Voltage clamping with a single microelectrode

A technique is described which allows neurons to be voltage clamped with a single microelectrode, and the advantages of this circuit with respect to conventional bridge techniques are discussed. In this circuit, the single micro electrode is rapidly switched from a current passing to a recording mode. The circuitry consists of: (1) an electronic switch; (2) a high impedance, ultralow input capacity amplifier; (3) a sample-and-hold module; (4) conventional voltage clamping circuitry. The closed electronic switch allows current to flow through the electrode. The switch then opens, and the electrode is in a recording mode. The low input capacity of the preamplifier allows the artifact from the current pulse to rapidly abate, after which time the circuit samples the membrane potential. This cycle is repeated at rates up to 10 kHz. The voltage clamping amplifier senses the output of the sample-and-hold module and adjusts the current pulse amplitude to maintain the desired membrane potential. The system was evaluated in Aplysia neurons by inserting two microelectrodes into a cell. One electrode was used to clamp the cell and the other to independently monitor membrane potential at a remote location in the soma.     

Influence of a swirl motion on the interaction between microalgal cells and environmental medium during ultrafiltration of a culture of Tetraselmis suecica

The role of the environmental medium of a marine microalga culture (Tetraselmis suecica) in membrane fouling phenomenon is quantified for two ultrafiltration units: one generating a classical tangential plane flow and another involving a swirling decaying flow induced by a tangential inlet. Compared to the plane unit, the swirling configuration increases the performances of the ultrafiltration process (augmentation of 20% in terms of limiting flux). Interactions between the culture medium and cells depend on the module design. Furthermore, experiments emphasize the significant role of the particles whatever the module design: more than 70% of the total hydraulic resistance could be due to the microalgal cells, while about 30% of membrane fouling is relevant to soluble materials.     

Comparison of microporous and nonporous membrane bioreactor systems for the treatment of BTEX in vapor streams

Increased regulatory constraints on industrial releases of atmospheric volatile organic compounds (VOCs) have resulted in an interest in using biofilters, bioscrubbers and air/liquid membranes for treatment of vapor phase waste streams. In this report, we describe the comparison of the use of two fundamentally different types of membrane module systems that allow the rapid diffusion of vapor phase aromatics and oxygen to an active biofilm for subsequent biodegradation. One system used a commercial membrane module containing microporous polypropylene fibers while the other used a nonporous silicone tubing membrane module for the delivery of substrate (a mixture of benzene, ethylbenzene, toluene, and xylenes [BTEX]) and electron acceptor (O₂). Tests of the systems under similar conditions with BTEX in the vapor feed stream showed significant performance advantages for the silicone membrane system. The average surface-area-based BTEX removal rate for the microporous membrane system over 500 h of operation was 7.88 μg h⁻¹ cm⁻² while the rate for the silicone membrane system was 23.87 μg h⁻¹ cm⁻². The percentages of BTEX removal were also consistently better in the silicone membrane system versus the microporous system. Part of the performance problem associated with the microporous membrane system appeared to be internal water condensation and possible plugging of the pores with biomass over time that could not be resolved with vapor phase backflushing. Journal of Industrial Microbiology & Biotechnology (2002) 28, 245–251 DOI: 10.1038/sj/jim/7000235 Received 17 August 2001/ Accepted in revised form 03 December 2001     

Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor

Surfactin and fengycin are lipopeptide biosurfactants produced by Bacillus subtilis. This work describes for the first time the use of bubbleless bioreactors for the production of these lipopeptides by B. subtilis ATCC 21332 with aeration by a hollow fiber membrane air–liquid contactor to prevent foam formation. Three different configurations were tested: external aeration module made from either polyethersulfone (reactor BB1) or polypropylene (reactor BB2) and a submerged module in polypropylene (reactor BB3). Bacterial growth, glucose consumption, lipopeptide production, and oxygen uptake rate were monitored during the culture in the bioreactors. For all the tested membranes, the bioreactors were of satisfactory bacterial growth and lipopeptide production. In the three configurations, surfactin production related to the culture volume was in the same range: 242, 230, and 188 mg l⁻¹ for BB1, BB2, and BB3, respectively. Interestingly, high differences were observed for fengycin production: 47 mg l⁻¹ for BB1, 207 mg l⁻¹ for BB2, and 393 mg l⁻¹ for BB3. A significant proportion of surfactin was adsorbed on the membranes and reduced the volumetric oxygen mass transfer coefficient. The degree of adsorption depended on both the material and the structure of the membrane and was higher with the submerged polypropylene membrane.     

