The Florida Institute of Phosphate Research was created in 1978 by the Florida Legislature (Chapter 378.

101, Florida- Statutes) and empowered to conduct research supportive to the responsible development of the states phosphate resources. The Institute has targeted areas of research responsibility. These are: reclamation alternatives in mining and processing, including wetlands reclamation, phosphogypsum storage areas and phosphatic clay containment areas; methods for more efficient, economical and environmentally balanced phosphate recovery and processing; disposal and utilization of phosphatic clay; and environmental effects involving the health and welfare of the people, including those effects related to radiation and water consumption. FIPR is located in Polk County, in the heart of the central Florida phosphate district. The Institute seeks to serve as an information center on phosphate-related topics and welcomes information requests made in person, by mail, or by telephone.

Research Staff Executive Director Paul R. Clifford Research Directors G. Michael Lloyd Jr. Jinrong P. Zhang Steven G. Richardson Gordon D. Nifong -Chemical Processing -Mining & Beneficiation -Reclamation -Environmental Services

Florida Institute of Phosphate Research 1855 West Main Street Bartow, Florida 33830 (863) 534-7160 Fax:(863) 534-7165 http://www.fipr.state.fl.us

BACTERIA AS FLOTATION REAGENTS FOR THE FLOTATION OF A DOLOMITIC PHOSPHATE ROCK FINAL REPORT

Ross W. Smith Principal Investigator with Manoranjan Misra, Rajendra K. Mehta, and Xiapeng Zheng

UNIVERSITY OF NEVADA RENO Reno, Nevada 89557

Prepared for FLORIDA INSTITUTE OF PHOSPHATE RESEARCH 1855 West Main Street Bartow, Florida 33830

Contract Manager: Patrick Zhang FIPR Contract Number: 94-02-l06R

January 1997

DISCLAIMER

The contents of this report are reproduced herein as received from the contractor. The opinions, findings and conclusions expressed herein are not necessarily those of the Florida Institute of Phosphate Research, nor does mention of company names or products constitute endorsement by the Florida Institute of Phosphate Research.

PERSPECTIVE
Patrick Zhang, Research Director - Beneficiation & Mining

With the depletion of the higher grade, easy-to-process Bone Valley deposits, the central Florida phosphate industry has been forced to move into the lower grade, more contaminated (mainly by dolomite) ore bodies from the Southern Extension. Although extensive resources have been directed at developing processes for separating dolomite from phosphate, no practical and economical approach is available to date, except for the IMC-Agrico heavy media process. In an effort to identify the most feasible technique for processing Florida dolomitic ores, FIPR has conducted an in-house research project to evaluate five flotation separation processes utilizing the same high dolomite pebble feed. Only one of the processes achieved successful removal of dolomite at a seemingly reasonable cost, but with a recovery of PZOS only about 60% from the original pebble feed. Flotation is generally the least expensive mineral processing technique. There are two basic approaches for separating dolomite from phosphate by flotation: floating phosphate while depressing carbonate, or floating carbonate while depressing phosphate. The former is restricted by the lack of carbonate depressants, and the latter is limited by poor selectivity of anionic collectors as well as the low efficiency of phosphate depressants. The use of microorganisms as flotation reagents is one of the new frontiers of bio-mineral processing. Research has established that mineral surface properties can be modified biologically. Some microorganisms may act as depressants, activators or collectors for different minerals under different conditions. Thiobacillus ferrooxidans was used as conditioning reagents for sphalerrite and galena; biosurface modification was studied in separating pyrite from coal; mycobacterium phlei was found to be an efficient hematite collector; and some bacteria were found to be effective flocculants for finely sized clay suspensions. The major goal of this project was to develop alternative, environmentally friendly, and cost effective flotation reagents for depressing dolomite and/or promoting phosphate in processing Florida phosphate deposits of the future. Potential benefits of research in this field to the State of Florida and its citizens as well as the phosphate industry may include: a) processing phosphate minerals in a more environmentally sound manner by replacing chemicals with microorganism; b) extending phosphate resources significantly by successful processing of the dolomitic deposits; and c) insulating the industry against dramatic price increases due to possible shortages of certain flotation reagents.

ABSTRACT The mineralogical characteristics of two Florida dolomitic phosphates were investigated by microscopic analysis, liberation degree analysis, and BET surface. area measurement. The growth of two bacteria, Mycobacteria phlei and Bacillus licheniformis JF-2, and study on some of their properties in aqueous solutions were conducted through electrokinetics, contact angle, and surface tension measurements. The experimental work on the micro-flotation of pure apatite and dolomite and flotation of the real Florida dolomitic phosphate pebble samples were also conducted using bacteria as both collector and depressant in anionic collector flotation. The results of the investigations and experiments indicated that apatite in the phosphate pebble samples has good liberation from other minerals and a very large surface area, which is the major reason for high reagent consumption and low flotation selectivity. The cell walls of both M.phlei and B.licheniformis JF-2 include very active biosurfactants, which can remarkably reduce the surface tension of the suspensions and increase the contact angle of minerals they adsorb onto in aqueous solution. The results of experiments indicate that M.phlei was markedly adsorbed on the dolomite and apatite surfaces. The bacterium demonstrated more affinity towards dolomite than apatite, and that B. licheniformis JF-2 has even more affinity than M.phlei for adhesion onto dolomite. Flotation tests revealed that both bacteria act as a depressant of dolomite during phosphate flotation using anionic collectors. A flotation concentrate less than 1% MgO content can be obtained from Florida phosphate pebble flotation.

ACKNOWLEDGMENTS

We are grateful to Dr. Malcolm J. Hibbard, professor of Department of Geological Science, University of Nevada Reno (UNR), for advice on mineralogical analysis and for help with obtaining the micrographs, to Dr. Ashok M. Raichur, Research Associate of the Department of Chemical and Metallurgical Engineering, UNR, for assistance on preparation of bacteria, and to Mr. Chan C. Lee, undergraduate in Chemical Engineering, UNR, for his help with phosphate analysis of the samples.

vi

EXECUTIVE SUMMARY This report summarizes the progress made during one and half years of work on the research project Bacteria as Flotation Reagents for the Flotation of a Dolomitic Phosphate Rock. The description of work done is in accordance with the proposal approved by The Florida Institute of Phosphate Research under the Contract Number of 94-02-106R. As Florida low dolomitic phosphate reserves become exhausted, the remaining Florida phosphate rock contains less phosphate and significantly more dolomite. It is generally difficult to obtain, from such materials, a phosphate concentrate containing less than the desired maximum magnesium content of 1% MgO. Microorganisms and products derived from them can function as modifying agents or even collectors in mineral flotation due to their selective adhesion onto specific minerals. The microorganism adhesion onto the minerals result from their specific metal-binding ability. The work on using the bacteria Mycobacterium phlei and Bacillus licheniformis JF-2 (whole cells and products derived from cell rupture) as flotation modifiers/collectors of dolomitic Florida phosphate rock was performed at the University of Nevada, Reno. The experimentation included mineralogical analysis of Florida phosphate pebble samples, properties analysis of the two bacteria in aqueous solutions, evaluation of the surface chemical nature of the microorganisms and their derivatives and the minerals present in dolomitic Florida phosphate rocks. The experimentation was performed through microflotation, electrokinetic, contact angle, adsorption and surface tension studies. At the same time experimental work on the flotation of actual dolomitic Florida phosphate rocks was performed using cells/cell products of the microorganisms both as modifiers in anionic flotation and as flotation collectors. The results of analysis and experimentation reveal that it is possible to use successfully microorganisms in Florida phosphate flotation. The three main mineralogical characteristics of phosphate minerals in Florida dolomitic phosphate rock, cryptocrystalline character, CO2 substitution, and porous structure, make it difficult to separate them from dolomite. Both bacteria studied exhibit high, negative, zeta potentials and strongly adsorb calcium and magnesium ions in the aqueous solutions depending on the pH values. The Florida sedimentary apatite, francolite, and sedimentary dolomite demonstrate different electrokinetic behavior from that of crystalline apatite and dolomite. The cells or soluble products from cells of the two microorganisms demonstrated very high surface activity to magnesium or calcium ions in the aqueous solution. In particular, Bacillus licheniformis JF-2 shows remarkable affinity to magnesium over calcium ions. The results of both micro flotation and actual sample flotation indicates that the two microorganisms can act as a dolomite depressant but not as a collector for phosphate or dolomite. Using fatty acid or diphosphonic acid as collector and bacteria as depressants in alkaline medium, a concentrate containing less than 1% MgO can be obtained at 60 -70% P,O, recoveries.

