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.     

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.     

Der Einfluß der Flockenbildung auf die Versorgung der Hefezellen mit Sauerstoff bei der Fermentation flüssiger Kohlenwasserstoffe

Floc formation, especially the influence of floe diameter variations on the total velocity of the process, was investigated in aerobic growth processes of yeast on the hydrocarbons of crude oil. The experimental results show that the diameter of the flocs is a function of the rheological properties of the fluids and the flow conditions. The floc diameter varies between 0,1 mm and a few millimeters. About 90% of the total yeast cells are situated in the interior of the flocs. Since oxygen must be transferred to all yeast cells their oxygen supply was studied. Thus, the yeast cells in the floc interior were not sufficiently supplied with oxygen, if the floc diameter reached a critical value. In such cases a decrease of the biomass formation rate was observed, although the dissolved oxygen concentration of the aquaeous fermentation medium was greater than zero. Therefore, aerobic microbial growth processes in multicomponent systems must be carried out without floc formation or under such conditions as cause very small floc diameters.     

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.     

絮凝颗粒粒度分布对自絮凝酵母SPSC01乙醇耐受能力的影响

利用激光聚焦反射式颗粒测量系统, 通过调节不同的搅拌速率, 得到了分批补料培养条件下粒度分布不同的四个絮凝酵母SPSC01颗粒群体, 进而对絮凝颗粒群体分布对乙醇耐受性进行了系统研究。经过6 h、20%乙醇的冲击, 颗粒粒度为100、200、300和400 mm的自絮凝酵母SPSC01的存活率分别为3.5%、26.7%、48.8%和37.6%。这表明不同粒度分布的絮凝颗粒群体乙醇耐受性具有明显差别, 在一定粒度范围内乙醇耐受性达到最高, 乙醇耐受性最高的酵母群体的乙醇得率系数85.5%, 比乙醇耐性最低的颗粒群体提高了7.2%。粒度为100、200和300 mm的自絮凝酵母颗粒群体总麦角固醇、游离麦角固醇及海藻糖含量与粒度大小成正相关, 但在粒度为400 mm的絮凝颗粒群体中总麦角固醇、游离麦角固醇及海藻糖含量呈下降趋势, 与其乙醇耐性低于300 mm絮凝颗粒的结果相一致。对细胞膜透性的研究表明, 颗粒粒度为300 mm的絮凝酵母颗粒细胞膜通透性(P′)最低, 分别仅为颗粒粒度为100 mm和200 mm颗粒群体的43%和52%, 表明粒度分布不同的絮凝颗粒群体乙醇耐性的差别与细胞膜透性密切相关。     

Enhancement of metabolic rates of yeast flocculent cells through the use of polymeric additives

The influence of several polymeric additives on specific glucose uptake rate of flocs of a S. cerevisiae strain — S. cerevisiae NRRLY 265 was studied. A special continuous membrane microreactor was used to measure glucose uptake on the presence of calcium and of the tested additives — two cationic polymers — bis(polyoxyethylene-bis(amine)) 20,000 and BPA 1,000 and one anionic polymer — Magna Floc LT25.An increase on glucose uptake rate was always observed when comparing with calcium bound flocs. For bis(polyoxyethylene-bis(amine)) 20,000 the increase was only 19% but for BPA 1,000 a value of more than 50% was observed. For Magna Floc LT25 a two fold increase was measured.The determination of floc size and porosity in the presence of the additives indicated that, on the basis of these parameters, it was not possible to explain the observed glucose uptake rates. The floc porosites in additive bound flocs were similar and 10% larger than for calcium bound flocs and glucose uptake rate was larger for the largest flocs — Magna Floc LT25 bound flocs were the largest followed by BPA 1,000, bis(polyoxyethylene-bis(amine)) 20,000 and calcium bound flocs. These values disagree with what should be expected in diffusion controlled processes.The calculation of intercellular floc distance indicated that polymeric additives act on the reduction of diffusional limitations by increasing the available flux area for glucose inside the flocs. By analysing different kinds of packings, it was also observed that the packing arrangement for yeast cells in flocs is close to the cubic packing. The simulation of this arrangement for the obtained floc sizes confirmed that the 10% increase in floc porosity is sufficient to explain the increase in the available flux area.     

Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS)

The roles of extracellular polymer substances (EPS) in the shear stability of aerobic and anaerobic flocs were investigated. Both pH and EDTA concentration had a significant effect on the floc stability. The sludge flocs became much weaker as the solution pH increase to above 10. Addition of 1 mM EDTA or more could cause considerable cell erosion and deflocculation of the anaerobic flocs, whereas more than 3 mM EDTA was needed to show its adverse effect on the stability of aerobic flocs. A fraction of the EPS, around 10 mg/g SS for the aerobic flocs and 15 mg/g SS for the anaerobic flocs, could be extracted by fluid shear when the dispersed mass concentration approached the equilibrium. This suggests that most of the dispersed particles were glued by a small amount of readily-extractable EPS fraction. In addition to the abundance of this EPS fraction, its proteins/carbohydrates ratio, about 0.22:1 for the aerobic flocs and 2.66:1 for the anaerobic flocs, also appeared to be an important factor governing the microbial floc stability. A lower content of the readily-extractable EPS fraction and a lower ratio of proteins/carbohydrates were responsible for the greater stability of microbial flocs. The total content of the EPS, however, did not show a direct correlation with the floc stability. A hypothesis about biological flocs with two distinct structural regions was proposed. The outer part contained dispersible cells loosely entangled by the readily-extractable EPS fraction. This part was layered and would become completely dispersed at an infinite shear intensity. On the other hand, the inner part contains biomass in a stable structure tightly glued by EPS, which could not be dispersed by shear except under unfavorable conditions.     

Size and settling velocity distributions of flocs in the Tamar estuary during a tidal cycle

Data extracted from video recordings of individual estuarine flocs near the estuary bed during the advance and retreat of the salt intrusion show changes in size and settling velocity distributions. The recordings were taken using INSSEV —IN Situ SEttling Velocity instrument. Size coupled with effective density variations due to both changes in floc structure and ambient salinity result in changes in the settling velocity during the tidal cycle. In particular, just after high water slack, the appearance of high settling velocity medium size flocs and individual particles suggest that the lower density flocs have been broken up by the intense vertical shear in the currents caused by the salt wedge intrusion. Current shear is shown to have a significant influence on floc effective density.     

Measuring Floc Structural Characteristics

A review is presented of a range of techniques for the structural characterisation of flocs. Flocs may be considered as highly porous aggregates composed of smaller primary particles. The irregular size and shape of flocs makes them difficult to measure and quantify. A range of different equivalent diameters are often used to define the floc size and allow comparison with other floc systems. The application of a range of floc sizing methods has been described. Microscopy is time consuming, requiring large sample size and considerable preparation but gives good information on floc shape and form. Light scattering and transmitted light techniques have been used to good effect to measure floc size on-line whilst individual particle sensors have limited applicability to measuring floc size. Fractal dimension can be measured using one of three major techniques: light scattering, settling and two dimensional (2D) image analysis. Light scattering is ideally suited for small, open flocs of low refractive index whilst settling may be applied to most floc systems of low porosity. 2D image analysis requires flocs to have good contrast between the solid in the floc and the background.     

Does a change in reactor loading rate affect activated sludge bioflocculation?

