The flotation processand related flotation cells are widely used for treating metallic and nonmetallic ores and in addition, it is receiving an ever widening application in other industries. A greater tonnage of ore is treated by flotation than by any other single process. Practically all the metallic minerals are being recovered by the flotation process and the range of non-metallics successfully handled is steadily being enlarged. In recent years the art of flotation has been successfully applied in other than the mining industry, such as flotation of wheat, and other industrial applications. As flotation reagents are further developed, the application of flotation will be more widespread.
The Sub-A Flotation Cellhas been applied to all types of flotation problems and these machines have continuously demonstrated their superiority. They have given very successful results through a wide range of problems, and their supremacy is fully proven by world-wide acceptance and application.The feature of the Sub-A is the design. The Sub-A incorporates all of the basic principles and requirements of the flotation process and these, coupled with the special and exclusive wear features, make it the ideal Flotation Cell.
Sub-A Flotation Cells have been developed over the intervening years since 1927 until today there are over 26,000 cells in operation. Flotation cells are standard equipment for an ever widening range of metallurgical and industrial problems. They are being used in plants of all types and sizes and they are giving excellent results at minimum cost at tonnages of a few tons up to 35,000 tons per 24 hours.
To take care of the wide range of problems confronting the flotation process, the Sub-As are built in a wide and flexible range of commercial sizes, from the No. 8 through the No. 12, No. 15, No. 18, No. 18 Special, No. 21, No. 21 Deep, No. 24 and the No. 30.
There is a particular size cell for every problem and tonnage, with each cellhaving incorporated into itsdesign features to take care of any condition. This is the basis on whichSub-A Cells have been designed. Standard cells are as follows:
The construction of the Sub-A Standard Flotation Cell is with double welded steel tank, alloy iron cell side liners, rubber bonded to steel bottom liners, impellers and diffuser wearing plates of molded rubber or alloy iron, individual cell pulp level control, rubber protected shafts and rubber sand relief bushings. To the standard cells the supercharging principle can be quickly adapted as all recent cells are furnished with automatic air seal and air bonnet to which low pressure air is easily connected. Variations from the standard cellallow the pulp to bypass through convenient ports which can be opened or closed while the unit is operating. This feature makes the Sub-A either the exclusive positive circulation unit or an open type cell.
Sub-A Flotation Cells are built with special design for work in acid and corrosive circuits. The construction of this typecellis similar to the standardSub-A except that the parts in contact with the pulp are of special materials and the tank itself is of wood construction. There is also a variation in design to take care of the special conditions usually associated with acid circuits and for convenience the acid-proof cells are built in 2-cell units.
The Sub-A Grain Peeling Flotation Cell is a special adaptation of the Sub-A for the peeling of wheat and other grains by flotation. The peeling cellis a continuous, straight-sided tank without spitzkasten overflow. This unit with its special features, loosens the outer brans. From the peeling unit feed passes to the Sub-A special grain Flotation Cell which is similar in design to the standard Sub-A, except for the special construction to meet the requirements of this type flotation. These peeling-Flotation Cells are built for a capacity of approximately 150 bushels per hour. The number of cells per unit and the number of units required for an installation is dependent upon the feed rate required and also upon the type of grain to be peeled.
The widespread success of the Sub-A Flotation Cell is attributed to the basic qualities of the design of this type Flotation Cell. Successful metallurgy results from the distinctive gravity flow feature, which assures positive circulation of all pulp fractions with reagents from cell to cell and hence results in high efficiency.
The pulp flows by gravity into each cell through the feed pipe, from which it drops directly on top of the rotating impeller below the stationary hood. As the pulp cascades over the impeller blades it is thrown outward and upward from the impeller and diffuser wearing plate by the centrifugal action of the impeller. The pulp is kept in complete circulation by the impeller action and as the flotation reaction takes place, the pulp is passed from cell to cell. Pulp overflows to each succeeding cell over an adjustable weir gate in the partition. This gate gives accurate control of pulp level as the pulp passes through the machine. To take care of coarse oversize each cell has a rubber sand relief opening in the partition weir casting which feeds oversize direct to the impeller of the next cell without short circuiting. Circulation within each cell itself and return of middlings is by means of adjustable openings in the hood above each impeller, although for normal operation these are kept closed, except for middling return.
Circulation in Sub-A Flotation cells is highly efficient due to the distinctive gravity flow feature. This method of pulp circulation assures the positive circulation of all pulp fractions with resultant maximum treatment of each and every particle. It is an established fact that the mechanical method of circulating material is the most positive and economical, particularly where the impelleris below the pulp. A Flotation Cell must not only be able to circulate coarse material (encountered in practically every mill circuit) but also must re-circulate and retreat the difficult middling products.
An alternate pulp flow is obtained from cell to cell by the side ports in the partitions. The ports are adjustable so that a portion of the pulp can pass from cell to cell through these ports and consequently bypass the impeller and weir overflow.
It is not essential to have each individual cell with separate weir gate control; however, for most installations this is recommended. An alternate arrangement is with gate control every two to four cells for pulp level control, and free pulp passage from cell to cell, by means of the ports, as well as cell to cell overflow. The arrangement is actually a grouping without sacrificing the positive circulation feature.
The passage of pulp through the cell and the action created in the impeller zone draws air down the stationary standpipe and from the partition along the feed pipe. This positive suction of air gives the ideal condition for average flotation and the action in the impeller zone thoroughly mixes the air with the pulp and reagents. As this action proceeds, a thoroughly aerated live pulp is produced and furthermore, as this mixture is ground together by the impeller action, the pulp is intimately diffused with exceedingly small air bubbles which support the largest number of mineral particles.
For particular problems the aeration in the Sub-A can be augmented by the application of Supercharging, whereby fully controlled air under low pressure is diffused into the pulp. This feature is accomplished by the introduction of air from a blower or turbo-compressor through the standpipe connection into the aerating zone where it is premixed with the pulp by the impeller action. This supercharging of the pulp creates a highly aerated condition which is maintained by the automatic seal in the cell partition. Supercharging is of particular advantage for low ratio of concentration and slow-floating ores.
Throttling of air in the Sub-A Flotation Cell is of benefit when suppressed flotation is required. This is accomplished by cutting off or decreasing the size of air inlet on the standpipe. Suppressed flotation finds its chief use in grain flotation, certain non-metallics and occasionally in cleaner or recleaner operations.
The aeration and mixing of the pulp with reagents all takes place in the lower zone of the cell. This thorough mixing, which is below the stationary hood, is to a considerable degree responsible for the metallurgical efficiency of the cell.
Supercharging Sub-A Flotation Cells by increasing the sub-aeration is obtained with a small volume of air at low pressure. The air bonnet and automatic air seal are integral parts of all standard Sub-A Cells; hence, to increase the aeration all that is required is a connection from the air bonnet to a source of air supply.
The aerated pulp, after leaving the mixing zone, passes upward by displacement to the central section of the cell. This is a zone of quiet and is free from cross currents and agitation. In this zone, the mineral-laden air bubbles separate from the worthless gangue and pass upward to the froth column without dropping their load, due to the quiescent condition. The gangue material follows the pulp flow and is rejected at the discharge end of the cell.
It is in the separation zone that effective aeration is essential and this is assured in the Sub-A as the air is broken up into minute bubbles. These finely diffused bubbles are essential for carrying a maximum load of mineral.
The mineral-laden bubbles move from the separation zone to the pulp level and are carried forward to the overflow lip by the crowding action of succeeding bubbles. To facilitate the quick removal of mineral-laden froth, Flotation Cellsare equipped with froth paddles. Froth removal can be further facilitated by the use of crowding panels which create a positive movement of froth to the overflow.
Concentrates produced by Sub-As are noted for their distinctive high grade and selectiveness. The spitzkasten built into Sub-A cells is partially responsible as it allows a quiescent zone just before froth removal in order that middling fractions may fall back into the pulp flow. Cells are built with single overflow as standard, but double overflow can be supplied.
These are several of the distinctive advantages obtained with the use of Sub-A Flotation Cells which are found in no other Flotation Cell. The combination of these several advantages is necessary to obtain successful flotation results.
Positive circulation of all pulp fractions from cell to cell is assured by the distinctive gravity flow principle of the Sub-A. No short circuiting can occur through the cell; hence, every particle is subject to positive treatment. In instances where successful metallurgy demands the handling of a dense pulp containing an unusually large percentage of coarse material, the sand relief opening aids in the celloperation. This opening removes from the lower part of the cell the coarse fractions and passes them through the feed pipe to the impeller of each succeeding cell. The sand relief openings assure the passage of slow floating coarse mineral to each impeller and therefore it is subject to the intensive mixing, aeration and optimum flotation condition of each successive cell. The finer pulp fraction passes over the weir or through the intermediate ports. The passage of the coarse fractions through each impeller eliminates short circuiting and thus, both fine and coarse mineral are subject to positive flotation.
A Sub-A cell will not choke up, even when material as coarse as one quarter inch is circulated. Choking cannot occur as the feed to each cell is to the top of the impeller. After a shut-down, it is not necessary to drain the Flotation Cell as the stationary hood with diffuser wearing plate protects the impeller and feed pipe from sanding-up. Even though the flotation feed is finely ground, coarse material occasionally gets into the circuit and if the Flotation Cell does not have the gravity flow feature, sanding and choke-ups will occur. This gravity flow principle of pulp circulation has made possible the widespread phenomenal success of a flotation cell between the ball mill and classifier. The recovery of mineral, as coarse and as soon as possible, in a high grade concentrate is now considered a requisite to a maximum metallurgical efficiency and hence Sub-A operators value its 24-hour per day service and freedom from shut-downs.
Middling products from Sub-As can be returned by gravity from any cell to any other cell in the average flotation circuit. The flexibility is possible without the aid of pumps or elevators. The middling pulp flows to the required cell and by means of a return feed pipe, falls directly on top of the impeller, assuring positive treatment and areation of the middling product without impairing the action of the cell. This feature, exclusive with Sub-A, is of particular advantage in circuits where several cleaning steps are required to bring middling products to final grade. The initial feed can also enter into the front or back of any cell through the return feed pipe.
Sub-A cells are under full control with normal operating conditions. The pulp level is maintained at the desired place with adjustable weirs. Aeration is controlled with flexible methods of air addition which allow variable aeration for different conditions. Any overloads or surges of coarse material from the grinding circuit are effectively taken care of with the sand relief openings, ports or quickly adjustable weirs. With these control features the operator has every opportunity to maintain his circuit in balance. Pulp fluctuations can be minimized and absorbed due to the control features.
Sub-A Flotation Equipment is metallurgically unsurpassed for the production of concentrates most suitable for subsequent thickening, filtering and smelting.The selectivity of Sub-A through all mesh sizes is one of the outstanding features of this Flotation Cell. Selectivity in Cells is not by chance, but results from the basic principle in design. The distinct gravity flow feature, coupled with the individual cell construction, controlled individually or in groups, and positive circulation through the cell, is to a large degree responsible for the recovery of coarse products by flotation. The advantage of positive circulation becomes obviously important with coarser grinds. A homogeneous and thoroughly mixed pulp is circulated at all times in each cell and there is no tendency towards. classification and segregation. Thorough mixing and aeration of all pulp fractions by positive circulation is the only means of obtaining selective flotation and metallurgical efficiency through all mesh sizes. The absence of pulp stratification prevents slime recovery from surface pulp or drift of heavy granular fractions through the cell. Selective flotation with Sub-A results in several major features, such as:
Recovery in flotation is of prime importance. In studying recoveries it is essential also to investigate thoroughly the intermediate products produced. It is a simple matter to make a high recovery or a low tailing if no thought is given to the nature of the concentrate produced or circulating load. Sub-A Flotation Cells will produce a high recovery, coupled with a high grade concentrate, low volume of middling, and a final concentrate most acceptable for subsequent treatment. The overall efficiency of this Flotation Cell will assure an equitable balance between recovery and nature of products produced.
Sub-A Flotation Cells have demonstrated that they alone produce products most acceptable for economic efficiency. In competitive tests where all phases of the operation are studied in thorough detail, it has been proven time and again that Sub-As show metallurgical advantages which contribute to the highest overall efficiency of an entire mining operation. Sub-A cells are:
(1) More selective through all mesh sizes. (2) Produce a coarser concentrate. (3) Produce a concentrate more acceptable to subsequent treatment. (4) Produce equal or higher recovery in conjunction with a higher grade concentrate and higher ratio of concentration.
A comparison of product assays does not give true and complete information with respect to the performance of a Flotation Cell. Product assays for two flotation machines operating in parallel could quite conceivably be identical, yet the physical characteristics of the products recovered and discarded would be entirely dissimilar. Wide differences which would be obvious in detailed investigation might not be indicated by a cursory examination. A detailed study of flotation concentrates shows that Sub-As recover the coarser more granular sulphides which parallel cells lose in the tailing. The higher recovery of coarse concentrate has been the story in every instance where Sub-A cells have been on a comparative basis. The use of Sub-A cells is responsible for the trend in concentration by flotation of coarse granular concentrates with minimum slimes. Higher recoveries have been possible in many instances by changes in grinding and removal of coarse primary concentrates. Recovery at a coarser grind means a decreased amount of slime mineral in the pulp. Absence of slime in concentrates is reflected in the analysis of the insoluble fraction. Sub-A cells always show a lower percentage of slime in concentrate due to selectivity and this means minimum refractories in subsequent treatment.
Screen analysis of products recovered and rejected clearly demonstrate the absence of sanding and segregation in Sub-A cells and the patented positive circulation principle assures balanced products.
The capacity of a flotation cell, treating any ore, depends upon facts and conditions which can best be determined by experience and test work. The pulp density and flotation contact period required materially affect the capacity of a Flotation Cell. With these factors known from previous work or test results, the size machine can be determined. Three conditions are factors in determining the proper size celland number of cells.
Flotation contact time required for the ore is one of the determining factors in calculating capacity. If an ore is slow floating and requires twelve minute treatment time, and another ore is fast floating and requires but six minute treatment, it is evident that a cellof only half the capacity is necessary in the last instance. Pulp density and specific gravity of dry solids control the cubic feet of pulp handled by the Flotation Cell, so are determining factors in calculating the flotation contact period. The Sub-A capacity recommendations are conservative figures which are based on years of actual field operation, treating many kinds of material.
The volume of the flotation cell must be known, as the volume in the Floatation Cell determines the time available for flotation of the values to take place. Therefore, the capacity of any Flotation Cell is dependent on the volume. All flotation cells having the same volume will have approximately the same capacity, with allowance made for horsepower, the efficiency of the impeller and aeration. As the flotation contact period is very important in any Flotation Cell, the actual cubical content of any machine should be carefully checked as well as accurate determinations on average pulp specifications.
Metallurgical results required from the floatation machine will have considerable bearing on the installed capacity. Several stages of cleaning may be required to give a high grade concentrate and this can be accomplished by the Sub-A, usually in one machine without resort to pumps for middling return. Results with cells of equal volume will not necessarily be equal because they may not be equally efficient. It may be easy enough to pass pulp through a Floatation Cell but to have a machine give a high-grade concentrate, to retreat middlings, and to give a low tailing, is an advantage obtained by use of Sub-As.
Under the table, problems are given to illustrate the methods of calculating the number of cells required. In order to secure the maximum positive treatment of the mineral, and to produce a high grade concentrate, it is best to have the necessary total volume divided into at least four cells and preferably six cells, each a separate cell, so that they may be used for roughing, cleaning, or recleaning purposes.
To determine the number of Sub-A cells requiredmultiply the proposed tonnage per day (24 hours) by the time (number of minutes necessary to float the mineral) then divide this product by the proper figure given in the table. This figure is secured by taking the size cellunder consideration (find the horizontal line giving the dilution of mill pulp and the vertical line giving the specific gravity of your ore); the figure will be at the point of intersection.For conservative estimates on a gold, silver, copper, or lead ore, use the tonnage capacities in the following table.