The modular nature of the β‐barrel assembly machinery,illustrated in Borrelia burgdorferi

Diderm bacteria have an outer membrane that provides defense against environmental factors including antibiotics. Understanding the process of outer membrane biogenesis is, therefore, of critical importance in order to envisage new treatments of these bacterial pathogens. Borrelia burgdorferi is the pathogen responsible for Lyme disease. Its outer membrane contains integral, β‐barrel proteins as well as swathes of externally exposed lipoproteins. Previous work has demonstrated that the β‐barrel assembly machine (BAM complex) in B. burgdorferi and other Spirochetes shares several similarities with the BAM complex in other bacterial lineages, such as the Proteobacteria that includes Escherichia coli. However, Iqbal et al. ( 2016 ) have identified the inner membrane protein TamB as a subunit of the BAM complex in Spirochetes. This latest study highlights the modular nature of the BAM complex, and suggests that in some bacterial lineages the BAM complex and translocation and assembly module (the TAM) function as a single unit.     

C6-Like and C3-Like Molecules from the Cephalochordate, Amphioxus, Suggest a Cytolytic Complement System in Invertebrates

The mammalian immune system has cytotoxic mechanisms, both cellular and humoral, that destroy the membrane integrity of target cells. The main effector molecules of these cytolytic mechanisms—perforin, used by killer lymphocytes, and the membrane attack complex (MAC) components of the complement system—share a unique module called the MAC/perforin module. Until now, both immunological cytotoxicity and the MAC/perforin module have been reported only in jawed vertebrates. Here, we report the identification of a protein containing the MAC/perforin module from the invertebrate cephalochordate, amphioxus (Branchiostoma belcheri), using expressed sequence tag (EST) analysis of the notochord. The deduced amino acid sequence of this molecule is most similar to the primary structure of human complement component C6 and is designated AmphiC6. AmphiC6 shares a unique modular structure, including the MAC/perforin module, with human C6 and other MAC components. Another EST clone predicts the presence of a thioester-containing protein with the closest structural similarity to vertebrate C3 (therefore designated AmphiC3). AmphiC3 retains most of the functionally important residues of vertebrate C3 and is shown by phylogenetic analysis to be derived directly from the common ancestor of vertebrate C3, C4, and C5. Only opsonic activity has been assigned to the invertebrate complement system until now. Therefore, this is the first molecular evidence for complement-mediated immunological cytotoxicity in invertebrates. Received: 24 August 2001 / Accepted: 12 November 2001     

Preparation of a mesoporous silica quorum quenching medium for wastewater treatment using a membrane bioreactor

Abstract

Various quorum quenching (QQ) media have been developed to mitigate membrane biofouling in a membrane bioreactor (MBR). However, most are expensive, unstable and easily trapped in hollow fibre membranes. Here, a sol-gel method was used to develop a mesoporous silica medium entrapping a QQ bacterial strain (Rhodococcus sp. BH4). The new silica QQ medium was able to remove quorum sensing signalling molecules via both adsorption (owing to their mesoporous hydrophobic structure) and decomposition with an enzyme (lactonase), preventing MBR biofouling without affecting the water quality. It also demonstrated a relatively long life span due to its non-biodegradability and its relatively small particle size (<1.0?mm), which makes it less likely to clog in a hollow fibre membrane module.     

The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection

Verticillium dahliae is a soil-borne fungus that causes vascular wilt on numerous plants worldwide. The fungus survives in the soil for up to 14 years by producing melanized microsclerotia. The protective function of melanin in abiotic stresses is well documented. Here, we found that the V. dahliae tetraspan transmembrane protein VdSho1, a homolog of the Saccharomyces cerevisiae Sho1, acts as an osmosensor, and is required for plant penetration and melanin biosynthesis. The deletion mutant ΔSho1 was incubated on a cellophane membrane substrate that mimics the plant epidermis, revealing that the penetration of ΔSho1 strain was reduced compared to the wild-type strain. Furthermore, VdSho1 regulates melanin biosynthesis by a signalling mechanism requiring a kinase-kinase signalling module of Vst50-Vst11-Vst7. Strains, ΔVst50, ΔVst7 and ΔVst11 also displayed defective penetration and melanin production like the ΔSho1 strain. Defects in penetration and melanin production in ΔSho1 were restored by overexpression of Vst50, suggesting that Vst50 lies downstream of VdSho1 in the regulatory pathway governing penetration and melanin biosynthesis. Data analyses revealed that the transmembrane portion of VdSho1 was essential for both membrane penetration and melanin production. This study demonstrates that Vst50-Vst11-Vst7 module regulates VdSho1-mediated plant penetration and melanin production in V. dahliae, contributing to virulence.     

