Parameters for the Operation of Bacterial Thiosalt Oxidation Ponds

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1985, p /85/9663-7$2./ Copyright 1985, American Society for Microbiology Vol. 5, No. 3 Parameters for the Operation of Bacterial Thiosalt Oxidation Ponds M. SILVER Biotechnology Section, Extractive Metallurgy Laboratory, Canada Centre for Mineral and Energy Technology, Ottawa, Ontario, Canada KIA OGJ Received 8 April 1985/Accepted 1 June 1985 Shake flask and ph-controlled reactor tests were used to determine the mathematical parameters for a mixed-culture bacterial thiosalt treatment pond. Values determined were as follows: Km and Vmax (thiosulfate), 9.83 g/liter and mg/liter per h, respectively; Ki (lead), 3.17 mg/liter; Ki (copper), 1.27 mg/liter; Qlo between 1 and 3 C, From these parameters, the required bioxidation pond volume and residence time could be calculated. Soluble zinc (.2 g/liter) and particulate mill products and by-products (.25 g/liter) were not inhibitory. Correlation with an operating thiosalt biooxidation pond showed the parameters used to be valid for thiosalt concentrations up to at least 2 g/liter, lead concentrations of at least 1 mg/liter, and temperatures of >2 C. Brunswick Mining and Smelting Corp. (BMS), Bathurst, N.B., operates a mine and mill processing 1, tonnes of a pyritic zinc-lead-copper-silver ore per day by a process which includes wet grinding, pulp aeration with SO2 addition, flotation, and upgrading with heating. Partially oxidized anions of sulfur-thiosulfate (S-SO32-), trithionate (3S-S- S32-), tetrathionate (3S-S-S-SO32-), and other polythionates (3S-Sn-SO32-), collectively known as thiosalts, are formed during the processing of the ore, mainly during flotation, aeration, and grinding (11). Conditions favorable for the formation and stability of the thiosalts exist throughout the processing circuit. Grinding and aeration are performed under alkaline conditions in which the stability of the thiosalts is increased; the addition of SO2, long processing times (3 h), high sulfide content, and small particle size of the ore contribute to thiosalt formation. Because of the stability and the solubility of their calcium salts, the thiosalts are resistant to conventional effluent treatment procedures that include lime addition, and they pass virtually unaltered into the receiving surface waters upon discharge of the mill effluents (1). The thiosalts are then oxidized to sulfate, presumably by thiobacilli, and thus contribute to the deterioration of the environment by decreasing the ph values of these waters to <3 (6). In 1976, the Canadian Centre for Mineral and Energy Technology (CANMET) initiated a program with the mining industry and other government agencies to assist in the resolution of environmental problems associated with thiosalt-containing mill effluents. Among methods of thiosalt destruction evaluated, biological oxidation was estimated to be the least expensive with regard to capital and operating costs (11). Laboratory studies (2, 6) and on-site tests (11) demonstrated the technical feasibility of this process; the optimum temperature of 32.5 C and the optimum concentrations of ammonium (1 mg/liter) and phosphate (1 mg/liter) were also defined (8). Heavy metals in solution, especially copper, lead, and silver, were found to inhibit bacterial thiosalt oxidation (8, 11). The inhibitory effects of the metals were not additive for the oxidation of thiosalts by an authentic strain of Thiobacillus thiooxidans. The BMS tailings pond system is similar to a large, substrate-limited, continuous-flow reactor with the substrate, the thiosalts, added at concentrations usually between.3 and 2 g/liter. Dissolved heavy metals are always 663 present in the mill effluents; typical concentrations are as follows, in milligrams per liter: copper,.1 to.75; lead,.3 to 2; and zinc, 1 to 2. The operating ph is approximately 2.5. Slurries of particulate matter, such as copper, lead, and zinc concentrations from the mill thickener overflows, are occasionally discharged into the system. These materials are believed to be inhibitory to the bacterial thiosalt oxidation process (11). The concentrations of the dissolved metals may be controlled by chemical removal, dilution, or the recycling of effluents containing