A collection of particles held together by different interparticle forces might eventually give rise to the formation of activated sludge flocs. This process is known as bioflocculation and is crucial for both conventional activated sludge systems and membrane bioreactors. Since industrial wastewater treatment plants generally face varying reactor loading rates due to varying production schemes in the facility, this paper investigates the impact of reactor loading rates on activated sludge bioflocculation. For this purpose, two reactors were initially operated at a nominal reactor loading rate (RLR) and afterwards changed to a high and low RLR. Based on the obtained results, it can be observed that sludge under low RLR conditions is prone to floc fragmentation due to an increase in water-soluble extracellular polymeric substances (EPS). The reactor under high RLR indicated increased floc erosion as a result of increased biomass concentration, which might imply more collisions between sludge flocs, releasing small sludge particles from the floc. In the high RLR reactor, no significant increase in EPS was observed. A distinction between the different (de)flocculation phenomena was made based on sludge volume index, effluent suspended solids and EPS data supplemented with microscopic image analysis.     

Effects of Ionic Strength on Bacterial Adhesion and Stability of Flocs in a Wastewater Activated Sludge System

The success of biological wastewater treatment is to a large extent governed by the ability of bacteria to induce floc formation, thereby facilitating the separation of particles from the treated water. We performed studies on the dynamics of floc stability, the desorption of cells from the flocs, and the reflocculation of detached material. The floc stability was affected by the ionic strength of the medium in a way that strongly suggests that the interactions between the floc components can be explained by the theory of Derjaugin, Landau, Verwey, and Overbeek (DLVO theory). At increasing concentrations of electrolytes, the stability of the flocs increased. However, above an ionic strength of about 0.1 the floc stability decreased, and it seems that at this high electrolyte concentration the DLVO theory cannot be applied. The reversibility of the electrostatic double-layer effects was experimentally shown by treating the sludge repeatedly with a low-ionic-strength solution until parts of the flocs detached. When salt was added at this point, flocs re-form, resulting in a dramatic decrease in the turbidity of the supernatant liquid. Both reflocculation and detachment of floc material were seen with calcium as well as with potassium. This finding clearly indicates that the reflocculation and destabilization of flocs were due to changes in double-layer thickness rather than bridging effects of multivalent ions such as calcium. The results indicate that the ionic strength may well be an important factor for the floc stability in wastewater in situ.     

Online monitoring and characterization of flocculating yeast cell flocs during continuous ethanol fermentation

Both intrinsic and observed kinetic investigations for those ethanol fermentations using self-flocculated yeast strains have been hindered by the lack of real online monitoring techniques and proper characterization methods for the flocs. An optical detecting technique, the focused beam reflectance measurement probe developed by Lasentec (Redmond, WA) was inserted into a fermentor to monitor the floc chord length distributions. Using a simulating system composed of the floc-buffer suspensions, the total floc chord length counts per second were directly correlated with the floc biomass concentrations so that the floc biomass concentrations can be in situ detected. Furthermore, a characterization method of the flocs was established by properly weighted treatments of the detected floc chord length distributions. When a real yeast floc ethanol fermentation system was detected during its intrinsic kinetic investigations in which the floc size needed to be controlled at a level of micrometer scale to eliminate inner mass transfer limitations, it was found and validated that CO(2) produced during fermentation exerted significant disturbances. By applying 1/length-weighted treatment, these disturbances were effectively overcome.     

Two parameters account for the flocculated growth of microbes in biodegradation assays

Microbes in activated sludge tanks mostly occur in flocs rather than in cell suspensions. Flocculation results in a limited supply of substrate to the bacteria inside the flocs, which reduces the biodegradation rate of organic compounds by several orders of magnitude. This article presents a simple two-parameter extension of growth models for cell suspensions to account for the ensuing reduction of the degradation rate. The additional parameters represent floc size at division and diffusion length. The biomass of small flocs initially increases exponentially at a rate equal to that of cell suspensions. After this first phase, the growth rate gradually decreases and finally the radius becomes a linear function of time. At this time flocs are large and have a kernel of dead biomass. This kernel arises when the substrate concentration decreases below the threshold level at which cells are just able to pay their maintenance costs. We deduce an explicit approximative expression for the interdivision time of flocs, and thereby for the growth of flocculated microbial biomass at constant substrate concentrations. The model reveals that the effect of stirring on degradation rates occurs through a reduction of the floc size at division. The results can be applied in realistic biodegradation quantifications in activated sludge tanks as long as substrate concentrations change slowly.     

