A comparison of two centrifuge techniques for constructing vulnerability curves: insight into the ‘open‐vessel’ artifact

A vulnerability curve (VC) describes the extent of xylem cavitation resistance. Centrifuges have been used to generate VCs for decades via static‐ and flow‐centrifuge methods. Recently, the validity of the centrifuge techniques has been questioned. Researchers have hypothesized that the centrifuge techniques might yield unreliable VCs due to the open‐vessel artifact. However, other researchers reject this hypothesis. The focus of the dispute is centered on whether exponential VCs are more reliable when the static‐centrifuge method is used rather than the flow‐centrifuge method. To further test the reliability of the centrifuge technique, two centrifuges were manufactured to simulate the static‐ and flow‐centrifuge methods. VCs of three species with open vessels of known lengths were constructed using the two centrifuges. The results showed that both centrifuge techniques produced invalid VCs for Robinia because the water flow through stems under mild tension in centrifuges led to an increasing loss of water conductivity. In addition, the injection of water in the flow‐centrifuge exacerbated the loss of water conductivity. However, both centrifuge techniques yielded reliable VCs for Prunus, regardless of the presence of open vessels in the tested samples. We conclude that centrifuge techniques can be used in species with open vessels only when the centrifuge produces a VC that matches the bench‐dehydration VC.     

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min^–1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 10⁶ cells ml^–1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process.     

Cycle-powered short radius (1.9M) centrifuge: exercise vs. passive acceleration

A human-powered short-arm centrifuge is described. This centrifuge could be used during spaceflight to provide +Gz acceleration while subjects performed exercise, thus supplying two forms of weightlessness countermeasures. Results from a study of cardiovascular responses while using the centrifuge are presented.     

Aquatic animals and use of centrifuge in space experiments]

There are some experiments which do not need a centrifuge apparatus in space, however, for other space experiments the use of centrifuge is indispensable as the control. The characteristics of these two types of space experiments are explained. Generally, the more quantitatively the phenomenon is analyzed, the greater the need of centrifuge apparatus becomes. The centrifuge in space can realize various gravity values ranging from 0 to 1 G, and this will extend the present biology to a more universal biology.     

Centrifuge Modeling of Cadmium Migration in Saturated and Unsaturated Soils

Researchers recently have come to recognize that geotechnical centrifuge can provide a powerful experimental tool for investigating the flow and transport of inorganic contaminants in soils. Inert, non-adsorptive inorganic compounds (e.g., sodium ions) rather than adsorptive heavy metals are often used as the chemical for the investigation of pollutant transport behavior in most centrifuge modeling. To overcome the shortcomings of this approach, centrifuge tests for the study of one-dimensional pollutant migration in both saturated and unsaturated soils were designed using an adsorptive pollutant (i.e., cadmium) and conducted at two centrifugal accelerations. In this study, the concentration profile of adsorptive cadmium along the depth of soil, the moisture content varying with the soil depth and the transport behavior of the cadmium in soils were investigated. The centrifuge results show that the cadmium concentration profiles are found to be similar for the centrifuge models performed at 15 g and 20 g and the one-dimensional moisture movement in the unsaturated soil can be reproducible. The validity of centrifuge modeling of adsorptive pollutants might be affected by the g-level that, in turn, determines the centrifuge testing time and affects the sorption equilibrium.     

The scale-down of an industrial disc stack centrifuge

The means are described whereby a disc stack centrifuge may be scaled-down by up to 10-fold of its separation capacity. The centrifuge separation characteristics so measured are suitable for direct scale-up predictions of centrifuge performance where only small volumes of particle suspension are available for study. Such an ability to scale-down is especially important in the processing of biological particles where for example, in the early stage of process development, there is often insufficient fermentation broth for fullscale studies. Scale-down is achieved by the reduction of the number of discs available for separation purposes and by the careful positioning of these discs in the overall disc stack. A combination of dye tracer and particle separation studies are used to optimise the disc stack configuration. The resulting grade efficiency curve is an accurate reflection of the curve for the full-scale centrifuge especially in the critical design region specifying centrifuge throughput for near complete particle recovery.     

Evaluation of a class III biological safety cabinet for enclosure of an ultracentrifuge

An evaluation of a special safety cabinet housing a high-speed centrifuge was made. The cabinet enclosed both the top access port and the drive and pumping machinery of the centrifuge. A titanium rotor was loaded with tubes containing a bacterial culture, weakened, and driven until rotor rupture occurred. There were several bent and broken components in the centrifuge, and bacteria leaked from the vacuum chamber. Although the forces were sufficient to displace the cabinet, none of the test bacteria were found outside the cabinet.     