intolerably high concentrations of these metals. Although the inhibitory effects of dissolved metals, determined by using synthetic effluent (8), were found to be valid in on-site pilot scale tests (M. Wasserlauf, personal communication), a more precise definition would allow the effluent treatment ponds to be designed and operated more efficiently and economically. The objective of the present investigation was, therefore, to examine the effect of thiosalt concentration and the inhibition of bacterial thiosalt oxidation by dissolved heavy metals and particulate copper, lead, and zinc concentrates in authentic BMS mill effluents, using a bacterial culture obtained from the BMS treatment pond system. The mathematical parameters determined in this study would be used to define the volume of the biological oxidation pond and the residence time required to decrease the thiosalt content to environmentally acceptable concentrations (approximately 1 mg/liter). The results of this investigation are applicable not only to effluents of the BMS operation, but also to the effluents of other mills which generate thiosalt-containing wastes. MATERIALS AND METHODS Bacteria. A mixed culture of thiosalt-oxidizing bacteria originating from a rock-filled thiosalt oxidation pond of the BMS was used in this investigation. This culture is similar to that used in the previous investigation (8) in that it consists mainly of T. thiooxidans, which oxidizes thiosulfate, tetrathionate, and elemental sulfur under acidic conditions. Cultivation on thiosulfate at neutral ph revealed that this culture also contained thiobacilli similar to T. thioparus. The sulfur-oxidizing bacteria in this culture were obligate autotrophs, which used thiosulfate, tetrathionate, and elemental sulfur but not glucose, sucrose, or glutamate as substrates, and were obliate aerobes, as no growth occurred

2 664 SILVER TABLE 1. Elemental analysis of particulates Concn (%, wt/wt) ElemenTailin s Zinc Lead Copper Bulk g concentrate concentrate concentrate concentrate Sulfur Iron Zinc Copper Lead anaerobically in the presence of nitrate. No iron-oxidizing bacteria were detected when this culture was inoculated into the 9K ferrous iron medium of Silverman and Lundgren (9). The mixed culture was maintained in BMS effluent, adjusted initially to ph 7, and supplemented with 2 g of thiosulfate, 1 mg of ammonium, and 1 mg of phosphate per liter. This medium was used as it preserved the mixed character of the culture. Inoculum was prepared for each experiment by growing the mixed culture in this medium initially adjusted to ph 3.5 for 2 h, at which time the ph had decreased to 2.5 to 3.. During the course of this investigation, the presence of both acidophilic and neutral thiosulfateoxidizing autotrophs was verified in this culture. Test medium. The test solution used throughout this investigation is a sample of effluent of the BMS mill selected for low concentrations of heavy metals. This solution, originally containing 1.25 g of total thiosalts (thiosulfate and polythionates) per liter of which 66% was thiosulfate, was aged for 2 months at '15 C for the depletion of the thiosalts and filtered to remove particulate material. Also present in the effluent were lead (.2 mg/liter), zinc (3 mg/liter), copper (<.1 mg/liter), iron (6 mg/liter), calcium (61 mg/liter), and magnesium (4 mg/liter). Before use, 1 mg each of ammonium and phosphate per liter and, where indicated, thiosulfate, particulates, and the chloride salts of copper and lead and the sulfate salt of zinc, were added, and the ph was adjusted with either 18 N H2SO4 or 1 N NaOH. The particulates used in this investigation were products or by-products of the BMS mill; their chemical analyses are shown in Table 1. The zinc, lead, and copper concentrates are the products of BMS and consist principally of the mineral sphalerite, galena, and chalcopyrite, respectively. The bulk concentrate contains all three minerals, whereas the waste product, the tailings, contains pyrite and quartz as principal constituents. Shake flask experiments. All shake flask experiments used throughout this investigation to determine the mathematical parameters of bacterial thiosalt oxidation were conducted as in the previous investigation (8) in 25-ml Erlenmeyer flasks containing 1-mi