PROBLEM 2How many No. 18 Sp. (3232) Sub-A Cells are required to treat 125 tons of lead-zinc ore per day, with treatment time 14 minutes for the lead, dilution 3 to 1, and with treatment time 16 minutes for the zinc, dilution 3 to 1, and sp. gr. 3.4?
Continuous 24-hour per day service depends upon the mechanical design and construction of a Flotation Cell. There is no unit so rugged, nor so well built to meet the demands of the process, as the Flotation Cell. The ruggedness of each cell is necessary to give long life and to meet the requirements of the process. Numerous competitive tests all over the world have conclusively proved the real worth of these cells to many mining operators who demand Maximum results at the lowest cost.
The location of the feed pipe and the stationary hood over the rotating impeller account for the simplicity of the Sub-A cell construction. These parts eliminate swirling around the shaft and top of the impeller, reduce power load, and improve metallurgical results.
Improvements in construction of Sub- A cells during the last ten years have been gradually made as a result of plant scale testing and through suggestions from the mining fraternity. Today the Sub-A is mechanically unexcelled with rugged construction, pressure cured wearing parts, heavy duty, dependable drives. The abrasive cell zone is protected with rubber bottom liners and hard iron or Decolloy side liners. The heavy duty shafts are also rubber protected so the entire abrasive zone is sheathed for protection against wear.
The Sub-A, with its distinctive advantages, is moderately priced, due to standardization and quantity production. There is a definite mechanical or metallurgical reason behind the construction of every part of the Sub-A as explained in the following specifications.
The tank for the Flotation Cell is made of heavy steel joints are electric welded both inside and out. Partition plates are furnished with gaskets and arranged for bolting to partition channels so that if necessary all of the plates can be changed at any time in the field to provide either a right or left hand machine. Right hand machine is standard and will be furnished unless otherwise noted.
Flotation Cells are also available in wood tank construction especially suitable for corrosive circuits. These machines can be supplied with modifications so that they are ideal for use in special applications.
All cells are placed at a common floor level and due to the gravity flow principle of Flotation Cells almost any number of cells can be used in any circuit at one elevation without the necessity of pumps or elevators to handle the flow from one cellto the next. Operation and supervision is thus simplified.
For export shipments all of the items for the Flotation Cell are packed, braced, and blocked inside of the steel tank so that minimum volume is required. Safe delivery of parts without damage is thus assured.
The shaft and bearings of the Sub-A are supported in an enclosed ball bearing housing designed to properly carry and maintain the rotating impeller. Both the upper and lower heavy duty, oversized, anti-friction bearings are seated in this housing, insuring perfect alignment and protection against dirt.
Bearings have grease seals to prevent grease or oil getting into the cells; lubrication is only needed about once in six months. Many thousands of these standard bearings are in daily use on Sub- A cells, giving continuous service and low horsepower.
The hood, which is located near the bottom of the cell, is an important part of the assembly as it serves a number of purposes. The vanes on this hood prevent swirling of the pulp in the cell, producing a quiet action in the central or separation zone. The hood also supports the stationary standpipe and the hood wearing plate. Aeration of the pulp takes place in the impeller zone just below the stationary hood. The wearing plate is bolted to the bottom of the hood and prevents the impeller from being buried by pulp when the Flotation Cell is shut down.
Data from large operations have shown that the life of rubber parts is from six to fifteen times longer than the life of hard iron wearing parts. The slightly greater cost of these parts is therefore more than offset by the longer life. The advantages gained not only in lower maintenance but also in reduction in horsepower (because of the lower coefficient of friction when using molded rubber impellers) make them most economical. Both receded disk and conical disk wearing parts are also available in special hard alloy iron.
The receded disk impellers and diffuser wearing plates have been proved in commercial installations for many years and are one of the important developments made in the Flotation Cell. The receded disk impellers and diffuser wearing plates are furnished with all machines unless otherwise specified. The advantages of these parts are as follows:
Agitation is intense in the agitation zone but elsewhere it is held at a minimum and at the same time the air is finely dispersed throughout the pulp so that the cell surface presents the appearance of a smooth and quiet blanket of froth, conducive to good flotation. Molded rubber parts are recommended due to their lighter weight, perfect balance, and longer life.
In keeping with a long established policy, it is possible to use these parts on any Flotation Cell irrespective of age, without the necessity of making any major changes, thus adhering to the standard policy of No yearly models but continually improving.
The conical disk impellers and wearing plates, as illustrated, are obtainable for all sizes of machines. The conical disk impellers and wearing plates have been used in Flotation Cells for many years but are rapidly being replaced by the receded disk impellers and diffusers for general purposes. Conical disk impellers are recommended for Unit Flotation Cells and applications such as treatment of dense pulp and coarse material. Diameters of all impellers have a definite relationship to cell sizes, thus insuring uniform circulation of the pulp.
The Sub-A was the first Flotation Cellto use the Multi-V-Belt Horizontal Drive, which has proved so successful. Sub-A Flotation Cells have been carefully designed to be driven either by a motor and V-belts or by V-belts to a main drive shaft. In the motor driven type the impeller shafts are driven by V-belts, sheaves, and vertical ball bearing motor. This type of motor drive is much more economical and desirable than a direct motor driven unit because it makes any speed range available and does not require a special motor shaft assembly.
The standard drive on all flotation machines of an even number of cells is one motor driving two cells through V-belt drives. If an odd number of cells is ordered, a drive which will prove most economical in first cost and provide the greatest operating efficiency will be furnished. Adjustment of belt tension is provided for in the motor mounting.
The paddleshaftdrive is taken generally from the last impeller shaft by means of V-belt drive to a speed reducer, which in turn drives the paddleshaft at slow speed. The No. 30 paddleshaft drive is from a gear motor. The quick removal of the mineral froth, in the form of a concentrate, increases the recovery; quick removal of this mineral froth is very important and when a high grade concentrate is desired, the rotating paddles can be regulated as desired.
Every Sub-A Cell is actually an individual flotation machine with its own pulp level, controlled by its weir overflow. Correct overflow normally requires this positive pulp level control in each cell even though this adjustment when once made is infrequently changed. There are three methods of regulating pulp level:
Weir Blocks, as illustrated, slide easily into place at the weir, and consist of wood slats held down by means of a steel wearing bar. On the smaller machines, especially, adjustment by this means is easy as the weir is readily accessible. Actual plant practice shows in the normal circuit that it is not necessary to change the pulp level frequently.
Handwheel operated weir gates can be provided, as illustrated, so that changes in pulp level in each individual cell can be accomplished by turning the handwheel which is located far above the froth level. Changes in level can be made quickly and easily with minimum effort.
Gear driven handwheel gates, as illustrated, can be provided and are especially useful on large size Flotation Cells. This arrangement brings the control of the pulp level out to the front of the machine making it unnecessary to reach over the froth lip. The use of a gear box with handwheel control reduces the effort required for raising or lowering the gate, and provides a method of quick and easy adjustment.
Cell liners fit easily into the cell and consist of four cast iron liners and a rubber bottom liner. This bottom liner consists of a rubber compound similar to that used on the molded rubber parts, firmly bonded to a steel backing so that it does not rip or blister. This liner is held in place at the edges by the side liners.
Cell drainage is through an easily accessible port at the back of each cell.A small recirculation gate is provided near the top of each cell so that if desired, a portion of the pulp can be removed from the middling zone and returned to the impeller for retreatment. This recirculation feature influences the production of high grade concentrates in some cases. A gate is provided for this recirculation opening so that an adjustment of the zone and amount of recirculation can be varied.
Flotation Cells are provided with openings in the partition plates for by-passing the pulp from cell to cell without the pulp circulating through each hood feed pipe. In normal operation these partition gates are left closed; however, this arrangement is advantageous when large tonnages are fed to the Flotation Cell. This arrangement also allows the machine to be operated in groups of cells with the same positive control and circulation applied to each group.
The impeller assembly, consisting of steel shaft, totally-enclosed spindle bearings, standpipe, air- bonnet, hood, wearing plate, and impeller, fits easily into the cell as a unit. Diffuser and receded disk impellers, as illustrated, are furnished but conical disk impellers can be used if desired. The hood rests on corners of the cell side liners and is provided with keystone plug plates in front and back with recirculation openings. These recirculation opening in the plug plates can be opened, closed or bushed to small sizes as desired but in normal operation are closed. The keystone plug plate can be removed to provide an opening to the impeller for the return of middlings or feed, by means of return feed pipe which is easily placed to fit between the hood and front or back plate of the cell. Openings are provided in front of each cell for the return of middlings into any cell by gravity or these openings can be used to introduce feed into the cell if desired.
Adjustment of the impeller is easily and quickly accomplished from the rear of the Flotation Cell by means of the threaded rod holding the end of the spindle bearing housing. Proper adjustment of the clearance between impeller and wearing plate is important, and is easily done by loosening the bolts holding the spindle bearing housing and raising or lowering the entire housing by means of the adjustment provided by this threaded rod. After the proper clearance is secured the housing is tightened in place. Guides on each bearing housing keep rotation of impeller in perfect alignment and make vertical adjustment easy.
This policy of continual improvement is the aim for advancement of the Sub-A. Experiences gained in field studies have shown the factor of safety to use on shafting, bearings, and other operating parts.
Miningpioneered and developed the method of producing molded rubber wearing parts in 1932. Experience in handling practically all types of abrasive pulps, and in circuitswith various types of flotation reagents, and oils, have facilitated the development of suitable rubber compounds to meet all conditions. The use of Sub-A Flotation with molded rubber wearing parts is extremely valuable to the operator, with the assurance of the lowest possible maintenance cost. Molded rubber wearing parts are still the leaders in the field, giving trouble-free service for much longer periods than any other make impeller. Molded rubber impellers have handled some very large tonnages and records of 4 and 5 years continuous operation are common.
These features, combined with the sturdy construction of the cells, oversize bearings, heavy duty shafting and rugged cell liners, are showing average maintenance costs including labor for installation of less than $0.001 per ton in many cases. Even under the most adverse conditions Sub-A Cells rarely show total repair cost in excess of $0.003 per ton.
Each Sub-A Cell is provided with an air bonnet on the shaft assembly so that low pressure air may be connected if desired. To assure complete diffusion of air in the pulp an automatic seal is built in each weir casting.
Feed may enter any cell of a Flotation Cell, through the front or back. The hand of the Sub-A may be easily changed in the field by reversing the position of the weir casting with plate and partition plate. The hood assembly is turned through 180 degrees and the feed liner is changed with the liner in the opposite segment.
The MAC flotation cell was developed by Kadant-Lamort Inc. It can save energy comparedto conventional flotation systems. The MAC flotation cell is mainly used in the flotation section of waste paper deinking pulping, for removal of hydrophobic impurities such as filler, ash,ink particles, etc. It can increase pulp whiteness and meet the requirements of final paper appearance quality. Table11.11 shows the features of MAC flotation cell. Kadants MAC flotation cell deinking system uses air bubbles to float ink particles to the cell surface for removal from the recycled material. The latest generation of the MAC cell deinking system incorporates a patented bubble-washing process to reduce power consumption and also fiber loss. It combines small, new, auto-clean, low-pressure injectors with a flotation cell. The function of injectors is to aerate the stock before it is pumped and sent tangentially to the top of the cell. The air bubbles collect ink particles in the cell and rise up to the top to create a thick foam mat that is evacuated because of the slight pressurization of the cell. The partially deinked stock then goes to a deaeration chamber and is pumped to the next stage. Here, the operation is exactly the same as for the first stage. This stage also has the same number of injectors and same flow (Kadant,2011). This operation is repeated up to five times for a high ink removal rate. Remixing of the air coming from downstream stages of the process helps the upstream stages and improves the overall cell efficiency. Adjustable and selective losses of fiberdepend on the application and technical requirements inks, or inks and fillers. The use of low-pressure injectors in the MAC flotation cell could save about 2530% of the energy used in conventional flotation systems (ECOTARGET,2009). The benefits of the MAC flotation cell are summarized in Table11.12.
Agitated flotation cells are widely used in the mineral processing industry for separating, recovering, and concentrating valuable particulate material from undesired gangue. Their performance is lowered, however, when part of the particulate system consists of fines, with particle diameters typically in the range from 30 to 100m. For example, it was observed difficult to float fine particles because of the reduction of middle particles (of wolframite) as carriers and the poor collision and attachment between fine particles and air bubbles; a new kinetic model was proposed .
As an alternative to agitated cells, bubble columnsused in chemical engineering practice as chemical reactorswere proposed for the treatment of fine particle systems. Flotation columns, as they came to be known, were invented back in the 1960s in Canada . The main feature that differentiates the column from the mechanical flotation cell (of Denver type) is wash water, added at the top of the froth. It was thought to be beneficial to overall column performance since it helps clean the froth from any entrained gangue, while at the same time preventing water from the pulp flowing into the concentrate. In this way, it was hoped that certain cleaning flotation stages could be gained.
Let us note that the perhaps insistence here on mineral processing is only due to the fact that most of the available literature on flotation is from this area, where the process was originated and being widely practiced. The effect of particle size on flotation recovery is significant; it was shown that there exists a certain size range in which optimum results may be obtained in mineral processing. This range varies with the mineral properties such as density, liberation, and so on, but was said to be of the order of 10100m .
Regulating the oxidation state of pyrite (FeS2) and arsenopyrite (FeAsS), by the addition of an oxidation or reduction chemical agent and due to the application of a short-chain xanthate as collector (such as potassium ethyl xanthate, KEX), was the key to selective separation of the two sulfide minerals, pyrite and arsenopyrite . Strong oxidizing agents can depress previously floated arsenopyrite. Various reagents were examined separately as modifiers and among them were sodium metabisulfite, hydrazinium sulfate, and magnesia mixture. The laboratory experiments were carried out in a modified Hallimond tube, assisted by zeta-potential measurements and, in certain cases, by contact angle measurements.
This conventional bench-scale flotation cell provides a fast, convenient, and low-cost method, based on small samples (around 2g), usually of pure minerals and also artificial mixtures, for determining the general conditions under which minerals may be rendered floatableoften in the absence of a frother (to collect the concentrate in the side tube) . This idea was later further modified in the lab replacing the diaphragm, in order to conduct dissolved air or electroflotation testssee Section 3.
Pyrite concentrates sometimes contain considerable amounts of arsenic. Since they are usually used for the production of sulfuric acid, this is undesirable from the environmental point of view. However, gold is often associated with arsenopyrite, often exhibiting a direct relationship between Au content and As grade. There is, therefore, some scope for concentrating arsenopyrite since the ore itself is otherwise of little value (see Fig.2.2). Note that previous work on pyrites usually concentrated on the problem of floating pyrite .
In the aforementioned figure (shown as example), the following conditions were applied: (1) collector [2-coco 2-methyl ammonium chloride] 42mg/L, frother (EtOH) 0.15% (v/v), superficial liquid velocity uL=1.02cm/s, superficial gas velocity uG=0.65cm/s, superficial wash water velocity uw=0.53cm/s; (2) hexadecylamine, 45mg/L; pine oil, 50mg/L; EtOH, 0.025%; uL=0.84cm/s; uG=0.72cm/s; uw=0.66cm/s; (3) Armoflot 43, 50mg/L; pine oil, 50mg/L; EtOH, 0.025%; uL=0.84cm/s; uG=0.71cm/s; uw=0.66cm/s . The pyrite (with a relatively important Au content of 21g/ton) was a xanthate-floated concentrate. The presence of xanthates, however, might cause problems in the subsequent cyanidation of pyrites when recovering their Au value, which perhaps justified the need to find alternative collectors. In general, the amines exhibited a behavior similar to that of the xanthates (O-alkyl dithiocarbonates). The benefit of the amine was in its lower consumption, as compared with the xanthate systems.