INTRODUCTION

PURPOSE OF STUDY The United States is the largest phosphate rock producer in the world. About 30% of the world production in 1990 was produced by the United States (Bartels and Gurr, 1994). This level of production has continued to the present. Florida accounts for approximately 80% of U.S. phosphate production (Harben, 1980). During the past 100 years the Florida phosphate industry has produced high quality products having a MgO content of less than 0.5%. Phosphate reserves and resources in Florida have the potential to continue production at a rate of about 40-55 million metric tons/year for hundreds of years (Sandvik, 1979). The Florida phosphate rock deposits are located in the states central and northern land pebble districts (Moudgil and Ince, 1991). The land-pebble phosphate district of Florida, also called the Central Florida phosphate district, is the major district where phosphate is produced. The reserves of phosphate in this district are being rapidly depleted and future production will shift to the southern extension (Bernard and Hall, 1980). The southern reserves have a lower phosphate concentration and a severe MgO contaminant, i.e., significant quantities of dolomitic carbonates (Lawver et al., 1982). The processing of these reserves will require special beneficiation techniques to produce concentrates containing less than the practical limit of about 1% MgO. Thus, for Florida phosphorus resource conservation, study on the dolomite separation processes is a very important item. As it is generally difficult to obtain from such materials a phosphate concentrate containing less than the desired maximum content of 1% MgO, many attempts have been made to study the separation processes. The past studies mainly focused on the flotation of dolomite from phosphate, and a dozen reagents have been found to be effective in depressing phosphate. The flotation of phosphate from dolomite is hard to perform due to the lack of the proper dolomite depressants. So far, only two main carbonate depressants, sodium silicate and sodium hydroxide, have been identified (Zhang, 1994). Development of the flotation of phosphate from dolomite, however, is also attractive because this process is considered to be simple, and would discharge the dolomite and quartz together. In order to further exploit Florida dolomitic phosphate pebble ores, the development of effective and economical dolomite depressants for the flotation of phosphate from dolomite appears to be essential. The Center for Mineral Bioprocessing and Remediation, University of Nevada-Reno, has been engaged in the development of dolomite depressants for anionic collector flotation in recent years. One approach has been using bacteria as depressants for dolomite. PRELIMINARY SURVEY Over the past years, five flotation processes have been developed to separate dolomite from phosphate or phosphate from dolomite. They are the TVA (Tennessee Valley Authority) phosphonic Process (Lehr and Hsieh, 1981) the UF (University of Florida) two-stage conditioning Process 2

(Moudgil and Chanchani, 1985), the Alabama non-conditioning Process (Anazia and Hanna, 1987), USBM (United States Bureau of Mines) Process (Davis, Liewellyn and Smith, 1984), and the IMC (International Minerals and Chemical Corporation) cationic Process (Snow, 1979). These processes require the secondary stage operation to separate the final phosphate from quartz or dolomite, and the change of conditions from alkaline to acidic pH which lead to their complexity. Many of the reagent systems developed are not attractive because they require high doses and are more expensive than the conventional reagent systems. Biomass, such as algae, fungi, and bacteria, has been long known to absorb metal ions from aqueous solution in some environmental and metallurgical processes applications (Brierley et al., 1986, Thompson, 1986). But there have been almost no studies and/or applications of them as mineral flotation reagents. Only one attempt has been tried to use a kind of bacterium as flotation collector (Smith et al., 1993).

Mycobacterium phlei is a bacterium that is not readily classified as being either gram
positive or gram negative because of its fatty acid surface. It is highly hydrophobic, and will produce a hydrophobic substance, mycobactin (Van Loosdrecht, 1987). It is non-pathogenic in humans and in all animals thus far tested and is easily and readily cultured (Laskin and Lechevalier, 1977). Not only is Mycobacterium phlei highly hydrophobic (contact angle 65-70 degree), it also is highly negatively charged with an isoelectric point (iep) at about pH 2.5. It should, thus, readily adhere onto a hydrophilic mineral surface if the mineral is of low negative, neutral or positive charge. The lipids of M.phlei may constitute more than 50% cell dry weight. Various saturated, monosaturated, and methyl-branched fatty acids are important components of these lipids, both in esterified form, and as precursors of long chain mycolic acids. It is reported that in the growth of M.phlei, fatty acid unsaturation increases with decreasing temperature, and consequently, fatty acid branching decreases and mean chain length increases as the temperature is reduced (Suutari and Laakso, 1993).

Bacillus licheniformis JF-2, isolated from oilfield injection brine (Jenneman et al., 1983), is
a gram-positive bacterium. It is able to grow and produce a very effective biosurfactant under both aerobic and anaerobic conditions at a very wide range of temperature and in the presence of high concentrations of salts. The surface tension of the medium in which it is cultured can be remarkably reduced from 70 to 74 mN/m to as low as 28 mN/m due to production of an anionic biosurfactant (Javaheri et al., 1985). Bacterial surfaces of this species are typically anionic and, therefore, interact with metal cations leading to metal binding. The metal binding can be partially attributed to the chemical properties of the metallic aqua ions involved. The cell walls of another gram positive bacterium, Bacillus subtilis, containing teichoic acid-peptidoglycans, are strongly anionic and bind metal avidly. In the case of B. subtilis, Mg* binds much more strongly than Ca*+ (Beveridge and Murray, 1976). This example implies that the metal binding of specific bacteria species is selective in nature, and that teichoic acid, which is present in amounts up to 70% dry weight of B.subtilis when grown in phosphate- and glucose-containing medium, is responsible for Mg*+ and not Ca* binding. Bacillus licheniformis JF-2 has a wall which contains teichuronic acid in combination with teichoic acid-peptidoglycan polymers. This added complexity makes the B.licheniformis wall a good comparison for the B.sublitis system. Experimental results indicate that teichoic and teichuronic

acids are the prime sites of the metal binding in B.licheniformis (Beveridge et al., 1982) The binding characteristics of these bacteria should, thus, contribute to bacterial adhesion to the minerals containing Ca2+ and Mg* at their surfaces. Further, it is probable that the selectivity of metal binding also occurs in mineral adhesion. APPROACH AND STRATEGY The objective of this research is to evaluate the effectiveness of two microorganisms,

Mycobacterium phlei and Bacillus licheniformis JF-2 as collectors or regulators for the flotation of,
Florida dolomitic phosphate rock. Data on the mineralogical characteristics of the Florida phosphate pebble samples, and the properties of the two bacteria in aqueous solution are needed in for the guidance of subsequent flotation tests. Flotation tests of the Florida dolomitic pebble samples were conducted using bacteria as reagents after flotation tests of pure minerals were initiated. The final target for flotation of Florida phosphate rock was to obtain a concentrate containing less than 1% MgO at a reasonable recovery of phosphate values. During the reporting period, experiments such as electrokinetic, contact angle, surface tension, and surface area measurements were conducted to help explain the mechanisms of reactions between bacteria and minerals.

METHODOLOGY MATERIALS AND THEIR PREPARATIONS 1. Pure Mineral Samples Unless otherwise stated, all the pure mineral samples were obtained from Wards Natural Science Establishment, Inc. Crystallized fluorapatite, Ca,[PO,],(F,Cl,OH), with blue-green color and massive shape, was from Ontario, Canada. Sedimentary apatite, francolite was from Florida,

U.S.A. Crystallized dolomite, CaMg(CO,),, excellently crystallized with large and gray cleavages, which was from Selasvann, Norway. Sedimentary dolomite was from Minnesota, U.S.A. Quartz, with milky appearance, was from Boulder County, Colorado, USA. All the mineral samples were first hand crushed, followed by grinding in a porcelain mortar with a pestle, and then screened to the desired size fraction. The different size fractions were used for the various measurements and tests. 2. Florida Dolomitic Phosphate Pebbles Two dolomitic phosphate pebble samples used in this study were obtained from the Florida Institute of Phosphate Research and IMC Agrico Company. The P,O, content of samples was analyzed using the spectrophotometric method adopted by the Association of Florida Phosphate Chemists. A Spectronic 21 UVD spectrophotometer, Bausch & Lomb Inc., USA, was used for P,O, analysis. Chemical analyses of MgO and CaO were performed using an ICP chemical analysis system. The particle size distribution and chemical analysis of these pebble samples are shown in Tables 2 & 3, where the content of head samples was calculated by material balance. The pebble samples were dried, crushed and ground to -35 mesh (-500 pm). The -35+150 (-500+106 urn) mesh size fraction was used as flotation feed while the -150 (-106 urn) mesh size fraction was removed after the wet screening. Tables 4 & 5 show the particle size and chemical analysis results for the -35 mesh (-500 pm) fraction.

TABLE

3: SIZE AND CHEMICAL

ANALYSIS

OF THE,# 2 SAMPLE

Analysis, %

Distribution, mo 64.28 14.52 CaO

% Ins01 43.50 16.58 1 18.63 1

49.14 19.51 120.59

1 -12+18 1 -18+28

1 21.3 1 4.21

24.18 21.45 17.79

1 2.62 1 2.56 ( 2.33 1 3.91

142.83 142.47 142.52 140.87

12.39 17.54 32.66 14.16

122.23 I 3.90 1 5.35 1 100.0

14.28

ITotal
TABLE

I 100.00

23.17

4: SIZE AND CHEMICAL

ANALYSIS

OF THE # 1 SAMPLE

AFTER

GROUND

Size, mesh

wt. %

An; lysis, %

Distribution, I Ins01 13.91 20.61 19.37 19.87

% Ins01 31.30 18.05 14.95 9.96 1 4.09 1 21.66 100.0

PA I MU

CaO 43.02 42.09 42.83 42.47

W5
37.16 13.27 11.76 7.61

MN
30.38 10.85 9.35 6.46 3.38
39.58

CaO 35.10 13.37 11.99 7.72 ) 4.32

I 27.5 1

-150+200

1 4.29

24.05 1 1.74 21.19 1 3.08 23.34 1 2.21

42.52 1 14.60 ~ 4.42 40.87 1 11.67 42.20 1 15.30 25.78 100.0

~100.0

100.0

Except for the -200 (-75 pm) mesh fractions, which are higher in MgO in both # 1 and # 2 ground samples and lower in insoluble material, the size fractions of the ground sample do not show significant differences in chemical and mineral compositions. After the -150 (-106 pm) mesh fractions were removed, the # 1 flotation feed contained 24.20 % P,O,, 1.87 % MgO, 42.73 % CaO and 16.88 % insoluble matter, and the # 2 flotation feed contained 24.27 % P20s, 3.09 % MgO, 40.74 % CaO and 15.8 % insoluble matter, about the same composition as the original pebble samples. The phosphate pebbles in samples were observed to be rounded black or gray pelleted grains, containing about 27-32 % P,O,, 10% insoluble matter and less than 0.2 % MgO. The dolomite and other carbonates occurred as liberated white, yellow or gray fragments, usually with sharp edges, which contained less than 3% P2O5, 10-15 % MgO and less than 5% insoluble matter. 3. Bacteria

