Capillary Bioreactor Research Articles

Multi-channel capillary bioreactor for hydrophobic VOC and CO2 abatement – Process intensification through silicone oil addition

A multi-channel capillary bioreactor devoted to the continuous abatement of hydrophobic volatile organic compounds (VOCs) by a bacterial and bacterial/microalgae consortium was investigated for 200 days. Toluene, α-pinene and hexane removal in the capillary bioreactor was up to 99 %, 98 %, and 55 %, respectively, which is remarkably high considering the low gas contact time of less than 1 second. Addition of silicone oil increased the removal efficiency (RE) of α-pinene within two days from 45 ± 6 % to 98 ± 2 %, probably through alleviation of biokinetic inhibition provided by the oil acting as buffer for the α-pinene and/or its metabolites. The RE of toluene increased after silicone oil addition over a period of about eight weeks from 81 ± 3 % to 99 ± 1 %, most likely via microbial adaptation. On the contrary, the removal of hexane did not increase following silicone oil addition, potentially due to the inhibition of hexane or its metabolites as the bioreactor was deliberately operated without replenishing the recirculation liquid. Interestingly, biomass adhered to the silicone oil phase rather than residing in the water phase. The bacterial diversity was substantially enhanced, and probably contributed to the observed stable performance of the capillary bioreactor. After the introduction of microalgae on day 150, lower CO2 concentrations at the outlet compared to the inlet were observed immediately. A net CO2 consumption was recorded, achieving complete carbon sequestration from the removed VOCs, along with additional CO2 removed from the inlet ambient air.

Hydrophobic air pollutants removal at one second gas contact in a multi-channel capillary bioreactor

Biological processes are increasingly applied for gas purification as a sustainable and economical alternative to conventional physical-chemical processes (chemical absorption, incineration, adsorption). Although biological gas treatment is accepted as an economical, safe, and reliable air pollution control technology, it faces important limitations when applied for the treatment of poorly water-soluble compounds due to mass transfer limitations. A twenty-five capillary channels bioreactor was studied to characterize mass transfer coefficients and the removal of hydrophobic air pollutants under segmented gas-liquid flow pattern. The removal efficiency of hexane, toluene and α-pinene vapors reached values up to about 75%, 99% and 75%, respectively, at a gas contact time of less than 1 s, which is at least one, but closer to two orders of magnitude shorter than conventional biological gas purification systems. The bioreactor displayed stable operation for 100 days and was robust against common upsets, which opens the new opportunities for expanding the application field of biological processes for air pollution control and the mitigation of greenhouse gases in dilute air streams. SynopsisThis study breaks through existing barriers of gas treating biotechnologies which allows expansion of the application field of more sustainable processes for air pollution control.

Population dynamics of a dual Pseudomonas putida–Pseudomonas fluorescens biofilm in a capillary bioreactor

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72 h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48 h. Even when the inoculation by P. putida was delayed for 24 h, P. fluorescens was eliminated from the capillary within 48 h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.

Multienzyme Cascade Bioreactor for a 10 min Digestion of Genomic DNA into Single Nucleosides and Quantitative Detection of Structural DNA Modifications in Cellular Genomic DNA.

Identification and quantification of chemical DNA modifications provide essential information on genomic DNA changes, for example, epigenetic modifications and abnormal DNA lesions. In this vein, it requires to digest genomic DNA strands into single nucleosides, facilitating the mass spectrometry analysis. However, rapid digestion of such supramacromolecule DNA of several millions Daltons (molecular weight) into single nucleosides remains very challenging. Here, we constructed an immobilized benzonase capillary bioreactor and further tandemly coupled with immobilized snake venom phosphodiesterase and alkaline phosphatase capillary bioreactor to form a novel three-enzyme cascade bioreactor (BenzoSAC bioreactor). In these constructions, the chosen enzymes were immobilized onto synthetic porous capillary silica monoliths. With the tailor-made porous structure and high immobilized capacity and high digestion rate of benzonase, genomic DNA of >99.5% can be digested into single nucleosides within only 10 min when passing through the BenzoSAC bioreactor by microinjection pump. In contrast, traditional digestion requires 8-24 h. By offline coupling this benzoSAC bioreactor with liquid chromatography-tandem mass spectrometry, we detected 5-hydroxymethylcytosine, a major oxidation product of the epigenetically crucial 5-methylcytosine, in genomic DNA isolated from ladder cancer (T24) cells. The newly synthesized BenzoSAC bioreactor and the proposed mass spectrometry detection are promising for fast identification and analysis of structural modifications in DNA.

Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides.

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.

Preparation of porous graphene oxide–poly(urea–formaldehyde) hybrid monolith for trypsin immobilization and efficient proteolysis

Porous graphene oxide–poly(urea–formaldehyde) (GO–PUF) hybrid monolith was prepared by the in situ polycondensation of the urea–formaldehyde prepolymer solution in the presence of GO sheets and ammonium chloride. The amide groups in the residues of asparagine and glutamine in trypsin was allowed to condense with the residual N-hydroxymethyl groups of PUF in the monolith at low pH to fabricate bioreactors for proteolysis. The morphologies and structures of the prepared materials were investigated by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and Raman spectroscopy. The results indicate that both GO–PUF hybrid and trypsin-immobilized GO–PUF hybrid mainly consist of irregular micrometer size flakes and pores that are interconnected to form 3D porous architectures. In addition, the feasibility and performance of the novel capillary bioreactors were demonstrated by the digestion and identification of bovine serum albumin, lysozyme, ovalbumin, and cytochrome c. The digestion time was significantly reduced to less than 10s. The digests were identified by mass spectrometry with results that were comparable to those obtained by 12-h conventional in-solution tryptic digestion.

Acetylcholinesterase immobilized capillary reactors coupled to protein coated magnetic beads: A new tool for plant extract ligand screening

The use of immobilized capillary enzyme reactors (ICERs) and enzymes coated to magnetic beads ((NT or CT)-MB) for ligand screening has been adopted as a new technique of high throughput screening (HTS). In this work the selected target was the enzyme acetylcholinesterase (AChE), which acts on the central nervous system and is a validated target for the treatment of Alzheimer’s disease, as well as for new insecticides. A new approach for the screening of plant extracts was developed based on the ligand fishing experiments and zonal chromatography. For that, the magnetic beads were used for the ligand fishing experiments and capillary bioreactors for the activity assays. The latter was employed also under non-linear conditions to determine the affinity constants of known ligands, for the first time, as well as for the active fished ligand.

A capillary bioreactor to increase methane transfer and oxidation through Taylor flow formation and transfer vector addition

The impact of two strategies to enhance the mass transfer of hydrophobic compounds, Taylor flow (or segmented flow) and the addition of an organic transfer vector (silicone oil), were investigated under abiotic and biotic conditions in a capillary bioreactor. The capillary bioreactor consisted of a capillary column (where Taylor flow was produced in a gas/liquid flow) and a gas–liquid separator at the outlet of the capillary column which was operated as a stirred tank with superficial aeration. It was shown that the system was limited by mass transfer and not by the biological reaction. Taylor flow in the capillary resulted in an increase of up to two orders of magnitude for the volumetric oxygen transfer coefficient (kLa) when compared to the coefficient for the gas–liquid separator, or values previously obtained in other turbulent contactors. The bioconversion rates of methane in the capillary column where found to be significantly higher than for conventional systems. Silicone oil addition increased kLa up to 38% in the gas–liquid separator, but reduced it with 38% in the capillary. Contrary to observations during abiotic kLa determinations, silicone oil addition increased the CH4 removal and O2 consumption by the methanotrophic consortium in both, gas–liquid separator and capillary. Increased gas flow rate gave an 19% increase in methane removal in the capillary bioreactor, an additional increase of 8% was obtained adding 5% of silicone oil at the same flow, while an additional increase of 47% was obtained adding 10% of silicone oil at the same flow with inoculum pre-adapted to transfer vector. The contribution of the capillary channel to the overall methane removal in the system was high considering that the volume of this channel was just 0.64% of the total volume in the bioreactor, indicating a good potential of further optimization of the reactor system.