The arsenic content of the pyrite was approximately 9% (from an initial 3.5% of the mixed sulfide ore). The material was sieved and the75m fraction was used for the laboratory-scale cylindrical column experiments. The effect on metallurgical characteristics of the flotation concentrate of varying the amount of ferric sulfate added to the pulp was studied; three collectors were used and their performance was compared (in Fig.2.2). Both hexadecylamine and Armoflot 43 (manufactured by Akzo) exhibited an increased recovery but a very low enrichment, whereas 2-coco 2-methyl ammonium chloride (Arquad-2C) showed a considerable enrichment; a compromise had to be made, therefore, between a high-grade and a low recovery.
Electroflotation (electrolytic flotation) is an unconventional separation process owing its name to the bubbles generation method it uses, i.e., electrolysis of the aqueous medium. In the bottom of the microcell, the two horizontal electrodes were made from stainless steel, the upper one being perforated. The current density applied was 300 Am2. It was observed that with lime used to control pH, different behavior was observed (see Fig.2.3). Pyrite, with permanganate (a known depressant) also as modifier, remained activated from pH 5.0 to 8.0at 80% recovery, while it was depressed at the pH range from 9.0 to 12.0. A conditioning of 30min was applied in the presence of modifier alone and further 15min after the addition of xanthate. The pure mineral sample, previously hand collected, crushed, and pulverized in the laboratory, was separated by wet sieving to the45 to+25m particle size range.
Pyrite due to its very heterogeneous surface, consisting of a mosaic of anodic and cathodic areas, presents a strong electrocatalytic activity in the anodic oxidation of xanthate to dixanthogen. It is also possible that the presence of the electric field, during electroflotation, affected the reactions taking place. In order to explain this difference in flotation behavior thermodynamic calculations for the system Fe-EX-H2O have been done . It was concluded that electroflotation was capable of removing fine pyrite particles from a dilute dispersion, under controlled conditions. Nevertheless, dispersed air and electroflotation presented apparent differences for the same application.
The size of the gas bubbles produced was of the order of 50m, in diameter . Similar measurements were later carried out at Newcastle, Australia ; where it was also noted that a feature of electroflotation is the ability to create very fine bubbles, which are known to improve flotation performance of fine particles.
In fact, the two electrodes of a horizontal electrodes set, usually applied in electroflotation, could be separated by a cation exchange membrane, as only one of the produced gases is often necessary . In the lower part/separated electrode, an electrolyte was circulated to remove the created gas, and in the meantime, increase the conductivity; hence having power savings (as the electric field is built up between the electrodes through the use of the suspension conductivity). Attention should be paid in this case to anode corrosion, mainly by the chloride ion (i.e., seawater).
Microorganisms have a tremendous influence on their environment through the transfer of energy, charge, and materials across a complex biotic mineralsolution interface; the biomodification of mineral surfaces involves the complex action of microorganism on the mineral surface . Mixed cationic/anionic surfactants are also generating increasing attention as effective collectors during the flotation of valuable minerals (i.e., muscovite, feldspar, and spodumene ores); the depression mechanisms on gangue minerals, such as quartz, were focused .
Another design of a flotation cell which applies ultrasound during the flotation process has been developed by Vargas-Hernndez et al. (2002). The design consists of a Denver cell (Koh and Schwarz, 2006) equipped with ultrasonic capabilities of performing ultrasound-assisted flotation experiments. This cell is universally accepted as a standard cell for laboratory flotation experiments. In Figure 35.25, a schematic of the Denver cell equipped with two power transducers is shown operating at 20kHz. The ultrasonic transducers are in acoustic contact with the body of the flotation cell but are not immersed in the same cell. Instead, they are submerged in distilled water and in a thin membrane that separates the radiant head of the transducer from the chamber body. The floatation chamber has a capacity of 2.7l and is also equipped with conventional systems to introduce air and mechanical agitation able to maintain the suspension of metallurgical pulp. In the upper part of the cell there is an area in which the foam is recovered for analysis by a process called skimming. The block diagram of Figure 35.25 further shows that the experimental system was developed to do ultrasonic-assisted flotation experiments. The transducers operate at 20kHz and can handle power up to 400W. In the Denver cell an acoustic probe, calibrated through a nonlinear system and capable of measuring high-intensity acoustic fields, is placed (Gaete-Garretn et al., 1993, 1998). This is done in order to determine the different acoustic field intensities with a spatial scanner during the experimentation. Figure 35.26 shows the distribution of ultrasonic field intensity obtained by a spatial scanner in the central area of the flotation chamber. The Denver cell with ultrasonic capabilities, as described, is shown in Figure 35.27. The obtained results were fairly positive. For example, for fine particle recovery it worked with metallurgical pulp under 325mesh, indicating floating particles of less than 45m, and the recovery curves are almost identical to those of an appropriate size mineral for flotation. This is shown in Figure 35.28, where a comparison between typical copper recovery curves for fine and normal particles is presented. The most interesting part of the flotation curves is the increase in recovery of molybdenum with ultrasonic power, as shown in Figure 35.29. The increase in recovery of iron is not good news for copper mines because the more iron floating the lower grade of recovery. This may be because the iron becomes more hydrophobic with ultrasonic action. According to the experts, this situation could be remedied by looking for specific additives to avoid this effect. Flotation kinetics shown in Figure 35.30 with 5 and 10W of acoustic power applied also show an excellent performance. It should be noted that the acoustic powers used to vary the flotation kinetics have been quite low and could clearly be expanded.
Figure 35.28. Compared recovering percent versus applied power in an ultrasonic-assisted flotation process in a Denver cell: (a) fine and ultrafine particles recovering and (b) normal particles recovering.
These experiments confirm the potential of power ultrasound in flotation. Research on assisted flotation with power ultrasound has been also carried out by Ozkan (2002), who has conducted experiments by pretreating pulp with ultrasound during flotation. Ozkhans objective was to recover magnesite from magnesite silts with particles smaller than 38m. Their results show that under ultrasonic fields the flotation foam bubbles are smaller, improving magnesite recovery rates. When Ozkhan treated magnesite mineral with a conventional treatment the beneficial effect of ultrasound was only manifested for mineral pretreatment. The flotation performed under ultrasonic field did not show improvement. This was because power ultrasound improves the buoyancy of clay iron and this has the effect of lowering the recovery of magnesite.
Kyllnen et al. (2004) employed a cell similar to Jordan to float heavy metals from contaminated soils in a continuous process. In their experiments they obtained a high recovery of heavy metals, improving the soil treatment process. Alp et al. (2004) have employed ultrasonic waves in the flotation of tincal minerals (borax Na O710 B4 H2O), finding the same effects as described above, i.e., that power ultrasound helps in the depression of clay. However, the beneficial effect of ultrasound is weakened when working with pulps with high mineral concentration (high density), probably due to an increase in the attenuation of the ultrasonic field. Safak and Halit (2006) investigated the action mechanisms of ultrasound under different flotation conditions. A cleaning effect on the floating particles was attributed to the ultrasonic energy, making the particles more reactive to the additives put in the metallurgical pulp. Furthermore due to the fact that the solid liquid interface is weaker than the cohesive forces of the metallurgic pulp liquids, it results in a medium favorable to creation of cavitation bubbles. The unstable conditions of a cavitation environment can produce changes in the collectors and even form emulsions when entering the surfactant additives. In general, many good properties are attributed to the application of ultrasound in flotation. For example, there is a more uniform distribution of the additives (reagents) and an increase in their activity. In fact in the case of carbon flotation it has been found that the floating times are shortened by the action of ultrasound, the bubble sizes are more stable, and the consumption of the reagents is drastically lowered.
Abrego Lpez (2006) studied a water recovery process of sludge from industrial plants. For this purpose he employed a flotation cell assisted by power ultrasound. In the first stage he made a flotation to recover heavy metals in the metallurgical pulp, obtaining a high level of recovery. In the second stage he added eucalyptus wood cones to the metallurgical pulp to act as an accumulator of copper, lead, nickel, iron, and aluminum. The author patented the method, claiming that it obtained an excellent recovery of all elements needing to be extracted. zkan and Kuyumcu (2007) showed some design principles for experimental flotation cells, proposing to equip a Denver flotation cell with four power transducers. Tests performed with this equipment consisted of evaluating the possible effects that high-intensity ultrasonic fields generated in the cell may have on the flotation. The author provides three-dimensional curves of ultrasonic cavitation fields in a Denver cell filled with water at frequencies between 25 and 40kHz. A warming effect was found, as expected. However, he states that this effect does not disturb the carbon recovery processes because carbon flotation rarely exceeds 5min. They also found that the pH of tap water increases with the power and time of application of ultrasound, while the pH of the carbonwaterreagentsludge mixture decreases. The conductivity of the metallurgical pulp grows with the power and time of application of ultrasound, but this does not affect flotation. The carbon quality obtained does not fall due to the application of ultrasound and the consumption of lowered reagents. They did not find an influence from the ultrasound frequency used in the process, between 25 and 40kHz. They affirmed that ultrasound is beneficial at all stages of concentration.
Kang et al. (2009) studied the effects of preconditioning of carbon mineral pulp in nature by ultrasound with a lot of sulfur content. They found that the nascent oxygen caused by cavitation produces pyrite over oxidation, lowering its hydrophobicity, with the same effect on the change of pH induced by ultrasonic treatment. Additionally, ultrasound decreases the liquid gas interfacial tension by increasing the number of bubbles. Similar effects occur in carbon particles. The perfect flotation index increases 25% with ultrasonic treatment. Kang et al. (2008) continued their efforts to understand the mechanism that causes effects in ultrasonic flotation, analyzing the floating particles under an ultrasonic field by different techniques like X-ray diffraction, electron microscopy, and scanning electron microscopy techniques. In carbon flotation it is estimated that ultrasonic preconditioning may contribute to desulfurization and ash removal (deashing) in carbon minerals. Zhou et al. (2009) have investigated the role of cavitation bubbles created by hydrodynamic cavitation in a flotation process, finding similar results to those reported for ultrasonic cavitation flotation. Finally, Ozkan (2012) has conducted flotation experiments with the presence of hard carbon sludge cavitation (slimes), encountering many of the effects that have been reported for the case of metallurgical pulp with ultrasound pretreatment. This includes improved flotation, drastic reduction in reagent consumption, and the possible prevention of oxidation of the surface of carbon sludge. A decrease in the ash content in floating carbon was not detected. However, tailings do not seem to contain carbon particles. All these effects can be attributed to acoustic cavitation. However, according to the author, there is a need to examine the contribution of ultrasound to the probability of particlebubble collision and the likelihood of getting the bubbles to connect to the particles. The latter effects have been proposed as causes for improvements in flotation processes in many of the publications reviewed, but there is no systematic study of this aspect.
In summary, power ultrasound assistance with flotation processes shows promising results in all versions of this technique, including conditioning metallurgical pulp before floating it, assisting the continuous flotation process, and improving the yields in conventional flotation cells. The results of ultrasonic floating invariably show a better selectivity and an increase, sometimes considerable, in the recovery of fine particles. Paradoxically, in many experiments an increase has been recorded in recovering particles suitable for normal flotation. These facts show the need for further research in the flotation process in almost all cases, with the exception perhaps of carbon flotation. For this last case, in light of the existing data the research should be directed toward scale-up of the technology.
The concentrate obtained from a batch flotation cell changes in character with time as the particles floating change in size, grade and quantity. In the same way, the concentrate from the last few cells in a continuous bank is different from that removed from the earlier cells. Particles of the same mineral float at different rates due to different particle characteristics and cell conditions.
The recovery of any particular mineral rises to an asymptotic value R which is generally less than 100%. The rate of recovery at time t is given by the slope of the tangent to the curve at t, and the rate of recovery at time t1 is clearly greater than the rate at time t2. There is a direct relationship between the rate of flotation and the amount of floatable material remaining in the cell, that is:
The process is carried out in a flotation cell or tank, of which there are two basic types, mechanical and pneumatic. Within each of these categories, there are two subtypes, those that operate as a single cell, and those that are operated as a series or bank of cells. A bank of cells (Fig. 8) is preferred because this makes the overall residence times more uniform (i.e., more like plug flow), rather than the highly diverse residence times that occur in a single (perfectly mixed) tank.
FIGURE 8. Flotation section of a 80,000t/d concentrating plant, showing the arrangement of the flotation cells into banks. A small part of the grinding section can be seen through the gap in the wall. [Courtesy Joy Manufacturing Co.]
The purpose of the flotation cell is to attach hydrophobic particles to air bubbles, so that they can float to the surface, form a froth, and can be removed. To do this, a flotation machine must maintain the particles in suspension, generate and disperse air bubbles, promote bubbleparticle collision, minimize bypass and dead spaces, minimize mechanical passage of particles to the froth, and have sufficient froth depth to allow nonhydrophobic (hydrophilic) particles to return to the suspension.
Pneumatic cells have no mechanical components in the cell. Agitation is generally by the inflow of air and/or slurry, and air bubbles are usually introduced by an injector. Until comparatively recently, their use was very restricted. However, the development of column flotation has seen a resurgence of this type of cell in a wider, but still restricted, range of applications. While the total volume of cell is still of the same order as that of a conventional mechanical cell, the floor space and energy requirements are substantially reduced. But the main advantage is that the cell provides superior countercurrent flow to that obtained in a traditional circuit (see Fig. 11), and so they are now often used as cleaning units.
Mechanical cells usually consist of long troughs with a series of mechanisms. Although the design details of the mechanisms vary from manufacturer to manufacturer, all consist of an impeller that rotates within baffles. Air is drawn or pumped down a central shaft and is dispersed by the impeller. Cells also vary in profile, degree of baffling, the extent of walling between mechanisms, and the discharge of froth from the top of the cell.
Selection of equipment is based on performance (represented by grade and recovery), capacity (metric tons per hour per cubic meter); costs (including capital, power, maintenance), and subjective factors.
Among all processing industries, only in the ore and mining industries is the accent more on wear resistance than corrosion. In mining industries, the process concerns material handling more than any physical or chemical conversions that take place during the refining operations. For example, in the excavation process of iron ore, conventional conveyer systems and sophisticated fluidized systems are both used [16,17]. In all these industries, cost and safety are the governing factors. In a fluidized system, the particles are transported as slurry using screw pumps through large pipes. These pipes and connected fittings are subjected to constant wear by the slurry containing hard minerals. Sometimes, depending on the accessibility of the mineral source, elaborate piping systems will be laid. As a high-output industry any disruption in the work will result in heavy budgetary deficiency. Antiabrasive rubber linings greatly enhance the life of equipment and reduce the maintenance cost. The scope for antiabrasive rubber lining is tremendous and the demand is ever increasing in these industries.
Different rubber compounds are used in the manufacture of flotation cell rubber components for various corrosion and abrasion duty conditions. Flotation as applied to mineral processing is a process of concentration of finely divided ores in which the valuable and worthless minerals are completely separated from each other. Concentration takes place from the adhesion of some species of solids to air bubbles and wetting of the other series of solids by water. The solids adhering to air bubbles float on the surface of the pulp because of a decrease in effective density caused by such adhesion, whereas those solids that are wetted by water in the pulp remain separated in the pulp. This method is probably the more widely used separation technique in the processing of ores. It is extensively used in the copper, zinc, nickel, cobalt, and molybdenum sections of the mineral treatment industry and is used to a lesser extent in gold and iron production. The various rubber compounds used in the lining of flotation cells and in the manufacture of their components for corrosive and abrasive duties are:
Operating above the maximum capacity can cause the performance of flotation cells to be poor even when adequate slurry residence time is available (Lynch et al., 1981). For example, Fig. 11.21 shows the impact of increasing volumetric feed flow rate on cell performance (Luttrell et al., 1999). The test data obtained at 2% solids correlates well with the theoretical performance curve predicted using a mixed reactor model (Levenspiel, 1972). Under this loading, coal recovery steadily decreased as feed rate increased due to a reduction in residence time. However, as the solids content was increased to 10% solids, the recovery dropped sharply and deviated substantially from the theoretical curve due to froth overloading. This problem can be particularly severe in coal flotation due to the high concentration of fast floating solids in the flotation feed and the presence of large particles in the flotation froth. Flotation columns are particularly sensitive to froth loading due to the small specific surface area (ratio of cross-sectional area to volume) for these units.
