Metabolic,physiological and kinetic aspects of the alcoholic fermentation of whey permeate by Kluyveromyces fragilis NRRL 665 and Kluyveromyces lactis NCYC 571

Optimum growth conditions for the fermentation of non-concentrated whey permeate by Kluyveromyces fragilis NRRL 665 have been defined. Use of 3.75 g yeast extract l^?1, a growth temperature of 38°C and a pH of 4.0 allowed a maximum productivity of 5.23 g ethanol l^?1 h^?1 in continuous culture with a yield 91% of theoretical. Complete batch fermentation of permeate with 100 g lactose l^?1 was possible with a maximum specific growth rate of 0.276 h^?1 without any change in ethanol yield. Fermentation of concentrated permeate resulted, however, in a general decrease of specific substrate consumption rate, demonstrated by the inability to completely convert an initial 90 or 150 g lactose l^?1 in continuous culture, even at dilution rates as low as 0.05 and 0.08 h^?1, respectively. The decrease could be related to substrate inhibition, to an increase in osmotic pressure caused by lactose and salts, and to ethanol inhibition of both alcohol and biomass yield. The decrease in specific productivity could be counterbalanced by use of high cell density cultures, obtained by cell recycle of K. fragilis. Fermentation of a non-concentrated permeáte at a dilution rate of 1 h^?1 resulted in a productivity of 22 g l^?1 h^?1 at 22 g ethanol l^?1. Cell recycle using flocculating Kluyveromyces lactis NCYC 571 was also tested. With this strain a productivity of 9.3 g l^?1 h^?1 at 45 g product l^?1 was attained at a dilution rate of 0.2 h^?1, with an initial lactose concentration of 95 g l^?1.     

中缝大核在臂旁内侧核区注射吗啡所致呼吸抑制效应中的作用

实验在33只浅麻醉、肌肉麻痹、人工呼吸及切断双侧颈迷走神经的家兔上进行。观察中缝大核区电解损毁或微量注射利多卡因对呼吸活动及臂旁内侧核区微量注射吗啡所致呼吸抑制效应的影响。结果是:电解损毀中缝大核区,使呼吸频率增加,膈神经放电的幅度和频率均无明显变化,而臂旁内侧核区微量注射吗啡抑制呼吸的程度减轻;中缝大核区微量注射利多卡因,则部分消除臂旁内侧核区微量注射吗啡的呼吸抑制效应。中缝大核旁网状结构电解损毁或微量注射利多卡因,不影响吗啡的呼吸抑制效应。上述结果提示,中缝大核区可能在脑桥臂旁内侧核区微量注射吗啡抑制呼吸的机制中起一定作用。     

Cohesive sediment transport: emerging issues for toxic chemical management

The association of many environmentally sensitive chemicals and their transformation products with mineral and organic substrates is of considerable importance for environmental monitoring, prediction and management purposes in rivers and their basins. Our understanding of these relationships is poor. This paper reviews processes of particular concern, including the physical behaviour of fine-grained (< 63 µm) sediment in freshwater; the role of flocculation as a transport vector; the processes that control freshwater flocculation including microbiological factors; the uncertainty in conventional sediment transport models for predicting pathways of sediment-associated chemistry; the relationship between suspended sediment and toxicity in the water column; and the partitioning of chemicals between the sediment, organic and water phase, including the significance of these in predicting chemical transport on suspended matter.     

Potential relationship between glutathione metabolism and flocculation in the yeast Kluyveromyces lactis

Reduced glutathione (GSH) is involved in biochemical and physiological processes in cells. Flocculation is an important mechanism in microorganisms. The present study concerned the potential relationship between GSH metabolism and flocculation. Two yeast strains, a flocculent (Kluyveromyces lactis 5c) and a nonflocculent (Kluyveromyces lactis 5a) strain, were used. The level of intracellular GSH measured during the growth period was significantly higher in the nonflocculent than in the flocculent strain; in contrast, the flocculent strain exhibited brighter staining of vacuoles than the nonflocculent strain when observed using epifluorescence microscopy. Compounds acting either on flocculation (EDTA, galactose) or on GSH metabolism (buthionine sulfoximine, and N-acetylcysteine) were tested on the flocculent strain during the growth period. Both EDTA and galactose fully inhibited flocculation and induced GSH overproduction of 58% and 153%, respectively. Buthionine sulfoximine decreased GSH level by 76% but had no effect on flocculation; N-acetylcysteine increased the GSH level and flocculation by 106% and 41%, respectively. Combination of EDTA and N-acetylcysteine produced similar effects than with each of them. Combination of galactose and N-acetylcysteine increased the GSH level but decreased flocculation. These results demonstrated that GSH homeostasis is linked to the flocculation mechanism. A hypothesis related to stress is given.     

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell–cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg® led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg® of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg® crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg® crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.     

Construction of biofilms with defined internal architecture using dielectrophoresis and flocculation

A novel approach was developed for the construction of biofilms with defined internal architecture using AC electrokinetics and flocculation. Artificial structured microbial consortia (ASMC) consisting of localized layered microcolonies of different cell types were formed by sequentially attracting different cell types to high field regions near microelectrodes using dielectrophoresis. Stabilization of the microbial consortia on the electrode surface was achieved by crosslinking the cells using the flocculant polyethyleneimine (PEI). Consortia of Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae were made as model systems. Also, more natural consortia were made of the bacteria Pseudomonas putida, Clavibacter michiganense, and Methylobacterium mesophilum, which are found together in consortia during biodegradation of metal-cutting waste fluids.     