Comparison of two size variants of a miniaturized multi-compartment three-dimensional (3D)-bioreactor system for primary human hepatocyte cultivation

Co-cultivation of primary human hepatocytes (pHH) with nonparenchymal liver cells in a perfused 3D-membrane capillary bioreactor (BR) enables more efficient long-term cell cultivation with maintenance of the physiological cell functions in comparison to standard 2D culture systems. Due to the restricted availability of primary human liver cells, miniaturization of the culture system while providing sufficient material for extra- and intracellular analyses is desirable for in vitro studies on hepatocyte physiology and metabolism. In this study two size variants of a miniaturized multi-compartment BR technology consisting of two or four capillary layers, each composed of medium perfusion and oxygenation capillaries were compared with respect to their metabolic activity. pHH from the same donors, (n=3) respectively, were cultivated in parallel in two- or four-layer BRs for 10 days with regular measurement of metabolic parameters. Cytochrome P450 (CYP) activities, including CYP1A2, CYP2B6, CYP2C9 and CYP3A4/5, as well as urea production showed no significant differences in the two different BR systems during the cultivation period. Equally aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release, measured to assess cellular injury, showed comparative concentrations in both systems. In addition, immunochemically assessed expression patterns of cytokeratin (CK) 19 as biliary marker, CK18 as hepatocyte/biliary marker and the mesenchymal marker vimentin confirmed the preservation of cellular identity within the two size variants of the BR technology. In conclusion both miniaturized BR systems are suitable for extended pHH culture while preserving specific metabolism. While the smaller model is useful for studies with small cell numbers, experiments requiring larger cell numbers for complex analyses should be performed with the four-layer model.

Capillary Monolithic Bioreactor of Immobilized Snake Venom Phosphodiesterase for Mass Spectrometry Based Oligodeoxynucleotide Sequencing

A capillary monolithic bioreactor of snake venom phosphodiesterase (SVP) was constructed to generate different single-nucleotide mass ladders of oligodeoxynucleotides for mass spectrometry (MS)-based sequencing by immobilization. The immobilization of SVP in the porous silica monolith significantly enhances its stability for prolonged and repeated applications. The constructed capillary bioreactor has the advantages of handling (sub)microliter DNA samples and having good permeability. Benefiting from its good permeability, DNA solutions can be directly injected into the sequential digestion bioreactor simply by hand pushing or a low-pressure microinjection pump. Moreover, the immobilization of SVP facilitates the elimination or repression of the metal adducts of oligodeoxynucleotides, improving the analytical performance of MS sequencing. By the application of capillary bioreactor of immobilized SVP, the sequence-specific modification of single-stranded oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr) was identified, demonstrating its promising applications in identification of sequence-specific damage, which may further our understanding of DNA damage caused mutagenesis.

Capillary bioreactors based on human purine nucleoside phosphorylase: A new approach for ligands identification and characterization

The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The KM value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255±29.2μM and 133±14.9μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC50=40.6±0.36nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay.

Dynamic 3D Culture Promotes Spontaneous Embryonic Stem Cell DifferentiationIn Vitro

Spontaneous in vitro differentiation of mouse embryonic stem cells (mESC) is promoted by a dynamic, three-dimensional (3D), tissue-density perfusion technique with continuous medium perfusion and exchange in a novel four-compartment, interwoven capillary bioreactor. We compared ectodermal, endodermal, and mesodermal immunoreactive tissue structures formed by mESC at culture day 10 with mouse fetal tissue development at gestational day E9.5. The results show that the bioreactor cultures more closely resemble mouse fetal tissue development at gestational day E9.5 than control mESC cultured in Petri dishes.

