Membrane bioreactors: Engineering aspects.

Membrane bioreactors have in-situ separation capability lacking in other types of immobilized cell reactors. This makes them very useful for certain systems. Enzyme reactions utilizing cofactors and hydrolysis of macromolecules are advantageous in membrane reactors. Anaerobic cell culture may be efficiently carried out in membrane cell recycle systems, while aerobic cultures work well in dual hollow fiber reactors. Animal and plant cells have much a better chance of success in membrane reactors because of the protective environment of the reactor and the small oxygen uptake rate of these cells.

Microencapsulation of microbial cells.

The high level of biocatalysts such as microbial cells and enzymes plays an important role in increasing the productivity of a bioreactor. The beads entrapped with microbial cells are not strong enough for long-term use. The small void space of polymer matrix and the leakage of cells limit a final cell loading in the beads. The recent success of encapsulating microbial cells makes it possible to prepare dense biocatalyst composed of recombinant microbial cells. In addition to encapsulating microbial cells, immobilization of animal and plant cells in capsules is also briefly described.

High density cell culture by membrane-based cell recycle.

Enhancement of productivity of a bioprocess necessitates continuous operation of bioreactors with high biomass concentrations than are possible in conventional batch, fedbatch or continuous modes of culture. Membrane-based cell recycle has been effectively used to maintain high cell concentrations in bioreactors. This review compares membranebased cell recycle operation with other such high density cell culture systems as immobilized cell reactors and reactors with cell recycle by centrifugation or gravity sedimentation. A theoretical of production of primary and secondary metabolites in membrane-based recycle systems is presented. Operation of this type of system is discussed with examples from aerobic and anaerobic fermentations.

Continuous production of 6-APA in an aqueous two-phase system.

An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate provided a favorable environment for bioconversion of penicillin G to 6-aminopenicillanic acid (6-APA). The recombinant E. coli cells containing penicillin acylase were partitioned in the phosphate-rich bottom phase, and the product 6-APA in the PEG-rich top phase, which protected the enzyme from deactivation for a longer period of time. The continuous production of 6-APA by recycling the bottom phase showed a slight decrease in enzyme activity from initial 80 mM of 6-APA production to 61 mM during the eight days of operation at a space velocity of 0.12 hours-1. This work showed a possibility that the aqueous two-phase whole-cell enzyme bioconversion could serve as an alternative to immobilized cells on solid matrices.

Shikonin production by extractive cultivation in transformed-suspension and hairy root cultures of Lithospermum erythrorhizon.

Effects of in situ extraction, fungal elicitation, a permeabilizing agent, and the oxygen transfer rate on shikonin production in transformed suspension and hairy root cultures of Lithospermum erythrorhizon were studied. Shikonin production with in situ extraction in transformed cell and hairy root cultures by n-hexadecane was 7.6 and 3 times higher than those of the control culture. Shikonin production of transformed L. erythrohizon increased with the enhanced gas exchange, and in situ extraction also increased sucrose consumption and shikonin production. The optimal volume of n-hexadecane in the hairy root culture was similar to that in the transformed cell cultures. In situ extraction at an earlier stage significantly enhanced shikonin production both in transformed cell and hairy root cultures. Dimethylsulfoxide used as a permeabilizing agent was harmful to cell growth and shikonin production, and permeabilizing was unnecessary when in situ extraction was applied. This occurred because with the solvent addition, most shikonin was spontaneously released from the cells and was dissolved in the solvent layer. The combined addition of n-hexadecane of the extract of the fungus Penicillium as an elicitor seemed to result in a higher production of shikonin both in cell suspensions and transformed root cultures. However, an increase of shikonin induction by fungal elicitation in a hairy root culture was not significant in comparison with that of normal cell cultures.

The relationship of aging to endotoxin shock and to production of TNF-alpha.

BACKGROUND: Aged people are considered prone to gram-negative bacteremia and septic shock. This relationship was tested in murine endotoxin shock. METHODS: Balb/c mice of various ages (1.4-13.4 months) were intraperitoneally injected with lipopolysaccharide (LPS), and rates of survival were observed. The production of TNF-alpha in vivo induced by LPS was measured. RESULTS: The survival rates were the smallest in the oldest and youngest groups. Production of TNF-alpha attained a maximum at 2 h after LPS injection and was smaller in the oldest group; it had a reciprocal relationship to survival rates in each group except the youngest group. CONCLUSION: Old and young mice had smaller rates of survival and greater production of TNF-alpha following endotoxin shock induced by LPS.

Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2.

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.

Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli.

Plasmids containing the Alcaligenes eutrophus poly(3-hydroxybutyric acid) (PHB) biosynthetic genes were constructed for the production of PHB in Escherichia coli and plasmid stability was investigated by repeated subculturing without antibiotic pressure. Both pSYL101 (high copy) and pSYL102 (medium copy) were unstable during the subcultures. Higher instability was observed when cells were accumulating PHB. Segregational instability was aggravated by the faster growth of plasmid-free cells and by appearance of non-dividing cells harboring large amount of PHB during the fed-batch culture. Two derivatives, pSYL103 and pSYL104, were then developed by cloning the parB locus of plasmid R1 into pSYL102 and pSYL101, respectively. They showed 100% stability even during PHB synthesis and accumulation over 110 generations. All four plasmids were structurally stable. The final cell mass, PHB concentration, and PHB per dry cell weight (P/X, w/w, %) of 101.4 g l-1, 81.2 g l-1, and 80.1%, respectively, were obtained in 39 h by high cell density culture of XL1-Blue (pSYL104). The final PHB concentration was lower using XL1-Blue (pSYL103), which suggested that high gene dosage was required for the synthesis and accumulation of PHB to a high concentration in E. coli.

Removal of hydrogen sulfide by Chlorobium thiosulfatophilum in immobilized-cell and sulfur-settling free-cell recycle reactors.

Bioconversion of hydrogen sulfide to elementary sulfur by the photosynthetic bacterium Chlorobium thiosulfatophilum was studied in immobilized-cell and sulfur-settling free-cell recycle reactors. The cells immobilized in strontium alginate beads excreted elementary sulfur and accumulated it as crystal in the bead matrices, which made it possible that the reactor broth remained clear and the light penetrated the reactor deeper than with the free cells. In comparison with the free cells, the immobilized cells required 30% less light energy at a H2S removal rate of 2 mM/(L.h) and showed an activity of 2.4 times that of the free cells. However, in 40 h after the reaction the deterioration of the H2S removal efficiency became significant due to the accumulation of sulfur in the beads. The scanning electron micrograph (SEM) and energy-dispersive X-ray spectrometer (EDS) studies showed that the sulfur in the beads existed within a layer of 0.4 mm from the bead surface. In the sulfur-settling free-cell recycle reactor, about 80% of the sulfur excreted by the free cells could be removed in a settler. The 4-L fed batch reactor with the settler improved the light transmission to result in a H2S removal rate of 3 mumol/(mg of protein.h), 50% higher than that without it. The settling recycle reactor was much better in the removal of H2S than the immobilized-cell reactor because the former was a continuous system with the constant removal of sulfur particles by settling and of spent medium by supplying fresh medium at the same rate as the filtering rate of the reactor broth, while the latter was essentially a batch system where toxic metabolites and produced sulfur could not be removed.

Desulfurization of diesel oils by a newly isolated dibenzothiophene-degrading Nocardia sp. strain CYKS2.

A dibenzothiophene (DBT)-degrading bacterial strain was isolated from dyeing industry wastewater and identified as Nocardia sp. CYKS2. The newly isolated bacterial strain Nocardia sp. CYKS2 was able to convert DBT to 2-hydroxybiphenyl (2-HBP) as the dead-end metabolite through a sulfur-specific pathway. Other organic sulfur compounds, such as thiophene derivatives, thiazole derivatives, sulfides, and disulfides were also desulfurized by Nocardia sp. CYKS2. In batch culture, 0.2 mM DBT was completely desulfurized in 60 h. After DBT was depleted, neither cell growth nor 2-HBP production was observed. When a model oil which DBT was dissolved in hexadecane was treated with growing cells, DBT was desulfurized from 10 mM to about 2 mM in 80 h. In this case, desulfurization rate was 0.279 mg-sulfur/(L-dispersion.h), which was about 2.5 times higher than that in the previous case of batch culture. When diesel oil was treated, the sulfur content decreased from 0.3 to 0.24 wt % in 48 h. A volumetric phase ratio of oil to water was 1/10 in this case. The sulfur decreased from 0.3 to 0.2 wt % in 48 h, when the volumetric phase ratio was 1/20. The desulfurization rates were 0.909 and 0.992 mg-sulfur/(L-dispersion.h), respectively.