Viscoelastic evaluation of topical creams containing microcrystalline cellulose/sodium carboxymethyl cellulose as stabilizer

The purpose of this study was to examine the viscoelastic properties of topical creams containing various concentrations of microcrystalline cellulose and sodium carboxymethyl cellulose (Avicel(R) CL-611) as a stabilizer. Avicel CL-611 was used at 4 different levels (1%, 2%, 4%, and 6% dispersion) to prepare topical creams, and hydrocortisone acetate was used as a model drug. The viscoelastic properties such as loss modulus (G"), storage modulus (G'), and loss tangent (tan delta) of these creams were measured using a TA Instruments AR 1000 Rheometer and compared to a commercially available formulation. Continuous flow test to determine the yield stress and thixotropic behavior, and dynamic mechanical tests for determining the linear viscosity time sweep data, were performed. Drug release from the various formulations was studied using an Enhancer TM Cell assembly. Formulations containing 1% and 2% Avicel CL-611 had relative viscosity, yield stress, and thixotropic values that were similar to those of the commercial formulation. The elastic modulus (G') of the 1% and 2% formulation was relatively high and did not cross the loss modulus (G"), indicating that the gels were strong. In the commercial formulation, G' increased after preshearing and broke down after 600 seconds. The strain sweep tests showed that for all formulations containing Avicel CL-611, the G' was above G" with a good distance between them. The gel strength and the predominance of G' can be ranked 6% > 4% > 2%. The strain profiles for the 1% and 2% formulations were similar to those of the commercial formulation. The delta values for the 1% and 2% formulations were similar, and the formulations containing 4% Avicel CL-611 had lower delta values, indicating greater elasticity. Drug release from the commercial preparation was fastest compared to the formulations prepared using Avicel CL-611, a correlation with the viscoelastic properties. It was found that viscoelastic data, especially the strain sweep profiles of products containing Avicel CL-611 1% and 2%, correlated with the commercial formulation. Rheological tests that measure the viscosity, yield stress, thixotropic behavior, other oscillatory parameters such as G' and G" are necessary tools in predicting performance of semisolids.     

Effect of different starvation conditions on the flocculation of Saccharomyces cerevisiae

AIMS: To study the effect of different starvation conditions on the flocculation of an ale brewing yeast of Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was assessed by a micro-flocculation technique (Soares and Mota 1997). Carbon-starved cells of a NewFlo phenotype strain did not lose flocculation during a 48 h period. Cells incubated only in the presence of fermentable carbon sources (glucose, galactose and maltose at 2%, w/v), showed a progressive flocculation loss. The incubation of cells in 4% (v/v) ethanol did not induce a flocculation loss. The simultaneous incubation of cells in the presence of 2% (w/v) glucose and 15 microg ml(-1) cycloheximide hindered flocculation loss. The presence of 0.1 mmol l(-1) PMSF or 10 mmol l-1 EDTA prevented partially or completely, respectively, the loss of flocculation in the presence of glucose. CONCLUSIONS: Fermentable sugars induced a flocculation loss, which seems to require de novo protein synthesis and the involvement of different proteases. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the role of nutrients on the physiological control of yeast flocculation.     

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.     

乌勇布拉克盐湖嗜盐古菌絮凝效果筛选及活性检测

【目的】从嗜盐古菌中筛选可产生生物絮凝剂的菌株,对发酵液、上清液、菌悬液、胞外聚合物的絮凝作用进行检测,筛选能够适应高盐废水处理,且具有广谱盐度及pH作用范围的微生物絮凝剂。【方法】以新疆乌勇布拉克干盐湖沉积物为研究对象,利用纯培养方法对嗜盐古菌进行分离,对絮凝菌株进行初筛及16S rRNA基因测序,构建系统进化树,初步判断菌株分类地位;复筛检测不同生物材料的絮凝效果;选择絮凝效果较好的生物材料,检测其盐度、pH的絮凝效果稳定性。【结果】采用纯培养方法共分离到28株嗜盐古菌,絮凝初筛共筛选出16株嗜盐古菌,分布于碱线菌属(Natrinema)、盐缓长菌属(Halopiger)和盐土生菌属(Haloterrigena)。菌株发酵液、上清液、菌悬液、胞外聚合物具有不同程度的絮凝效果。菌株A279-1、A133、RP33、NGA0064、RM-152、A389的发酵液、上清液的絮凝效果较好,其中菌株A389的发酵液絮凝率为61.06%,上清液为67.92%。所有菌株菌悬液的絮凝率达到80%以上。菌株所产胞外聚合物表现出较好的絮凝效果,菌株RM-152所产胞外聚合物的絮凝率最高,达89.86%,其次是A389 (81.53%)。菌株A389所产胞外聚合物的产量最大,达12.53 g/L,具有广泛的盐度和pH适应性。【结论】乌勇布拉克干盐湖沉积物中蕴含丰富的可产生微生物絮凝剂的嗜盐古菌资源。嗜盐古菌菌株发酵液、上清液、菌悬液及胞外聚合物均具有良好的絮凝作用,尤其是胞外聚合物表现出较好的絮凝效果,具有广谱的盐度和pH耐受性。嗜盐古菌所产生物絮凝剂的发现对于后续高盐废水功能材料开发具有重要应用价值。