Mycobacterium phlei
The freeze-dried culture of microorganism, M. phlei, was first transferred to a rehydration medium (both supplied by Carolina Biological Supply Company) and incubated at 35C for 48 hours. The M. phlei was grown in a culture medium consisting of the following: 10 g/l D-(+)glucose, 1 g/l beef extract, 1 g/l yeast extract, and 2 g/l enzymatic hydrolyzed casein. The above materials were supplied by Sigma Chemical Company, St. Louis, MO. The culture medium was sterilized at 121C for 25 minutes in a Spectroline Model 750 autoclave. The sterilized culture medium was cooled and the incubated bacteria were inoculated into the medium. Culturing was carried out in 250 mL flasks continuously shaken at 150 rpm at 35C in a G24 environmental incubator shaker (New Brunswick Scientific Co. Inc., NJ, USA). After about 30 hours of culturing, the M. phlei suspension was centrifuged using an IEC Model K centrifuger (International Equipment Company, MS, USA), then filtered using a 0.45 pm Millipore filter paper, washed twice and then 7

resuspended in distilled water. Sterilized glassware and distilled water were used throughout the investigation. The concentration of bacteria in aqueous solution was obtained by filtering a certain amount of bacterial suspension and weighing the dried residue on the filter. However, in the adsorption measurements, the cell accounting method was used and then converted to ppm dry weight. The soluble fraction of a bacterium is usually about 40% of the total cell weight. Study of the properties of the soluble fraction helps elucidate the mechanism of reaction of a bacterium with mineral particles. The soluble fraction of M.phlei was obtained using an ultrasonic vibration method. About 100 ml of M.phlei whole cell suspension (concentration about 2,000 ppm) was sonicated (Branson Sonic Power Co., CT, USA) to release the soluble fraction in the bacteria. The soluble . fraction of M.phlei was separated from the solid cell mass by centrifugation and filtration. The separate suspensions of the whole cells, cell wall materials and soluble fractions were stored in the refrigerator at most for a week before use. After this time period the suspensions appeared to deteriorate. However, M.phlei in rehydration medium or in culturing medium can be kept in the refrigerator for months without evidence of decay. M.phlei in culturing medium saved in this way can be used to produce the second or third generation of bacteria. Although it appeared that such subsequent generation of bacteria had the same characteristics as the first, in the present investigation only the first generation was used.

Bacillus licheniformis JF-2

The freeze-dried culture of the microorganism, Bacillus licheniformis JF-2, ATCC 39307, was first transferred to a medium including 10.0 g of sucrose, 50.0 g of NaCl, 1.0 g of (NH,),SO,, 0.25 g of MgSO,, 10.6 g of K,HPO,, 5.3 g of KH*PO,, and 10.0 ml of trace salts solution in the 1.0 liter of deionized water. The trace salt solution consists of 1,000 ppm of Na,EDTA, 3,000 ppm of MnSO,*H,O, 100 ppm of FeSO,.7H,O, 100 ppm of CaCl,.H,O, 100 ppm of CoCl,*GH,O, 100 ppm of ZnSO,*7H,O, 10 ppm of CuSO,*SH,O, 10 ppm of A1K(S0,),.12H20, 10 ppm of H,BO,, and 10 ppm of Na,Mo0,*2H20. The culture medium was sterilized at 121C for 25 minutes in a Spectroline Model 750 autoclave. The sterilized culture medium was cooled and the Bacillus licheniformis JF-2 was inoculated into the medium. Culturing was carried out in 250 mL flasks continuously shaken at 150 rpm at 40C in a G24 environmental incubator shaker (New Brunswick Scientific Co. Inc., NJ, USA). After about 48 hours of culturing, Bacillus licheniformis JF-2 suspensions were centrifuged using an IEC Model K centrifuger (International Equipment Company, MS, USA). Then the residues were suspended in deionized water. Sterilized glassware and distilled water were used throughout the investigation. The production of the soluble fraction of B. licheniformis JF-2 was similar to that of M.phlei. The only difference in this case was the use of 0.2 pm filtration paper instead of 0.45 pm filtration paper. 4. Chemicals Purified sodium oleate, obtained from Fisher Scientific Co., was used as collector in the micro flotation experiments. In addition, a new phosphate collector, diphosphonic acid, whose main 8

compounds are disodium salt of 1-hydroxyoctylidene 1, 1-diphosphonic acid, DPA, synthesized by the Center of Mineral Bioprocessing and Remediation, University of Nevada Reno, was also used in flotation experiments. ACS certified grade KOH, HNO,, Na,CO,, and Na,SiO, were used for pH adjustment or pulp modification in measurements, micro flotation and actual sample flotation. Tall oil, No. 154 (obtained from Westvaco Chemicals Co.) plus kerosene was used as collector mixture in the bench scale flotation tests. All measurements and micro flotation experiments with the pure mineral samples were conducted using deionized water with more than 17 MQcm resistivity value. Tap water was employed for bench scale flotation tests. EXPERIMENTAL METHODS AND PROCEDURES 1. Mineralogical Measurements for Florida Dolomitic Pebble Samples For understanding the reaction between bacteria/reagents and minerals, mineralogical measurements were conducted. Included were XRD (X-Ray Diffraction) analysis using a XRG 3 100 X-ray diffraction meter (Philips Co.) and Jade X-ray Pattern Processing software (Materials Data, Inc.), microscopic analysis of Florida phosphate pebble samples to evaluate the relationships among phosphate minerals and associated minerals, and analysis of the liberation degree for phosphate minerals, carbonates, and silicates after grinding. The surface areas of pebble samples also were measured using the BET method. 2. Electrokinetic Measurements for Minerals and/with Bacteria Electrokinetic potentials of mineral samples and bacterial suspensions were measured by electrophoresis using a Laser Zee Meter (Model 501, Pen Ken Inc., USA). In the experimentation 5 mg samples of minerals, of -38 pm (-400 mesh) size, were aged overnight in 50 ml of deionized -3 water with 10 mol/l KNO,. The pH of suspension was adjusted 3 minutes before making the measurements. In the case of bacteria, 200 pl of about 2,000 ppm of bacteria suspension was used to make the same 50 ml of suspension. When studying the minerals with the soluble fraction, 5 mg of a mineral sample was put into the suspension with the soluble bacterial fraction at a given concentration. All experiments were conducted at the temperature of about 22&2C. 3. Surface Tension Measurements for Soluble Bacteria Surface tension measurements, by the du Nouy ring method, were conducted using a Model 215 Autotensiomat Surface Tension Analyzer (Fisher Scientific Company, USA). A platinumiridium ring, with 53.79 of R/r ratio and 6.005 cm of mean circumference, was used. The variations of the surface tension for the soluble fractions of bacteria were investigated as a function of pH and concentration of the soluble fraction of bacteria. The apparent surface tension was converted into the absolute surface tension by using a correction factor, F, which is given by: y = y,F F = 0.725 + (O.O1425y$(2r~R)~p)* - 1.679r/R + 0.04534 where y is absolute surface tension, ya is the apparent surface tension, R is the radius of the ring, r is the radius of the wire of the ring, and is the density of water. 4. Adhesion Measurements Adhesion measurements were performed using one gram of mineral particles in 100 ml of 9

deionized water. The mixture of minerals and water was put into a cone beaker and shaken in a G24 environmental incubator shaker for five minutes after pH was adjusted. After shaking, the mineral particles were allowed to settle for two minutes and the supernatant was taken out for measuring the remaining bacterial concentration. The measurement of bacterial concentration in the supernatant was completed by cell counting. The number of the cells counted was then converted to weight percentage (ppm). 5. Contact Angle Measurements for Minerals with Soluble Bacteria Contact angle measurements were conducted using an A-100 Contact Angle Goniometer (Rame-Hart Inc., USA). Mineral samples of approximate dimensions 2 x 2 x 1.5 cm were molded with an epoxy resin. The surface of each sample was polished using 1,000 and 4,000 grits SiC polishing paper followed by fine polishing with 0.05 pm alumina suspensions. The samples were rubbed over the wet polishing cloth, and washed with distilled water before each measurement. Contact angles of pure apatite and dolomite were measured using the captive bubble technique (Misra, Miller and Song, 1984). In this method an air/mineral/water perimeter of contact was produced at a horizontal mineral surface. This technique involves immersing the freshly polished sample into an optical glass cell containing water and then attaching an air bubble to the mineral surface. The holder was raised to a fixed distance until the area of contact between the bubble and a solid surface was seen to contract. The holder was then gently tapped to avoid any slackness in the bubble. The angle at each side of the bubble was noted, and the five measurements were made for each condition. All the measurements were made after three minutes under the conditions of the test although no significant variation was observed for extended time. The cell was cleaned with chromic acid and rinsed with deionized water for each experiment to prevent the samples from contamination. All measurements were carried out at 22*2C. 6. Pure Mineral Flotation The apatite and dolomite flotation experiments were performed using a Hallimond tube. In carrying out the micro flotation experiments, three grams of the ground mineral (-200+400 or 100+200 mesh) were added to 150 ml of deionized water and conditioned for 3 minutes in a beaker. The pH was adjusted to the desired value with HNO, and KOH. Bacteria were added and conditioned for 5 minutes before collectors were added and conditioned for three minutes. All contents were transferred to the Hallimond tube and conditioned for two more minutes. In the case of flotation of sedimentary phosphate and dolomite, a shorter Hallimond tube and 100 ml of water were used to float concentrates, and 10 ml of water were used to condition in a beak. Flotation was completed in 3 minutes. Pure mineral flotation tests were divided into three categories, single mineral flotation, 1:1 mixed mineral flotation, and 2:1 mixed mineral flotation. 7. Florida Dolomitic Phosphate Pebble Flotation In bench scale tests, a 200 g sample (dry basis) was conditioned for 5 minutes in 200 ml of tap water. A Denver D-12 flotation machine (Denver Equipment Co., CO, USA) was used. Agitation speed was set at approximately 1,000 rpm. Bacteria were added and conditioned for 7 minutes after Na,CO, and/or Na,SiO, were added. After collectors were added and agitation, the pulp was diluted to 40 wt% solid with tap water, and phosphate values were recovered as froth products. 10