Continuous ethanol production from concentrated wood hydrolysates in an internal membrane-filtration bioreactor.

Continuous culture for the production of ethanol from wood hydrolysate was carried out in an internal membrane-filtration bioreactor. The hydrolysate medium was sterilized at a relatively low temperature of 60 degrees C with the intention of reducing the formation of inhibitory compounds during the sterilization. The maximum ethanol concentration and productivity obtained in this study were 76.9 g/L and 16.9 g/L-h, respectively, which were much higher than those (57.2-67 g/L and 0.3-1.0 g/L-h) obtained in batch cultures using hydrolysate media sterilized at 60 degrees C. The productivity was also found to be much higher than that (6.7 g/L-h) obtained in a continuous cell retention culture using a wood hydrolysate sterilized at 121 degrees C. These results show that the internal membrane-filtration bioreactor in combination with low-temperature sterilization could be very effective for ethanol production from wood hydrolysate.

Production of a desulfurization biocatalyst by two-stage fermentation and its application for the treatment of model and diesel oils.

For the production of oil-desulfurizing biocatalyst, a two-stage fermentation strategy was adopted, in which the cell growth stage and desulfurization activity induction stage were separated. Sucrose was found to be the optimal carbon source for the growth of Gordonia nitida CYKS1. Magnesium sulfate was selected to be the sulfur source in the cell growth stage. The optimal ranges of sucrose and magnesium sulfate were 10-50 and 1-2.5 g x L(-1), respectively. Such a broad optimal concentration of sucrose made the fed-batch culture easy, while the sucrose concentration was maintained between 10-20 g x L(-1) in the actual operation. As a result, 92.6 g x L(-1) of cell mass was acquired by 120 h of fed-batch culture. This cell mass was over three times higher than a previously reported result, though the strain used was different. The desulfurization activity of the harvested cells from the first stage culture was induced by batch cultivation with dibenzothiophene as the sole sulfur source. The optimal induction time was found to be about 4 h. The resting-cell biocatalyst made from the induced cells was applied for the deep desulfurization of a diesel oil. It was observed that the sulfur content of the diesel oil decreased from 250 mg-sulfur x L-oil(-1) to as low as 61 mg-sulfur x L-oil(-1) in 20 h. It implied that the biocatalyst developed in this study had a good potential to be applied to a deep desulfurization process to produce ultra-low-sulfur fuel oils.

Velocity field of pulsatile flow in a porous tube.

This paper describes velocity fields for fully developed periodic laminar flow in a rigid tube with a porous wall. We obtained an analytical solution of the flow by the linear approximation of the Navier-Stokes equation. Unlike the previous works with a constant seepage rate along the axis, we used a wall velocity which contained hydraulic permeation constant Lp. The axial velocity profile shows a local maximum velocity near the wall at a large Womersley number alpha. This suggests that concentration polarization in porous tubular membrane may be reduced at high frequencies if a membrane device is operated under pulsatile flow conditions. The magnitude of wall permeation velocity decreases linearly along the tube axis because the damping of the pressure difference between the inside and the outside of the tube is very small.

Preliminary evaluation of the correlation between in vitro release and in vivo bioavailability of two aminophylline slow-release tablets.

An in vitro dissolution study has been carried out with a USP rotating paddle apparatus using the stepwise pH change method. The dissolution kinetics of two different matrix-type aminophylline slow-release (SR) tablets were compared. both aminophylline SR tablets show similar release rates and slow release behavior. The bioavailability of these two aminophylline SR tablets was also evaluated in eight healthy male Chinese volunteers and correlated with in vitro dissolution results by moment analysis. The preliminary results suggest that there is no evidence indicating that the two different matrix types of aminophylline SR tablets are not bioequivalent, and a good in vitro-in vivo correlation for these aminophylline SR tablets may therefore be presumed.

Angiogenic induction and cell migration in an orthopaedically expanded maxillary suture in the rat.

