New concepts for rapid yeast settling. II. pH switching with an inert powder

A new technique is outlined for the rapid settling of yeast cells in fermentation media. The technique involved the addition of dense, inert particles (nickel powder) to a yeast suspension (Saccharomyces cerevisiae) at pH 4.5 and a rapid change of pH to 8.0-9.0. When the pH was changed large flocs formed immediately and settled rapidly, leaving a clear supernatant. On returning the pH to 4.5 the flocs were destroyed. This technique gave larger flocs and higher settling rates than the constant pH method, and much lower nickel/yeast ratios were required. Good flocculation also occurred in a fermentation medium. The technique was used to recycle yeast cells to a semicontinuous ethanol fermentation. Application of the technique to this and similar systems is discussed. The factors affecting yeast/inert powder flocculation are also discussed and a model is proposed to explain the observed experimental behavior for flocculation with a rapid change in pH.     

Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.     

Harvesting Euglena fracilis cells with a nontoxic flocculant

Practical criteria for the usefulness of an algal separation process for laboratory routine are effectiveness and time consumption. We tested the feasibility of a flocculation procedure for harvesting a large volume of Euglena gracilis culture. This procedure turned out to be a technically viable system for avoiding tedious centrifugation and preserving Euglena flagellar apparatus integrity.Euglena cultures were treated with chitosan, a byproduct derived from chitin, the major component of the exoskeleton of crustaceans. Since chitosan carries a positive chrge, it functions as a polycationic coagulating agent by adsorbing onto particles in suspension and by bridging into agglomerates or flocs. A 96–98% reduction in the number of suspended cells was obtained in the cultures with 200 mg/l of chitosan at pH 7.5.     

Flocculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation.

The phenomena of flocculation and floc formation by Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum lipoferum Sp59b (ATCC 29707) were studied in aerobic liquid cultures. Carbon sources representative of various entry pathways in combination with various nitrogen sources induced flocculation in both species of azospirilla. Noticeably, the combination of fructose and nitrate was the most effective in terms of floc yields. Phase-contrast microscopic observations revealed a transition in cell morphology from freely motile, vibrioid cells to nonmotile, highly refractile encysting forms during the formation of flocs. The nonmotile forms in flocs appeared to be entangled within a fibrillar matrix, and the cells were highly resistant to desiccation. Dried flocs kept for almost 6 months still maintained the highly refractile encysting forms, and their viability was confirmed by pellicle formation and acetylene reduction in semisolid malate medium. Electron microscopic observations of the desiccated flocs revealed the presence of cell forms containing abundant poly beta-hydroxybutyrate granules within a central body and surrounded by a thick layer of exopolysaccharides. The latter were characterized by alkali and acid digestion, crude cellulase hydrolysis, and calcofluor staining. It was concluded that the overproduction of exocellular polymers induces the flocculent growth and is associated with the concomitant transformation of vegetative cells to the desiccation-resistant encysting forms under limiting cultural conditions.     

Deoxyribonuclease-susceptible Floc Forming Pseudomonas sp.

A bacterium belonging to Pseudomonas which was isolated from activated sludge formed flocs in glycerol-containing medium. The flocs were deflocculated by deoxyribonuclease treatment in the presence of magnesium ions. Flocs were also deflocculated by 2 m NaCl, heating at temperatures higher than 50°C, and at pH below 1 or above 11. The observations suggest that deoxyribonucleic acid is directly involved in the association of cells and that ionic bonds are responsible for the flocculation of cells.     

Modeling brewers' yeast flocculation

Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.     

New concepts for rapid yeast settling. I. Flocculation with an inert powder

A novel technique for settling microorganisms has been described. The technique involves adding a dense, inert powder to a suspension of microorganisms under conditions where flocculation of the microorganism with the inert poweder occurs. The flocs formed are small and relatively dense and settle rapidly. Suspensions of Saccharomyces cerevisiae yeast have been flocculated with several different inert seed materials achieving rapid settling and separations of up to 99.9%. Nickel powder was used as a seed material for most experiments described here, and iron sand showed promise as a cheaper seed for large-scale use. The degree of flocculation and cell separation obtained depended largely on the seed concentration and the components in solution. Temperature and pH had little effect. When the method was initially applied to a practical fermentation, flocculation was poor because of inhibiting compounds in the fermentation medium, but modification of the technique produced good flocculation in the medium.     

Recovery of bacteria from growth media by starch-dependent floc formation

Addition of starch to suspensions of Escherichia coli K-12 resulted in the formation of bacterial flocs. The flocculation was dependent on the high expression of a receptor for starch (maltoporin) on the surface of the bacterium. Factors influencing floc formation were investigated and optimal conditions for flocculation based on cell density, starch concentration, time, and pH established. As quantitated by a sedimentation assay, over 80% of bacteria in a culture could be removed by settling without centrifugation in 3 h under optimal conditions. Floc formation was evident with bacteria containing wild-type maltoporin but was faster and occurred to a greater extent with strains expressing a high-affinity allele (lamB1400) of the starch receptor. Bacteria could be harvested by floc formation directly in growth medium under defined conditions of maltoporin expression and medium composition. These results demonstrate the effectiveness of starch-dependent aggregation in the harvesting of cells, using an inexpensive, biologically acceptable agent to induce flocculation.     