total volume of the previously described test media. Incubation was at 3 C with agitation at 3 cycles per min, and 1. ml of the mixed culture of thiosaltoxidizing bacteria in the early maximum stationary phase was used as inoculum. Sulfuric acid (18 N) was used to adjust the ph of each flask to immediately after inoculation. For sampling, 7-ml portions were withdrawn during the course of each experiment, the ph was determined, and the sample was frozen and maintained at - 1 C until analyzed. Oxidation rates were calculated during linear decrease of thiosulfate concentrations and do not include lag time. ph-controlled reactor experiments. The reactors used were 3-liter glass reaction kettles, each fitted with a four-hole APPL. ENVIRON. MICROBIOL. cover, immersed in a constant-temperature bath. Temperature was controlled to ±1 C with an electric immersion heater or refrigerated coolant circulated through a cooling coil or both. The ph of the reaction mixture was maintained at 2.5 (±.2) by a glass electrode ph-stat which activated a solenoid valve controlling the entry of the feed solution into the reaction kettle. The volume of the reaction mixture was maintained at 1 (±.1) liter by a syphon tube, and mixing and aeration were accomplished by bubbling 1 liter of air per min through a fritted-glass gas dispersion tube. Where indicated, =1 liter of porcelain "O" rings (2.75 cm in diameter by 2.5 cm in length) was used as packing. At the initiation of each experiment, 1 liter of test solution inoculated with 1 ml of thiosalt-oxidizing bacteria in the early maximum stationary phase and adjusted to ph 3.75 (±.1) was placed in the reaction kettle. When the ph of the reaction mixture decreased to below 2.5, the feed solution, which is the test solution adjusted to ph 7.1 (±.2), was fed into the reactor, supplying fresh substrate and increasing the ph. Each test was conducted for a minimum of 6 days, during which steady-state conditions existed for a minimum of 4 days. The steady state was manifested by constant thiosalt concentrations in the effluents and by constant flow rates through the reactors, from which the rates of thiosalt oxidation were calculated. Effluent from the reaction kettles, the volume of which was determined at least twice daily and samples of which were submitted for analysis as described above after ph determination, was collected in a refrigerated incubator chamber maintained at.5 (±.1) C. Chemical analysis. Thiosalts were determined by the method of Makhija and Hitchen (4) and are expressed as milligrams of thiosulfate per liter; appropriate corrections were applied for the presence of heavy metals as required. All metals were analyzed by atomic absorption spectrophotometry. Treatment of data. A regression analysis computer program was used to calculate the rates of thiosalt oxidation from the shake flask and ph-controlled reactor experiments. The same program was used to fit the lines for the calculation of the mathematical parameters Ki,, Vmax, K;, and Qlo and to analyze the data for correlation of the results of this investigation with the results of the BMS pilot plant test. The rates of thiosalt oxidation (v), in milligrams per liter per hour, were calculated from the shake flask experiments, using the formula: -A[S] v = (1) and from the ph-controlled reactor experiments by the formula: ([Sin - [S]out)(V) v= / +(1+V) t where the thiosalt concentration ([SI) is expressed as milligrams of S23 per liter, the time (t) is expressed in hours, and the effluent volume (V) is expressed in liters. From these rates, the Vmax and Km values can be calculated by using the formula: 1 Km 1 _ = maxs Va+ (3) V' Vmax[S] Vmax The parameters V1max and Km can then be used to calculate the theoretical reaction rate (v) at any substrate concentration ([S]), using the formula: (2)