Immobilization enzyme fluorescence capillary analysis for determination of lactic acid

A new method for the determination of lactic acid based on the immobilization enzyme fluorescence capillary analysis (IE-FCA) was proposed. Lactic dehydrogenase (LDH) was immobilized on inner surface of a capillary with glutaraldehyde, and an immobilized enzyme lactate capillary bioreactor (IE-LCBR) was formed for the determination of lactic acid. After nicotinamide adenine dinucleotide (NAD +) is mixed with lactic acid solution, it was sucked into the IE-LCBR and was detected at λ ex 353 nm/ λ em 466 nm. Optimized conditions are as follows: the temperature is 38 °C; the reaction time is 15 min; the concentrations of Tris buffer (pH 8.8) and NAD + are 0.1 mol L −1 and 4 mmol L −1, respectively; the concentration of LDH used for immobilization is 15 kU L −1. The concentration of lactic acid is directly proportional to the fluorescence intensity measured from 0.50 to 2.0 mmol L −1; and the analytical recovery of added lactic acid was 99–105%. The minimum detection limit of the method is 0.40 mmol L −1 and sensitivity of the IE-CBR is 4.6 F mmol −1 L −1 lactate. Its relative standard deviation (R.S.D.) is ≤2.0%. This IE-FCA method was employed for determination of lactate in milk drink.

Determination of various alcohols based on a new immobilized enzyme fluorescence capillary analysis

A novel method for the determination of ethanol in tequila based on the immobilized enzyme fluorescence capillary analysis (IE-EFCA) has been proposed. Alcohol dehydrogenase (ADH) was immobilized in inner surface of a capillary and an immobilized enzyme capillary bioreactor (IE-ECBR) was formed. After nicotinamide adenine dinucleotide (NAD +) as an oxidizer is mixed with alcohol sample solution, it was sucked into the IE-ECBR. The fluorescence intensity of the mixed solution in the IE-ECBR was detected at λ ex = 350 nm and λ em = 459 nm. The experimental conditions are as follows: The reaction time is 20 min; temperature is 40 °C; the concentrations of phosphate buffer solution (pH 7.5) and NAD + are 0.1 mol L −1 and 5 mmol L −1, respectively; immobilization concentration of ADH is 10 U L −1. The determination range of ethanol is 2.0–15.0 g L −1 ( F = 10.44C + 6.6002, r > 0.9958); its detection limit is 1.11 g L −1; and relative standard deviation is 1.9%. IE-EFCA method is applicable for the determination of the samples containing alcohol in medicine, industry and environment.

Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor

Microorganisms that colonize surfaces, biofilms, are of significant importance due to their role in medical infections, subsurface contaminant remediation, and industrial processing. Spatially resolved data on the distribution of biomass within a capillary bioreactor, the heterogeneity of the biofilm itself and the impact on transport dynamics for a Staphylococcus epidermidis biofilm in the natural growth state are presented. The data demonstrate the ability of magnetic resonance microscopy to study spatially resolved processes in bacterial biofilms, thus providing a basis for future studies of spatially resolved metabolism and in vivo clinical detection.

Growth of tumor-derived activated T cells for the treatment of advanced cancer.

