chlorpyrifos remediation. Six biotic and abiotic factors (substrate-loading rate, slurry phase pH, slurry phase dissolved oxygen (DO), soil water ratio, temperature and soil micro flora load) were evaluated by design of experimental (DOE) methodology employing Taguchi's orthogonal array (OA). The selected six factors were considered at two levels L-8 array (2^7, 15 experiments) in the experimental design. The optimum operating conditions obtained from the methodology showed enhanced chlorpyrifos degradation from 283.86µg/g to 955.364µg/g by overall 70.34% of enhancement. In the present study, with the help of few well defined experimental parameters a mathematical model was constructed to understand the complex bioremediation process and optimize the approximate parameters upto great accuracy.

The influence of nickel on the bioremediation of multi-component contaminated tropical soil: microcosm and batch bioreactor studies.

Large petrochemical discharges are responsible for organic and inorganic pollutants in the environment. The purpose of this study was to evaluate the influence of nickel, one of the most abundant inorganic element in crude oil and the main component of hydrogen catalysts for oil refining, on the microbial community structure in artificially petroleum-contaminated microcosms and in solid phase bioreactor studies. In the presence of metals, the oil biodegradation in microcosms was significantly delayed during the first 7 days of operation. Also, increasing amounts of moisture generated a positive influence on the biodegradation processes. The oil concentration, exhibiting the most negative influence at the end of the treatment period. Molecular fingerprinting analyses (denaturing gradient gel electrophoresis--DGGE) indicated that the inclusion of nickel into the contaminated soil promoted direct changes to the microbial community structure. By the end of the experiments, the results of the total petroleum hydrocarbons removal in the bioreactor and the microcosm were similar, but reductions in the treatment times were observed with the bioreactor experiments. An analysis of the microbial community structure by DGGE using various markers showed distinct behaviors between two treatments containing high nickel concentrations. The main conclusion of this study was that Nickel promotes a significant delay in oil biodegradation, despite having only a minor effect over the microbial community.

Whole cell three phase bioreactors allow for effective production of fatty acid alkyl esters derived from microalgae lipids

Lipids of the microalgae Botryococcus braunii and Nannochloropsis gaditana were used in a novel whole cell three-phase bioreactor (WCTB) using Rhizopus oryzae as biocatalyst for biodiesel (fatty acid alkyl esters, FAAE) production. The effects varying running conditions (the microalgae lipid, type of acyl acceptor, temperature, nutrient availability, alcohol to lipid ratio, and agar surface area exposed to gas-phase alcohol of varying convective fluxes) on the FAAE yield were studied and optimal conditions for the reactor identified. We found that the complexity of carbon source used (lipids) was related to the efficiency of the process, since it is used for both, growth and FAAE production, hence N. gaditana lipids were a more suitable raw material for FAAE production compared to B. braunii lipids due their higher content of fatty acid. The optimal operational conditions for our work were 20°C and 8:1 alcohol to lipid volumetric ratio, where the best results was obtained using N. gaditana lipids as raw material and ethanol as acyl acceptor, reaching a FAAE yield of 92%. The results of our work indicate that both mass transfer process and the availability of nutrients are the most important variables in the performance of WCTB, where the hyphae have an important role in mass transfer since they may acts as ‘pipeline’, connecting different phases with the different components of the three phase bioreactor. These results should be considered as key design factors for developing the three phase bioreactor configuration.

Reduced pressure gas phase bioreactor as a tool for stereoselective reduction catalyzed by alcohol dehydrogenase from Parvibaculum lavamentivorans

This study presents an immobilized alcohol dehydrogenase reaction in the gas phase under reduced pressure. The catalytic ability (activity, stereoselectivity and productivity) of alcohol dehydrogenase from Parvibaculum lavamentivorans was investigated as a function of various factors for process design. The reduction of 4-methyl-2-pentanone to (R)-4-methyl-2-pentanol with concomitant regeneration of cofactor using 2-propanol as cosubstrate was employed as a model reaction. Under reduced pressure the catalytic ability of the immobilized enzyme was efficiently exhibited, similar to that under standard pressure. On the other hand, the dependence of the water and substrate activities as well as the reaction temperature on the catalytic ability of the immobilized enzyme remained almost the same, compared to the standard pressure. Based on the kinetic parameters (maximum reaction rate and Michaelis constant) estimated, the enzyme activity was found to be effectively maintained under reduced pressure. These findings indicate that the reduced pressure gas phase reactor is capable of proceeding enzyme reaction for chiral compound production. However, as the future scope, the reaction system which proceeds with the catalytic ability of enzyme enhanced more in the reduced pressure condition needs to be widely investigated.

Substrate-Dependent rRNA Production in an Airborne Bacterium

Bacteria become aerosolized by a variety of processes and remain in the atmosphere for days. Many studies have characterized bacteria in air, but little is known regarding whether airborne bacteria are active. Here, we show that a bacterial strain previously isolated from air, Sphingomonas aerolata NW12, exhibited metabolic activity while in the airborne state. Airborne cells incubated in rotating gas phase bioreactors in the presence of known growth substrates acetic acid and ethanol in the gaseous phase had higher rRNA to rRNA gene content than airborne cells without growth substrates. This demonstrates that this species of airborne bacteria has a differential metabolic response to gaseous substrates in the aerial environment with an increasing rRNA content, suggesting that the cells have the potential to produce proteins while aloft.

