A modeling study of anaerobic biofilm systems: II. Reactor modeling

A rigorous steady-state model of anaerobic biofilm reactors taking into account acid-base and gas-phase equilibria in the reactor in conjunction with detailed chemical equilibria and mass transfer in acetate-utilizing methanogenic biofilms is presented. The performances of ideal completely stirred tank reactors (CSTRs) and plug-flow reactors, as well as reactors with nonideal hydraulic conditions, are simulated. Decreasing the surface loading rate increases the acetate removal efficiency, while decreasing the influent pH and increasing the buffering capacity improves the removal efficiency only if the bulk pH of the reactor shifts toward more optimal values between 6.8 to 7.0. The reactor can have negative or positive removal efficiencies depending on the start-up conditions. The respiration coefficient plays a critical role in determining the minimum influent pH required for reactor recovery after failure. Having multiple CSTRs-in-series generally increases the overall removal efficiency for the influent conditions investigated. Monitoring of the influent feed quality is critical for plug-flow reactors, becasue failure of the initial sections of the reactor may cause a cascading effect that may lead to a rapid reactor failure. (c) 1995 John Wiley & Sons, Inc.     

Lipid specificity for membrane mediated partial unfolding of cytochrome c

In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes. From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c. Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme. Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E. coli cardiolipin. Experimnts with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.     

The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

The rapid accumulation of sequence data generated by the various genome sequencingprojects and the generation of expressed sequence tag databases has resulted in the need forthe development of fast and sensitive methods for the identification and characterisation oflarge numbers of gel electrophoretically separated proteins to translate the sequence data intobiological function. To achieve this goal it has been necessary to devise new approaches toprotein analysis: matrix-assisted laser desorption and electrospray mass spectrometry havebecome important protein analytical tools which are both fast and sensitive. When combinedwith a robotic system for the in-gel digestion of electrophoretically separated proteins, itbecomes possible to rapidly identify many proteins by searching databases with MS data. Thepower of this combination of techniques is demonstrated by an analysis of the proteins presentin the myofibrillar lattice of the indirect flight muscle of Drosophila melanogaster. Theproteins were separated by SDS-PAGE and in-gel proteolysis was performed bothautomatically and manually. All 16 major proteins could quickly be identified by massspectrometry. Although most of the protein components were known to be present in theflight muscle, two new components were also identified. The combination of methodsdescribed offers a means for the rapid identification of large numbers of gel separatedproteins.     

High rate anaerobic digestion of a petrochemical wastewater using biomass support particles

Anaerobic digestion of wastewater from a dimethyl terephthalate plant was studied in continuously stirred tank reactors with plastic net biomass support particles (BSP) at a level of 20% (v/v). The experimental results showed that the BSP system could treat the wastewater at a hydraulic retention time as low as 1.5 d, organic loading as high as 20 kg COD/m3/d and at acidic feed pH as low as 4.5 with 95% COD reduction and biogas production of about 8l/l/d, while the control system without support particles could not treat the wastewater above a 5-d hydraulic retention time, 5 kg COD/m3/d organic loading and a feed pH of 6.0. Thus, augmentation of BSP upgraded the performance of the conventional suspended growth system to an equivalent level to advanced reactors.     

Localization of the enzymes involved in H2 and formate metabolism inSyntrophospora bryantii

Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacteriumSyntrophospora bryantii contained high hydrogenase activities (8.5–75.8 µmol · min^–1 mg^–1 protein) and relatively low formate dehydrogenase activities (0.04–0.07 µmol · min^–1 mg^–1 protein). The K _M value and threshold value of the hydrogenase for H₂ were 0.21 mM and 18 µM, respectively, whereas the K _M value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 µM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO₂ reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H₂ is formed intracellularly while formate is formed at the outside of the cell.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

Dissociation of photoreceptors from whole heads of the fruit fly,Drosophila melanogaster

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple smash technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.     

Characterization of (Na+ + K+)-ATPase liposomes: II. Effect of α-subunit digestion on intramembrane particle formation and Na+,K+-transport

The effect of the protein structure of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ on its incorporation into liposome membranes was investigated as follows: the catalytic α-subunit of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ decrease progessively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight α-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with intact protein structure and digested, non functional enzyme consisting of fragments of the α-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$.     

Proteolytic cleavages of cytochalasin B binding components of Band 4.5 proteins of the human red blood cell membrane

The putative hexose transport component of Band 4.5 protein of the human erythrocyte membrane was covalently photolabelled with [³H]cytochalasin B. Its transmembrane topology was investigated by electrophoretically monitoring the effect of proteinases applied to intact erythrocytes, unsealed ghosts, and a reconstituted system. Band 4.5 was resistant to proteolytic digestion at the extracellular face of the membrane in intact cells at both high and low ionic strengths. Proteolysis at the cytoplasmic face of the membrane in ghosts or reconstituted vesicles resulted in cleave of the transporter into two membrane-bound fragments, a peptide of about 30 kDa that contained its carbohydrate moiety, and a 20 000 kDa nonglycosylated peptide that bore the cytochalasin B label. Because it is produced by a cleavage at the cytoplasmic face and because the carbohydrate moiety is known to be exposed to the outside, the larger fragment must cross the bilayer. It has been reported that the Band 4.5 sugar transporter may be derived from Band 3 peptides by endogenous proteolysis, but the cleavage pattern found in the present study differs markedly from that previously reported for Band 3. Minimization of endogenous proteolysis by use of fresh cells, proteinase inhibitors, immediate use of ghosts and omission of the alkaline wash resulted in no change in the incorporation of [³H]cytochalasin B into Band 4.5, and no labelling of Band 3 polypeptides. These results suggest that the cytochalasin B binding component of Band 4.5 is not the product of proteolytic degradation of a Band 3 component.     

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.