3 VOL. 5, 1985 BACTERIAL THIOSALT OXIDATION POND PARAMETERS 665 Vmax[S] V = ~~~~~~~~(4) Km + [S] Heavy-metal cation inhibition is generally regarded to be of the noncompetitive type; the inhibition constant (K1) can thus be calculated by using the equation: V] 1 1 Ki Km 5 v Vmax Vmax[S] and the degree of inhibition equation: (i) can be calculated with the V] Ki + K~~~+[J] [I] 6 where [I] is the concentration of the heavy-metal inhibitor. The effects of temperature were examined by using the Arrhenius equation: a ln v = C -- T (7) where In v is the natural logarithm of the reaction rate, C is the intercept on the temperature axis 1T, and a is the slope. The slope is equal to the natural logarithm of the proportional change of activity with temperature. The temperature coefficient, Qlo, can be calculated either from the Arrhenius equation, or by using the Van't Hoff equation in the form: log Qio = 1 P2 log- T2-T1 vi (8) where v1 and v2 are the temperatures T1 and T2. rates of thiosalt oxidation at ~~~~(6) RESULTS Shake flask tests. (i) Determination of Vmax and Km values. The Vmax and Km values for thiosulfate oxidation by the mixed culture of thiosalt-oxidizing bacteria were determined from double-reciprocal plots of thiosulfate oxidation rates against thiosulfate concentration. With 1 thiosulfate concentrations (.5 to 4.8 g/liter), a linear reciprocal plot was obtained from which the Vmax value of mg/liter per h and the Km value of 9.83 g/liter were calculated. The lag phase was <6 h at thiosulfate concentrations of approximately.5 g/liter, and approximately 5 h at thiosulfate concentrations of 4 g/liter. Final ph values ranged from 2.5 to <2., with the lowest ph values being attained with the highest thiosulfate concentrations. Thiosulfate was oxidized at approximately 2 mg/liter per h in the absence of bacteria. (ii) Determination of Ki values for lead and copper. The Ki values of thiosulfate oxidation were determined by plotting the reciprocal of the thiosulfate oxidation rates in the presence of either lead or copper against the six concentrations used of each of these heavy metals. A straight line was obtained with both of these inhibitors, from which the Ki values for lead was calculated to be 3.17 mg/liter and that for copper was calculated to be 1.27 mg/liter. (iii) Effect of zinc and particulates on bacterial thiosalt oxidation. Bacterial thiosalt oxidation rates in the presence of.25 g of each of the particulate fractions, zinc, copper, lead, and bulk concentrates and tailing slimes, or.2 g of zinc added as zinc sulfate per liter ranged from 99 to 12% of the theoretical rates, demonstrating that these materials do not inhibit bacterial thiosalt oxidation. Atomic absorption analysis of the reaction mixture supernatant revealed soluble iron concentrations of 7 to 9 mg/liter and soluble lead concentrations of 1 to 4 mg/liter, demonstrating that very little of the particulate material was solubilized. No additional zinc or copper was extracted from the particulates as shown by soluble zinc concentrations (3 to 4 mg/liter) and soluble copper concentrations (<1 mg/liter, which are identical to the concentrations of zinc (3 mg/liter) and copper (<1 mg/liter) of the test medium. ph-controlled reactor tests. (i) Effect of thiosalt concentration and packing. The rate of thiosalt oxidation in an unpacked pfh-controlled reactor corresponds to the rate calculated from the shake flask data, using equation 4 and the parameters Vmax = mg/liter per h and Km = 9.83 g/liter to a thiosalt concentration of approximately.7 g/liter (Fig. 1). Above this concentration, washout of bacteria limits the reaction rate. Inclusion of porcelain rings in the reactors allows the thiosalt oxidation rate to correspond to the rate calculated for thiosulfate concentrations up to at least 2 g/liter. Thiosalt-oxidizing bacteria attach to the porcelain rings and thus washout is minimized. The association of the thiosalt-oxidizing bacteria with the packing can be demonstrated by incubating the porcelain rings, either with or without thorough rinsing, in an Erlenmeyer flask containing the test solution and observing thiosalt oxidation. The bacteria and colloidal sulfur were also observed microscopically to be released from rinsed rings by washing with.2% Tween 8. (ii) Inhibition of bacterial thiosalt oxidation by lead and copper. Figures 2 and 3 show that bacterial thiosalt oxidation is inhibited by lead and copper to concentrations of approximately 2 mg of each of these heavy metals per liter, w THEORETICAL RATE OF THIOSALT OXIDATION // O25 // Wzo/~ ~~~~~~~~ "-35-5/- /* ~~~TIOAL /A I N g (gil) THIOSALT CONCENTRATIO FIG. 1. Effect of thiosalt concentration on bacterial thiosalt oxidation in ph-controlled reactors with or without porcelain ring packing.