Sizing and counting of saccharomyces cerevisiae floc populations by image analysis, using an automatically calculated threshold

A standardized image analysis method has been developed permitting determination of the number of yeast flocs and their size distribution. The method includes image grabbing, image enhancement, automatic determination of the appropriate threshold, curve fitting of the areahistogram, determination of the mean single floc area and its standard deviation, and floc counting. The extension of the method to other applications is immediate and straightforward. Two Saccharomyces cerevisiae floc Populations (with ages of 48 and 72 h) were analyzed. The results showed a variation around the mean of 9%-12% for the single floc mean area, 6%-7% for the number of single flocs, and 5%-6% for the total number of flocs. Aggregates of two flocs (doublets) and three flocs (triplets) were enumerated. The correctness of the method was checked by analyzing the parameters of interest as a function of the threshold. The constant correlation between the parameters and the threshold showed the validity and consistency of the method. (c) 1996 John Wiley & Sons, Inc.     

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.     

Intrinsic kinetics of continuous growth and ethanol production of a flocculating fusant yeast strain SPSC01

The intrinsic kinetics of continuous yeast cell growth and ethanol production for a self-flocculating fusant yeast strain SPSC01 was investigated by means of mechanically dispersing the flocs and correspondingly established floc size distribution on-line monitoring technique using the focused beam reflectance measurement system, through which the floc intra-particle mass transfer limitation was effectively eliminated, but its ethanol formation metabolism was not affected. Modified kinetic models were developed, which can be used to predict the continuous kinetic behaviors of SPSC01, especially when low dilution rates are applied and limiting substrate concentrations are undetectable and almost all kinetic models developed previously are failed in predicting corresponding kinetic behaviors. Both substrate and product inhibitions reported for freely suspended yeast cell ethanol production were also observed for SPSC01 when high gravity media were fed and relatively high levels of residual sugar and ethanol presented. Model parameters were evaluated through numerical calculation method and validated by experimental data mu = 0.584C(s)/0.155 + C(s) + C(2)(s)/160.7(1 -P/125)(3.68) + 0.004 for growth, nu = 1.998C(s)/0.427 + C(s) + C(2)(s)/366.7(1- P/125)(1.72) + 0.060 for ethanol production These intrinsic kinetic models can be further used to develop the observed kinetic models that quantitatively correlate the impact of the self-flocculating yeast cell size distributions on their apparent rates for yeast cell growth, substrate uptake and ethanol production and optimize the ethanol production process.     

Modelling diffusion-reaction phenomena in yeast flocs of Saccharomyces cerevisiae

Limitations in the diffusion of substrates into the flocs will condition cell metabolic behaviour, having obvious consequences on growth and product formation. Polymeric additives have been used aiming the reduction of those limitations. The knowledge of the concentration profiles and metabolic fluxes of glucose and oxygen inside the flocs would bring valuable information about the?conditions under which a fermentation should run. Direct measurement of such profiles is rather difficult but their simulation has been performed and is presented in this work. Calculations were made for different possible sizes of the yeast flocs, considering also the presence or absence of a polymeric additive. Only a small percentage of the cells in the flocs metabolise glucose oxidatively due to severe oxygen limitations. The presence of the polymeric additive increases the ratio of cells operating under respiratory metabolism over those under fermentative metabolism: from 0.4% to 5.7% without additive to 1.2% to 8.5% with additive, depending on the bulk glucose concentration. Also, based on this data, it is possible to justify the yeast floc natural shape.     

Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up.