RESULTS

MINERALOGICAL MEASUREMENTS Phosphate most commonly is derived from the mineral apatite, which is chemically described as Ca,(F,Cl,OH)(PO,),. Carbonate, CO3, can partly substitute for PO4 forming carbonate apatite called francolite. On the other hand, cellophane is a name given to cryptocrystalline apatite found in phosphate rock in fossil form (Bartels and Gurr, 1994). The apatite of all of the phosphate deposits of Florida is francolite (carbonate fluorapatite) (Cathcart, 1989) and/or cellophane. Both characteristics of carbonate substitution and cryptocrystalline structure make it much more difficult to separate phosphate values from gangue material containing a high concentration of magnesium. Two dolomitic phosphate pebble samples, obtained from The Florida Institute of Phosphate Research and The IMC Agrico Company, were used in this study. 1. Carbonate Content All of the apatite from Florida phosphate contains some carbonate. The substitution of carbonate for phosphate is usually expressed as the content of CO,. The CO2 content of the apatite is difficult to measure because of included very small particles of calcite and dolomite in an apatite particle. An investigation has demonstrated that the measurement of an x-ray diffraction pattern, of the difference in degrees 28, of the 410 and 004 peaks of francolite could be used to estimate its CO2 content, and therefore, the approximate degree of substitution (Gulbrandsen, 1970). According to the scatter diagram, which shows the relationship of CO2 content and A28, the difference of diffraction angles of the 410 and 004 crystal plans, the samples 1 and 2 contain about 3.5% and 4% CO2, corresponding to the A28 value of 1.3 and 1.2 respectively (Figures 1 and 2). 2. Optical Microscopy Observations The shape of phosphate intra clusters is very distinctive. Within each size fraction, most phosphate grains of the sample are highly rounded (Figure 3), although some of them are irregular shaped (Figure 4). Most gangue minerals, such as carbonate minerals and quartz, are rounded or bounded by a phosphate matrix. It is believed that the different colors of phosphate matrices are caused by differing iron concentration. The colors vary from deep dark to brown. The dark color represents a greater iron content. Some phosphorite of one color were wrapped by the others of different colors. The inclusions in apatite are very important economically since the inclusions are to contain magnesium. The magnesium in the apatite structure cannot be removed by beneficiation. From the photographs taken under the polarized microscope, most of the dolomite exists as aggregation of microcrystalline materials (Figure 5). Their shapes are mostly irregular. They contact phosphate matrices with an instinct boundary. However, some fine crystalline dolomite is also found in phosphate matrices. Quartz is the major part of insoluble materials in the ore samples. Most quartz, exhibiting an 11

angular shape and a size of 0.1 to 0.5 mm, are mounted in phosphate matrixes (Figure 6). Some of them are believed to be less than 5 pm in size (Figure 7). This quartz is very hard to separate from phosphate by mechanical methods. There is also some macrocrystalline quartz to contact with phosphorite which can be easily separated under grinding. 3. Grinding and Liberation After grinding, the pebble phosphate samples demonstrate a good liberation of the associated minerals. The degree of liberation of phosphate is estimated as much as 95% in the each size range of -35+48 (-425+300 pm), -48+65 (-300+212 pm), -65+100 (-212+150 pm), -100+150 (-150+106 p), -150+200 (-106+75 pm), and -200 (75 pm) mesh. It is seen that most quartz has been liberated from phosphate matrixes except of very fine dispersed grains of quartz (Figures 8 and 10). Dolomite brakes as very fine particles, which lead a higher MgO content, 3.08% and 7.98%, in the -200 mesh (75 pm) fraction of the samples 1 and 2, compared to the MgO content in the original samples, 2.21% and 4.07%. 4. Specific Surface Area An important factor affecting mineral flotation is the specific surface area of minerals. A larger surface area will lead to greater reagent consumption. Also, like the effect of fine particle size, the larger specific surface area will cause the loss of selectivity. Data on the specific surface area of the two Florida pebble samples were obtained using nitrogen adsorption. The surface area of crystallized apatite was also measured for comparison. From the data listed in Table 6, it is seen that the specific surface area of francolite is much more than that of crystalline apatite. The difference in surface areas for the finest size (-400 mesh) and the coarsest size (-65+100 mesh) of the francolite is about double. The difference between sedimentary and crystalline apatite is more than ten times. Thus, the Florida phosphate minerals contain considerable porosity.

FIGURE

3: ROUNDED DOUBLE

PHOSPHATE, PHOSPHATE

WRAPPED (POLARIZED,

QUARTZ, AND FIELD WIDTH

DOUBLE 3.5 MM)

AND

FIGURE

4: IRREGULAR SHAPE OF PHOSPHATE (35X48 MESH, FIELD WIDTH 0.8 MM)

16

FIGURE

5: AGGREGATION OF DOLOMITE IN FLORIDA PHOSPHATE PEBBLE ( DOLOMITE AND QUARTZ CLUSTER, FIELD WIDTH 0.8 MM)

17

FIGURE

6: PHOSPHATE (POLARIZED,

WITH QUARTZ FIELD WIDTH

INCLUSIONS 3.5 MM)

18

FIGURE

7: PHOSPHATE (POLARIZED,

WITH TINY QUARTZ GRAINS FIELD WIDTH 3.5 MM)

INCLUSIONS

19

FIGURE

8: FLORIDA PHOSPHATE (35X48 MESH, FIELD

PEBBLE AFTER WIDTH 0.8 MM)

GROUND

20

FICURE9:FLORIDAPHOSPHATEPEBBLEAFTERCROU1\1:D (65X100 MESH, FIELD WIDTH 0.8 MM)

FIGURE

10: FLORIDA (100x150

PHOSPHATE MESH, FIELD

PEBBLE WIDTH

AFTER 0.8 MM)

GROUND

22

liberation of particles, but because of the large surface area of the phosphate minerals, carbonate substitution in apatite and the crypocrystalline structure of the minerals. ELECTROKINETIC MEASUREMENTS The zeta potential of francolite and sedimentary dolomite as a function of pH, with and without lOA and lo5 M Ca(II) or Mg(II) are shown in Figures 11 through 14. In both cases, addition of 10m4 M of Ca(II) or Mg(II) caused the isoelectric point (IEP) of the two mineral samples to shift to more basic values. The electrokinetic properties of both francolite and sedimentary dolomite are quite different from that of crystallized apatite and dolomite. In the case of crystallized apatite and dolomite, IEPs of two minerals are at about pH 5.5 and 7 respectively. But the IEPs of both francolite and sedimentary dolomite are below pH 2, which is rather comparable to the IEPs of many silicate minerals. However, unlike the variation of zeta potential with increasing pH for silicates, the zeta potential of the two minerals display an increase in zeta potential at moderately alkaline pH values. Probably dissolved cations from the minerals readsorb again as pH is increased. When 10m3 M Ca(II) was added, the zeta potential of francolite remains positive regardless of pH. The experimental data implies that calcium and magnesium species can adsorb onto both the mineral surfaces over the whole pH range. The sharp increase of adsorption of cations at higher pH is due to the adsorption of calcium and magnesium hydroxy complexes. A significant phenomenon is that the zeta potential of sedimentary dolomite drops and rises as pH is increased. On the other hand, the electrokinetic behavior of M.phlei, shown in Figures 15 and 16, is somewhat similar to that of quartz and francolite in aqueous solution. At lower pH values, calcium species adsorb more onto the bacterium than magnesium species. Above pH 11, magnesium species adsorbed onto bacteria give rise to the much more positive potentials. The results of electrokinetic measurements for M.phlei indicate that calcium and magnesium ion species can absorb onto M.phlei, and adsorption is dependent on pH and concentration of calcium and magnesium species. The electrokinetic behavior of B. licheniformis JF-2 in aqueous solutions was similar to that of M.phlei (Figures 17 and 18). However, its zeta potential values are about 10 mV more negative than that of M.phlei, especially over the basic range. This indicates that the cell walls of B.licheniformis JF-2 are more negatively charged than the cell walls M.phlei. The Figures 1.9 and 20 show zeta potentials of both minerals as a function of pH in the presence and absence of the water soluble fraction derived from M.phlei and B.licheniformis JF-2. Addition of the water soluble fractions greatly decreased the zeta potentials on the minerals. Thus, the water soluble fraction adsorbed onto mineral surfaces seems to be responsible for the decreasing zeta potentials. The water soluble fraction of B.licheniformis JF-2 led to more negative zeta potentials of minerals than that of M.phlei. SURFACE TENSION MEASUREMENTS The water soluble fractions of both M.phlei and B.licheniformis JF-2 at concentrations of 10 ppm and greater decreases the surface tension of water (Figure 21). Maximum surface tension