Total containment of a continuous-flow zonal centrifuge

A continuous-flow, high throughput, turbine-driven zonal centrifuge has been contained for use with biologically hazardous materials. The centrifuge and all ancillary equipment are enclosed in three sealed cabinets that are maintained at negative pressure, decontaminated with ethylene oxide, and provide cooling for the process fluids. Sample handling is semiautomated and remotely controlled for ease of operation and includes an automated decontamination system. The centrifuge subsystems can be decontaminated prior to an engineer servicing the machine, and apparatus is provided for absolute filtration of the centrifuge turbine exhaust.     

The total containment of a batch-type zonal centrifuge.

A batch-type zonal centrifuge has been modified and totally contained for use with biologically hazardous materials. A sealed cabinet encloses the centrifuge and the ancilliary equipment. It is operated with a flow of filtered air when the zonal system is on, decontaminated with ethylene oxide, and maintained at a negative pressure throughout. The centrifuge subsystems can be drained, flushed, and decontaminated with ethylene oxide before an engineer services the machine. The sample handling system within the cabinet is remotely controlled.     

8. Tube Inserts as Aids in Preparative Ultracentrifugation

ABSTRACT

Conventional preparative ultracentrifugation was modified by using inserts in the centrifuge tubes, causing a reduction of the time necessary to centrifuge a protein at reduced rotor velocity from suspension.     

Tube Inserts as Aids in Preparative Ultracentrifugation

Abstract

Conventional preparative ultra centrifugation was modified by using inserts in the centrifuge tubes, causing a reduction of the time necessary to centrifuge a protein at reduced rotor velocity from suspension.     

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.     

Feasibility Study of Using Centrifuge for Investigating LNAPL Migration in Unsaturated Soils

Centrifuge modeling appears potentially useful for studying geo-environmental problems such as pollutant migration in subsurface systems. In this study, the “modeling of models” technique was used to validate the feasibility of using a geotechnical centrifuge to model the transport behavior of light non-aqueous phase liquids (LNAPLs) in unsaturated soils. All the experiments were conducted to simulate a gasoline spill from a leaking underground storage tank (UST) and the subsequent subsurface migration of the gasoline. When the gravity in the centrifuge reached the desired g-level, the gasoline was released from the UST and then it migrated in the unsaturated soil corresponding to a prototype time equivalent of one year. After the centrifuge tests, soil samples were collected using sampling tubes and the concentrations of individual constituent in the LNAPL were directly measured by means of gas chromatograph analysis. Results obtained from the centrifuge tests at different g-levels show that similar migration patterns are found for LNAPL transport in unsaturated porous media. The location of the peak concentration and the behavior of lateral spreading can be adequately described. In addition, centrifuge test data show that the migration pattern of LNAPLs is related to the soil type and the physical properties of individual constituents in the LNAPLs.     

Evaluation of a single-use disk stack centrifuge for improved efficiency and sustainability at 1000 L GMP manufacturing scale

Direct depth filtration is an established technology for single-use harvest operation. Advantages of direct depth filtration include familiarity with depth filtration in downstream processes and simplicity of the operation. Drawbacks include low capacity, large footprint, labor-intensive set-up, high water use, and high waste in the form of discarded filters. Single-use centrifugation is emerging as an alternative to depth filtration for the single-use harvest step. Within the single-use centrifugation space, disc stack centrifugation represents the newest entrant. In this study, we evaluated the performance of the GEA kytero single-use disc stack centrifuge to clarify two monoclonal antibody-producing cell culture fluids. The separation performance of the GEA kytero centrifuge varied between the two cell culture fluids, with differences in centrate turbidity and centrate filterability measured. A comparison was then performed to determine resource savings, compared to direct two-stage depth filtration, when using a GEA kytero centrifuge to harvest a 1000 L bioreactor. The analysis concluded that replacement of the first stage of depth filters with a GEA kytero centrifuge has the potential to decrease the required second stage depth filtration area by up to 80%. The decrease in depth filter area resulting from the use of the GEA kytero would result in a decrease in the harvest step footprint, a decrease in buffer volume required to prime and rinse depth filters, and a decrease in the volume of plastic waste. An economic comparison of the GEA kytero single-use centrifuge against a direct depth filtration step found that for a 1000 L harvest step, the GEA kytero centrifuge may reduce costs by up to 20% compared with two-stage direct depth filtration.     

Centrifugal dewatering of acid casein curd: effect of casein manufacturing and centrifugation variables on curd compression in a laboratory centrifuge

Data relevant to curd compression in a horizontal, solid bowl decanter centrifuge have been obtained by studying the dewatering of acid casein curd in a batch laboratory centrifuge. Analysis of curd compression under centrifugal force predicts a moisture content gradient in the dewatered curd from a maximum at the curd-liquid interface to a minimum at the centrifuge bowl wall. This moisture content gradient was also measured experimentally, and its practical implications are discussed. Increases in centrifugal force, centrifugation time, and centrifugation temperature all caused a marked de crease in dewatered curd moisture content, whereas in creases in precipitation pH and maximum washing temperature caused a smaller decrease in dewatered curd moisture content.     