4 666 SILVER APPL. ENVIRON. MICROBIOL..5 THEORETICAL.3 RATE OF THIOSALT OXIDATION _ I I I I i I6 LEAD CONCENTRATION ( mg / L) FIG. 2. Inhibition of bacterial thiosalt oxidation by lead in phcontrolled reactors packed with porcelain rings. The ratio Vi/Vo is the thiosalt oxidation rate in the presence of inhibitor divided by the uninhibited thiosalt oxidation rate. corresponding to the rates predicted with the Ki values of these inhibitors. Above these concentrations, however, the inhibition is only about 3% of that predicted theoretically for lead and 55% of that predicted for copper. (iii) Effect of temperature. The effect of temperatures between 5 and 3 C on the rate of bacterial thiosalt oxidation is shown in Fig. 4. The data obtained from aerated flask tests (8) of thiosulfate and tetrathionate oxidation by bacteria obtained from BMS are included. These data have been redrawn on an Arrhenius plot; the slope of the line is.665 from which a Qio value of 1.95 is calculated. Calculation of Qlo values from the intermediate temperatures yield Qlo values between 1.93 and 2.1. These values are valid at temperatures between 1 and 3 C. Below 1 C, the rate of bacterial thiosalt oxidation decreases more rapidly with decreasing temperature. DISCUSSION From the results of this investigation, the mathematical parameters Km and V1max were calculated to be 9.83 g/liter and mg/liter per h. These parameters were then used to calculate the theoretical rate of thiosulfate oxidation, using equation 4. At a thiosulfate concentration of 2 g/liter, the theoretical rate of oxidation is mg/liter per h at 3C. Results of a previous investigation (8), using synthetic mill effluent, demonstrated oxidation rates of 4 1 mg/liter per h at a thiosulfate concentration of 2 g/liter and 5 ± 2 mg/liter per h at a tetrathionate concentration of 2 g/liter. The differences between the results of these rates may be due to either experimental error or the partial conversion of thiosulfate to tetrathionate in the presence of traces of copper ions in the authentic mill effluent (7). Also to be considered is the instability of thiosulfate below ph 4.5; thus, the removal of thiosulfate from the reaction mixture is the result of both biological oxidation and acid hydrolysis. Comparison of the inhibition constant (Ki) values of authentic T. thiooxidans (8) with those of the mixed culture of thiosalt-oxidizing bacteria originating from the BMS operation shows that the mixed culture is more resistant to inhibition by lead of copper. The Ki values for lead and copper of the authentic culture oxidizing thiosulfate in synthetic effluents are 2. and.46 mg/liter, respectively, whereas the Ki values for lead and copper of the mixed culture oxidizing thiosulfate in authentic BMS mill effluent are 3.17 and 1.27 mg/liter per h, respectively. The inhibition of thiosulfate oxidation in synthetic medium by the mixed culture reaches a maximum at approximately 1 mg of lead per liter and at approximately 5 mg of copper per liter (8). In the ph-controlled reactors, the inhibition of thiosulfate oxidation by the mixed culture is a maximum at lead or copper concentrations of 2 mg/liter. The maximum inhibition, relative to the theoretically predicted values, is 3% in the presence of lead and 55% in the presence of copper. Thus, the inhibition of thiosulfate oxidation in the presence of concentrations of <2 mg of lead or copper per liter can be defined by equation 6, using Ki values of 3.17 and 1.27 mg/liter for lead and copper, respectively. At concentrations of these heavy metals of >2 mg/liter the inhibition can be predicted at 3% for lead and 55% for copper. Neither soluble zinc nor particulate concentrates or tailings cause the rate of thiosalt oxidation to decrease, indicating the inhibition of thiosalt oxidation, when slurries enter the biooxidation ponds, is due to such heavy metals as copper or lead dissolved in the slurry water. The slight increase in the thiosalt oxidation rates in the presence of particulates may be due to adherence of the thiosaltoxidizing bacteria to the surface of freshly ground particulate 1. COPPER.9 THEORETICAL RATE OF THIOSALT U(IDATION Q.8.7- > _.2.I C COPPER CONCENTRATION (mg/l FIG. 3. Inhibition of bacterial thiosalt oxidation by copper in ph-controlled reactors packed with porcelain rings. The ratio Vi/Vo is the thiosalt oxidation rate in the presence of inhibitor divided by the uninhibited thiosalt oxidation rate.