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin‐like protein

Various aerolysin‐like pore‐forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin‐like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β‐prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high‐mannose glycans triggers drastic conformational changes of the aerolysin module in a pH‐dependent manner, ultimately resulting in the formation of a membrane‐bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore‐forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish‐specific defense molecule.     

Sequence analysis of Arabidopsis thaliana E/ANTH-domain-containing proteins: membrane tethers of the clathrin-dependent vesicle budding machinery

Summary. The epsin N-terminal homology (ENTH) domain is a conserved protein module present in cytosolic proteins which are required in clathrin-mediated vesicle budding processes. A highly similar, yet unique module is the AP180 N-terminal homology (ANTH) domain, which is present in a set of proteins that also support clathrin-dependent endocytosis. Both ENTH and ANTH (E/ANTH) domains bind to phospholipids and proteins, in order to support the nucleation of clathrin coats on the plasma membrane or the trans-Golgi-network membrane. Therefore, E/ANTH proteins might be considered as universal tethering components of the clathrin-mediated vesicle budding machinery. Since the E/ANTH protein family appears to be crucial in the first steps of clathrin-coated vesicle budding, we performed data base searches of the Arabidopsis thaliana genome. Sequence analysis revealed three proteins containing the ENTH signature motif and eight proteins containing the ANTH signature motif. Another six proteins were found that do not contain either motif but seem to have the same domain structure and might therefore be seen as VHS-domain-containing plant proteins. Functional analysis of plant E/ANTH proteins are rather scarce, since only one ANTH homolog from A. thaliana, At-AP180, has been characterized so far. At-AP180 displays conserved functions as a clathrin assembly protein and as an α-adaptin binding partner, and in addition shows features at the molecular level that seem to be plant-specific. Correspondence and reprints: Cell Biology, Heidelberg Institute for Plant Sciences, Im Neuenheimer Feld 230, 69120 Heidelberg, Federal Republic of Germany.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

Continuous production of extracellular ultrafine calcite particles by the marine coccolithophorid alga Pleurochrysis carterae

A new procedure for the production of ultrafine calcite particles by the marine coccolithophorid alga Pleurochrysis carterae is reported. During continuous culture, calcite particles (coccoliths) were detached from the cell surface by optimized air-bubbling, which greatly reduced the damage associated with previous sonication methods. Detached calcite particles could be continuously recovered directly from the culture medium using a nylon mesh membrane filtration module. Cells remained viable and continued to produce coccoliths during culture. The optimum productivity of ultrafine calcite particles was 18 mg/l per day. These results demonstrate the potential for a continuous system for the photosynthetically driven removal of CO₂ and its fixation into ultrafine inorganic calcite particles. Correspondence to: T. Matsunaga     

Control of ethanol production and monitoring of membrane performance by mass-spectrometric gas analysis in the coupled fermentation-pervaporation of whey permeate

A coupled fermentation-pervaporation process was operated continuously with on-line mass spectrometric gas analysis monitoring of product accumulation on both the upstream and the downstream sides of the membrane. Efficient coupling of the fermentation with pervaporation was attained when a steady state of ethanol production and removal was achieved with whey permeate containing high concentrations of lactose (>8%) or by controlled lactose additions that also compensated for loss of liquid due to pervaporation. The combined system consists of a tubular membrane pervaporation module, directly connected to a stirred fermentor to form one circulation loop, kept at 38°C, with both units operating under computer control. Mass spectrometric gas analysis of the CO₂ gas evolved in the fermentor and the ethanol and water in the pervaporate on the downstream side of the membrane enabled us to follow the production of ethanol and its simultaneous removal. Membrane selectivity was calculated on-line and served to monitor the functioning of the membrane. Batch-wise-operated fermentation-pervaporation with Candida pseudotropicalis IP-513 yielded over 120 gl^–1 of concentrated ethanol solution using supplemented whey permeate containing 16% lactose. A steady state lasting for about 20 h was achieved with ethanol productivity of 20 g h^–1 (approx. 4 g l^–1 h^–1). Membrane selectivity was over 8. Controlled feeding of concentrated lactose suspension in the whey permeate (350 g l^–1) resulted in the continuous collection of 120–140 g l^–1 of ethanol pervaporate for 5 days, by which time salt accumulation hampered the fermentation. Medium refreshment restored the fermentative activity of the yeast cells and further extended the coupled process to over 9 days (200 h), when reversible membrane fouling occurred. The membrane module was exchanged and the combined process restarted. Correspondence to: Y. Shabtai     