Comparison of of techniques for measuring +Gz tolerance in man.

Two objective methods and one subjective method for measuring +Gz tolerance (inertial vector in a head-to-foot direction) were compared on the human centrifuge. Direct eye-level blood pressure (Pa), blood flow velocity in the superficial temporal artery (Qta), and subjective visual symptoms were used to determine tolerance to rapid onset acceleration (1 G/s) on the USAFSAM human centrifuge. Seven "relaxed" subjects with extensive centrifuge experience were exposed to gradually increasing +Gz plateaus until the subject reported 100% loss of peripheral centrifuge gondola lights (PLL) and 50% loss of central light (CLD); viz., blackout. Zero forward Qta occurred 6 s (range 4-9 s) before subjective blackout and when mean eye-level blood pressure had reached 20 +/- 1 mmHg (SE). The results of this study indicate that flow changes in the superficial temporal artery reflect flow changes in the retinal circulation during +Gz stress.     

Modeling industrial centrifugation of mammalian cell culture using a capillary based scale-down system

Continuous-flow centrifugation is widely utilized as the primary clarification step in the recovery of biopharmaceuticals from cell culture. However, it is a challenging operation to develop and characterize due to the lack of easy to use, small-scale, systems that can be used to model industrial processes. As a result, pilot-scale continuous centrifugation is typically employed to model large-scale systems requiring a significant amount of resources. In an effort to reduce resource requirements and create a system which is easy to construct and utilize, a capillary shear device, capable of producing energy dissipation rates equivalent to those present in the feed zones of industrial disk stack centrifuges, was developed and evaluated. When coupled to a bench-top, batch centrifuge, the capillary device reduced centrate turbidity prediction error from 37% to 4% compared to using a bench-top centrifuge alone. Laboratory-scale parameters that are analogous to those routinely varied during industrial-scale continuous centrifugation were identified and evaluated for their utility in emulating disk stack centrifuge performance. The resulting relationships enable bench-scale process modeling of continuous disk stack centrifuges using an easily constructed, scalable, capillary shear device coupled to a typical bench-top centrifuge.     

Direction finding by hornets in a vertically rotating centrifuge

In a vertically rotating centrifuge, the direction of the resultant gravitational and centrifugal forces is constantly changing. Hornets placed in such a centrifuge will build their combs in the direction of the resultant only if the centrifuge is stopped every day and left in the same position for at least half an hour, because during the cessation of motion, they presumably “learn” some geometrical cues which enable them to determine the preferred angle of building. Hornets can detect and respond to a centrifugal force as small as 0·18% of the earth's gravitational force. At a rotational rate of 1/8 of a revolution per minute there was no comb construction whatsoever and hornet mortality rate was 100% within three days.     

Effects of gravitropic stress on the development of the primary root of lentil seedlings grown in space

Root growth and cell differentiation were analysed in lentil seedlings grown (1) in microgravity (F microg), (2) on the 1 x g centrifuge (F1 x g), (3) in microgravity and placed on the 1 x g centrifuge for 4 h [F(microg + 1 x g)], (4) on the 1 x g centrifuge and placed in microgravity for 4 h [F(1 x g + microg)]. In microgravity, there were strong oscillations of the root tip, even when the seedlings were grown first on the 1 x g centrifuge [F(1 x g + microg)]. In the [F(microg + 1 x g)] sample, the roots grown in microgravity were oblique with respect to the 1 x g acceleration when the seedlings were placed on the centrifuge. They were therefore gravistimulated. However, root length was similar in the 4 samples after 29 h of growth and growth rate of the root was the same between 25 h and 29 h although it appeared to be slightly greater in the [F(microg + 1 x g)] sample. Cell elongation was analysed as a function of the distance from the root cap junction. Cell length was similar in the seedlings grown in microgravity or on the 1 x g centrifuge. The transfer from the 1 x g centrifuge to microgravity [F(1 x g + microg)] did not modify cell elongation in the roots. Cell length in the roots which were grown in microgravity and gravistimulated [F(microg + 1 x g)] was different from that observed in microgravity but this was only due to gravistimulation. Thus, gravity does not have an effect on cell elongation when the roots are strictly oriented in the vertical position but it does as soon as the root tip deviates from this orientation.     

Biohazards Assessment in Large-Scale Zonal Centrifugation

A study was conducted to determine the biohazards associated with use of the large-scale zonal centrifuge for purification of moderate risk oncogenic viruses. To safely and conveniently assess the hazard, coliphage T₃ was substituted for the virus in a typical processing procedure performed in a National Cancer Institute contract laboratory. Risk of personnel exposure was found to be minimal during optimal operation but definite potential for virus release from a number of centrifuge components during mechanical malfunction was shown by assay of surface, liquid, and air samples collected during the processing. High concentration of phage was detected in the turbine air exhaust and the seal coolant system when faulty seals were employed. The simulant virus was also found on both centrifuge chamber interior and rotor surfaces.