Theoretical studies indicate that loading capacity (i.e., carrying capacity) of the froth, which is normally reported in terms of the rate of dry solids floated per unit cross-sectional area, is strongly dependent on the size of particles in the froth (Sastri, 1996). Studies and extensive test work conducted by Eriez personnel also support this finding. As seen in Fig. 11.22, a direct correlation exists between capacity and both the mean size (d50) and ultrafines content of the flotation feedstock. The true loading capacity may be estimated from laboratory and pilot-scale flotation tests by conducting experiments as a function of feed solids content (Finch and Dobby, 1990). Field surveys indicate that conventional flotation machines can be operated with loading capacities of up to 1.52.0t/h/m2 for finer (0.150mm) feeds and 56t/h/m2 or more for coarser (0.600mm) feeds. Most of the full-scale columns in the coal industry operate at froth loading capacities less than 1.5t/h/m2 for material finer than 0.150mm and as high as 3.0t/h/m2 for flotation feed having a top size of 0.300mm feeds.
Froth handling is a major problem in coal flotation. Concentrates containing large amounts of ultrafine (<0.045mm) coal generally become excessively stable, creating serious problems related to backup in launders and downstream handling. Bethell and Luttrell (2005) demonstrated that coarser deslime froths readily collapsed, but finer froths had the tendency to remain stable for an indefinite period of time. Attempts made to overcome this problem by selecting weaker frothers or reducing frother dosage have not been successful and have generally led to lower circuit recoveries. Therefore, several circuit modifications have been adopted by the coal industry to deal with the froth stability problem. For example, froth launders need to be considerably oversized with steep slopes to reduce backup. Adequate vertical head must also be provided between the launder and downstream dewatering operations. In addition, piping and chute work must be designed such that the air can escape as the froth travels from the flotation circuit to the next unit operation.
Figure 11.23 shows how small changes in piping arrangements can result in better process performance. Shown in Fig. 11.23 is a column whose performance suffered due to the inability to move the froth product from the column launder although a large discharge nozzle (11m) had been provided. In this example, the froth built up in the launder and overflowed when the operators increased air rates. To prevent this problem, the air rates were lowered, which resulted in less than optimum coal recovery. It was determined that the downstream discharge piping was air-locking and preventing the launders from properly draining. The piping was replaced with larger chute work that allowed the froth to flow freely and the air to escape. As a result, higher aeration rates were possible and recoveries were significantly improved.
Some installations have resorted to using defoaming agents or high-pressure launder sprays to deal with froth stability. However, newer column installations eliminate this problem by including large de-aeration tanks to allow time for the froth to collapse (Fig. 11.24a). Special provisions may also be required to ensure that downstream dewatering units can accept the large froth volumes. For example, standard screen-bowl centrifuges equipped with 100mm inlets may need to be retrofitted with 200mm or larger inlets to minimize flow restrictions. In addition, while the use of screen-bowl centrifuges provides low product moistures, there are typically fine coal losses, as a large portion of the float product finer than 0.045mm is lost as main effluent. This material is highly hydrophobic and will typically accumulate on top of the thickener as a very stable froth layer, which increases the probability that the process water quality will become contaminated (i.e., black water).
This phenomenon is more prevalent in by-zero circuits, especially when the screen-bowl screen effluent is recycled back through the flotation circuit, either directly or through convoluted plant circuitry. Reintroducing material that has already been floated to the flotation circuit can result in a circulating load of very fine and highly floatable material. As a result, the capacity of the flotation equipment can be significantly reduced, which results in losses of valuable coal. Most installations will combat this by ensuring that the screen-bowl screen effluent is routed directly back to the screen bowl so that it does not return to the flotation circuit. The accumulation of froth on the thickener, which tends to be especially problematic in by-zero circuitry, is also reduced by utilizing reverse-weirs and taller center wells, as this approach helps to limit the amount of froth that can enter into the process water supply. Froth that does form on top of the clarifier can be eliminated by employing a floating boom that is placed directly in the thickener (Fig. 11.24b) and used in conjunction with water sprays. The floating boom can be constructed out of inexpensive PVC piping, and is typically attached to the rotating rakes. The boom floats on the water interface and drags any froth around to the walkway that extends over the thickener, where it is eliminated by the sprays.
Column cells have been developed over the past 30 years as an alternative to mechanically agitated flotation cells. The major operating difference between column and mechanical cells is the lack of agitation in column cells that reduces energy and maintenance costs. Also, it has been reported that the cost of installing a column flotation circuit is approximately 2540% less than an equivalent mechanical flotation circuit (Murdock et al., 1991). Improved metallurgical performance of column cells in iron ore flotation is reported and attributed to froth washing, which reduces the loss of fine iron minerals entrained into the froth phase (Dobby, 2002).
The Brazilian iron ore industry has embraced the use of column flotation cells for reducing the silica content of iron concentrates. Several companies, including Samarco Minerao S.A., Companhia Vale do Rio Doce (CRVD), Companhia Siderrgica Nacional (CSN), and Mineraes Brasileiras (MBR), are using column cells at present (Peres et al., 2007). Samarco Minerao, the first Brazilian producer to use column cells, installed column cells as part of a plant expansion program in the early 1990s (Viana et al., 1991). Pilot plant tests showed that utilization of a column recleaner circuit led to a 4% increase in iron recovery in the direct reduction concentrate and an increase in primary mill capacity when compared to a conventional mechanical circuit.
There are also some negative reports of the use of column cells in the literature. According to Dobby (2002), there were several failures in the application of column cells in the iron ore industry primarily due to issues related to scale-up. At CVRD's Samitri concentrator, after three column flotation stages, namely, rougher, cleaner, and recleaner, a secondary circuit of mechanical cells was still required to produce the final concentrate.
Imhof et al. (2005) detailed the use of pneumatic flotation cells to treat a magnetic separation stream of a magnetite ore by reverse flotation to reduce the silica content of the concentrate to below 1.5%. From laboratory testing, they claimed that the pneumatic cells performed better than either conventional mechanical cells or column cells. The pneumatic cells have successfully been implemented at the Compaia Minera Huasco's iron ore pellet plant.
This chapter presents a novel approach to establish the relationship between collector properties and the flotation behavior of goal in various flotation cells. Coal flotation selectivity can be improved if collector selection is primarily based on information obtained from prior contact angle and zeta potential measurements. In a study described in the chapter, this approach was applied to develop specific collectors for particular coals. A good correlation was obtained between laboratory batches and large-scale conventional flotation cells. This is not the case when these results are correlated with pneumatic cell trial data. The study described in the chapter was aimed at identifying reasons for the noncorrelation. Two collectors having different chemical compositions were selected for this investigation. A considerable reduction in coal recovery occurred at lower rotor speeds when comparing results of oxidized and virgin coal. The degree to which a collector enhances flocculation in both medium- and low-shear applications and also the stronger bubble-coal particle adherence required for high-shear cells must, therefore, all be taken into consideration when formulating a collector for coal flotation.
The 10th International Flotation Conference (Flotation '21) is organised by MEI in consultation with Prof. Jim Finch and is sponsored by Promet101, Maelgwyn Mineral Services, Magotteaux, Gold Ore, CiDRA Minerals Processing, Hudbay Minerals, Senmin, Clariant, BASF, Eriez, Nouryon, Festo, Newmont,Cancha, Zeiss,FLSmidthand Kemtec-Africa.
Flotation circuits are a common technology for the concentration of a broad range of minerals and wastewater treatments. Froth flotation is based on differences in the ability of air bubbles to adhere to specific mineral surfaces in a solid/liquid slurry. Particles with attached air bubbles are carried to the surface and removed, while the particles that are not attached to air bubbles remain in the liquid phase. The concept is simple, but the phenomena are complex because the results depend on what happens in the two phases (froth and pulp phases) and other phenomena, such as particle entrainment. In flotation, several parameters are interconnected, which can be classified into chemical (e.g., collectors, frothers, pH, activators, and depressants), operation (e.g., particle size, pulp density, temperature, feed rate and composition, and pulp potential), equipment (e.g., cell design, agitation, and air flow), and circuit (e.g., number of stages and configuration). If any of these factors is changed, it causes (or demands) changes in other parts, and studying all of the parameters simultaneously is impossible. Conversely, there is not a model that includes all variables; most of the models are empirical and use only a few variables.
In the literature, various methodologies for flotation circuit design have been proposed, with most using optimization techniques. In these methodologies, the alternatives are presented using a superstructure, a mathematical model is developed, and an algorithm is used to find the best option based on an objective function. The differences between these methodologies depend on the superstructure, the mathematical representation of the problem, and the optimization algorithm. However, one problem with these methods is that the recovery of each stage must be modeled, and because the recovery of each stage is a function of many variables and is difficult to model, the results are usually debatable.
Flotation circuits are commonly used for the concentration of a broad range of minerals; such circuits also used in wastewater treatment (Rubio et al. 2002). Froth flotation is based on the differences in the ability of air bubbles to adhere to specific mineral surfaces in a solid/water slurry. Then particles with or without air bubbles attached are either carried to the surface and removed or left in the liquid phase. The current method used to design these circuits is based on seven steps (Harris et al., 2002): 1) conduct a mineralogical examination in conjunction with a range of grinding tests; 2) conduct a range of laboratory scale batch tests and locked cycle tests; 3) create a circuit design based on scale-up laboratory kinetics; 4) perform a preliminary economic evaluation of the ore body; 5) pilot-plant test the circuit design; 6) evaluate the economy; 7) design a full-scale plant. This procedure has several problems: 1) the design of the circuit is made in step three based on rule-of-thumb scale-up from laboratory data; therefore, the design depends heavily on the designer's experience; 2) building laboratory and pilot plants are costly and time consuming; therefore, the designed circuit cannot be analyzed in depth; 3) other aspects, such as system dynamics, are not considered in the design process.
Froth flotation design and operation are complex tasks because various important parameters are interconnected. The parameters can be classified into four types of components, as shown in Fig. 1 If any of these factors is changed, other parts of the design will also be changed. It is impossible to study all of the parameters at the same time. For example, if six parameters are selected for study in a four-stage circuit, over eight million tests are required for a two-level fractional experiment design. In addition, for any given number of stages, there are several potential circuit configurations. For example, if six flotation stages are considered, more than 1,400 potential circuit configurations will be possible. Therefore, the design will not be analyzed in depth.
In this work, a new methodology that integrates the first five design steps outlined previously is presented. The objectives are 1) to better orient the goals of the laboratory tests; 2) to reduce the number of laboratory and pilot-plant testing, and achieving lower cost and execution times; 3) to design the flotation circuit systematically, and 4) to expedite the design process. The methodology, inspired by the work of d'Anterroches and Gani (2005), has three design steps: 1) definition and analysis; 2) process synthesis; and 3) final design. Four tools are used in this procedure: 1) laboratory testing, 2) process group contribution, 3) sensitivity analysis, and 4) reverse simulation (Fig. 2).
The example of flotation circuit without grinding considered the concentration of copper ore. The feed to the circuit corresponded to 6 t/h of chalcopyrite (33% of copper), 12 t/h of chalcopyrite slow (16% of copper), and 300 t/h of gangue. The superstructure considers five flotation stages. If all interconnection was allowed, there were over 3 million circuit structure alternatives. However, if origin-destination matrices were used to eliminate nonsense and redundant alternatives, the number of feasible flotation circuits was 6912. The procedure utilized for the postulation of a superstructure and the formulation of the mathematical programming model was the one utilized by Calisaya et al. (2016), which corresponded to a MINLP. The variables with uncertainties corresponded to the stage recoveries of the chalcopyrite, chalcopyrite slow, and gangue. The stage recoveries were difficult to model as there is not a model that can be used under all flotation circuit structures included in the superstructure. Here, the stage recoveries were represented by values obtained from the uniform distribution. Under these conditions, the design problem is a MILP.
After the optimal flotation circuit configuration is determined, the water integration problem is addressed. This problem has not been analyzed before because the main concern in mineral processing has been the recovery and the product grade. Water can be recycled from tail or concentrate dewatering operations. However, only some of the water recovered in these operations can be recycled because it affects the flotation behavior.
Recently, El-Halwagi et al. (2004) have used the concept of clustering for process design based on property integration. Property integration is defined as a functionality-based holistic approach to the allocation and manipulation of streams and processing units, which is based on tracking, adjusting, assigning, and matching functionalities throughout the process. Here the methodology developed by El-Halwagi et al. (2004) was used to design the water integration problem.
The overall problem definition given by El-Halwagi et al. (2004) can be adapted as follows: "Given a flotation circuit with certain sources (process streams and water streams) and flotation units along with their properties (pH, solid concentration, oxygen concentration) and constraints, it is desired to develop graphical techniques that identify optimum strategies for allocation and interception that integrate the properties of sources, sinks, and interceptors so as to optimize a desirable process objective (minimum usage of fresh water, maximum utilization of water recycled from tail and concentrate dewatering operations, minimum cost of slaked lime while satisfying the constraints on properties and flow rate for the sinks".
The objective of the flotation circuit used by the mining concentration plant in El Salvador is to obtain a concentrate rich in Cu and Mo. The influence of the input factors on the variability of the grade and recovery of Cu and Mo in the final concentrate is analyzed. The input factors to consider are the kinetic constants of each species in each stage, maximum recoveries of each species in each stage, residence time of the pulp in each stage, number of cells in the R and RS stages, solids concentration in each stage, froth depth height on the columns, and superficial air and water rates in the column. In total, there are 47 input factors, and uncertainty ranges were taken from Yianatos et al. (2005) and Yianatos and Henriquez (2006). Based on the current operating conditions of the El Salvador plant, variations of 15% and 3% for kinetic constants and maximum recoveries, respectively, were considered. In the case of the solids concentration and the froth depth, variations of 10% were considered. In the case of superficial air and water rates, variations of 11% and 12%, respectively, were considered. The variation in the number of cells in R and CS were 7-9 and 8-10, respectively. The variation of the residence time in the R and CS stages was 10%. Finally, the residence time in C varied by 7%.
Figure 2 shows that input factors 10 (kmax molybdenite in C), 11 (kmax of pyrite in C), 25 (surface flow), 27 (Rmax chalcopyrite in R), 30 (Rmax molybdenite in R), and 42 (Rmax molybdenite in CS) are the main sources of variability of Cu and Mo recoveries and grades. Input factor 10 affects the Mo grade and recovery, input factor 11 affects the Mo and Cu grades, input factor 25 affects both the Cu and Mo grades and recoveries, input factor 27 affects the Cu recovery, and input factors 30 and 42 affect the Mo grade and recovery. From these results, it is clear that six of the 47 input factors are the key variables for the Cu-Mo grade and recovery.
There are various methods available for cleaning fine coal, of which froth flotation has become the most common practice. Froth flotation depends on differences in surface properties between coal and shale. Air bubbles are generated within an aqueous suspension of fine raw coal with a solids concentration of less than 10%. The hydrophobic coal particles attach to the air bubbles and are buoyed to the top of the froth flotation cell where they are removed as froth. The hydrophilic shale particles remain as a suspension and are removed over the tailings weir. The property of hydrophobicity is imparted to coal particles by the addition of a collector like diesel oil. This facilitates the attachment of coal to air bubbles in preference to gangue particles. The aircoal attachment is made stable by the addition of a frothing agent like pine oil. Successful flotation is governed by different factors like oxidation and rank of coal, flotation reagents, agitation and aeration, particle size and pulp density, flotation machine, conditioning time, and pH of the pulp.