23

depression occurs at a soluble M.phlei concentration of about 1,000 ppm, and at a soluble B. licheniformis JF-2 concentration of about 400 ppm. Also, the surface tension of soluble M.phlei increases rapidly with the increase in pH (Figure 22). These phenomena seem to be attributed to the characteristics of soluble compounds of the cell walls from bacteria. Maybe polar groups with the carboxyl, hydroxyl and phosphate functional groups at the cell walls of bacteria have changed when pH increases. ADHESION MEASUREMENTS The Figures 23 through 26 show the experimental results of adhesion of two bacteria on both dolomite and phosphate minerals. The adhesion of M.phlei to dolomite happened at a very low level of the bacteria concentration. The saturated adhesion amount was 7 - 8 mg/g dolomite when the bacterial concentration was more than 300 ppm. For B. licheniformis JF-2, the saturated adhesion of bacteria onto apatite, at 400 ppm of bacterial concentration, was 5- 6 mg/l (Figure 24). Francolite and sedimentary dolomite behaved differently. Concentration at saturated adhesion dramatically increased to about 2,000 ppm in the case of sedimentary dolomite, and to 5,000 ppm in the case of francolite. But the same adhesion comparative properties as crystallized minerals, i.e., B. licheniformis JF-2 over M.phlei on dolomite, and less M.phlei on phosphate, were obtained. CONTACT ANGLE MEASUREMENTS The contact angles on dolomite and apatite increase with concentration of soluble M.phlei and B.licheniformis JF-2 (Figure 27). It is, thus, indicated that while bacteria decrease the surface tension of the solution/air interface, they also decrease the interfacial tension of the air/mineral interface and leads to a larger contact angle. It is evident that both M.phlei and B. licheniformis JF-2 adhere onto the mineral surfaces, and that the increase of contact angles of the minerals is due to the hydrophobic property of the soluble fractions of bacteria. Figure 28 demonstrates the dependence of contact angles of both minerals on pH in the presence of M.phlei. PURE MINERAL FLOTATION 1. Single Mineral Flotation with M.phlei as Collector The Figure 29 shows the flotation of -200+400 (-75+38 pm) mesh apatite and dolomite as a function of M.phlei concentration at pH 8.8 and 9.5. However, if particle size is greater than 200 mesh there is no significant flotation of either mineral. Quartz cannot be floated even when the 200+400 mesh sample was used. The similar results using B.licheniformis JF-2 as collector were also obtained. 2. Single Mineral Flotation with Bacteria as Depressants When larger size mineral particles (-100+200 mesh, -150+75 pm) are floated using other anionic collectors, both M.phlei and B. licheniformis JF-2 acts as depressants. Two collectors, sodium oleate and diphosphonic acid, were studied, and their collecting abilities as a function of pH are 24

shown Figure 30. It is seen that diphosphonic acid, DPA, shows different collecting characteristics from sodium oleate for the four minerals. At a given pH value, the collecting ability of DPA for apatite is more than for dolomite, whether crystallized or sedimentary minerals. However, the collecting ability of sodium oleate for phosphates is less than for dolomites. The Tables 7 and 8 show flotation results for the two minerals using sodium oleate as collector and M.phlei as depressant. The results indicate that M.phlei is a strong depressant for

containing minerals when oleate is used as collector. But depression of dolomite is much stronger than that of apatite. At pH 9.7, only 10 ppm dosage of M.phlei can lead to about 30% depression of dolomite and 15% depression of apatite. When 50 ppm M.phlei was used, the floated fraction of dolomite flotation decreased by 80% and that of apatite flotation by 50% at pH 9.7. At pH 11.2, the depression effect of M.phlei decreased for both minerals, although the amount of dolomite depressed was still more than that of apatite. When DPA was used as flotation reagent, M.phlei performs the same depressing function for dolomite and apatite. The Figure 31 shows the depressing effect of two microorganisms for dolomite flotation when sodium oleate was used as collector. Bacteria successful depressed flotation of dolomite within very wide pH range. At the basic pH range, say over pH 10, the flotation recovery of dolomite deceased mainly due to competition of OK cations but not bacteria. B.licheniformis JF-2 demonstrated an advantage over M.phlei in depressing, which is in correspondence with the results of electrokinetic, adsorption, and surface tension experiments. Figure 32 shows the depressing effect of two microorganisms for apatite flotation when sodium oleate was used as collector. The depressing effect of microorganisms on apatite was less than on dolomite. When DPA was used as collector, this general trend of difference was still kept, but depressing effect of bacteria on dolomite was better because of the good selectivity of DPA between two minerals (Figures 33 and 34).

26

10.0 -

w A

without Mg(NO,), with lOA M Mg(NO,), with low3 M Mg(NO,),

> E
-10.0 -

-30.0 -

0 -50.0' 0 I
I 2 I I
I

IO

12

PH
FIGURE 12: ZETA POTENTIAL OF FRANCOLITE Mg(NO,),
AS A FUNCTION

OF PH WITH AND WITHOUT

28

20.0 1
t

I a
0 n

10.0

t-

without Ca(NO& with IO4 M Ca(NO& with 1Om3 M Ca(NO,),

-30.0

a v

-40.0

-50.0 I -60.0 0 I I 2 I I 4 I I 6 I I 8 I I 10 I I 12 I I 14

PH
FIGURE 13: ZETA POTENTIAL A FUNCTION OF SEDIMENTARY DOLOMITE Ca(NO& AS OF PH WITH AND WITHOUT

29

20.0

I 0

I O
n

10.0 -

without Mg(NO& with 10e4 M Mg(NO& with 10e3 M Mg(NO,),

0.0 -

-10.0 -

-20.0 -

-30.0 -

-40.0 -

-50.0 I I 2 I I 4 I I 6 I I .8 I I IO, I I 12 I 14

-60.0 0

PH
FIGURE 14: ZETA POTENTIAL A FUNCTION OF SF$DIMENTARY DOLOMITE Mg(NO,), AS OF PH WITH AND WITHOUT

30

20.0

I
2

.e 0

10.0

n .

without Ca(NO,), with lOA M Ca(NO,), with 10s3 M Ca(NO,),

> E .co g -5 a. m -55 N

0.0

-10.0

-20.0

-30.0

-40.0 0 2 4 6 8 IO 12 14

PH
FIGURE 15: ZETA POTENTIAL OF Mphlei AS A FUNCTION Ca(NO& OF PH WITH AND WITHOUT

31

24.0 A4n n

I @
0
I

1
-

without Mg(NO,), with IO4 M Mg(NO,), with 10e3 M Ma(NOA

fx f\

-I

0.0

-10.0

-20.0

-30.0

-40.0

10

12

14

PH
FIGURE 16: ZETA POTENTIAL OF PH WITH OF Mphlei AS A FUNCTION Mg(NO,),

AND WITHOUT

32

10.0

I
l 0

without Ca(NO&
with IO4 M Ca(NO& with 10D3 M Ca(NO&

0.0 -

-10.0 -

-20.0 -

-30.0 -

-40.0 -

-50.0 -

-60.0'

10

12

14

PH
FIGURE 17: ZETA POTENTIAL FUNCTION OF B. Zicheniformis JF-2 AS A Ca(NO&

OF PH WITH AND WITHOUT

33

I u.u I l 0

0.0 -

without Mg(NO,), with IO4 M Mg(NO,), with 10e3 M Mg(NO,),

-10.0 -

-20.0 -

-30.0 -

-v \
l

-40.0 -

-50.0 I 6 I 8 I I 10 I I 12 I 14

-60.0 0

I 2

I 4

PH
FIGURE 18: ZETA POTENTIAL OF B. licheniformis JF-2 AS A FUNCTION OF PH WITH AND WITHOUT M&NO,),

34

0.0

l
0

2.

without bacteria with 200 ppm soluble M.ph/ei with 200 ppm soluble B./icheniformis

I I I

I I
JF-2

10

12

PH
FIGURE 19: ZETA POTENTIAL OF FRANCOLITE AS A FUNCTION BACTERIA

OF PH WITH AND WITHOUT

SOLUBLE

35

I .
\

I a
0

without bacteria with 200 ppm soluble M.ph/ei

10

12

PH
FIGURE20:ZETAPOTENTIALOFSEDIMENTARYDOLOMITEASA FUNCTI~NOFPHWITHANDWITH~UTS~LUBLEBA~TERIA

36

80.0

70.0

60.0

50.0

40.0

30.0 0 I 2 3 4 5 6 7

Ln(concentration),
FIGURE 21: SURFACE TENSION

ppm
AS A FUNCTION BACTERIA OF

OF WATER OF SOLUBLE

CONCENTRATION

37

80.0

65.0

60.0 0 2 4 6 8 IO 12 14

PH
FIGURE 22: SURFACE TENSION OF SOLUBLE OF PH

Mphlei SUSPENSION

(50 ppm) AS A FUNCTION

38

10.0

8.0 -

0 0

a
0

with Mphlei with B./icheniformis JF-2

10

12

14

16

Concentration
FIGURE 23: AMOUNT A FUCTION

of bacteria, x10* ppm

ADHERING ON DOLOMITE AT PH 9 AS

OF BACTERIA

OF THEIR CONCENTRATION

39

10.0

8.0

6.0

4.0 I

with M.phlei with B. iicheniformis 2.c I

-

J F-2

0.c 0 2 4 6 8 10 12 14 16

Concentration
FIGURE 24: AMOUNT A FUCTION OF THEIR

of bacteria, x10* ppm

ADHERING ON APATITE AT PH 9 AS CONCENTRATION

OF BACTERIA

40

60.0

~ 3 E -iz ii5 5 z 2 E 6

50.0

40.0

30.0

20.0
0

with M.phlei with Blicheniformis

JF-2

10.0

0.0 0 IO 20 30 40 50 60 70 80 90 100

Concentration
FIGURE 25: AMOUNT OF BACTERIA OF THEIR AS A FUCTION

of bacteria,
ADHERING CONCENTRATION

x10* ppm
DOLOMITE AT PH 9

ON SEDIMENTARY

41

60.0

I,,

~ 32 E -ii!'L a, % E s E Q,

50.0

40.0

30.0

20.0 with f3.licheniformi.s 10.0 JF-2

0.0 0 10 20 30 40 50 60 70 80 90 100

Concentration
FIGURE 26: AMOUNT AS A FUCTION

of bacteria, x10* ppm

ADHERING ON FRANCOLITE AT PH 9

OF BACTERIA

OF THEIR CONCENTRATION

42

75.0 -

11111~

ll1lll~

llllr

l
0

m cl 65.0 -

apatite with M.phfei dolomite with M.ph/ei apatite with BJicheniformis JF-2 dolomite with BJicheniformis JF-2