Extracellular K5 polysaccharidic antigen recovery performed by different cell separation technologies

The K5 polysaccharidic antigen obtainable from a strain of Escherichia coli is the non-sulphated precursor in heparin biosynthesis; K5 is composed by two components, 16000 and 1500 Da, their ratio depending on the fermentation conditions. In this study an assessment was made of how various cell-culture filtrate separation technologies affected the components themselves and their ratio. A dynamic membrane filtration technology, a tubular ceramic module for tangential flow microfiltration and the standard discontinuous centrifugation were comparatively employed to perform the separation. Experiments carried out on E. coli cultures containing the two components in different ratios showed that the DMF system is a suitable technology for K5 isolation.     

1H, 13C and 15N resonance assignments of the rhodanese domain of YgaP from Escherichia coli

Rhodanese domain is a ubiquitous structural module commonly found in bacterial, archaeal and eukaryotic cells. Growing evidence indicates that rhodanese domains act as the carrier of reactive sulfur atoms by forming persulfide intermediates in distinct metabolic pathways. YgaP, a membrane protein consisting of a rhodanese domain and a C-terminal transmembrane segment, is the only membrane-associated rhodanese in Escherichia coli. Herein, we report the resonance assignments of ¹H, ¹³C and ¹⁵N atoms of rhodanese domain of YgaP. Totally, chemical shifts of more than 95% of the atoms were assigned.     

Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000×g, followed by 90,000×g and finally at 150,000×g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000×g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000×g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO₂ fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.     

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida,PmHS1

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

     

Bovine serum albumin-hemoglobin fractionation: significance of ultrafiltration system and feed solution characteristics

This work investigates the fractionation of similar molecular weight proteins bovine serum albumin (69 kD) and bovine hemoglobin (67 kD) by ultrafiltration. Three different membranes, viz. regenerated cellulose, poly(sulfone) and surface modified poly(acrylonitrile), each with a nominal molecular cutoff rating of 100 kD, were examined. The experiments were conducted in dead end, crossflow and vortex flow filtration modes and the separation was studied as a function of feed pH and ionic strength. Under similar system hydrodynamics, the surface modified poly(acrylonitrile) membrane displayed the highest resolution with minimum membrane fouling. The separation could be improved further by operating at low applied pressure (40 kPa) and high mass transfer (> 20 × 10^–6 m/s) in a vortex flow module. Under these conditions, the highest separation factor of 40 was obtained at the pI of hemoglobin.     

Characteristics of cross-flow filtration of pullulan broth

Cross-flow filtration of culture broth from Aureobasidium pullulans, which elaborates pullulan, was done with a thin channel-type module and microfiltration membranes made of different materials and with different pore sizes. Various factors affecting the results of the filtration were studied. The specific resistance of the microbial cake was found to be higher than that of bakers yeast, the cells of which are about the same size as an A. pullulans cell, and resistance increased with cultivation time. The flux and transmission of pullulan through the membrane decreased with cultivation time as the specific resistance increased. The flux and transmission ] of pullulan depended on the structure and pore size of the membrane and also on the pH of the broth. With a polysulphone membrane with a nominal pore size of 2.0 m, transmission was nearly 100% with negligible leakage of cells and the flux was high when the pH of the broth was adjusted to 2.0.On leave from Hayashibara Co., Ltd., Amase-minamimachi, Okayama 700 Japan Correspondence to: K. Nakanishi     

Ethanol fermentation coupled with complete cell recycle pervaporation system: Dependence of glucose concentration

Summary A membrane bioreactor system comprised of a fermenter and a flat pervaporation module was developed for continuous ethanol fermentation by Saccharomyces cerevisiae. In order to obtain the guidelines for high sugar concentration fermentation, the dependence of glucose concentration on the coupled system was investigated. Fed by 158 and 290g glucose/l, the improvement in productivity was obtained with 1. 58 and 1. 86 times, and the ethanol yield was 0. 45 and 0. 395, respectively. With the fermentation proceeding, the permeate flux decreased but the selectivity kept unaltered.