Preparation and performance of immobilized yeast cells in columns containing no inert carrier

Schizosaccharomyces pombe was cultivated in a medium of glucose (10 g/L) malt extract (3 g/L), yeast extract (3 g/L), and bactopeptone (5 g/L) to form flocs. More than 95% of the cell population were flocculated. Variation in glucose concentration (from 10 to 100 g/L) did not affect flocculation. Yeast extract helped induce flocculation. Application of the immobilized yeast for the continuous production of ethanol was tested in a column reactor. Soft yeast flocs (50-200 mesh) underwent morphological changes to heavy particles (0.1-0.3 cm diameter) after continuously being fed with fresh substrates in the column. Productivity as high as 87 g EtOH L(-1) h(-1) was obtained when a 150 g/L glucose medium was fed. The performance of this yeast reactor was stable over a two-month period. The ethanol yield was 97% of the theoretical maximum based upon glucose consumed.     

Yeast flocculation: factors affecting the measurement of flocculence

The physical meaning of the residual absorbance of a yeast suspension after flocculation and settling has been investigated. Starting with a dispersed suspension, agitation accelerates flocculation by increasing the probability of collision between particles. As flocculation advances, agitation also breaks the flocs. A stationary state is reached when flocculence (tendency to flocculate) is counterpoised by agitation. If the intensity of agitation is maintained constant, the free cell concentration reflects the flocculence, provided the stationary state is reached. The residual absorbance, determined after settling of the flocs, is a measure of the free cell concentration and represents an adequate parameter to characterize yeast flocculence.     

Involvement of Ca2+ in the flocculation of Kluyvera cryocrescens KA-103

Kluyvera cryocrescens KA-103 showed a dispersed growth in Ca²⁺-free Polypepton medium, but formed flocs on addition of a sufficient concentration of Ca²⁺ to the bacterial cell suspension. Therefore, calcium adsorption properties and flocculation conditions were investigated using bacterial cells cultured in the Ca²⁺-free Polypepton medium. The bacterium required 1.5 mM Ca²⁺ or more for good flocculation (F>90%), but a cooperative effect of Na⁺ and Ca²⁺ on good flocculation was observed at lower concentrations of Ca²⁺. The Langmuir adsorption isotherm was used to describe the adsorption of Ca²⁺ by the bacterial cells.     

Flocculation and its effect on the vertical transport of fine-grained sediments

Recent experimental and theoretical work on flocculation and settling speeds of flocs is reviewed. On the basis of this work, an accurate and computationally efficient model of the aggregation and disaggregation of fine-grained sediments is proposed. This model is then used to predict flocculation times and steady-state floc sizes for a wide range of environmental conditions. The predicted flocculation times are smaller, sometimes by as much as two orders of magnitude, than those predicted by mono-disperse theory. The model is also used to show that the disaggregation of flocs due to increased shear near the sediment-water interface may be a possible mechanism for the increased concentrations often observed near this interface.     

Quantification of brewers' yeast flocculation in a stirred tank: effect of physical parameters on flocculation

Quantification of yeast flocculation under defined conditions will help to understand the physical mechanisms of the flocculation process used in beer fermentation. Flocculation was quantified by measuring the size of yeast flocs and the number of single cells. For this purpose, a method to measure floc size and number of single cells in situ was developed. In this way, it was possible to quantify the actual flocculation during fermentation, without influencing flocculation. The effects of three physical parameters, floc strength, fluid shear, and yeast cell concentration, on flocculation during beer fermentation, were examined. Increasing floc strength results in larger flocs and lower numbers of single cells. If the fluid shear is increased, the size of the flocs decreases, and the number of single cells remains constant at approximately 10% of the total cells present. The cell concentration also influences flocculation, a reduction of 50% in cell concentration leads to a decrease of about 25% in floc size. The number of single cells decreases in linear proportion to the cell concentration. This means that, during yeast settling at full scale, the number of single cells decreases. The results of this study are used in a model for yeast flocculation. With respect to full scale fermentation the effect of cell concentration will play an important role, for flocculation and sedimentation will occur simultaneously leading to a quasi steady state between these phenomena. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 190-200, 1997.     

Genetic control of flocculation inEscherichia coli

Summary Escherichia coli cells form flocs or aggregates by overproducing type 1 pili. When thepil operon is placed under the control of atac orlac promoter-operator sequence, the bacterial cells can be induced to form flocs by adding isopropyl--d-thiogalactopyranoside to the culture medium. This phenomenon of genetically induced flocculation can aid in the downstream processing of biological products. This paper describes the construction of two artificially controlled plasmids which cause cell flocculation. Cell aggregates 50 m in mean diameter were obtained 1 h after the cells were induced.     