45.0 -

35.0 t IO0

I IO

l111111 IO2

IIIII

IO3

Concentration
FIGURE 27: EFFECT OF SOLUBLE ANGLE

of soluble bacteria
FRACTION OF BACTERIA

ON CONTACT

OF MINERALS

43

75.0

I 0

apatite with M.phlei dolomite with M.phlei aDatite with B./icheniformis JF-2 apatite dolomite with BJicheniformis JF-2

65.0

55.0

45.0

35.0 2 4 6 8 10 12

Concentration
FIGURE 28: CONTACT ANGLE OF 350 ppm SOLUBLE

of soluble bacteria
OF MINERALS FRACTION AS A FUNCTION OF BACTERIA

44

90.0
80.0 -

I)

I I111111(
l
0

111111~

Illlll~

I111111~

I cl

apatite with whole cell, pH 8.8 dolomite with whole cell, pH 9.5 apatite with soluble part, pH 8.8 dolomite with soluble part, pH 9.5

70.0 60.0 50.0 40.0 30.0 20.0 10.0

II
10-1.0

l1lllll l()O.O

I111111 1

llllll I

IIIrlrl

O'*O

02.0

103-0

M.ph/ei concentration,
FIGURE 29: FLOTATION AS A FUCTION

ppm

RESULTS OF 200x400 MESH MINERALS OF Mphlei CONCENTRATION

45

IO I IO'
2

I
3

II

II1

8 9102

Collector 0
n A +
0

concentration,

mg/l

q
A

apatite with DPA, pH 8.5 dolomite with DPA, pH 8.5 cellophane with DPA, pH 8.0 sedimentary dolomite with DPA, pH 8.0 apatite with oleate, pH 9.8 dolomite with oleate, pH 9.8 cellophane with oleate, pH IO sedimentary dolomite with oleate, pH 10 OF MINEXALS AS A FUNCTION

FIGURE

30: FLOTATION

OF COLLECTOR

CONCENTRATION

46

100.0

80.0 s x5 -2 a 0 Yc 0 .% It 60.0

40.0

20.0 -

a 0

without bacteria with 50 ppm M.ph/ei with 50 ppm Blicheniformis

JF-2
I I

0.0 6

10

12

PH
FIGURE 31: FLOTATION OF DOLOMITE AS A FUNCTION OF PH WITH 40 MG/L SODIUM OLEATE

47

80.0

60.0
e 0 n

without bacteria with 50 ppm M.phlei with 50 ppm Bhheniformis I I 8 ,. I

JF-2 I IO I 12

40.0 6

PH
OF APATITE AS A FUNCTION OF PH WITH 80 MG/L SODIUM OLEATE

FIGURE 32: FLOTATION

48

60.0

without bacteria with 50 ppm M.phlei with 50 ppm B.licheniformis

JF-2

60.0 s -lif 5 u5 .H IL 20.0 40.0

0.0

FIGURE 33: FLOTATION

OF DOLOMITlil

AS A FUNCTION ACID

OF PH WITH 50 MG/L DIPHOSPHONIC

49

80.0 s z 5 rc E .2 LL 40.0
0 0

60.0

without bacteria with 50 ppm M.phlei with 50 ppm f3.licheniformi.s

JF-2

20.0 2

IO

12

PH
FIGURE 34: FLOTATION OF APATITE AS A FUNCTION ACID OF PH WITH 50 MG/L DIPHOSPHONIC

50

3. Mixed Mineral Flotation with Bacteria as Depressants The results of 1:1 dolomite/apatite mixed mineral flotation with M.phlei are shown in Table 8. It can be seen, from the data, that M.phlei acted as depressant for dolomite and also for apatite. Addition of M.phlei, however, led to a greater decrease in recovery of MgO than of P2O5 when oleate was used as collector. Note that 50 ppm M.phlei decreased recovery of P2O5 by about 35%, but recovery of dolomite by about 50%, and increased the grade of P2O5 in the float at pH 9.5. Also, at lower and higher pH values, there are decreased MgO and increased P,O, recoveries. When DPA was used as collector, best results were obtained at pH 7. When 50 ppm M.phlei was added, the grade of P,O, in the froth product increased from 29.39% to 33.26% and MgO decreased from 5.4% to 3.41%, with a decrease of P,O, recovery by 18.75% and a decrease of MgO recovery by 54.70%, when 50 ppm M.phlei was added. The results of 1:1 mixed mineral flotation indicate that M.phlei can more markedly depress dolomite than apatite. A decrease of MgO and increase of P,O, in the flotation concentrate can be obtained using M.phlei as depressant at a sacrifice in recovery of P2O5. Also the results again show that DPA has better collecting selectivity for apatite flotation than sodium oleate. The results of 2:1 dolomite/apatite mixed mineral flotation with both M.phlei and B.licheniformis JF-2 are shown in Table 10. The results show that B.licheniformis JF-2 was better than M.phlei in depressing dolomite, regardless of the collector used. A concentrate, with a grade of 37% P,O,, less than 1% MgO, and the recovery of 95%, was obtained when using DPA as collector without any bacterium as depressant at pH 6.8. Addition of bacteria decreased the recovery of P,O, and increased the grade of P,O, in the concentrate at a lower MgO content. The results of Flotation of 2:1 mixture of Florida francolite and Minnesota sedimentary dolomite were poorer than that of 2:1 mixture of crystalline apatite and dolomite. It is seen, from the Table 11, that oleate flotation did not reach the target of the proper level of both P,O, and MgO. Use of DPA improved the results, and a concentrate containing 29% P,O, and nearly 1% MgO was obtained at a recovery of 63% P,O,.

51

TABLE 9: FLOTATION

RESULTS OF 1:l MIXTURE OF APATITE WITH AND WITHOUT MPHLEI

Mphlei
mgll

AND DOLOMITE

Collector reagent oleate mg/l 40

pH

Item

Wt, % 86.65 13.35 40.85 59.15 82.85 17.15 57.08 42.92 95.32 4.68 48.73 5 I .27 42.89 57.11 34.75 65.25 32.04 67.96 16.45 83.55 48.29 51.71 34.67 65.33

Analysis, p&4 mo

Distribution, p*05 87.27 12.76 45.52 54.48 85.04 14.96 64.12, 35.88 92.57 7.43 46.35 53.65 63.83 36.17 55.83 44.17 45.76 54.24 24.89 75.11 71.28 28.72 57.91 42.09

% ME30 86.06 13.94 36.18 63.82 80.66 19.34 50.03 49.97 98.00 2.00 51.11 48.89 21.96 78.04 13.68 86.32 18.33 81.67 8.01 91.99 25.30 74.70 11.46 88.54

9.5

float sink

20.05 19.03 22.19 18.34 20.44 17.37 22.37 16.65 19.34 31.6 18.94 20.84 29.63 12.61 3 1.99 13.48 28.44 15.89 30.13 17.90 29.39 11.06 33.26 12.83

10.24 10.77 9.13 11.12 10.04 11.63 9.04 12.01 10.61 4.40 10.81 9.83 5.28 14.09 4.06 13.64 5.90 12.39 5.02 11.35 5.40 14.89 3.41 13.98

oleate

40

50

9.5

float sink

oleate

40

11.0

float sink

oleate

40

50

11.0

float sink

oleate

40

7.0

float sink

oleate

40

50

7.0

float sink

DPA

40

9.5

float sink

DPA

40

50

9.5

float sink

DPA

40

10.8

float sink

DPA

40

50

10.8

float sink

DPA

40

7.0

float sink

DPA

40

50

7.0

float sink

52

TABLE

10: FLOTATION DOLOMITE

RESULTS OF 2:l MIXTURE OF APATITE WITH AND WITHOUT BACTERIA

AND

Collector reagent oleate I mg/l 60 I

Bacteria mg/l 0

pH

Item I Wt,

Analysis, % I Distribution,

% I

w5
10.8 float 1 57.50 1 28.31 )
I I I

MgO
4.05 )
I

ho,
70.22

MgO
34.81 65.19 100.00 18.48 81.52 100.00 14.72 85.28 100.00 8.77 91.23 100.00 6.99 93.01 ) 100.00 I 6.53 1 93.47 1 100.00 1

sink 1 42.50 1 16.24 1 10.26 1 29.78 feed oleate 60 Mphlei 50 10.8 float sink feed oleate 60 JF-2 50 10.8 100.00 43.74 56.26 100.00 23.18 30.48 17.98 23.45 6.69 2.87 9.81 6.77 100.00 56.85 43.15 100.00

float 1 40.56 1 32.65 1 sink 1 59.44 ( 17.59 1 feed 100.00 59.70 40.30
I

2.43 1 55.88 9.61 1 44.12 6.70 0.99 15.28 100.00 95.32 4.68
I I

23.70 37.67 2.74

I

DPA

100

6.8

float sink feed

100.00

23.59

6.75

100.00 92.15 7.85 100.00

DPA

100

Mphlei 50

6.8

float 1 56.83 I 38.41 ( sink feed 43.17 100.00 4.31 23.69

0.82 I 14.38 6.67

DPA

100

JF-2 50

6.8

53

TABLE 11: FLOTATION RESULTS OF 2:l MIXTURE OF FRANCOLITE SEDIMENTARY DOLOMITE WITH AND WITHOUT BACTERIA
Collector reagent oleate mg/l 60

AND

r
60

Bacteria mg/l 0

PH

Item

Wt, % 62.53 37.47

Analysis, %

Distribution,

WA
21.41 21.29

MgO w,

mo

10.5

float sink

5.62 1 62.66 1 62.28 1 5.68 1 37.34 1 37.72 1 5.64 1 100.00 ( 100.00 I 4.53 1 60.03 1 43.52 1 7.05 1 39.97 ) 56.48 1 5.67 1 100.00 I 100.00 I 4.21 I 7.45 I 58.99 1 38.33 I 41.01 I 61.67 I

feed ) 100.00 1 21.37 1 oleate Mphlei 100 10.5 float 1 54.47 1 23.67 1 siti 1 45.43 1 18.90 1

feed 1 100.00 1 21.48 1 oleate 60 JF-2 100 DPA 100 0 8.0 10.5 float 1 52.38 1 23.91 I sink I 47.62 I 18.28 I feed I 100.00 1 21.23 I float I 52.53 I 28.17 I