In 1994, we reported on a series of patients treated with T-cell therapy (Study #1). This paper (Study #2) is an update of our experience through 1999 in the production of tumor-derived activated cells (TDAC), also called tumor-infiltrating lymphocytes (TIL), from tumor biopsies. TDAC were successfully grown in medium containing Interleukin-2 from 75% of the 366 tumor biopsies tested. There was no significant difference in success (growth to 1 x 10(9) cells) comparing primary and metastatic tumors. Many of the tumors were shipped to the laboratory by overnight delivery from distant sites. Success rate did decrease with the length of time for tumor transport in excess of 24 hours. Certain additional cytokines were tested when cultures did not grow. Interleukin-4 was beneficial in the development of 1 of 4 TDAC cultures which did not grow with IL-2 alone. In order to produce TDAC to treat patients, cells were grown in gas permeable plastic bags or in artificial capillary bioreactor cultures. Approximately 1 x 10(9) were seeded from an initially successful "feasibility study" to bulk produce cells for treatment. Harvest was carried out after about 3 weeks. Sixty-three patients were treated at least once with a minimum of 1 x 10(10) TDAC given by intravenous infusion. On the average, the number of cells per treatment was 3 x 10(10) with a viability of 87%. TDAC cultures contained T cells with variable ratios of CD4 to CD8 cells. Secreted granulocyte-monocyte colony stimulating factor, interferon gamma and tumor necrosis factor alpha were measured in TDAC conditioned medium. Only 34 patients received the full course of 4 TDAC treatments. The cells were well tolerated with mild fever and dyspnea. Partial responses were observed in 8 patients, including the dramatic regression of scalp nodules in a patient with renal cancer. These results showed that therapeutic amounts of TDAC can be produced in cell culture in a reasonable and cost-effective manner. The cells were well tolerated and responses were seen in renal and melanoma patients resistant to IL-2 with bulky, advanced cancer.

Comparative Evaluation of Different Membranes for the Construction of an Artificial Liver Support System

During the past decades, many technological improvements have been made in the construction of extracorporeal liver support systems. Among these achievements, membranes of artificial capillary system, used as substrates of hepatocyte growth, aroused our interest in their application for the construction of bioreactors. The present paper studied the comparison of hepatocyte growth and function on six different membranes. Four of them are cellulose based membranes, Cuprophan, Hemophan, Cellulose acetate, and Bioflux; two are synthetic polymer SPAN and Polysulphone. Human hepatoma cell line SMMC-7721, with moderately differentiated hepatocyte-specific functions, was inoculated into the hollow fiber cartridges. These cells were allowed to attach and to grow over these membranes. It was found that there existed differences in hepatocyte immobilization and growth among these membranes. They influenced the growth and functions of hepatoma cells in vitro to some extent. These results show that membrane is an important factor in the construction of capillary membrane bioreactors for artificial liver support.

Growth of Tumor Derived Activated T-Cells for the Treatment of Cancer

This report describes the production of Tumor Derived Activated Cell cultures (TDAC, also called tumor infiltrating lymphocytes) from patient tumor biopsies and our preliminary experience growing these cells to therapeutic levels using artificial capillary bioreactor cultures. TDAC were successfully grown in medium containing Interleukin 2 from 80% of the 113 tumor biopsies tested. There was no significant difference in success (growth to 1 x 10(9) cells) comparing primary and metastatic tumors. Many of the tumors were shipped to the laboratory from distant sites. Success rate did decrease with the length of time for tumor transport. Interleukin 4 was beneficial in the development of 1 of 4 TDAC cultures which did not grow with IL-2 only. Seventy-seven bioreactor cultures were initiated for 31 patients. On the average, 1.9 x 10(9) TDAC were inoculated per bioreactor; 3.3 x 10(10) were harvested in 22 days. Twelve liters of medium were required per 1 x 10(10) TDAC produced. TDAC cultures contained T cells with variable ratios of CD4 to CD8 cells. Secreted granulocyte monocyte colony stimulating factor, interferon gamma and tumor necrosis factor were measured in the bioreactor cartridge conditioned medium. Twenty three patients were evaluated. Partial responses were observed in 4 patients including a dramatic remission of scalp nodules in a patient with renal cancer. Results showed that therapeutic amounts of TDAC cells may be produced in a reasonable and cost effective manner using artificial capillary bioreactor cultures.