Effects of superficial gas velocity on process dynamics in bioreactors

Present work analyzes the flow hydrodynamics and mass transfer mechanisms in double Rushton and CD-6 impeller on wide range (0.0075–0.25 m/s) of superficial gas velocity (vg) in a gas-liquid phase bioreactor by employing computational fluid dynamics (CFD) technique. The volume averaged velocity magnitude and dissipation rate are found higher with increasing superficial gas velocity. Higher relative power draw (Pg/P0) is predicted in CD-6 than the Rushton impeller but no significant difference in volume averaged mass transfer coefficient (kLa) observed between these two types of impeller. The ratio of power draw with mass transfer coefficient has been found higher in CD-6 impeller (25–50 %) than the Rushton impeller.

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.

Reactivity of Immobilized Alcohol Dehydrogenase Containing Polyvinyl Alcohol in a Gas Phase Bioreactor

Reactivity of Immobilized Alcohol Dehydrogenase Containing Polyvinyl Alcohol in a Gas Phase Bioreactor

Hexane biodegradation in two-liquid phase bioreactors: High-performance operation based on the use of hydrophobic biomass

An innovative operation mode in two-liquid phase bioreactors (TLPB) for the treatment of volatile organic compounds (VOC) was investigated. This mode was based on confining the biocatalytic activity exclusively in the non-aqueous phase (NAP) by using hydrophobic microorganisms. The TLPB was implemented in a 2.5L stirred tank reactor using 10% (v/v) of silicone oil as NAP and hexane as model VOC. A stable elimination capacity (EC) of 21.0±2.5gm−3h−1 (corresponding to a removal efficiency of 80%) was recorded for 26 days. The accumulation of inhibitory metabolites resulted in drastic drops in the elimination capacity (EC) and an unstable performance of the system, hexanol being identified as potential inhibitory metabolite. Aqueous culture broth exchange by fresh mineral salt medium at a dilution rate of 0.2day−1 allowed maintaining a high and sustained VOC removal performance. Dissolved oxygen concentration measurements revealed that the oxidative metabolism was strongly stimulated by the aqueous broth exchange. The temporary blockage of the gas/water/NAP transfer pathway for O2 highlighted the paramount role of this pathway on the performance of the TLPB based on hydrophobic microorganisms.

Enhanced catalytic performance of immobilized Parvibaculum lavamentivorans alcohol dehydrogenase in a gas phase bioreactor using glycerol as an additive

The efficacy of glycerol as an additive at enzyme immobilization on the catalytic performance (activity, stability, enantioselectivity and productivity) of immobilized (R)-specific Parvibaculum lavamentivorans alcohol dehydrogenase in a continuous gas phase reactor was investigated. The enantioselective reduction of 4-methyl-2-pentanone with concomitant NADH regeneration by 2-propanol as co-substrate was used as a model reaction. Non-porous glass beads were used as immobilization carriers. The immobilized alcohol dehydrogenase performance was markedly enhanced by the addition of glycerol. Interestingly, the enantioselectivity was stabilized during the reaction progress. The optimal glycerol amount, thermodynamic activity of water in the feed gas phase, and reaction temperature were obtained at 10mg/g-glass carriers, 0.8 and 313K in terms of the productivity, respectively. As a function of the thermodynamic activity of substrate in the feed gas phase, it was also optimized at 0.103 for 2-propanol and 0.250 for 4-methyl-2-pentanone. Compared to the glycerol-free immobilized enzyme system, the productivity of the chiral compound was ca. 15-fold higher, on the other hand, based on the kinetic analysis the maximum reaction rate was found to be about 2-times higher, while the Michaelis constant for 2-propanol was about 2-fold lower and that for 4-methyl-2-pentanone decreased slightly. These findings indicate that the gas phase enzyme reaction exhibits an excellent catalytic performance in the presence of additive glycerol for chiral compound production, however needs to optimize the additive amount and reaction conditions for effective industrial operation.

Key Role of Microbial Characteristics on the Performance of VOC Biodegradation in Two-Liquid Phase Bioreactors

Despite being studied for over 20 years, little is known about the mechanisms underlying the treatment of volatile organic compounds (VOCs) from industrial off-gases in two-liquid phase bioreactors (TLPBs). Recent reports have highlighted a significant mismatch between the high abiotic mass transfer capacity of TLPBs and the low VOC biodegradation rates sometimes recorded, which suggests that a process limitation might also be found in the microbiology of the process. Therefore, this study was conducted to assess the key role of microbial characteristics on the performance of VOC biodegradation in a TLPB using three different hexane degrading consortia. When silicone oil 200 cSt (SO200) was added to the systems, the steady state hexane elimination capacities (ECs) increased by a factor of 8.7 and 16.3 for Consortium A (hydrophilic microorganisms) and B (100% hydrophobic microorganisms), respectively. In the presence of SO200, Consortium C supported a first steady state with a 2-fold increase in ECs followed by a 16-fold EC increase after a hydrophobicity shift (to 100% hydrophobic microorganisms), compared to the system deprived of SO200. This work revealed that cell hydrophobicity can play a key role in the successful performance of TLPBs, and to the best of our knowledge, this is the first report on hydrophobic VOC treatment with exclusive VOC uptake within a nonbioavailable non aqueous phase. Finally, an independent set of experiments showed that metabolite accumulation can also severely inhibit TLPB performance despite the presence of SO200.