5.75 I 100.00 I 100.00 I 1.50 1 69.31 1 13.95 I 30.69 1 86.05 1 100.00 63.86 ! 36.14 100.00

sink 1 47.47 ( 13.80 ( 10.24 ( feed DPA 100 Mphlei 100 8.0 float sink feed DPA 100 JF-2 100 8.0 float 100.00 48.67 51.33 100.00 46.34 21.35 5.65 1.20 9.93 5.68

100.00
10.28 89.72 100.00

28.69
15.40 21.87 29.12

1.18 1 63.20 9.64 I 5.72

9.56

sink 1 53.66 ) 14.64 1 feed 100.00 21.35

36.80 1 90.44 I 100.00 100.00

54

FLORIDA DOLOMITIC PHOSPHATE PEBBLE FLOTATION The selective flotation of Florida dolomitic phosphate pebble samples was more difficult than that of natural mineral samples because of the composition and similarity of the surface characteristics of phosphate and carbonate minerals. The addition of bacteria increased the P,O, grade of the concentrate and decreased MgO content in it when tall oil/kerosene or sodium oleate was used as collectors (Tables 12 and 13). The optimum pH value seems to be around 10.6 for both #1 and #2 samples. Flotation concentrates with less than 1% MgO can be obtained from the #l phosphate sample at this pH value with 62.07% recovery of P,O,. However, the target level of a concentrate for the #2 sample was far from satisfactory. In order to decrease the concentrate content of MgO to less than 1%, the recovery of P 2O5 is 50% or less, which is unacceptable for commercial applications. From the test data of actual sample flotation, other modifiers may have positive effect on the depression of dolomite. It is seen that N%CO, demonstrated some depression of MgOcontaining carbonates. Also, there may be a synergistic effect of bacteria with other regulators. The depression behavior of Na&O, on dolomite and possible synergistic effect using bacteria with other regulators is now under investigation by Department of Chemical and Metallurgical Engineering, University of Nevada Reno. When oleate was used as collector, the grades of obtained concentrates went up, and the contents of MgO went down. B.licheniformis JF-2 still had an advantage over M.phlei in enhancing grade and lowering MgO content. Using combination of oleate and B.licheniformis JF-2, a concentrate with 28.48% P,O,, less than 1% MgO at a recovery of 60% P,OS was obtained. DPA demonstrated better performances with or without bacteria. The concentrate with a grade of 29-30% P,O, and recovery 60- 65% can be obtained from the # 1 sample at the same level of less than 1% MgO content (Table 14). For the # 2 sample, use of oleate was hard to perform well enough. When using DPA, it was possible to obtain a concentrate containing 30.01% P,O,, less than 1% MgO at a recovery of 56.39% recovery (Table 15).

55

TABLE

12: FLOTATION

RESULTS OF THE # 1 FLORIDA USING TALL OIL AS COLLECTOR

PHOSPHATE

PEBBLE

Collector kg/ton tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5

Regulator kg/ton Na,SiO,, 1

Bacteria kg/ton 0

pH

wt.,

Analysis, %
m

Distribution, Insol 8.93 33.72 16.90 8.23 30.14 17.05 8.15 31.91 18.02 8.16 33.21 17.45 7.98 27.26 16.88 7.21 27.50 17.38 P,O, 73.06 26.94 100.0 66.98 33.02 100.0 65.00 35.00 100.0 70.37 29.63 100.0 62.07 37.93 100.0 55.87 44.13 100.0 MgO 68.06 31.94 100.0 52.33 47.67 100.0 51.38 48.62 100.0 41.35 58.65 100.0 27.93 72.07 100.0 25.88 74.12 100.0

% Insol 35.83 64.17 100.0 28.83 71.17 100.0 26.44 73.56 100.0 29.43 70.57 100.0 25.46 74.54 100.0 20.69 79.31 100.0

MgO 1.88 1.86 1.87 1.65 2.23 1.88 1.67 2.22 1.90 1.23 2.96 1.87 0.98 2.95 1.89 0.96 2.74 1.85

9.2

C T F

67.83 32.17 100.0 59.74 40.26 100.0 58.46 41.54 100.0 62.92 37.08 100.0 53.85 46.15 100.0 49.87 50.13 100.0

26.03 20.24 24.17 27.13 19.85 24.20 27.15 20.58 24.42 26.98 19.28 24.12 27.89 19.89 24.20 27.57 21.67 24.61

Na,SiO,, 1

MphleiO

9.2

C T F

.25

Na,SiO,, 1

JF-2 0.25

9.2

C T F

Na,SiO,, 1 NGQ, 2.5 Na,SiO,, 1 Na,CO,, 2.5 Na,SiO,, 1 NW&, 2.5

10.6

C T F

M.phleiO
.25

10.6

C T F

JF-2 0.25

10.6

C T F

* c: concentrate,

T: tail, F: feed

56

TABLE 13: FLOTATION

RESULTS OF THE # 2 FLORIDA PHOSPHATE USING TALL OIL AS COLLECTOR

PEBBLE

Collector kg/ton tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5 tall oil, 2.5 kerosene 0.5

Regulator kg/ton Na$iO,, 1

Bacteria kg/ton
0

PH

wt., %I

Analysis, %

Distribution

% I
Ins01 I

I PA I mo I Ins01 I w, I M@
9.2 C 1 75.69 1 25.78 1 2.92 1 9.13 1 79.60 1 71.52 T 1 24.31 1 20.57 1 3.62 1 37.21 1 20.40 1 28.48 F 1 100.0 1 24.51 1 3.09 1 15.96 1 100.0 1 100.0

100.0 37.95 62.05 100.0 34.37 65.63 100.0 39.37 60.63 100.0

Na,,SiO,, 1

M.phlei

9.2

C T i;

65.87 34.13 100.0 63.28 36.72

26.34 20.37 24.30 26.81 19.65

2.66 3.94 3.10 2.62 3.85

9.03 28.49 15.67 9.25 30.44

71.39 28.61 100.0 70.16 29.84

56.58 43.42 100.0 54.01 45.99

0.25

N+SiO,, 1

JF:2 0.25

C T

F 1 100.0 1 24.18 1 3.07 1 17.03 1 100.0 1 100.0

Na$iO,, 1 KOH
0

10.6

NqSiO,, 1 KOH

M.phlei

0.25

10.6

C T F

59.38 40.62 100.0 55.89 44.11 100.0

27.49 19.26 24.15 27.62 20.82 24.62

2.24 4.13 3.01 2.19 4.32 3.13

8.13 27.93 16.17 7.39 26.59 15.86

67.60 32.40 100.0 62.70 37.30 100.0

44.22 55.78 100.0 39.11 60.89 100.0

29.85 70.15 100.0 26.04 73.96 100.0

Na$iO,, 1 KOH

JF-2

C T F

* C: concentrate, I

57

TABLE 14: FLOTATION RESULTS OF THE # 1 FLORIDA PHOSPHATE USING OLEATE AND DPA AS COLL&ZTORS

PEBBLE

Collector kg/ton oleate

2

Regulator kg/ton Na,SiO,, 1 NC%

2.5

Bacteria kg/ton 0

PH
10.6

wt.,

Analysis, % PA 27.72 19.25 24.36 MgO 1.21 2.90 1.88 Insol 7.95 30.93 17.07

Distribution,

100.0 1 100.0 1 100.0 1 61.38 1 28.46 1 20.93 1 38.62 1 71.54 ( 79.07 ( 100.0 I 100.0 I 100.0 I 60.26 1 26.69 1 19.82 )

oleate
2

Na,SiO,, 1 Na,C%
2.5

Mphlei 0.25

10.6

C 1 52.74 T ( 47.26 F 1 100.0

28.19 1 1.02 1 7.43 19.79 ( 2.86 ( 3 1.32 24.22 1 1.89 1 18.72 28.48 1 0.97 1 6.94 19.69 1 2.79 1 29.43 24.19 I 29.13 I 1.86 I 17.92 0.95 I 7.39

oleate
2

Na,SiO,, 1 Na,CQ,
2.5

JF-2
0.25

10.6

C 1 51.18 T 1 48.82 F ) 100.0

39.74

I 73.31

I 80.18

DPA
2

Na,SiO,, 1

7.2

C 1 54.23 T ) 45.77 F 1 100.0

18.49 ) 2.98 ) 29.41 24.26 ( 1.88 ( 17.47

34.88 ) 72.60 ) 77.06 1 100.0 I 100.0 I 100.0 I 63.47 1 25.63 36.52 100.0
I I

DPA
2

Na,SiO,, 1

Mphlei 0.25

7.2

C 1 51.21 T 1 48.79 F 100.0 48.16

29.97 1 0.93 1 8.13 18.10 1 2.82 1 28.42

23.09 1 76.91
I I

74.37 1oo;o

100.0
I

DPA
2

Na,SiO,, 1

JF-2

7.2

tC

T ) 47.84 F ( 100.0 1 24.31 1 1.87 1 16.98 C: concentrate, T: tai 1, F: feet

58

TABLE

15: FLOTATION RESULTS OF THE # 2 FLORIDA PHOSPHATE USING OLEATE AND DPA AS COLLECTORS

PEBBLE

Collector kg/ton oleate 2

Regulator kg/ton Na,SiO,, 1 NaPA, 2.5 Na,SiO,, 1 NaPA, 2.5 Na,SiO,, 1 NaPA, 2.5 Na,SiO,, 1

