Comparison of glucose fermentation by suspended and gel-entrapped yeast cells: An in vivo nuclear magnetic resonance study

Phosphorus-31 nuclear magnetic resonance ((31)P NMR) was used to compare the anaerobic metabolism of glucose by suspended and gel-entrapped Saccharomyces bayanus cells. The fermentation of glucose was carried out in a reaction system with continuous circulation through the NMR sample tube. The intracellular pH and the levels of some phosphorylated compounds were the levels of some phosphorylated compounds were noninvasively monitored by (31)P NMR while glucose, fermentation products, and biomass were determined by analytic techniques comparisons showed that no significant differences are observed in the relative concentrations in the spectra, but distinct profiles for the variation of both intracellular and extracellular pH are found. The internal pH of immobilized cells is maintained at a constant value throughout the fermentation as opposed to freely suspended cells for which a steady decrease in the internal pH occurs. A faster and stronger acidification is also observed in the external medium of the assays with suspended cells. Furthermore, higher yields for ethanol and biomass production and lower yields of fermentation by-products are obtained with immobilized cells. It is concluded that the higher intracellular pH achieved in the presence of the gel matrix had a regulatory effect on the metabolism which favored the ethanol production pathway. (c) 1993 John Wiley & Sons, Inc.     

Growth measurements of terrestrial microbial species by a continuous-flow technique

A continuous nutrient flow system has been developed to measure microbial activity in soil with various concentrations of added substrate. The system consists of a thin soil layer through which substrate was added continuously over periods up to 4.5 days. Substrate utilization was determined by effluent analysis. Respiration was measured manually by injecting a sample into a gas chromatograph or automatically by coupling the growth chamber to a computer-controlled gas sampling valve. This permitted respiratory CO₂ to be measured by the gas chromatograph at intervals selected by the investigator. Software controlling the valve and gas chromatograph not only automated gas phase sampling, but also provided a scan of CO₂ evolution and a preliminary data summary. This included the date and time of sample, peak height, and percent CO₂ in the gas phase. Data for growth on glucose using a microbial population native to a California annual grassland soil demonstrated that the direct cell count and respiratory techniques for biomass estimation give comparable results. This procedure provides the potential for detailed analyses of substrate utilization in studies of the growth and maintenance of soil microorganisms.     

微流控振荡流PCR快速检测单增李斯特氏菌

一种高通量振荡流PCR微流控技术已被成功开发,借此来快速检测食品致病菌-单增李斯特氏菌(L.monocytogenes).该PCR微流控装置主要由基于LabView的温度控制与采集系统、三个铜加热块以及聚四氟乙烯(PTFE)毛细管等构成.在该微流控装置上,三个铜块维持PCR反应所需要的三个温度,而包埋在铜块沟槽中的PT...     

Microbiological hydrogen (H2) thresholds in anaerobic continuous-flow systems: Effects of system characteristics

Hydrogen (H₂) concentrations that were associated with microbiological respiratory processes (RPs) such as sulfate reduction and methanogenesis were quantified in continuous-flow systems (CFSs) (e.g., bioreactors, sediments). Gibbs free energy yield (ΔǴ ~ 0) of the relevant RP has been proposed to control the observed H₂ concentrations, but most of the reported values do not align with the proposed energetic trends. Alternatively, we postulate that system characteristics of each experimental design influence all system components including H₂ concentrations. To analyze this proposal, a Monod-based mathematical model was developed and used to design a gas–liquid bioreactor for hydrogenotrophic methanogenesis with Methanobacterium bryantii M.o.H. Gas-to-liquid H₂ mass transfer, microbiological H₂ consumption, biomass growth, methane formation, and Gibbs free energy yields were evaluated systematically. Combining model predictions and experimental results revealed that an initially large biomass concentration created transients during which biomass consumed [H₂]_L rapidly to the thermodynamic H₂-threshold (≤1 nM) that triggerred the microorganisms to stop H₂ oxidation. With no H₂ oxidation, continuous gas-to-liquid H₂ transfer increased [H₂]_L to a level that signaled the methanogens to resume H₂ oxidation. Thus, an oscillatory H₂-concentration profile developed between the thermodynamic H₂-threshold (≤1 nM) and a low [H₂]_L (~10 nM) that relied on the rate of gas-to-liquid H₂-transfer. The transient [H₂]_L values were too low to support biomass synthesis that could balance biomass losses through endogenous oxidation and advection; thus, biomass declined continuously and disappeared. A stable [H₂]_L (1807 nM) emerged as a result of abiotic H₂-balance between gas-to-liquid H₂ transfer and H₂ removal via advection of liquid-phase.     

Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.     

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~ 27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency > 90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.     

An automated continuous-flow dynamic dialysis technique for investigating protein-ligand binding

An automated, continuous-flow dynamic dialysis technique has been developed to investigate protein-ligand binding. The method depends on a comparison of the diffusion of the low molecular mass ligand, in the presence and absence of protein, through a semipermeable membrane. The ligand passes from the sample compartment of a dialysis cell into the sink compartment through which a constant flow of eluting buffer is maintained. Digitized spectrophotometric determinations of the ligand concentration in the eluting buffer at successive, equally spaced time intervals, punched onto paper tape, provide the primary data (normally about 1000 data points). A mathematical treatment of the data based on a model of the diffusion system, whereby the protein-ligand binding isotherm may be evaluated, is discussed. The validity of the method is demonstrated from studies of the binding of phenol red to bovine serum albumin (BSA) at 15, 20, and 25°C. The method yields a large number of points on the binding isotherm (usually several hundred) which, in terms of a Scatchard model, provide values for the number of binding sites on the BSA molecule and binding constants for the phenol red-BSA interaction. The results obtained are consistent with values reported in the chemical literature but which are based on much scantier data.     

Experimental Approach to the Role of Different Ecological Types of Protozoa in the Activated-sludge System

Relationship between bacteria and ciliates was investigated in continuous cultures with and without the recycling of flocculated biomass. Monocultures of ciliates of various ecological types were used together with mixed cultures of bacteria or bacteria and flagellates. The effect of ciliates on the distribution of flocculated and dispersed, biomass was tested with respect to characterizing an optimum mixed culture for activated-sludge treatment. Monoprotozoal populations were not successful in stabilizing the morphology of a bacterial community in a system with recycling of flocculated biomass. Stabilization was observed (with particular ecological types of ciliates) in the system with a high residence time without recycling and/or in the system with the recycling inoculated by both flagellates and ciliates.     

连续流动式PCR微流控装置的研究

介绍了基于薄膜加热器的新型连续流动式PCR微流控装置的设计与制作;讨论了退火温度、PCR反应试剂(引物、Mg2 、dNTPs以及Taq DNA聚合酶)浓度以及PCR溶液的流动速度等对连续流动式PCR反应的影响;结果发现反应试剂影响连续流动式微流控PCR扩增的行为不同于它们影响传统PCR的行为,在较宽的浓度范围内都不会引起非特异性扩增。除此之外,在15 min内能成功对249 bp的人类β-肌动蛋白基因进行扩增,扩增速度比传统PCR快;通过低热容量的薄膜加热器来维持三个温度区带的恒温,完成33个循环的连续流动式PCR扩增能量消耗小于0.0088 kW.h,比传统PCR仪低得多,新研制的PCR微流控装置有可能成为便携式装置。