The purpose was to examine the effect of an angiogenic factor on cell migration patterns and osteoblast histogenesis during the 96 h following orthopaedic expansion of the anterior maxillary suture. Fifty rats were divided into four groups: (1) a control group that received only angiogenic induction via injection of 5 ng/g body wt recombinant human endothelial-cell growth factor; (2) an experimental group that received orthopaedic expansion and angiogenic induction; (3) a sham group that received orthopaedic expansion and normal saline injection; and (4) a baseline group that received no expansion or injection. The experimental and sham groups were subdivided to conduct experiments over 1, 2, 3 or 4 days. The anterior portion of each maxilla was dissected free and demineralized. Sections (4 microns thick) were cut from every block and stained with Mayer's haematoxylin and eosin. Cell migration was analysed using a previously established cell-kinetics model. The osteoprogenitor cells were divided into four categories according to nuclear volume: A cells (40-79 microns3), B cells (80-119 microns3), C cells (120-169 microns3) and D cells (> or = 169 microns3 A' cells are the portion of the A cell population that responds to osteogenic stimulus. As previously defined in periodontal ligament, the reciprocal association of a decreasing number of less differentiated (A + A) cells and an increasing number of C + D cells, as a function of distance from the nearest major blood vessel, was consistently found in all groups. This suggests a vascularly oriented gradient of progressively more differentiated osteoprogenitor cells. Also, A + A' cells were predominately located within 20 microns of the nearest major blood vessel whereas the C + D cells were found at a distance > 30 microns from the nearest major blood vessel. These results suggest that the A'-->C shift occurs 20-30 microns from the nearest major blood vessel. In the angiogenic induction groups, the numbers of committed osteoprogenitors (A + A') were significantly higher than in the sham group at day 1. At day 3, the numbers of preosteoblasts (C + D) in angiogenic sutures were significantly higher than in the sham groups. This enhancement of preosteoblast population strongly suggests the possible role of activated pericytes in expanded sutures as a source of osteoprogenitor cells.

Bench-scale production of acrylamide using the resting cells of Brevibacterium sp. CH2 in a fed-batch reactor.

Effects of various organic acids and salts on the stabilization of nitrile hydratase were investigated. The stability of the nitrile hydratase of Brevibacterium CH2 during storage was greatly enhanced by the addition of n-butyric acid. Effects of temperature, pH, and concentrations of acrylonitrile and n-butyric acid on acrylamide production by the resting cells were also investigated. Acrylamide production per unit dry weight of the cells increased 1.33 times by the addition of 0.05% n-butyric acid. A 20% acrylamide solution was successfully produced in a bench-scale reactor (12 l) with only a trace amount of salts after 10 h of hydration reaction under optimum reaction conditions without using an isotonic substrate. The conversion yield was nearly 100%, and acrylic acid as a by-product was not produced. Final acrylamide production of 400 g g-1 cells and productivity of 20 g/(g cells l-1 x h-1) were obtained.

Ethanol production using concentrated oak wood hydrolysates and methods to detoxify.

Ethanol production from concentrated oak wood hydrolysate was carried out to obtain a high ethanol concentration and a high ethanol yield. The effect of added inhibitory compounds, which are typically produced in the pretreatment step of steam-explosion on ethanol fermentation, was also examined. p-Hydroxybenzoic aldehyde, a lignin-degradation product, was the most inhibitory compound tested in this study. Compounds with additional methyl groups had reduced toxicity and the aromatic acids were less toxic than the corresponding aldehydes. The lignin-degradation products were more inhibitory than the sugar-derived products, such as furfural and 5-hydroxymethylfurfural (HMF). Adaptation of yeast cells to the wood hydrolysate and detoxification methods, such as using charcoal and overlime, had some beneficial effects on ethanol production using the concentrated wood hydrolysate. After treatment with charcoal and low-temperature sterilization, the yeast cells could utilize the concentrated wood hydrolysate with 170 as well as 140 g/L glucose, and produce 69.9 and 74.2 g/L ethanol, respectively, with a yield of 0.46-0.48 g ethanol/g glucose. In contrast, the cells could not completely utilize untreated wood hydrolysate with 100 g/L glucose. Low-temperature sterilization, with or without charcoal treatment, was very effective for ethanol production when highly concentrated wood hydrolysates were used. Low-temperature sterilization has advantages over traditional detoxification methods, such as using overlime, ion exchange, and charcoal, because of the reduction in the total cost of ethanol production.