Conventional mechanically agitated flotation machines use relatively shallow rectangular tanks, whereas column cells are usually tall vessels with heights normally varying from 7 to 16m as per requirement. Column cells do not use mechanical agitation and are typically characterised by an external sparging system, which injects air into the bottom of the column cell. The absence of intense agitation promotes higher degrees of selectivity. Modern flotation machines are high-intensity equipment designed to create very small bubbles and higher flotation rates. Smaller bubbles are generated by intensive mixing of pulps with air so that fast collisions between particles and bubbles take place. Microcel machines work with forced air, whereas the Jameson cell works with induced air. These machines are particularly suitable for coal flotation (Lynch et al., 2010).
Hydrodynamic analyses have shown that the use of air bubbles smaller than typically generated by conventional flotation machines can improve fine coal recovery. The selectivity also increases as smaller bubbles rise more slowly through the pulp, leaving the high ash impurities at the bottom. One disadvantage of flotation is that efficiency reduces for size range below 100m. To overcome this constraint, a new stirrerless cone-shaped flotation cell was developed in Germany (Bahr, 1982), now called the Pneuflot. This cell uses a novel aeration technique in which minute bubbles are introduced into the slurry before it reaches the cell. The upper particle size is restricted to 300m.
Flotation circuits are simple for Indian coals. Concentrates can be produced in one stage of flotation, and recleaning of the products may not be generally necessary. In the case of highly oxidised coal, two-stage flotation may be required to be incorporated, with rougher cells and cleaner cells. The circuit consists of a number of banks depending upon the total quantity to be handled, whereas each bank can have four to eight cells. For smooth operation of the system, proper operation and control are necessary.
It is observed that the existing flotation circuits in India are not working well. There is substantial loss of coal substance along with tailings. The causes of poor performance can be attributed to the following major factors as stated by Haldar (2007):
In addition, the quality of coal fines has deteriorated and other parameters have changed. Lower seam coals of inferior quality are now supplied. The concept of treating fine coal may need changes. The floatability tests are illustrated in Fig. 8.8; it is possible to produce clean coal with ash% of 17%18% ash with sufficient yield.
A model for the design of flotation circuits under uncertainty has been presented. Uncertainty is represented by scenarios that include changes in the feed grade and in the metal price. The model allows the operating conditions (residence time and mass flows of each stream) and flow structure (tail and concentrate stream of cleaner and scavenger stage) to be changed for each scenario while the fixed design (size of cells in flotation stages) for all scenarios is maintained. The model can be modified to include other uncertainties and other adaptive variables.
To solve the two-stage stochastic model, two solution strategies were proposed. The results show that the use of average values for the stochastic parameters leads to an inefficient design and hence a decrease in the profits made in the process.
Finally, it can be concluded that the use of stochastic programming can be a beneficial tool in the design of a metallurgical process, specifically the copper flotation process. The optimal configuration is capable of adapting to uncertainty, leading to an increase in the company profits
The simplest way of smoothing out grade fluctuations and of providing a smooth flow to the flotation plant is by interposing a large agitated storage tank (agitator) between the grinding section and the flotation plant:
Any minor variations in grade and tonnage are smoothed out by the agitator, from which material is pumped at a controlled rate to the flotation plant. The agitator can also be used as a conditioning tank, reagents being fed directly into it. It is essential to precondition the pulp sufficiently with the reagents (including sometimes air, Section 12.8) before feeding to the flotation banks, otherwise the first few cells in the bank act as an extension of the conditioning system, and poor recoveries result.
Provision must be made to accommodate any major changes in flowrate that may occur; for example, grinding mills may have to be shut down for maintenance. This is achieved by splitting the feed into parallel banks of cells (Figure 12.53). Major reductions in flowrate below the design target can then be accommodated by shutting off the feed to the required number of banks. The optimum number of banks required will depend on the ease of control of the particular circuit. More flexibility is built into the circuit by increasing the number of banks, but the problems of controlling large numbers of banks must be taken into account. The move to very large unit processes, such as grinding mills, flotation machines, etc., in order to reduce costs and facilitate automatic control, has reduced the need for many parallel banks.
Some theoretical considerations have been introduced (Section 12.11.2), but there is a practical aspect as well: if a small cell in a bank containing many such cells has to be shut down, then its effect on production and efficiency is not as large as that of shutting down a large cell in a bank consisting of only a few such cells.
Flexibility can include having extra cells in a bank. It is often suggested that the last cell in the bank normally should not be producing much overflow, thus representing reserve capacity for any increase in flowrate or grade of bank feed. This reserve capacity would have to be factored in when selecting the length of the bank (number of cells) and how to operate it, for example, trying to take advantage of recovery or mass pull profiling. If the ore grade decreases, it may be necessary to reduce the number of cells producing rougher concentrate, in order to feed the cleaners with the required grade of material. A method of adjusting the cell split on a bank is shown in Figure 12.54. If the bank shown has, say, 20 cells (an old-style plant), each successive four cells feeding a common launder, then by plugging outlet B, 12 cells produce rougher concentrate, the remainder producing scavenger concentrate (assuming a R-S-C type circuit). Similarly, by plugging outlet A, only eight cells produce rougher concentrate, and by leaving both outlets free, a 1010 cell split is produced. This approach is less attractive on the shorter modern banks. Older plants may also employ double launders, and by use of froth diverter trays cells can send concentrate to either launder, and hence direct concentrate to different parts of the flowsheet. An example is at the North Broken Hill concentrator (Watters and Sandy, 1983).
Rather than changing the number of cells, it may be possible to adjust air (or level) to compensate for changes in mass flowrate of floatable mineral to the bank. To maintain the bank profile at Brunswick Mine, total air to the bank was tied to incoming mass flowrate of floatable mineral so that changes would trigger changes in total air to the bank, while maintaining the air distribution profile (Cooper et al., 2004).
Consider that the circuit discussed in section 2 corresponds to a flotation circuit in which a material composed of gangue (specie 2) and a valuable species (species 1). The operating conditions of this circuit are such that the transfer functions for species 1 are, TR1=0.75, TS1=0.85, TC1 =0.73, and for specie 2 are TR2=0.25, TS2=0.40, TC2=0.30. Each stage corresponds to flotation bank with 5, 8 and 7 cells in the stages R, S and C, respectively. The transfer function for each stage is given by (Cisternas et al., 2006)
Under the conditions of Figure 2, we have thatR1 ( TR1, 0. 86 ,0.73 ) TR1 and R2 ( TR2, 0 40, 030) TR2 . Also, as TR1=0.75 for species 1 and TR1 =0.25 for species 2, we have the following overall recoveriesR1(0.75, 0.86, 0.73)=0.94 and R2(0.25, 0.40, 0.30 =0.143 (i.e., 94% for species 1 and 14.3% for species 2.) A sensitivity analysis can help us to improve the operational conditions of the stages involved in the process, and, therefore, reduce the percentage of gangue recovery without affecting too much the percentage recovery of useful material. The recovery and sensitivity of the above circuit are given by the equations 1 to 4. Then, setting the transfer functions TSj and TCj in the eq.2, the behavior of global recovery sensitivity with respect to the transfer function TRjcan be studied as shown in figure 3. Figure 3a shows Rj/TRj versus TRj for values of (TSj,TCj) close to the species 2. It can be seen that sensitivity increases as the value of TRjincreases. The opposite behavior is shown in Figure 3b which corresponds to values of (TSj,TCj) near species 1. This means that the behavior of the sensitivity is inversely between the valuable specie and the gangue.
Figure 4a shows RjTijversus Tijfor values of (TRj,TSj,TCj)close to species 2.We can observe that for species 2, the highest sensitivity to the global recovery is given in ( 0.25 , 0.4, TC2) and ( TR, 0.40, 0.3 ), which indicates that it is more sensitive to the transfer functions in the stage C (TC2 ) and R (TR2 ). Looking at the graphs in Figure 4b, we can observe that for species 1, the highest sensitivity in the global recovery is given in (0.75,TS1, 0.73), which indicates that it is more sensitive to the transfer function in the stage S (TS1). The same analysis can be performed for the other derivatives in equations 3 and 4. Performing the sensitivity analysis, it is possible to reach the following conclusions: 1) for species 1, the highest sensitivity to the global recovery for the stages R, S and C are given in ( 0.75 , TS1, 0.73 ),( 0.75 ,TS1, 0.73 )-( 0.75 , 0.86 , TC)and (0.75,TS 1, 0.73)-(0.75,0.86,TS 1), respectively. On the other hand, for species 2, the highest sensitivity to the global recovery for the stages R, S and C are given in (0.25, 0.4,TC2)-(TR2, 0.4,0.3),(0.25, 0.4,TC2)-(TR2, 0.4,0.3) and (TR2, 0.4, 0.3), respectively.
With this information about the behavior of global recovery, the sensitivity with respect to the transfer functions, we can intuitively change the values of the transfer functions of species 1 and 2 in the stages where they are most influential. Then, by reverse simulation with equation 5, it is possible to determine new designs (N) and/or operating conditions(kijand/or ) to achieve a better system performance. Thus, by changing the number of cells at 11 and 9 for stages S and C respectively, it is possible to obtain the following results: R1(0.75, 0.80, 0.68)=0.91 and R2(0.25, 0.40, 0.20)=0.077.
The self-tuning control algorithm has been developed and applied on crusher circuits and flotation circuits  where PID controllers seem to be less effective due to immeasurable change in parameters such as the hardness of the ore and wear in crusher linings. STC is applicable to non-linear time-varying systems. It however permits the inclusion of feed forward compensation when a disturbance can be measured at different times. The STC control system is therefore attractive. The basis of the system is
The disadvantage of the set-up is that it is not very stable and therefore in the control model a balance has to be selected between stability and performance. A control law is adopted. It includes a cost function CF, and penalty on control action. The control law has been defined as
A block diagram showing the self-tuning set-up is illustrated in Figure20.26. The disadvantage of STC controllers is that they are less stable and therefore in its application, a balance has to be derived between stability and performance.
A hybrid cell is defined here as a galvanic electrochemical generator in which one of the active reagents is in the gas phase. Hybrid cells occupy an intermediate position between the closed galvanic cells described in the remainder of this book, where operation is confined to reactants added to the cell at manufacture, and fuel cells in which both cathodic and anodic reactants are supplied continuously (usually in gaseous form) from sources external to the cell. Hybrid cells take advantage of both battery and fuel cell technologies. The most common example of a primary hybrid cell, namely the metalair system, was considered in Chapter 3. In mechanically rechargeable cells, the spent metal anode is substituted by a new electrode at the end of the discharge. Here, some typical rechargeable hybrid cells are described.
The implantable hybrid cell in which body oxygen is reduced on a cathode and a sacrificial anode is oxidized has been extensively studied as a long term power source for cardiac pacemakers (1-4). This laboratory undertook a corrosion study of aluminum and zinc metals to be used as anodes; in-vivo and in-vitro galvanostatic polarization measurements were conducted on these two metals (5,6). The in-vivo corrosion rates are found to be reduced by one order of magnitude over the in-vitro values as seen in Figures 1 and 2.
Aluminum in both sets of experiments exhibits a clear passivity region of constant anodic current density with a breakdown potential Eb at approximately 0.7000 V (S.C.E.); the zinc polarization curves do not show any region of passivity. However the linear polarization technique used to determine R pol, and from which the corrosion current is approximated by
Organic compounds which absorb on metal surfaces have been known to suppress both anodic and cathodic processes. Cystine has been shown previously by Svare et al to reduce the rate of dissolution of copper in human blood (8). Aragon and Hulbert reported the inhibition effects of the bovine plasma for a titanium aluminum alloy (9).
We consider a sequence of applications of hybrid cell-based finite-element models. These models are based on treating cells as individual particles, like in particle models. The behavior of the cells (regarding division, mutation, differentiation, death, migration, mechanicalchemical activity) depends on the environment they are in. The cells themselves influence their environment as well. Parameters like chemical concentrations and mechanical strains are modeled by the finite-element method. The applications involve cancer, wound healing, wound contracture, and the regeneration of a vascular network from preexisting blood vessels (angiogenesis). The current article is merely descriptive and does not highlight the mathematical details, such as the exact description of partial and stochastic differential equations involved in the studies.
Somatic nuclear reprogramming has also been demonstrated in hybrid cells between pluripotent ES/EG and somatic cells, which also restores pluripotency in somatic nuclei. These studies indicate that not only the oocytes, but also pluripotent ES/EG cells, must contain appropriate factors to reprogram the somatic nucleus. Reprogramming of somatic nuclei in ES/Gsomatic cell hybrids is, however, relatively less complex compared to its transplantation into oocytes. This is because the somatic nucleus in the oocyte has to be reprogrammed to recapitulate the entire program of early development to the blastocyst stage. It is important to note that this donor somatic nucleus has to be reprogrammed to generate pluripotent epiblast cells, as well as the highly differentiated trophectoderm cells. The latter should be viewed as a transdifferentiation event, because somatic nuclei of diverse origin must direct differentiation of highly specialized trophectoderm cells after only a few cleavage divisions. Indeed, in some respects, this transdifferentiation event is more striking as a reprogramming event. By comparison, reprogramming of somatic nuclei in ES/EGsomatic cell hybrids is less complex, as there is restoration of pluripotency without the necessity to recapitulate early events of development.
Although EG and ES cells on the whole have similar effects on somatic nuclei, there is at least one critical difference between them. Using EGthymocyte hybrid cells, it was shown that the somatic nucleus underwent extensive reprogramming resulting in the erasure of DNA methylation associated with imprinted genes, and the inactive X-chromosome was reactivated. The somatic nucleus also acquired pluripotency, as judged by the activation of the Oct4 gene, and the hybrid cells could differentiate into all three germ layers in chimeras. This study shows that EG cells, apart from conferring pluripotency to somatic nucleus, retained a key property found only in germ cells, which is the ability to erase parental imprints and, indeed, induce genome-wide DNA demethylation. Experiments using ESthymocyte hybrid cells gave similar results, including the restoration of pluripotency to somatic nuclei, as shown by the activation of the Oct4GFP reporter gene, and for the ability of these cells to differentiate into a variety of cell types. Unlike EG cells, however, ES cells do not cause erasure of imprints from somatic nuclei. Furthermore, in ESEG hybrids, EG cells can induce erasure of imprints from ES cells, which shows that EG cells have dominant activity for the erasure of imprints and DNA demethylation. However, from these studies it is clear that DNA demethylation activity, at least for the erasure of imprints present in EG cells, is not essential for restoring pluripotency to somatic cells. It is possible to use this system to design cell-based assays in search of key reprogramming factors.
The ability of ES/EG cells to restore pluripotency in somatic nuclei is significant because it also opens up possibilities to identify the molecules involved in reprogramming somatic nuclei. Such studies are difficult with mammalian oocytes, partly because they are small compared to amphibian oocytes, and it is difficult to collect large numbers of them. More importantly, as discussed earlier, oocytes are complex cells containing factors essential for pluripotency, as well as for the early development and differentiation of trophectoderm cells. By contrast, pluripotent ES/EG cells are relatively less complex, and, more importantly, they can be grown indefinitely in vitro. Thus, they can provide a considerable source of material for analysis. For example, it is possible to use nuclear extracts from ES/EG cells to examine reprogramming of somatic nuclei, as described in one experimental approach. The availability of relatively large amounts of nuclear extracts from ES/EG cells also makes it possible to undertake biochemical studies to identify the key reprogramming factors.