Nucleic acids, lipid, and colloidal particulate material can be selectively flocculated from a yeast cell homogenate by the cationic polymer polyethyleneimine (PEI). Flocculation can occur from a crude homogenate, a homogenate clarified centrifugally, or by the prior use of sodium tetraborate (borax). Flocculation from a homogenate previously clarified by the use of borax is best suited for large-scale operation. The supernatant obtained following centrifugation is effectively free of nucleic acid, lipid, and particulate material with essentially 100% soluble enzyme recovery. Enzyme specific activity increases by approximately 45% compared to a zero PEI control.     

Novel membrane bioreactor: Able to cope with fluctuating loads, poorly water soluble VOCs, and biomass accumulation

We determined the effect of different mixing intensities on the performance, methanogenic population dynamics, and juxtaposition of syntrophic microbes in anaerobic digesters treating cow manure from a dairy farm. Computer automated radioactive particle tracking in conjunction with computational fluid dynamics was performed to quantify the shear levels locally. Four continuously stirred anaerobic digesters were operated at different mixing intensities of 1,500, 500, 250, and 50 revolutions per min (RPM) over a 260-day period at a temperature of 34 +/- 1 degrees C. Animal manure at a volatile solids (VS) concentration of 50 g/L was fed into the digesters daily at five different organic loading rates between 0.6 and 3.5 g VS/L day. The different mixing intensities had no effect on the biogas production rates and yields at steady-state conditions. A methane yield of 0.241 +/- 0.007 L CH(4)/g VS fed was obtained by pooling the data of all four digesters during steady-state periods. However, digester performance was affected negatively by mixing intensity during startup of the digesters, with lower biogas production rates and higher volatile fatty acids concentrations observed for the 1,500-RPM digester. Despite similar methane production yields and rates, the acetoclastic methanogenic populations were different for the high- and low-intensity mixed digesters with Methanosarcina spp. and Methanosaeta concilii as the predominant methanogens, respectively. For all four digesters, epifluorescence microscopy revealed decreasing microbial floc sizes beginning at week 4 and continuing through week 26 after which no microbial flocs remained. This decrease in size, and subsequent loss of microbial flocs did not, however, produce any long-term upsets in digester performance.     

Physical and hydrodynamic properties of flocs produced during biological hydrogen production

Dense flocs readily form in continuous culture bioreactors used for hydrogen production, but the fractal and hydrodynamic properties of these flocs have not been previously analyzed. We therefore examined the size distribution, fractal dimension, and hydrodynamic properties of flocs formed in a continuous flow, well-mixed reactor treating synthetic wastewater at a fixed condition of a 4.5 h hydraulic detention time (23 degrees C, pH 5.5). The reactor was operated for a total of 3 months at three different organic loading rates (27, 53, and 80 g-COD/L-d) with influent glucose concentrations of 5, 10, and 15 g-COD/L. At all three loading rates the removal of glucose was nearly complete (98.6-99.4%) and biomass was produced in proportion to the organic loading rate (0.86 +/- 0.11, 2.40 +/- 0.26, and 4.59 +/- 1.55 g/L of MLVSS in the reactor). Overall conversion efficiencies of glucose to hydrogen, evaluated on the basis of a maximum of 4 mol-H2/mol-glucose, increased with organic loading rates in the order 17.7%, 23.1%, and 25.6%. The gas contained 56.1 +/- 4.9% hydrogen, with the balance as carbon dioxide. No methane gas was detected. Under these conditions, flocs were produced with mean sizes that increased with organic loading, in the order 0.12 cm (5 g-COD/L), 0.35 cm (10 g-COD/L), and 0.58 cm (15 g-COD/L). As the average floc size increased, the flocs became on average denser and less fractal, with fractal dimensions increasing from 2.11 +/- 0.17 to 2.48 +/- 0.13. Floc porosities ranged from 0.75-0.96, and resulted in aggregate densities that allowed little intra-aggregate flow through the floc. As a result, average settling velocities were not appreciably larger than those predicted by Stokes' law for spherical, impermeable flocs. Our results demonstrate that dense, relatively impermeable flocs are produced in biohydrogen reactors that have settling properties in reasonable agreement with Stokes' law.