5 VOL. 5, 1985 BACTERIAL THIOSALT OXIDATION POND PARAMETERS 667 TEMPERATURE -.5. N o 1. F F-.6 H '.5 t C )> -2.5 / TEMPERATURE (-C) FIG. 4. Effect of temperature on bacterial thiosalt oxidation in ph-controlled reactors packed with porcelain rings with the thiosalt supplied as thiosulfate () and in aerated flask tests with the thiosalt supplied as thiosulfate (@) or tetrathionate (). Symbols: Closed, results of this investigation; open, date from reference 8. material (1), which effectively increases the concentration of these bacteria in the reaction mixture. Similar to other metabolic activities of autotrophic thiobacilli (3), the Qlo value for bacterial thiosalt oxidation is approximately 2. In the ph-controlled bacterial thiosalt oxidation tests, this relationship is shown to be valid at between 1 and 3 C. At temperatures of <1 C, the reaction rate decreases more rapidly with decreasing temperature. To examine the validity of the predictive procedures determined in this study, the thiosalt oxidation rates of a rock-filled and aerated thiosalt biooxidation pond at BMS are compared with the theoretical rates at various temperatures (Fig. 5). The theoretical thiosalt oxidation rates are calculated by equation 4, using the mill effluent thiosalt concentrations. The theoretical inhibition by lead concentrations of <2 mg/liter is calculated with equation 6 or is assumed to be 3% of the predicted value for lead concentrations of >2 mg/liter. For this graph, the effects of temperature have been disregarded, although the recorded temperatures of the biooxidation pond ranged from -1 to 23 C. For temperatures of between 2 C and 23 C, the actual rates of thiosalt oxidation in the biooxidation pond at BMS appear to correlate with the rate calculated from the parameters determined in this investigation at 3 C. Data obtained when the operating temperature of the rock-filled pond was between +1 and -1 C were always lower than predicted, indicating that the correlation between the predicted and actual rates of thiosalt oxidation are invalid at temperatures of <2C. The actual rates of thiosalt oxidation in the BMS biooxidation pond at operating temperatures of between 2 and 23 C are shown in Fig. 5 by crosses. These are compared with the rates of thiosalt oxidation predicted by the mathematical parameters determined in this study at 1, 2, and 3 C and at the average operating temperature of the BMS biooxidation pond, 13.5C. The rates obtained at <2C (circles) are considerably lower than those at the higher temperatures. These correlations indicate that the BMS biooxidation pond operates more efficiently at temperatures above 2 C than predicted by the use of the mathematical parameters determined in this laboratory study, which includes the effect of temperature. Better correlation is attained when the effects of temperature are disregarded. The BMS biooxidation pond has been in operation since autumn 198, over which time a large population of thiobacilli, immobilized on the rock packing, has developed. This large population has thus had time to become acclimatized to the chemical and physical

6 668 SILVER APPL. ENVIRON. MICROBIOL. F 25 3 z + / 2 o ix t ~;+ + I I-~~~~~~~~~~~ cn o s lo Is 252 ED + U E o- - --'-Io ~ PREDICTED RATE OF THIOSALT OXIDATION ( mg/ L /h) FIG. 5. Correlation of the rate of bacterial thiosalt oxidation calculated from the mathematical parameters determined in this investigation at various temperatures with the actual rate of thiosalt oxidation in the BMS pilot plant biooxidation pond at temperatures between 2 and 23 C (+) and at temperatures between -1 and +1C (). The average operating temperature of the BMS biooxidation pond was 13.5C. conditions of the pond. A possible explanation for these rates is that a sufficiently large population is present so that no decrease in activity is apparent above the critical temperature of 2 C. This large acclimatized and immobilized population may also cause the biooxidation pond to be more resistant to other stresses, such as those caused by the influx of heavy metals. The mathematical parameters Km and Vmax and the Ki values for lead and copper, determined in this investigation, can be used to calculate the size of the oxidation pond required for a given set of operating conditions, namely, mill flow volumn rate and thiosalt and inhibitor concentrations. The dilution rate, D (in hours-'), can be determined from the equation: D Vmax [S] Km + [SI As the dilution rate can also be expressed as (9) D = flv (1) the required volume of the reaction pond (V) can be calculated for a given flow rate (f). Similarly, the