Enhanced acetone-butanol fermentation using repeated fed-batch operation coupled with cell recycle by membrane and simultaneous removal of inhibitory products by adsorption

A novel acetone-butanol production process was developed which integrates a repeated fed-batch fermentation with continuous product removal and cell recycle. The inhibitory product concentrations of the fermentation by Clostridium acetobutylicum were reduced by the simultaneous extraction process using polyvinylpyridine (PVP) as an adsorbent. Because of the reduced inhibition effect, a higher specific cell growth rate and thus a higher product formation rate was achieved. The cell recycle using membrane separation increased the total cell mass density and, therefore, enhanced the reactor productivity. The repeated fed-batchoperation overcame the drawbacks typically associated with a batch operation such as down times, long lag period, and the limitation on the maximum initial substrate concentration allowed due to the substrate inhibition. Unlike a continuous operation, the repeated fed-batch operation could beoperated for a long time at a relatively higher substrate concentration without sacrificing the substrate loss in the effluent. As a result, the integrated process reached 47.2 g/L in the equivalent solvent concentration (including acetone, butanol, and ethanol) and 1.69 g/L . h in the fermentor productivity, on average, over a 239.5-h period. Compared with a controlled traditional batch acetone-butanol fermentation, the equivalent solvent concentration and the tormentor productivity were increased by 140% and 320%, respectively. (c) 1995 John Wiley & Sons Inc.     

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall.

Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients.

The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments.

Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.     

Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts.     

Enzymatic and Microbial Preparation of d-Xylulose from d-Xylose

A high-d-xylulose mixture (d-xylose-d-xylulose = 33:67) was prepared from the cold ethanol extract of preisomerized d-xylose solution (d-xylose-d-xylulose = 77:23). Fusarium oxysporum f. sp. lini and Aspergillus niger were demonstrated to preferentially utilize d-xylose in the mixture of d-xylose and d-xylulose. Chromatographically pure d-xylulose was thus obtained in 90% yield. A high-d-xylulose mixture was also incubated with Rhodotorula toruloides, Klebsiella pneumoniae, Candida utilis, or Mucor rouxii.d-Xylose and d-xylulose were simultaneously consumed. When borate was added to the mixture, a d-xylulose-borate complex was formed, and it could be used to protect d-xylulose from being utilized.     

Glycoproteins from human colonic adenocarcinoma. Isolation and characterization of cell surface carcinoembryonic antigen from a cultured tumor cell line.

Alterations in cell surface glycoproteins have been implicated in malignancy. We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma. Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase. The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein- and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days.     

Formaldehyde incorporation by a new methylotroph (L3)

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

O-glycosylation of intact and truncated ribophorins in brefeldin A- treated cells: newly synthesized intact ribophorins are only transiently accessible to the relocated glycosyltransferases

Ribophorins I and II are type I transmembrane glycoproteins of the ER that are segregated to the rough domains of this organelle. Both ribophorins appear to be part of the translocation apparatus for nascent polypeptides that is associated with membrane-bound ribosomes and participate in the formation of a proteinaceous network within the ER membrane that also includes other components of the translocation apparatus. The ribophorins are both highly stable proteins that lack O-linked sugars but each contains one high mannose N-linked oligosaccharide that remains endo H sensitive throughout their lifetimes. We have previously shown (Tsao, Y. S., N. E. Ivessa, M. Adesnik, D. D. Sabatini, and G. Kreibich. 1992. J. Cell Biol. 116:57-67) that a COOH-terminally truncated variant of ribophorin I that contains only the first 332 amino acids of the luminal domain (RI332), when synthesized in permanent transformants of HeLa cells, undergoes a rapid degradation with biphasic kinetics in the ER itself and in a second, as yet unidentified nonlysosomal pre-Golgi compartment. We now show that in cells treated with brefeldin A (BFA) RI332 molecules undergo rapid O-glycosylation in a multistep process that involves the sequential addition of N-acetylgalactosamine, galactose, and terminal sialic acid residues. Addition of O-linked sugars affected all newly synthesized RI332 molecules and was completed soon after synthesis with a half time of about 10 min. In the same cells, intact ribophorins I and II also underwent O-linked glycosylation in the presence of BFA, but these molecules were modified only during a short time period immediately after their synthesis was completed, and the modification affected only a fraction of the newly synthesized polypeptides. More important, these molecules synthesized before the addition of BFA were not modified by O-glycosylation. The same is true for ribophorin I when overexpressed in HeLa cells although it is significantly less stable than the native polypeptide in control cells. We, therefore, conclude that soon after their synthesis, ribophorins lose their susceptibility to the relocated Golgi enzymes that effect the O-glycosylation, most likely as a consequence of a conformational change in the ribophorins that occurs during their maturation, although it cannot be excluded that rapid integration of these molecules into a supramolecular complex in the ER membrane leads to their inaccessibility to these enzymes.     

Energy transfer in complexes of E. coli single-stranded DNA-binding protein with single-stranded poly-(2-thiouridylic acid)

The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR). Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases. Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place. The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U. The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp. The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.     

Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.