Toxicological evaluation for bioremediation processes of TNT-contaminated soil by Salmonella mutagenicity assay

This research was performed to evaluate the toxicity for composting and slurry phase bioreactor processes of TNT (2,4,6-trinitrotoluene)-contaminated soils by Salmonella mutagenicity assay. For composting, the percentage reductions of final composts in strain TA98 and TA100 with S9 metabolic activation were 90.3–93.7% and 96.7–97.5%, respectively. For slurry phase bioreactor processes, the percentage reductions of final residuals in strain TA98 and TA100 with S9 metabolic activation were 95.0% and 99.1% for anaerobic, 96.2% and 99.2% for anaerobic/aerobic, and 96.6 and 97.4% for anaerobic treatment. Slurry phase treatment showed higher mutagenicity reduction than composting. It was implied that slurry phase treatment was a more effective process than composting in reducing toxicity. This research has the advantage of speed and ease of performance in comparison to testing of other higher life forms due to the shorter life cycles of microorganisms.

Immobilized Ketoreductase-Catalyzed Reduction of 4-Methyl-2-Pentanone in a Continuous Gas Phase Bioreactor

気相媒体下において固定化ケトン還元酵素を触媒とした4-メチル-2-ペンタノンの連続エナンチオ選択的反応を2-プロパノールを基質としたNADH再生反応を通じて行い，固定化酵素の反応活性，安定性，エナンチオ選択性に対する水分活性，基質活性ならびに反応温度の影響を実測した．なお，固定化担体として非多孔性ガラス粒子を用い，その表面に酵素と補酵素を固定化した．本系において高いエナンチオ選択性を満足する最適反応条件は，水分活性0.9と反応温度303–313 Kであった．また，固定化酵素の安定性及びエナンチオ選択性は，基質活性に依存せず，ほぼ一定の値を示した．一方，速度論解析を行い，速度定数（Vmax=190 [μmol/min/g-protein], Km,4-methyl-2-pentanone=0.405 [−], Km,2-propanol=0.140 [−]）を推算した．

Continuous Stereoselective Reduction Catalyzed by Thermophilic Alcohol Dehydrogenase in a Gas Phase Bioreactor

The present study investigates continuous gas phase stereoselective reduction catalyzed by commercial thermophilic alcohol dehydrogenase. The alcohol dehydrogenase and cofactor β-nicotinamide adenine dinucleotide phosphate were simultaneously immobilized on non-porous spherical glass beads. The reduction of 4-methyl-2-pentanone to (S)-4-methyl-2-pentanol with concomitant cofactor regeneration by 2-propanol was employed as a model reaction. According to the differing buffer pH dependence of the enzyme activity on both substrates, the maximal reactivity was obtained at a pH of 7.5 used for immobilized enzyme preparation. Owing to the higher stereoselective production, the water activity in the feed gas phase and reaction temperature were optimized at 0.9 and 333 K, respectively.

Petroleum‐contaminated soil remediation in a new solid phase bioreactor

AbstractBACKGROUND: Oil spills are a frequent source of environmental contamination. As a consequence, remediation of soils, waters and sediments is a great challenge in this area of research. This study aims at using a new type of soil bioreactor (13 L bench‐scale and 800 L pilot‐scale) to treat tropical soil contaminated with petroleum. Additionally, it includes the evaluation of the effectiveness of two auxiliary techniques: bulking agent addition (sawdust) and biostimulation using two different nitrogen sources (sodium nitrate and urea).RESULTS: The best result in bench‐ and pilot‐scale bioreactors were reached when using urea as a nitrogen supplement and bulking agent addition. Removal of 20 to 35% of total petroleum hydrocarbon (TPH) was achieved within 42 days. The molecular fingerprinting performed with 16S‐PCR analysis associated with denaturing gradient gel electrophoresis (DGGE) was used to evaluate changes in the pattern of the bacterial community for all experimental conditions tested. The results revealed that the use of urea caused a smaller change in the dominant bacterial community structure than the treatments using nitrate, showing that this analysis can be a useful complementary tool to evaluate the impact of treatment strategies applied to hydrocarbon‐contaminated soil.CONCLUSIONS: These new solid phase bioreactors showed satisfactory results in the tropical soil bioremediation process, proving that the homogenization system interferes with crude oil biodegradation efficiency. This new technology can be used as an isolated treatment as well as in association with other classically employed bioremediation technologies. Copyright © 2010 Society of Chemical Industry