Simultaneously with the development of techniques for producing hybrid cells, John Gurdon and his colleagues demonstrated that a nucleus obtained from a differentiated cell of a tadpole, when transferred to an enucleated frog oocyte, supported the development of an adult frog. The general conclusion from these studies was that all differentiated cells retain full genetic potential for development and that the differentiated states of functionally distinct cell types must therefore arise from differential regulation of gene activity. What, then, would be the result of combining the genomes from different cell types in a single hybrid cell?
In 1965, Henry Harris had described the behavior of different nuclei in heterokaryons formed between various cell types. The most striking of these combinations was the fusion of HeLa cells with mature chicken erythrocytes in which, unlike mammalian erythrocytes, the chromatin is condensed and the nucleus is inactive. In the resulting heterokaryons, the chicken erythrocyte nuclei became active for both DNA and RNA synthesis and re-expressed chicken-specific genes. This result was confirmed, and shown to be the result of cytoplasmic factors. For example, chicken erythrocyte nuclei were found to be reactivated when introduced into enucleated cytoplasm. Furthermore, the reactivated chicken nucleus in cells reconstituted with fibroblast cytoplasm supported synthesis of chicken globin.
Attempts to discern general rules from the fusion of cells of different phenotypes, however, proved difficult. Despite the results with erythrocyte heterokaryons, an early conclusion was that the fusion of cells expressing two distinct states of differentiation frequently resulted in the loss of those differentiated functions in the hybrid cells. For example, hybrids formed between fibroblasts and melanomas did not produce melanin, and globin synthesis was not inducible in hybrids between fibroblasts and Friend erythroleukemia cells. On the other hand, hybrids between human leukocytes and mouse liver tumor cell lines sometimes expressed liver-specific proteins from the human genome contributed by the fibroblasts. Similarly, rat hepatomamouse lymphocyte or fibroblast hybrids often expressed mouse albumin. In other studies, hybrids between neuroblastoma cells, which exhibit a variety of neural features, and mouse L-cells, which are long-established, immortalized fibroblastoid cells, retained at least some of the neural features of the neuroblastoma parental cells, notably their electrical activity.
Apart from attempting to investigate the control of specific differentiated states, Harris and his colleagues, in particular, sought to use cell hybridization to establish the mode of genetic control of the transformed state of cancer cells, in particular whether the genetic changes that underlie the transformed state of tumor cells were the result of the loss or gain of gene function. In a conclusion that presaged the identification of tumor suppressor genes, they reported that malignancy acted as a recessive trait at the cellular level, as fusion of malignant cells with nonmalignant partners resulted in the formation of nonmalignant hybrid cells in which malignancy reappeared with subsequent chromosome loss. Clearly, such a result is not always the case, as might be inferred from our current knowledge of oncogenes, as well as tumor suppressor genes. Perhaps the most notable exception to this rule was the formation of immortal and tumorigenic hybridomas that produced monoclonal antibodies following the fusion of terminally differentiated plasma cells and a lymphoid cell line.
Generally, it seemed that hybrids of cells with distinct phenotypes did not express a hybrid phenotype. Rather, they tended to express genes associated with one or other of the parent cells, but not both. For example, mouse hepatomaFriend erythroleukemia hybrids were described, which continued expressing liver functions, but in which globin expression was extinguished. Furthermore, in some cases, the gene expression typical of one parental phenotype was activated from the genome of the other parental cell. However, no clear rules emerged as to which phenotype would predominate.
The mechanisms by which one phenotype predominates over another in such hybrids largely remains poorly understood. In some cases, at least, the genome of one contributing nucleus retained the capacity for reactivation of its tissue-specific genes, even when extinguished in the initial hybrid cells. For example, in Chinese hamster fibroblastrat hepatoma hybrids, rat liver functions were extinguished, only to reappear in some subclones on subsequent passage, possibly because of the loss of particular chromosomes. This result implies a stable modification to the genome responsible for the maintenance of its epigenetic state and not erased in hybrid cells. In other situations, experiments with phenomena such as imprinting and X-inactivation indicate that DNA methylation and histone acetylation can play a role in the heritable regulation of gene activity. Similar mechanisms are likely to play a role in the maintenance of a stable differentiated phenotype and might underlie results such as these.
On the other hand, dynamic regulatory factors must also be important, because the early heterokaryon experiments clearly suggest that any repression of gene activity can be overcome by diffusible factors. Perhaps the earliest and clearest identification of a factor that can play a role in the dynamic regulation of gene activity was the discovery of the helixloophelix transcription factor, MyoD. The presence of MyoD alone, introduced into a cell by transfection with appropriate expression vectors, is sufficient to activate muscle-specific genes from several distinct cell types. MyoD is also subject to positive autoregulation so that, once expressed in a cell, it tends to maintain its own expression, thus establishing a dynamic system for maintenance of the muscle-differentiated state.
Nevertheless, and not surprisingly, even this story is not so simple. In some cells, MyoD is not sufficient to activate muscle gene expression; other somatic cell hybrid experiments show that MyoD itself is subject to negative regulators specified by loci elsewhere in the genome. If these patterns of regulation also apply to other key regulatory genes, it would not be unexpected that the outcome of fusion experiments between distinct types of differentiated cells would depend on the parental cells, the interactions of structural modifications to chromatin and DNA, and the dynamic, diffusible regulatory factors pertinent to those cells.
Baniasadi et al. (2013) developed a dual-cell hybrid photocatalytic system for enhanced hydrogen evolution, as shown in Fig. 5.64. The system is a kind of photoelectrochemical cell, having an anion exchange membrane and a specific catholyte. A sulfur-based photochemical system is used as the catholyte with stirred ZnS nanoparticulate photocatalysts and dissolved electron donors.
The photocatalysts were pure-grade 99.99% ZnS 325 mesh nanoparticulate from Alpha Aesar with used sodium sulfide electron donors given as 3% w/v in an aqueous solution. The aqueous solution is alkalinized to pH 13.2 using sodium hydroxide in both the photoreactor and dual-cell. The reactions occurring in the cathode compartment will be of two kinds. First is the water reduction that is catalyzed heterogeneously at the photocatalysts surface and at the electrode surface. The two reactions are
The obtained polarization curve of the cell is shown in Fig. 5.65. Since the delivered electrons to the reaction site are supplied by both photons and power supply, hydrogen evolution takes place in a hybrid manner. Both hydrogen and oxygen reactions initiate with higher rate constants, but the production rate becomes steady after almost 20min of operation and the production rate decreases to an almost constant value as shown in Fig. 5.66.
A techno-economic study was also conducted for the aforementioned hybrid cell (see, for example, Fig.2.8), applying the software tool SuperPro Designer (from Intelligen). The study showed that the capital investment of the plant would be of the order of US$ 0.92M (2003 prices). The operating costs are about $ 1.76M per year. The processing rate is 68.83106kg/year of influent and the unit processing cost is 0.0256 $/MT of influent; detailed tables were published. The total revenues from water recycling and reuse are $ 68,800 per annum. It is noted that this only accounted for the environmental costs and did not consider any credit due to copper recovery. The cost of the combined process was not the mere addition of the individual processes costs: the required membrane surface, which is a major cost factor, is lower in the hybrid process since flotation removed the majority of solid particles, and therefore a smaller membrane area is required to yield the same permeate .
In Table2.2, a brief account is given on costs from selected (from the very few available) papers in the recent literature; noting that the general trend, at least according to our opinion, is to avoid giving an idea on costs, but meanwhile speaking on low-cost effective processes. Finally, in an extensive review , a discussion on electroflotation costs can be traced, including some comparison with relative processes for effluent treatment.
To induce cell fusion, or indeed electrical breakdown of the cell membrane, a field strength of at least 10 times more than is typically used in DEP separations is required. Hybrid cells from electrofusion are viable, which suggests that cells having undergone exposure to normal DEP forces are not damaged. Further evidence includes the exclusion of trypan blue from dielectrophoretically separated erythrocytes and the successful culture of various cell types including yeast cells and CD34+ cells.
The fluid flow during a DEP separation procedure produces a maximum shear stress on the cell of around 3 dyn cm2. T-lymphocytes and erythrocytes have been reported to be able to withstand a shear stress some 50 and 500 times this value, respectively. Therefore, almost insignificant levels of shear stress are experienced by these cells in DEP chambers.
The conductivity of suspending medium used is normally much below that of a normal physiological medium, however, as long as the osmolarity is of the right value, osmotically sensitive cells can be investigated. This is achieved by additives such as sucrose at 280 mM, which has little effect on the conductivity. An alternative approach has been to use sub-micrometer electrodes which minimize heating effects enabling the use of normal physiological strength media.
Hybridoma cells are artificially developed through fusion of an immortal myeloma cell (such as Sp2/0 or NS0) and a mortal antibody-producing lymphocyte . The hybrid cells are selected in HAT medium (hypoxanthine, aminopterine, and thymidine) where only fused cells can survive. The hybridoma technology revolutionized modern biotechnology when it was developed in the 1970s. Before that time, antibodies were isolated from blood serum of immunized animals. These antibodies were polyclonal, with low specificity and variations in quality. With the hybridoma technology, highly specific, monoclonal antibodies could be produced in virtually unlimited quantity.
The first hybridoma-derived antibodies were murine. As such, they contained immunogenic epitopes that cause safety issues and diminished efficacy. This is called Human Anti-Mouse Antibody (HAMA) response . To improve the safety profile of the antibody drugs chimeric and humanized antibodies were developed. Chimeric antibodies are produced through fusing murine variable regions (VL+VH) into a human Fc backbone. Chimeric antibodies are less immunogenic compared with murine antibodies, but the murine Fabs still contain some sequences that might cause unwanted side effects . Humanized antibodies consist of mouse CDRs grafted onto a human framework. Humanized antibodies are significantly less immunogenic compared with mouse and chimeric antibodies but the humanization procedure is labor intensive.
The majority of antibodies that enter clinical trials today are humanized or fully human , and the preferred strategy is to use fully human antibodies (developed through methods such as phage display) and transgenic mice that are engineered to produce human antibodies [99,100]. Transgenic mice have enabled using hybridoma technology without any risk for HAMA responses. Fully-human monoclonal antibodies not only have a better safety profile, but they also have the additional advantage of being very specific for their therapeutic targets.
In 1965 Harris and Watkins reported that inactivated Sendai virus caused the hybridization of a mixed population of human HeLa cells and mouse Ehrlich ascites tumor cells. The result of the fusion was a mixed population of hybrid cells (called heterokaryons) that were genetically unstable. Figure 7 indicates the sequence of events during fusion showing the cytoplasmic fusion of two dissimilar cells followed by the hybridization of the nuclei of the two cells. After a period of growth the heterokaryons tended to lose some of their genetic material and become stable hybrids retaining some of the phenotypic characteristics of each parental cell. The method turned out to be an extremely valuable tool for biological research. In 1969 Harris showed that when normal cells were fused with malignant cells the malignant phenotype was not always retained. This was the first direct evidence for the existence of human suppressor genes, derived from the normal cells and that could result in suppression of the tumorigenic characteristics. These genes whose products include the retinoblastoma protein and p53 are now well characterized in terms of their role in malignancies.
The cell hybridization technique has also been useful in developing an understanding of cell differentiation and gene regulation. For example, the normally quiescent genetic material of highly differentiated cells can be reactivated following fusion with cells actively engaged in protein synthesis. This was shown by Harris in the late 1960s by the fusion of a cell population of chicken erythrocytes and growing HeLa cells.
Cell fusion has also been used extensively in human chromosome mapping. The heterokaryons resulting from the fusion of human and mouse cells are genetically unstable and tend to lose human chromosomes randomly. This eventually gives rise to a mixed population of stable hybrids from which individual cell clones can be isolated. Many of these clones may contain single human chromosomes. It is the association of a particular chromosome in an isolated cell clone with a selected measurable phenotypic characteristic such as an enzyme activity that allows the gene of that enzyme to be assigned to a specific human chromosome. With the use of this technique, many human genes have been assigned to particular chromosomes (known as chromosome mapping).
It was this same technique of cell fusion that Kohler and Milstein used in their work reported in 1975 that allowed the creation of stable hybrid cells from the hybridization of antibody-secreting B-lymphocytes with transformed myelomas. The resulting cells retained two important phenotypic characteristics from the parentsthe ability for infinite growth (from the myeloma) and the ability to synthesize antibody (from the lymphocyte). The original objective of this work was to study somatic mutation as a mechanism for antibody diversity. This is the ability of B-lymphocyte to go through a maturation process following initial contact with an antigen to produce antibodies of increasing affinity. However, the application of the cell fusion technique to produce antibody-producing cells with an infinite growth capacity had a major impact on the ability to produce large quantities of antibodies that could be used for a variety of functions both in biological research and also as medically important products. The term hybridoma was derived in 1976 by Herzenberg and Milstein to describe a homogeneous clone of these antibody-producing hybrid cells. The term monoclonal antibody refers to the secreted product of the cells. Unlike antibodies derived from blood samples (polyclonal), the monoclonal antibodies from a single hybridoma are molecularly homogeneous and have a specific affinity for a particular antigen.
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Presents the two-stage recognition strategy for copper flotation rougher condition recognition.Summarizes the visual features and their extraction methods.Presents the multi-source data fusion framework of process parameters and visual features.The feed ore types are divided into four classes and the identification model are established.Establishes the rougher working condition identification model based on BT-SVM method.
Due to the complex process of copper flotation and the frequently diversified conditions of ore sources, it is difficult to identify rougher flotation conditions and maintain the stability of production process. By deeply analyzing the characteristics of the copper flotation process, the recognition system for working conditions in copper rougher is established and the cascaded recognition method is presented. At the first stage, the recognition model is built to identify feeding ore types based on fusion information of froth image local colour features and process parameters. At the second stage, the asymmetry binary tree SVM multi-class classification method with working condition priority rating (WCP-BTSVM) is used to recognize copper rougher flotation conditions. As demonstrated in the industrial experiment, the proposed method can relatively accurate identify the working conditions in copper rougher and thus can provide a solid foundation for decision-making in follow-up process control.
Envelope glycoproteins from genetically-divergent virus families comprise fusion peptides (FPs) that have been posited to insert and perturb the membranes of target cells upon activation of the virus-cell fusion reaction. Conserved sequences rich in aromatic residues juxtaposed to the external leaflet of the virion-wrapping membranes are also frequently found in viral fusion glycoproteins. These membrane-proximal external regions (MPERs) have been implicated in the promotion of the viral membrane restructuring event required for fusion to proceed, hence, proposed to comprise supplementary FPs. However, it remains unknown whether the structurefunction relationships governing canonical FPs also operate in the mirroring MPER sequences. Here, we combine infrared spectroscopy-based approaches with cryo-electron microscopy to analyze the alternating conformations adopted, and perturbations generated in membranes by CpreTM, a peptide derived from the MPER of the HIV-1 Env glycoprotein. Altogether, our structural and morphological data support a cholesterol-dependent conformational plasticity for this HIV-1 sequence, which could assist cell-virus fusion by destabilizing the viral membrane at the initial stages of the process.
During the early phase of the replication cycle, the Human Immunodeficiency Virus type-1 (HIV-1) particle fuses its lipid envelope with the plasma membrane of the CD4+ target cell1,2. The reaction is triggered after engagement of the envelope glycoprotein (Env) with the cell receptor (CD4)/co-receptor (CXCR4 or CCR5), a specific recognition process that activates further refolding of the metastable native Env. Fusion activity of Env depends on the presence of the fusion peptide (FP), a conserved sequence located at the N-terminus of the transmembrane subunit gp41 (reviewed in Ref.3,4). Following fusion triggering, the FP is propelled towards the target cell membrane and embeds therein due to its hydrophobic character1,2. Subsequently, helical regions within gp41 ectodomains refold into an energetically stable, trimeric 6-helix bundle (6-HB), whose formation brings together the merging membranes: the plasma membrane of the target cell, and the lipid envelope of the virus (Fig.1, see also Supplementary Fig. 1)5,6,7.
Designation of the HIV-1 CpreTM sequence used in this study. (a) Schematic displaying the general organization of the HIV-1 Env glycoprotein in virions (pre-fusion state). (b) Diagram showing the constituents of the gp41 subunit ectodomain and transmembrane anchor. Functional domains designated within its sequence include FP, fusion peptide; NHR and CHR, amino- and carboxy-terminal helical regions, respectively; MPER, membrane-proximal external region; TMD, transmembrane domain (see also Supplementary Fig. S1). The MPER-TMD region contains epitopes for the recognition of several broadly neutralizing HIV antibodies as indicated. The CpreTM sequence that derives from this region is shown below.
In analogy to FPs, insertion into the viral membrane of a C-terminal gp41 sequence known as the Membrane-Proximal External Region (MPER), is postulated to further contribute to the overall process of membrane merger (Fig.1, see also Supplementary Fig. 1)8,9,10,11,12,13. This sequence, sitting at the bottom of the Env complex is exceptionally enriched in aromatic residues that promote interactions with the membrane interface (reviewed in Ref.14,15,16,17). MPER insertion may be disruptive for the viral membrane, since peptides and constructs derived from its sequence have been shown to exert virucidal effects18,19,20,21. The discovery that a number of antibodies targeting MPER can block membrane activity and infection, further underlines the importance of this region for the fusogenic function of the Env glycoprotein (reviewed in Ref.16,22).
There is structural evidence to support that the carboxy-terminal MPER sequence can combine with transmembrane domain (TMD) residues of gp41 to form a continuous helix, at least in one of the conformational states that are accessible to the pre-fusion Env complex23,24,25,26,27,28,29,30. A peptide that spans this helix (CpreTM, Fig.1b), has been shown to induce lipid bilayer restructuring upon partitioning into cholesterol (Chol)-enriched virus-like membranes21,31,32,33. However, it remains to be established whether structurefunction relationships displayed by the standard N-terminal FP of gp41, also apply to this MPER-derived C-terminal sequence.
Cumulative experimental work using synthetic surrogates and model membranes has delineated physiologically relevant aspects of the FP function (reviewed in Ref.4,34,35). High-resolution NMR studies reveal the adoption of continuous -helical structures in membrane mimics36,37, whereas the combination of Infrared (IR) and Solid-State Nuclear Magnetic Resonance (SS-NMR) spectroscopy demonstrates that the -helical conformation can convert into oligomeric strand structures, a transition promoted by peptide density and the increase of the Chol concentration in the membrane38,39,40. Attenuated Total Reflectance (ATR)-IR studies further indicate that monomeric FP -helices penetrate into lipid bilayers in an oblique angle41,42, while its oligomeric -strand counterparts appear to associate with the main axis forming a 90 angle with respect to the membrane normal43.
Thus, regarding its conformational behavior, a hallmark of membrane-bound HIV FP appears to be its plasticity, which enables the transition from inserted -helices, tilted relative to the bilayer normal, into extended -strands, lying almost parallel to the membrane plane3,4,39,40,41,43,44,45,46,47. Studies in model systems suggest that these alternating conformations of the FP sequence can disrupt the lipid bilayer, breaching its permeability barrier and/or inducing aggregation and inter-bilayer mixing of lipids40,44,47,48,49,50. Moreover, the membrane-inserted FP appears to modulate the elastic properties of the bilayer and facilitate the formation of the non-bilayer lipid intermediates required for fusion51,52,53,54. In this context, the HR1 and HR2 helical domains within the ectodomain of gp41 are conceived as a mechanical device that brings the host-cell plasma membrane, primed for merger by the inserted FP, into contact with the viral membrane (Supporting Fig. S1).
In this work, we combine conventional IR spectroscopy, two-dimensional correlation IR spectroscopy (2D-COS-IR), and ATR-IR, to analyze the conformation and orientation adopted by the CpreTM peptide upon reconstitution into lipid bilayers. In line with the notion that the sequences flanking the TMD anchors of fusion glycoproteins are endowed with a degree of conformational plasticity55,56,57, our data reveal that the CpreTM helix can adopt membrane-inserted -helical structures that convert primarily into an extended -strand conformation in Chol-rich membranes. Occurrence of the extended conformation lying parallel to the membrane plane correlates with the induction of vesicle fusion as visualized by cryo-electron microscopy (cryo-EM) of vitrified specimens. Thus, we conclude that CpreTM bound to membranes displays structural features of canonical FPs, and propose a structure-based mechanistic model that couples CpreTM helix unfolding to membrane merger during the HIV-1 fusion cascade.
Before establishing the membrane-bound conformations of the CpreTM peptide, we analyzed the secondary structure adopted in a medium that mimics the low-polarity of lipid bilayers. As a reference we employed the published NMR structure of monomeric CpreTM in buffer containing 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)24. Figure2a displays the superposition of the calculated models for the CpreTM structure in HFIP, and a representative single model (left and right panels, respectively). All calculated models were consistent with a predominantly -helical geometry, with evidence for disordered regions limited to the COOH and NH2 extremities. Panel b displays the circular dichroism (CD) spectra obtained for CpreTM in buffers with increasing HFIP content, and the quantitative analysis of the secondary structure composition58 (left and right panels, respectively). Consistent with peptide aggregation in solution, -strands and turn/coil structures dominated the spectra at the lowest HFIP concentration (2.5% v/v), and diminished upon decreasing polarity. At the highest HFIP concentration where monomers are expected to be favored (25% v/v), the -helix contribution was predominant (ca. 65%), whereas only a residual signal from peptide aggregation remained. In these samples, components attributable to the disordered conformations and turns amounted to ca. 30%.
Structural analysis in a low-polarity medium. (a) NMR structure of the CpreTM peptide solved in 25% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (v/v) (PDB ID: 2MG2). (b) Left: CD spectra obtained at 25C in media containing increasing amounts of HFIP as indicated in the panel; Right: secondary structure fractions estimated from CDPro83. Meansthe standard deviation for the fraction values estimated with CONTIN-LL, CDSSTR, and SELCON3 are plotted. Black dots, red triangles and green squares depict the fractions of helix, strand and the sum of turns+unordered conformations, respectively. (c) IR spectrum in the amide I region obtained in buffer containing 25% HFIP (v/v). The absorption band was decomposed into different components. The original spectrum and the sum of the band components are superimposed and indistinguishable. The inset displays the secondary structure assignation for the main components (bands labeled with numbers 1 to 5) and the area percentages (rounded off to the nearest integer).
Matching those findings, band decomposition of IR spectra obtained in 25% HFIP identified a majority of amide-I vibrational modes arising from helical conformers, with bands centered at 1665cm1 (310-helix), 1655cm1 (-helix) and 1630cm1 (-helix solvated)59,60,61 amounting to ca. 56% of the total band area (Fig.2c). Besides, a band centered at 1642cm1 (ca. 34%) was ascribed to disordered coil structures, whereas that at 1677cm1 was attributed to turns (ca. 9%). Again, consistent with the monomeric state of the peptide (Panel a), the contribution of extended-aggregated conformations was negligible in these samples (<1%).
We next reconstituted CpreTM in membranes by co-mixing it with lipids in organic solvent, followed by gentle evaporation and hydration (see Materials and Methods). Figure3 compares the IR spectrum of CpreTM in solution with that obtained after reconstitution in lipid bilayers made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phophocholine (POPC) (top and bottom panels, respectively). The amide-I region of the IR spectrum in solution displayed a prominent band centered at 1622cm1, which together with high-frequency absorption in the 16801690cm1 region, denoted that a majority of peptide chains were unfolded/aggregated. In contrast, upon reconstitution in POPC bilayers, the maximum shifted to 1654cm1, whereas the contribution of the 1620cm1 band was irrelevant. In these samples predominant helical conformers amounted to ca. 70%. Besides, in comparison with the absorption band components measured in HFIP (Fig.2c), the contribution of turns and disordered chains decreased, whereas the amide-I band became overall narrower. These spectral variations reflect a reduction in the conformational space accessible to the CpreTM chain upon reconstitution in lipid bilayers, consistent with the majority of the membrane-associated peptide adopting a canonical -helical conformation.
Reconstitution of CpreTM in lipid bilayers. Top and bottom IR spectra display respectively the amide I components measured in buffer or after reconstitution of the peptide in POPC lipid bilayers (Peptide-to-lipid ratio, 1:50). Insets in both panels display the secondary structure assignation for the main band components and their area percentages.
Cholesterol (Chol) is a major lipid of the HIV membrane required for virion infectivity62,63,64,65,66. Therefore, we analyzed the conformation adopted by CpreTM reconstituted in membranes containing increasing Chol concentrations. Figure4a displays the series of raw and deconvolved IR spectra as a function of Chol content in membranes (left and right panels, respectively). A shoulder centered at ca. 1620cm1 could be already discerned in the samples that contained low Chol, which evidenced an initial accumulation of extended chains. Samples containing the highest Chol concentrations displayed a more conspicuous band centered at 1622cm1, consistent with a -strand-like conformation dominating the secondary structure of the membrane-bound peptide under these conditions.
Conformations adopted by CpreTM as a function of the Chol content in membranes. (a) Amide I region IR spectra of CpreTM reconstituted in membranes (peptide-to-lipid ratio, 1:50) that contained increasing Chol concentrations as indicated in the panels. Raw and deconvolved spectra are shown (left and right panels, respectively). (b) 2D-COS IR analysis. Synchronous (top) and asynchronous (bottom) correlation map contours of the raw (left) and deconvolved (right) IR spectra obtained with increasing Chol concentrations are shown. Red peaks correspond to positive correlations and blue peaks to negative ones.
To get more insight into the CpreTM conformational changes induced by the membrane Chol concentration, we next performed the 2D-correlation analysis of the IR spectra in the corresponding amide I band region67,68,69 (Fig.4b). We note that relevant effects detected on the 2D maps often reflect subtle changes in the relative contents of the amide I band components. Therefore, in addition to the -strand band that dominates in samples containing high concentrations of Chol (centered at ca. 1620cm1), the analysis also revealed the evolution of the rest of the spectral components i.e., bands centered at ca. 1675, 1660, 1650, 1642 and 1635cm1.
In the synchronous () 2D maps of CpreTM (Fig.4b, top panels), autopeaks indicated simultaneous changes in the bands composing the amide-I spectrum. In the 2D maps of the raw spectra (left), autopeaks were found centered at 1650 and 1620cm1, whereas the single cross-relation negative peak 1620/1650cm1 reflected that both vibrations were affected in-phase by Chol, the first component augmenting in intensity, the second diminishing. Higher resolution was attained using the map based on the deconvolved spectra (right). Particularly, all helical components were evidenced as autopeaks centered at ca. 1635cm1, 1655cm1 and 1665cm1, which could be observed together with cross-relation negative peaks 1620/1635cm1, 1620/1655cm1 and 1620/1665cm1.
The corresponding asynchronous () maps reflected the sequential order of events induced by the increase of Chol (Fig.4b, bottom panels)67,68,69. The asynchronous peaks were positive (red contours) if the change in the first frequency occurred accelerated with respect to that in the second one, and negative (blue contours) if delayed. The positive correlation peak 1655/1665cm1 detected in the raw-spectra maps, suggests the formation of less stable short regions deviating from canonical -helicity and adopting 310-helical geometries, whereas the negative one at 1620/1665cm1 supports the conversion of the 310-helix intermediates into extended strands.
This pathway was also apparent in the map based on deconvolved spectra. In this case, an additional positive correlation peak was found for the pair 1635/1665cm1. It is known that partial solvation of -helical structures can give rise to low-frequency bands centered at ca. 16351630cm1 because of the cross-hydrogen bonds that can be formed with water60,61 (see also Fig.2c). Thus, we attribute the CpreTM absorption mode at 1635cm1 to a fraction of the helical structure not buried in the membrane, i.e., exposed to solvent and/or in contact with interfacial polar moieties. The positive correlation found at 1635/1665cm1 suggests that these solvated helices also unfold adopting 310-helical geometries, whereas the negative one at 1635/1655cm1 would be consistent with the buried helical fraction unfolding more readily than the solvent-exposed one upon increasing the Chol concentration.
Additional positive peaks were observed at 1655/1675cm1 and 1635/1675cm1, and a negative peak found at 1620/1675cm1. This indicates that -turns can also act as intermediates of the helix-to-strand unfolding process. In conclusion, upon increasing the Chol content in the membrane, -turns/310-helical regions seem to be produced at the expense of the canonical -helical conformations, and these intermediates appear to convert into extended strands.
To establish whether the C-terminal region accounted for the tendency of MPER to adopt extended conformations in membranes, we also analyzed the effect of Chol on the conformation adopted by NpreTM, a peptide overlapping with the aromatic-rich N-terminal stretch of CpreTM, but lacking its TMD residues (Supplementary Fig. S2a). Following CpreTMs trend, NpreTM reconstituted in POPC membranes adopted a main helical conformation (Supplementary Fig. S2b). However, when reconstituted in POPC:Chol (1:1) membranes, -strands did not dominate the overall conformation of the NpreTM peptide, supporting the implication of the membrane-buried CpreTM TMD residues in the conformational conversion promoted by Chol.
The previous results support the efficient lipid bilayer reconstitution of the CpreTM peptide as an -helix, and the possibility of its transitioning to extended structures in Chol-enriched membranes. Using ATR-IR spectroscopy, we next determined the tilt of these alternating CpreTM conformations relative to the membrane normal. ATR-IR absorbance spectra were measured using perpendicular and parallel polarized light (Fig.5a). From these spectra, the experimental average dichroic ratios were determined, and order parameters S and tilt angles calculated (Table 1)70,71,72.
Angle of insertion of CpreTM main conformations as determined by ATR-IR. (a) Top: Comparison of ATR-IR spectra in the amide I region of CpreTM reconstituted in POPC or POPC:Chol (1:1) membranes (left and right panels, respectively) (peptide-to-lipid mole ratio, 1:50). Bottom: a similar comparison was made in the CH2/CH3 stretching region of the spectra. The main orientations of peptide and acyl chains are inferred from the ratio of peak areas recorded with incident light polarized parallel (||) and perpendicular () to the membrane normal (calculated values for the order parameters and tilt angles are displayed in Table 1). (b) Models for the membrane-associated structures and orientations adopted by CpreTM in POPC and POPC:Chol (1:1) membranes (left and right panels, respectively).
According to the tilt angle inferred from the dichroic ratios, the longitudinal axis of the CpreTM helix formed an angle of 53 with the POPC lipid bilayer normal (Fig.5b). Angles of a comparable magnitude (ca. 50) have been reported in the literature for the HIV-1 and SIV FPs inserted into lipid bilayers42,46. Thus, our ATR-IR data support that, similarly to the N-terminal FP, the C-terminal Env sequence covered by CpreTM could insert in a tilted orientation into the membrane at some stage during the fusion process. Conversely, the CpreTM -strands oriented almost parallel to the membrane plane in the POPC:Chol (1:1) sample (angle of ca. 90 with respect to the normal) (Fig.5b), also in accordance with data reported in the literature for the N-terminal FP under fusogenic conditions39,43,44.
Despite the differences in the attained conformation, the reconstituted CpreTM peptide incorporated to the same extent and quantitatively into vesicles containing different concentrations of Chol (Fig.6a). In contrast, Cryo-EM images revealed different morphologies for the peptide-containing vesicles, suggesting that the adopted conformations induced distinct patterns of membrane destabilization (Fig.6b,c and Supplementary Fig. S3). Untreated control samples displayed spherical vesicles with heterogeneous sizes ranging in mean diameter from ca. 100 to 200nm (Fig.6b,c, bottom panels). The -helical peptide did not alter the overall morphology or size of POPC vesicles when incorporated at a 1:50 peptide-to-lipid dose (Fig.6b,c, top left panels). In contrast, an increase of extended conformations in the peptide-treated samples (Chol-containing membranes) correlated with a significant increase of the mean vesicle size (Fig.6b,c, top center and right panels). Particularly, in peptide-containing POPC:Chol (1:1) samples, massive aggregation and vesicle sizes in the range of 500nm could be observed.
Vesicle morphology changes induced by the different conformations adopted by CpreTM in membranes. (a) Vesicle flotation analysis. Incorporation to vesicles of CpreTM was verified after ultracentrifugation in a sucrose gradient (peptide-to-lipid mole ratio, 1:50). The presence of the peptide in the floated and non-floated fractions of the gradient and in the original sample (input) was revealed by Western Blot analysis after Tris-Tricine SDS-PAGE separation. Fluorescence emission spectra below reveal the presence of Rhodamine-labeled vesicles in the collected fractions. The amount of extended -strand adopted by the peptide in the samples is indicated below the panels (values determined in the spectra of Fig.5a after band decomposition). (b) Vesicle morphology by cryo-EM. Images obtained for vesicles containing reconstituted peptide are shown in the top-panels. Bottom panels display images of vesicles devoid of peptide. (c) Vesicle size distribution as determined from the diameters measured in cryo-EM images. The sizes of oval-shaped vesicles were determined by measuring their major axes. Conditions as those in the previous panel.
The Supplementary Figure S3 displays more detailed views of the effects exerted by CpreTM on vesicle morphology. The peptide reconstituted in POPC membranes did not affect the stability of the vesicle samples, whereas its inclusion into POPC:Chol membranes induced tight vesicle-vesicle contacts (lipid bilayer aggregation) and increased the mean diameter of the vesicles (membrane fusion). Notably, POPC:Chol (1:1) vesicles containing the reconstituted NpreTM control peptide displayed a pattern of tight bilayer aggregation, which did not result in an increase of the mean vesicle size (Supplementary Fig. S2c). Thus, it appears that completion of the fusion process required the presence of the CpreTM TMD residues.
Lipid aggregates with spongy morphology also accumulated sporadically in certain areas of CpreTM-containing POPC:Chol vesicles (Supplementary Fig. S3, bottom panels). The occurrence of lipid aggregates reminiscent of non-lamellar arrangements suggested that, following an FP-like fashion4,51,52,53,54, the CpreTM peptide could also facilitate the formation of highly curved lipid structures involved in membrane merger35. However, control experiments using 31P-NMR failed to detect evidence for the promotion of this type of non-lamellar arrangements in peptide-containing POPC:Chol (1:1) membranes (Supplementary Fig. S4). Moreover, an inspection by Atomic Force Microscopy (AFM) of supported lipid bilayers containing reconstituted peptide revealed that CpreTM disrupted the lipid continuity of the solvent-accessible membrane monolayer, and increased the amount of force required to break the bilayer (Supplementary Fig. S5). Thus, it appears that inclusion of CpreTM at doses leading to vesicle fusion did not facilitate membrane deformation by increasing curvature or reducing bilayer stiffness35.
Studies in model systems support a predominant -helical conformation for monomeric forms of the membrane-bound HIV FP, which appear to insert tilted relative to the membrane normal42,43,44,48,73. In addition, the membrane-inserted FP helix can undergo conformational changes leading to the formation of extended -strands, which have been associated with the perturbation of the lipid bilayer architecture and the promotion of lipid mixing during membrane fusion40,43,44,48,49,50,74,75. Such conformational plasticity would be at odds with the stagnant -helical conformation generally assumed for the MPER-TMD sequences of gp41 in the context of the Env glycoprotein24,27,28,29,76,77,78. However, challenging the existence of a single MPER conformation after biogenesis, several studies support that a substantial portion of the membrane-embedded MPER can exist in an extended conformation during the gp41 refolding process that accompanies fusion activation (Fig.7a)7,24,79. Epitope peptides resolved in complex with antibody fragments also suggest that partly extended MPER chains can be targeted by certain antibodies80,81. In a more general sense, it has been argued that sequences flanking viral glycoprotein TMD helices might unfold adopting -strand conformations, and contribute to the promotion of the fusion process by imparting negative curvature to the bilayer55.
Proposed role for CpreTM sequence in an extended conformation during the process of Env-mediated HIV fusion. (a) High-resolution structures of MPER sequences displaying sections of extended chain conformation. PDB accession numbers used to render the panel together with the sequence ranges covered are displayed below. (b) Mechanistic model: Compact (1) and open (2) conformations of the Env glycoprotein are postulated to interchange spontaneously before engagement with receptor/co-receptor (one Env monomer is depicted for simplicity). The cartoon representing the gp41 subunit is based on the pre-fusion X-ray structure (PDB accession code: 4TVP) and illustrates the relative positions of the most important constituents in the ectodomain (same color code as in previous Fig.1). Upon activation of the fusion cascade, helical regions reposition to interact with each other initiating the assembly of the 6-HB, whereas the main axis of the complex align with the membrane plane (3). This process is proposed to be facilitated by the extension of the MPER chain connecting the enlarging 6-HB to the TMD. Insertion of the MPER connection into the viral membrane in an extended conformation may prime it for fusion (*), creating poorly solvated spots to facilitate initial interbilayer contacts and/or generating a lipid bridge between the merging membranes.
Here, we employed IR spectroscopy approaches to analyze the conformations accessible to the membrane-proximal CpreTM sequence reconstituted in membranes. Together, the spectroscopy results confirm that the reconstitution process results in a membrane-inserted CpreTM -helix, which is partially exposed to solvent and orients in an oblique angle with respect to the membrane plane. Chol appears to induce conformational changes leading to the formation of -strands that lie mainly parallel to the membrane plane. The occurrence of CpreTM extended chains is associated with the destabilization of the lipid bilayers, as suggested by cryo-EM imaging and AFM characterization.
The model displayed in Fig.7b integrates these findings in a general model of HIV-1 gp41-induced membrane fusion. The prefusion Env complex may alternate compact (1) with more open conformations (2), and it is likely that in these structures the helices spanning the MPER-TMD sequence could kink at different positions. Recent structural studies support that, at least in one of those conformational states, a straight and continuous CpreTM helix inserted in a subtle angle would be the target to antibodies exerting broad and potent neutralization29,30.
Subsequent activation of the fusion process involves the refolding of the gp41 helical domains HR1 and HR2 (depicted in cyan and yellow colors, respectively) to initiate the formation of a compact 6-HB. Establishment of the extensive helix-helix hydrophobic interactions between HR1 and HR2 implies the relocation of the helical sections and the reorientation of the complex main axis with respect to the membrane (3). We infer that the initial formation of the 6-HB likely requires extension of the C chain at sections joining the emerging complex to the membrane-inserted sequences. These extended hydrophobic chains, most prominently at FP and MPER areas, could associate with membrane surfaces helping to overcome repulsive hydration and electrostatic forces, as the cell and viral membranes approach pulled by the growing 6-HB hairpin. Furthermore, our data suggest that the CpreTM sequence could also break lipid continuity of the viral membrane external monolayer (Supplementary Fig. S5), generating poorly solvated hydrophobic spots where the initial contacts could be established between the approaching bilayers.
Overall, the experimental data presented in this study support the notion that a similar conformational plasticity underpins the membrane activity of the FP and CpreTM region during the initial (and transient) stages of HIV-1 fusion, but caution that effects of these sequences on the elastic properties of membranes involved in the process might be different. In this regard, we note that the present work provides no hints as to how the membrane-inserted structures of the FP or MPER evolve at later stages of the fusion process. It has been argued that the FP could first assemble as -sheets on membrane surfaces, but later convert into -helices to complete fusion38. Thus, at least theoretically, it is possible that at later stages of the fusion process the CpreTM sequence also attains secondary structures and membrane topologies that differ from those described in this work. Such alternative conformations might allow completion of the 6-HB structure and/or modulate the elastic properties of the membrane to facilitate fusion54.
The peptide sequence derived from the gp41 MPER-TMD region, KKK-NWFDITNWLWYIKLFIMIVGGLV-KK (CpreTM) (Fig.1) was produced by solid-phase synthesis using Fmoc chemistry as C-terminal carboxamides and purified by HPLC (estimated purity 97%). 1-palmitoyl-2-oleoyl-sn-glycero-3-phophocholine (POPC) and cholesterol (Chol) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). N-(lissamine Rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) was from Thermo Fisher Scientific (Waltham, Massachusetts, USA). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Monoclonal antibody 4E10 (MAb4E10), kindly donated by D. Katinger (Polynum Inc., Vienna, Austria), and rabbit anti-human-IgG-HRP (Santa Cruz Biotechnologies) were used to reveal the membrane-bound peptide.
To prepare CpreTM-containing vesicles, adequate amounts of lipids and peptide were mixed in organic solvent prior to the production of the liposomes as described82. Briefly, phospholipid and cholesterol were dissolved in chloroform:methanol 1:2 (vol:vol) and mixed with CpreTM (dissolved in 100% ethanol) at a peptide-to-lipid molar ratio of 1:50. The mixture was dried under a N2 stream followed by 2h vacuum pumping to remove traces of organic solvents. Subsequently, the dried lipid films were subjected to 2h of gentle hydration with H2O using a N2 gas bubbler to facilitate dispersion of the dried lipid-peptide film in PBS buffer. Next, the multilamellar vesicles were bath sonicated (1h, 55C) and subjected to 15 freeze and thaw cycles to obtain unilamellar vesicles. Finally, effective incorporation of the peptide to the vesicles was ensured by peptide flotation coupled to that of lipid vesicles after ultracentrifugation of the samples in a sucrose gradient as described32.
Circular dichroism (CD) measurements were carried out on a thermally-controlled Jasco J-810 circular dichroism spectropolarimeter calibrated routinely with (1S)-(+)-10-camphorsulfonic acid, ammonium salt. CpreTM stock samples dissolved in DMSO, were lyophilized and subsequently dissolved in an aqueous buffer (2mM Hepes, pH, 7.4) at 0.03mM concentration with 2.5%, 10% or 25% (v:v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Spectra were measured in a 1mm path-length quartz cell equilibrated at 25C. Data were taken with a 1nm band-width, 100nm/min speed, and the results of 20 scans per sample were averaged. Quantitative analysis of the spectra was carried out using the CDPro software83, as previously described58.
Infrared spectra were recorded in a Thermo Nicolet Nexus 5700 (Thermo Fisher Scientific; Waltham, MA) spectrometer equipped with a mercury-cadmium-telluride detector using a Peltier based temperature controller (TempCon, BioTools Inc., Wauconda, IL) with calcium fluoride cells (BioCell, BioTools Inc., Wauconda, IL). CpreTM-containing samples were lyophilized and subsequently prepared at 3mg (peptide)/mL in D2O buffer (PBS). A 25l sample aliquot was deposited on a cell that was sealed with a second cell. Reference windows without peptide were prepared similarly. Typically 370 scans were collected for each background and sample, and the spectra were obtained with a nominal resolution of 2cm1. In the HFIP samples solvent contribution was subtracted from the original spectra before the data analysis to allow a reliable comparison between spectra.
Data treatment and band decomposition of the original amide I have been described elsewhere59. In brief, the number and position of bands were obtained from the deconvolved (bandwidth=18 and k=2) and the Fourier derivative (power=3 and breakpoint=0.3) spectra. The baseline was removed before starting the fitting procedure and initial heights set at 90% of those in the original spectrum for the bands in the wings and for the most intense component, and at 70% of the original intensity for the rest of bands. An iterative process followed, in two stages. (i) The band position of the component bands was fixed, allowing widths and heights to approach final values; (ii) band positions were left to change. For band shape a combination of Gaussian and Lorentzian functions was used. The restrictions in the iterative procedure were needed because initial width and height parameters can be far away from the final result due to the overlapping of bands, so that spurious results can be produced. In this way, information from band position, percentage of amide I band area and bandwidth were obtained for every component. Using this procedure the result was repetitive. Mathematical accuracy was assured by constructing an artificial curve with the parameters obtained and subjecting it to the same procedure again. The number of bands was fixed on the basis of the narrowing procedures. The molar absorption coefficient for the different bands was assumed to be similar and within a+/3% error.
To obtain the 2D-COS-IR maps, the Chol content was used to induce spectral fluctuations and to detect dynamic spectral variation in the secondary structure of CpreTM. Rendering of the two-dimensional synchronous and asynchronous spectra has been described previously69.
ATR-IR spectra were measured in a Bruker Tensor 27 spectrometer equipped with a mercury-cadmium-telluride detector using a BioATRCell II micro-ATR unit. 20l of the lipid mixtures containing peptide were dried on the surface of the ATR Ge crystal by flowing dried air into the infrared spectrometer chamber during 5h. For spectra acquisition, the polarized mirror (Pike Technologies) was adjusted to 0 and 90, to generate incident light oriented parallel and perpendicular to the lipid normal, respectively. 150 IR spectra at 2cm1 resolution were collected under each condition and averaged. The dichroic ratio of the amide I bond absorption was computed for parallel (0) versus perpendicular (90) polarized incident light relative to the membrane normal and was employed to calculate the peptide orientation as discussed previously71,84,85.
As initial screen to determine the optimal concentration, samples were first imaged by negative stain electron microscopy. 8 L aliquots were adsorbed onto glow-discharged carbon coated copper grids, and negatively stained with 1% uranyl formate. Specimens were imaged with a JEM-1230 transmission electron microscope (JEOL Ltd. Tokyo) using an Orius SC1000 (40082672 pixels) cooled slow-scan CCD camera (Gatan Inc.) at the equivalent nominal magnification of 20000x, and defocus values between -2 and -5m. Selected samples were then vitrified and imaged using a JEM-2200FSC transmission electron microscope (JEOL Ltd. Tokyo) equipped with a field emission gun (FEG) operated at 200kV and an in-column omega energy that helped us to record images with improved signal-to-noise ratio (SNR) by zero-loss filtering, using an energy selecting slit width of 30eV centered at the zero-loss peak of the energy spectra. Digital images were recorded under low dose conditions using a 4K x 4K UltraScan 4000 charge-coupled device (CCD) camara (Gatan Inc.) at the equivalent nominal magnification of 50000x, and defocus values between -1.5 and -4m. 4 L aliquots were applied onto Quantifoil R 2/2 on 300 mesh cooper grids and C-flat R 1.2/1.3 on 400 mesh cooper grids plasma cleaned with air for 5s using a PDC-002-CE plasma cleaner (Harrick Plasma). Grids were blotted and plunge frozen in liquid ethane with an automated Leica EM GP2 automatic plunge freezer (Leica Microsystems GmbH, Wetzlar).
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This study was supported by the Spanish MCIU (Grants RTI2018-095624-B-C21; MCIU/AEI/FEDER, UE to JLN and BA; and PID2019-111096GA-I00; MCIU/AEI/FEDER, UE to AC) and Basque Government (Grant: IT1196-19). Technical assistance from MI Collado and M Carril with 31P-NMR measurements and data processing is greatly acknowledged.
I.D.-A., J.T. and J.L.N. conceived the experiments; I.D.-A., J.T., I.T., A.C., I.U.-B., J.L.R.A., .B.A. and J.L.N. designed them; I.D.-A. and J.T. prepared samples and determined structures by Infrared Spectroscopy; I.T., J.T. and I.U.-B. prepared and analyzed samples by Cryo-Electron Microscopy; A.C. and J.T. prepared and analyzed samples by Atomic Force Microscopy. J.L.N. wrote the paper with input from I.U.-B. and B.A., and all authors reviewed it.
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de la Arada, I., Torralba, J., Tascn, I. et al. Conformational plasticity underlies membrane fusion induced by an HIV sequence juxtaposed to the lipid envelope. Sci Rep 11, 1278 (2021). https://doi.org/10.1038/s41598-020-80156-w