Bacteria kg/ton 0

PH
10.6

wt., %
PA C T 72.18 27.82

Analysis, % MgO 2.57 4.69 Insol 8.97 33.01

Distribution, P,O, 81.38 18.62 MgO 58.71 41.29

% Insol 41.35 58.65

27.68 16.43

oleate 2

M.phlei

0.25

oleate 2

JF-2 0.25

DPA 2

F
DPA 2 Na,SiO,, 1
Mphlei

100.0 57.28 42.72 100.0 45.83 54.17 100.0

24.70 29.12 19.05 24.82 30.01 19.63 24.39

3.12 1.05 5.99 3.16 0.98 4.93 3.12

16.12 6.93 28.75 16.25 7.10 24.60 16.58

100.0 67.20 32.80 100.0 56.39 43.61 100.0

100.0 19.03 80.97 100.0 14.40 85.60 100.0

100.0 24.43 75.57 100.0 19.63 80.37 100.0

7.2

C T F

0.25

DPA 2

Na,SiO,, 1

JF-2

7.2

C T F

C: concentrate,

T: I .I, F: fee

59

CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS The following conclusions are drawn from the experimental results and analysis of these results based on the phenomena and potential mechanisms. 1. Florida phosphate in a dolomitic pebble sample can be called as francolite based on its carbonate substitution or collophane due to its cryptocrystalline formation from sedimentation. The difficulty of separation of phosphate from dolomite is due to its cryptocrystalline character, carbonate substitution and huge specific surface area. 2. In the aqueous solutions, Florida phosphate in a dolomitic pebble sample, as sedimentary dolomite, has a high negative charge on its surface. Its IEP is at about pH 2, which is much different from the crystalline apatite. The calcium and magnesium ions, adding or getting rid of minerals surface, adsorb on the surface of the phosphate, and lead to fewer negative potentials or positive potentials, especially at the high pH range. 3. The gram-positive bacteria, M.phlei and B.licheniformis JF-2, have higher negative charge in the aqueous solutions, and adsorb calcium and magnesium ions on their cell walls. B.licheniformis JF-2 has a higher zeta potential at very wide pH range than M.phlei does, and has more affinity to magnesium than calcium ions, especially at the pH values of 10 to 12. The soluble fraction from bacterias cell is strongly adsorbed onto the surface of minerals to cause more negative charges on the minerals.

4. Both M.phlei and B.licheniformis strongly adhere to the surface of dolomite and phosphate,
but they seem to have more affinity for dolomite than for phosphate. The differences between adsorption of calcium and magnesium ions on the cell wall of bacteria, and between adhesion of bacteria onto the surface of apatite and dolomite may depend upon the chemical composition of the cell walls and their functions. 5. Both bacteria are poor collectors for phosphate pebble flotation because of their weak collecting ability. However, they may have some potential in the flotation of fine particles or used with other collectors or frothers. 6. Both bacteria, soluble fraction or whole cells, can be used for depression of dolomite in anionic flotation for phosphate when using either sodium oleate or diphosphonic acid as collectors. 7. When the commercial collector, tall oil with kerosene, was used for flotation of the # 1 dolomitic pebble sample at pH 10.6, a concentrate of about 28% P,O, with less than 1% MgO at recovery of about 60% P,O, can be obtained. When the same reagent system was employed for the more dolomitic pebble sample, the same quality of the concentrate cannot be obtained except at a P,O, recovery of less than 60%. Use of DPA with bacteria improves the P,O, grade of the concentrate at

60

about 60% of P,O, recovery. For the low dolomitic pebble sample, a concentrate with 30% P,O, and less than 1% MgO at a P2O5 recovery of 60-65% can be obtained. For the high dolomitic pebble, a concentrate with 30% P,O, and less than 1% MgO at P,O, recovery of 55% can be obtained. RECOMMENDATIONS The mechanisms of adsorption of calcium and magnesium ions onto the selected microorganisms and the adhesion of bacteria onto the target minerals are still not very clear, even though there is a direct relationship between the adsorption and adhesion. The better selectivity of B.licheniformis JF-2 adhesion may be due to its cell wall composition which has fewer fatty acid groups than M.phlei. There is still a lack of enough evidence to show that the makeup of the cell wall of B.licheniformis JF-2 is similar to that of the cell wall of B.sublitis. Further useful work may be to try B.sublitis or other B.licheniformis strains or to change culture conditions for growth of M.phlei or B. licheniformis JF-2. In some instances cultural conditions such as temperature, trace salt or energy sources in media, and aerobic or anaerobic growth, can change the composition of the cell wall and various functions in the cell walls. Fatty acids as collectors are cheap, but of poor selectivity for phosphate over dolomite. Fatty acid flotation results using bacteria as dolomite depressant are far from satisfactory because of low P,05 recovery. A study on pH adjustors may find data of significance. One of the possible adjusting chemicals is Na,CO,. We did not obtain enough results on the effect of Na,CO, in this study, and further work should be done to investigate how it affects the flotation. A new collector, diphosphonic acid, DPA, is superior for flotation of phosphate from dolomite. Both bacteria and DPA work well in pure mineral and real sample flotation. The work on study of DPA as collector should be extended to combine it with work of bacteria as depressants. For flotation of Florida phosphate rock, including dolomitic phosphate ores, there are several specific rules in terms of some operation parameters to follow. The operation factors, such as clay removal, sizing of feed, and high percent solids conditioning, are very critical in some circumstances. The further work should include searching optimum operating factors and the obtaining the combination of these parameters.

61

REFERENCES Anazia, I., and Hanna, J., New flotation approach for carbonate phosphate separation, Minerals and Metallurgical Processing, Vol.4, No.4, Nov. 1987, p. 196-202 Bartels, J.J. and Gurr, T.M., Phosphate rock, Industrial Minerals and Rocks, 6th Edition, Senior Editor, D.D. Carr, SME, Littleton, Colorado, 1994, p.751-764 Bernard, J.P. and Hall, R.B., Comparative analysis of the central Florida phosphate district to its southern extension, Mining Engineering, 1980, p.1256-1261 Beveridge, T.J. and Murray, R.G.E., Uptake and retention of metals by cell walls of Bacillus subtilis, Journal of Bacteriology, Vol.127, 1976, p.1502-1518 Beveridge, T.J., Forsberg, C.W. and Doyle, R.J., Major sites of metal binding in Bacillus licheniformis walls, Journal of Bacteriology, Vol.150, No.3, 1982, p.1438-1448 Brierley, J.A., Goyak, G.M. and Brierley, C.L., Considerations for commercial use of natural products for metals recovery, Immobilization of Ions by Bio-sorption, Edited by Eccles, H. and Hunt, S., Ellis Horwood, Chichester, 1986, p.105-167 Cathcart, J.B., Economic geology of the land pebble phosphate district of Florida and its southern extension, Florida Phosphate Deposits, Field Trip Guidebook T178, 28th International Geological Congress, Tampa to Jacksonville, Florida, June 30-July 7, 1989, p. 18-38 Davis, B.E., Liewellyn, T.O. and Smith, C.W., Continuous beneficiation of dolomitic phosphate rocks, USBM, RI 8903, 1984 Gulbrandsen, R.A., Relationship of carbon dioxide content of apatite of the phosphoria formation to regional facies, U.S. Geological Survey Professional Paper 700-B, 1970, p.B9-B13 Harben, P., "Where is Floridas phosphate industry going ?", Industrial Minerals, No. 148, Jan. 1980, p.48-55 Javaheri, M., Jenneman, G.E., McInerney, M.J. and Knapp, R.M., Anaerobic production of a biosurfactant by Bacillus licheniformis JF-2, Applied and Environmental Microbiology, Vol.50, 1985, p.698-700 Jenneman, G.E., McInerney, M. J., Knapp, R.M., Clark, J.B., Ferro, J.M., Revus, D.E. and Menzie, D.E., A halotolerant, biosurfactant-producing Bacillus species potentially useful for enhanced oil recovery, Development of Industrial Microbiolo gy, Vol.24, 1983, p.485-492 Laskin, A.I. and Lechevalier, H.A., Handbook of Microbiology, Second Edition, Volume 1, Bacteria,

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CRC Press, 1977, p.288 Lawver, J.E., Wiegel, R.L., Snow, R.B. and Hwang, C.L., Phosphate reserves enhancement by beneficiation, Mining Congress Journal, V.68, 1982, p.27-31 Lehr, J.R. and Hsieh, S.S., U.S. Patent 4 287 053, 1981 Misra, M, Miller, J.D. and Song, Q.Y., The effect of SO2 in the flotation of sphalerite and chalcopyrite, Flotation of Sulphide Minerals, Edited by Forssberg, E., 1984, p.175-196 Moudgil, B.M. and Chanchani, R., Selective flotation of dolomite from francolite using two-stage conditioning, Minerals and Metallurgical Processing, Feb., 1985, p. 19-25 Moudgil, B. M. and Ince, D., Effect of sodium chloride on flotation of dolomite from apatite, Mineral & Metallurgical Processing, V.8, No.3, Aug., 1991, p.139-143 Sandvik, Dr.P.O.; U.S. phosphates-abundant resources will last for hundreds of years, Engineering and Mining Journal, V.180, No.10, Oct., 1979, p.99-101 Smith, R.W., Misra, M., and Chen, S., Adsorption of a hydrophobic bacterium onto hematite: Implications in the froth flotation of hematite, Journal of Industrial Microbiology, No.11, 1993, p.6367 Snow, R.E., U.S. patent 4 144 969, 1979 Thompson, R., Trace Metal Removal from Aqueous Solution, The Royal Society of Chemistry, 1986 Van Loosdrecht, M.C.M., Lyklema, J., Norde, W., Schraa, G. and Zehnder, A.J.B., Electrophoretic mobility and hydrophobicity as a measure to predict the initial steps of bacterial adhesion, Applied and Environmental Microbiology, Vol.53, 1987, p. 1898-1901 Zhang, J., Phosphate beneficiation-challenges and opportunities, Separation Processes: Heavy Metals, Ions and Minerals, Edited by M. Misra, 1994, p. 167-183

63