絮凝菌株Shinella sp. xn-1对铜绿微囊藻的絮凝效果

【目的】研究絮凝功能细菌xn-1对有害水华藻——铜绿微囊藻的絮凝效果,以期为有害水华的治理提供新的选择。【方法】采用涂布划线法从藻际分离纯化絮凝功能微生物;基于16S r RNA基因序列确定进化地位;通过不同金属离子确定絮凝机制;梯度醇沉法获得絮凝物质;以酶标仪测定絮凝效率。【结果】菌株xn-1确定属于申氏杆菌属(Shinella),且命名为Shinella sp.xn-1。在添加Ca~(2+)作为促凝剂的条件下对铜绿微囊藻表现出高效的絮凝效果,其絮凝效果主要来源于胞外上清,而表现出高效的絮凝效果所需要的胞外上清添加量为3.0%。从胞外上清中获得的絮凝物质以0.5 g/L的添加量作用于藻细胞后表现出高效的絮凝效果,且随着处理时间增加,絮凝团的体积增大。【结论】Shinella sp.xn-1通过分泌胞外絮凝物质对铜绿微囊藻表现出高效的絮凝效果,在絮凝作用下藻细胞聚集在一起形成大体积的絮凝团,该研究有利于治理有害水华。     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.     

The use of laminar tube flow in the study of hydrodynamic and chemical influences on polymer flocculation of Escherichia coli

The optimization of microbial flocculation for subsequent biomass separation must relate the floc properties to separation process criteria. The effects of flocculant type, dose, and hydrodynamic conditions on floc formation in laminar tube flow were determined for an Escherichia coli system. Combined with an on-line aggregation sensor, this technique allows the flocculation process to be rapidly optimized. This is important, because interbatch variation in fermentation broth has consequences for flocculation control and subsequent downstream processing. Changing tube diameter and length while maintaining a constant flow rate allowed independent study of the effects of shear and time on the flocculation rate and floc characteristics. Tube flow at higher shear rates increased the rate and completeness of flocculation, but reduced the maximum floc size attained. The mechanism for this size limitation does not appear to be fracture or erosion of existing flocs. Rearrangement of particles within the flocs appears to be most likely. The Camp number predicted the extent of flocculation obtained in terms of the reduction in primary particle number, but not in terms of floc size. (c) 1992 John Wiley & Sons, Inc.     

Improved separation of bacteria with a dual flocculant hydrocol™ system

Summary Polyelectrolyte flocculants are inefficient at flocculating Z. mobilis and B. subtilis in a complex medium, when administered singly or when combined with an oppositely charged polymer. However, when polymer dosing is followed by bentonite addition, particularly when chitosan constitutes the soluble polymer, effective flocculation occurs. High broth clarities can be attributed to chitosan interaction with cells, extracellular polymer and bentonite.     

Flocculation behavior and mechanism of an exopolysaccharide from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913

Flocculation behavior and mechanism of the exopolysaccharide secreted by Pseudoalteromonas sp. SM9913 (EPS SM9913), a psychrophilic bacterium isolated from 1855m deep-sea sediment, has been studied in this paper. EPS SM9913 showed a peak flocculating activity of 49.3 in 1g/L kaolin suspension with 4.55mmol/L CaCl2 and the optimum pH range of 5-8. It appears that the flocculating activity of EPS SM9913 was stimulated by Ca2+ and Fe2+. This study found that EPS SM9913 showed a better flocculation performance than Al2(SO4)3 at salinity of 5-100 per thousand or temperatures of 5-15 degrees C. In addition, this EPS was effective to flocculate several other suspended solids. The measured zeta-potentials, the size of flocs formed during the flocculation process and the surface profile of flocs revealed by scan electron micrograph suggest that bridging is the main flocculation mechanism of the studied EPS. Deacetylation of EPS SM9913 resulted in a significant decrease in its flocculating activity indicating that the large number of acetyl groups in EPS SM9913 played an important role in its flocculation performance.     

DNA as a Flocculation Factor in Pseudomonas sp.

A component responsible for flocculation was extracted from Pseudomonas strain C-120 by treating the cells with 3 M guanidine hydrochloride. The guanidine hydrochloride-extracted cells were reflocculated, not only with the guanidine hydrochloride extract but with DNA prepared from various bacteria. The reconstituted flocs were deflocculated by deoxyribonuclease or guanidine hydrochloride which indicated that the reconstituted flocs closely resembled natural flocs. In reconstitution experiments using Escherichia coli DNA at different molecular weights, it was found that DNA with a molecular weight higher than about 6 × 10⁶ was required to flocculate the guanidine hydrochloride-extracted cells. Heat-denatured DNA did not flocculate the guanidine hydrochloride-extracted cells. DNA with a high molecular weight was detected in the guanidine hydrochloride extract. It was concluded that the component involved in flocculation of this organism was highly polymerized double stranded DNA.