residence time (TR), which is the reciprocal rate, can be calculated by using the equation: TR = Vlf (11) Thus, a dilution rate of.36/h or a biooxidation pond volume of liters for each liter of mill effluent per hour containing 1 g of thiosalt per liter is required. This is valid for operation at temperatures of >2C. At temperatures below 2 C, either a larger biooxidation pond would be required or a supplimentary method of thiosalt oxidation must be used. Oxidation of thiosalts by using hydrogen peroxide with a ferrous ion catalyst has been shown to be technically feasible (5), although reagent requirements are too costly for the treatment of all of the thiosalts in the mill effluents. This method is recommended as a supplementary or backup process to decrease thiosalt concentrations to acceptable levels when operational conditions limit the effectiveness of bacterial thiosalt oxidation. The pilot plant biooxidation pond at BMS contains a volume of 15,195 liters. By using the mathematical parameters determined in this investigation, this volume should be adequate to treat 3,88 liters of mill effluent per hour containing no lead and 1 g of thiosalt per liter at temperatures above 2 C. At lead concentrations above 2 mg/liter, this capacity is decreased by 3% to 2,666 liters/h. Data from BMS shown in Fig. 5 indicate that residual thiosalt concentrations of <1 mg/liter were achieved consistently in mill effluents with thiosalt concentrations up to 1 g/liter at flow rates of 2,7 liters/h or less. Flow rates of >7,2 liters/h did not permit acceptably low thiosalt concentrations in the biooxidation pond effluents except as very low thiosalt concentrations in the mill effluents. Thiosalt oxidation at intermediate flow rates was inconsistent; thiosalt oxidation rates corresponded to only 7% of the values predicted from the mathematical parameters defined in this investigation. These inconsistencies correlate either with great variations in the thiosalt concentrations of the mill effluents, which disturb the steady-state growth characteristics of the bacteria, or with congestion of the biooxidation pond with particulate material. The particulate matter impedes the flow and introduces dissolved copper and other heavy metals which inhibit bacterial thiosalt oxidation in the biooxidation pond. ACKNOWLEDGMENTS I thank M. P. Cauley for technical assistance. Thiosalt determinations were done by M. Leaver and E. Cyfracki of the Analytical Chemistry Section, CANMET, and R. Sutarno aided in the statistical analysis of the results. J. E. Dutrizac and and B. H. Lucas of CANMET and M. Wasserlauf of the Noranda Research Centre, Point Claire, Que., gave advice and encouragement, and S. Samson of BMS Corp. provided data for the pilot plant thiosalt biooxidation pond and the mixed culture of thiosalt-oxidizing bacteria. LITERATURE CITED 1. Francis,. J The disposal of thiosalt effluents from the Brunswick Mining and Smelting Corp. No. 12 mine: a preliminary assessment of a pipeline to the sea. Div. Rep. MRP/MSL (TR). CANMET, Energy, Mines and Resources Canada, Ottawa. 2. Guo, P. H. M., and B. E. Jank Oxidation of thiosalts in base metal mining industry effluents using a rotating biological contactor. Environment Protection Service, Environment Canada, Ottawa. 3. Lundgren, D. G., and M. Silver Ore leaching by bacteria. Annu. Rev. Microbiol. 34: MakhiJa, R., and A. Hitchen Determination of polythionates and thiosulphate in mining effluents and mill circuit solutions. Talanta 25: Rolia, E Oxidation of thiosalts with hydrogen peroxide. Div. Rep. MRP/MSL (TR). CANMET, Energy, Mines and Resources Canada, Ottawa. 6. Schmidt, J. M., and K. Conn Abatement of pollution from mine waste waters. Can. J. Min. 9: Schmidt, M Reactions of the sulfur-sulfur bond, p In B. Meyer (ed.), Elemental sulfur: chemistry and physics. John Wiley & Sons, Inc., New York. 8. Silver, M., and. Dinardo Factors affecting oxidation of

7 VOL. 5, 1985 BACTERIAL THIOSALT OXIDATION POND PARAMETERS 669 thiosalts by thiobacilli. Appl. Environ. Microbiol. 41: Silverman, M. P., and D. G. Lundgren Studies on the chemoautotrophic iron bacterium Ferrobacillusferrooxidans. I. An improved medium and a harvesting procedure to secure high cell yields. J. Bacteriol. 77: Wakao, N., M. Mishina, Y. Sakurai, and H. Shiota Bacterial pyrite oxidation. III. Adsorption of Thiobacillus ferrooxidans cells on solid surfaces and its effect on iron release from pyrite. J. Gen. Appl. Microbiol. 3: Wasserlauf, M., and J. E. Dutrizac CANMET's project on the chemistry, generation and treatment of thiosalts in milling effluents. Can. Min. Q. 23: