Mineral leaching of low-grade laterite ores using “‘bioacids” by molasses fungal metabolism P.G.Taeferis Abstract — Factory beet molasses media were used as cheap sources of sucrose for microbial leaching of Greek nickeliferrous laterites using strains of Penicillium and As- pergillus sp. Nickel recoveries of up to 62% and cobalt, recoveries of up to 50% were obtained by leaching in a two hase system in which bioacids were produced by fungal ‘metabolism in molasses ferrocyanide pretreated media. The addition of potassium ferrocyanide to the molasses sub- straies prior to leaching was found to have a beneficial effect by increasing the level of citric acid bioproduction, thus improving the leaching efficiency of the fermentation liquid. The metals known to interfere with citric acid production (ie., Fe, Cu,Ca.and Mn) were those most efficiently precipi- tated. Considering the much lower commercial price of ‘molasses in the industrial market compared to sucrose and. slucose carbohydrate media, molasses could prove to be a Promising alternative, despite the cost of the required pre- treatment. Introduction Extensive low-grade laterite deposits are found in central and northern Greece. These deposits are now being treated by a pyrometallurgical process for the production of {ferronickel. The very low nickel content (<1%) of the Greek deposits and the rising costs of fuel and electrical energy since the energy crisis of 1973 have had a severe impact on theproduction economics (Panagiotopoulos and Kontopoulos, 1988). Therefore, new energy saving and environmentally safe methods to recover the nickel from these deposits are needed. Microbial leaching of nonsulfide ores (including latrites) isa less energy intensive altemative that has recently been Baining interest. For these ores, heterotrophic leaching is necessary. A number of investigators have demonstrated the ‘mineral leaching ability of heterotrophic bacteria and fungi via processes of dissolution, mainly through secretion of organic chelating acids or other extracellular products by fermentative metabolism (Karavaiko, etal, 1988: Smith and Misra, 1991; Ehrlich and Holmes, 1986). No fungi, however, capable of direct enzymatic attack on a mineral have been reported (King, 1985). Microbiologically produced organic acids with particular leaching effectivenessarecitric and oxalic acids. These acids are produced by many fungi during their metabolism of carbohydrate substrates (Groudev and Gencev, 1978; Rossi MINERALS AND METALLURGICAL PROCESSING 1978; Silverman and Munoz, 1971; Dave, et al, 1981; Bosecker, 1985; Tzeferis, et al, 1991). Commercial proces sors who produce carboxylic acids from sugars have indi- ccated thatthe species of the genus Aspergillus and Peni lium sp. are the most efficient producers of these acids (Crueger and Crueger, 1990) However, at present, heterotrophic leaching appears tobe less amenable to commercial operations than autotrophic leaching, mainly because of the need to maintain microbial activity by providing organic carbon and energy sources. ‘The cost of refined sugars such as glucose and sucrose (used as the main bioleaching nutrients) is prohibitively expensive. ‘Thus, it is of considerable importance to find inexpensive substrates for the growth of acid-producing microorganisms suitable for leaching. The use of organic waste products containing hydrocarbons orby-products from the food, paper and agriculture industries could be useful alternatives. Cane and beet molasses, by-products of the sugar indus- try, have been reported as cheap, altemative sources of sucrose for effective heterotrophic leaching. Manganese bioleaching studies by the US Bureau of Mines showed that bby using molasses as the sole nutrient source, more than 95% of the manganese was extracted in flask, column and lab- scale heap experiments (Noble et a, 1991). The biological removal of iron from quartz sands, kaolins and clay was effected in shake flasks by leaching with organic acids produced as aresultof the cultivation of Aspergillus niger sp. inmolasses based media (Groudev,etal., 1985). Toexamine the effect of alternative carbon sources on bioleaching of Greek laterites, Leak, et al. (1991) used molasses from Appleford Industries at 150 g/L without pretreatment, A respectable maximum level of citric acid (9 to 25 g/L) was produced, but nickel recoveries were low. Also, an undesir- able high concentration of oxalic acid was produced, possibly because of the relatively high concentrations of iron con- tained in the molasses used. ‘The objective ofthis research was to examine the effect of_ using “bioacids” produced by the fungal metabolism of factory-grade domestic molasses on bioleaching of Greek low-grade laterite ores. In addition, the effect of molasses- P.G. Tzeferis is a metallurgical engineer with the Deparment of Minerals and Matorials Science, National Technical University, Ath ‘ens, Piraeus, Greece. SME nonmesting paper 93-682. Manuscript Dec. 6, 1998. Discussion ofthis peer-reviewed and approved paper isinvited and mustbe submitted, in duplicate, prior to Nov. 80, 1984. sucusr 04 +potassium-ferrocyanide pretreatment on acid production and leaching effectiveness was examined. Materials and methods Laterite ore The ore, provided by General Mining and Metallurgical LARCO S.A. (Larymna, Greece), was a typical low-grade Timonitic laterite containing: 0.73% Ni, 0.043% Co, 13.52% Fe (19.36% Fe,Os), 0.3% Mn, 41.50% SiOz, 2.08% MgO, 3.42% CaO, 2.17% Cr,03, and 18.43% Al,O3. The loss on ignition (1000°C for 30 min) was 9.5%. Before leaching, ore samples were crushed and ground to <150 mesh (1061). Mineralogical (XRD) and Electron be Analysis (EMA) showed that the ore consisted of nickel chlorite (the main nickeliferrous matrix mineral), hematite, quartz, chromite, tac, illite, diaspore and calcite in small amounts. Molasses ‘The molasses mash was obtained from the Hellenic Sugar Industry S.A... The as-received mash had the following average chemical composition: 22% sucrose, 47.1% total carbohydrate, 0.34% rafinose, 0.27% reducing sugars, 11.4% ash, 3.85% KO, 1.70% NazO, 1.7% total nitrogen (Kjeldahl) and 22.0% water. The mash also contained 13.0 mg/100 ml total Fe, 11.5 mg/100 ml Ca, 1.23 mg/100 ml Cu, and 0.96 mg/100 ml Mn. Microorganisms, medium and nutritional considerations. A wide variety of Aspergillus (codes A, Al, A2, A3) and Penicillium (codes P, P1, P2, P6, P24, P14) sp. were used. Microorganisms were obtained from the English (Common- ‘wealth Mycological Institute, London), German (Institute of Geosciences and Natural Resources, Hannover) and Greek (Technical Educational institute, Athens) collections. Also, one Penicillium strain (code F1) was isolated from an ac- lively laterizing Greek ore. This strain had characteristics likely to be beneficial to the leaching process. All strains were screened for their ability to: + produce organic chelating acids, and ‘+ tolerate high nickel and hydrogen ion concentrations, as in an actual leaching process. To investigate acid production, methyl-red indicator was added to the solid plates at a concentration of 0.3 g/L. For investigating the nickel tolerance, nickel chloride at various concentrations was used in the agar plates. Organisms which seemed promising after the screening process were used in the bioleaching experiments, ‘The following molasses based substrate was used for ‘growth and bioleaching experiments: 150.0 g/L. molasses, 1.8 g/L (NH,)HPO«, 1.95 g/L Cuy* (as CuCl - HO) and pH 5.0. Tron was not added because the iron content of the molas- ses was already high (13.0 mg/100ml). It has long been known that the optimal iron requirement for the production of citric acid is dependent, among other factors, on the starting material used, and, if optimal concentrations are exceeded, there may be a toxic effect (Perlman, et al., 1946; Shu and Johnson, 1948; King, 1985) Copper was added to counterbalance the inhibitory effect ofthe iron presentin the molasses (Noyes, 1969; King, 1985). ‘The concentration of added copper was determined by an ‘optimization procedure which was conducted by means of a factorial analysis to study the reversal by copper of the inhibition of citric acid formation by the amount of ferric iron contained (Tzeferis, 1991). All fungus strains used prior to leaching were adapted to the nickel concentration (up to 4000 ppm) by progressive acclimatization. Mash preparation Ferrocyanide treatment Inall tests, the mash was prepared from molasses diluted ‘with water to one-quarter strength and then centrifuged to remove the insoluble material. In the case of the ferrocyanide treated samples, the mash was adjusted to pH 6.0, autoclaved at 121°C for 20 min, and then treated with ferrocyanide immediately upon removal, from the sterilizer at 100°C. This technique was used to obtain a fermentable mash and to efficiently remove soluble minerals and metals known to interfere with citric acid production (Clark, et al., 1965 ). Varying ferrocyanide amounts were added to determine the optimum concentration for citric acid production. Measurements of metals by ‘Atomic Absorption Spectrophotometry (AAS) were also made to determine which metals, physiologically important to citric acid production (i.e., Fe, Mn, Cu, Ca and Zn) were precipitated by ferrocyanide treatment. Leaching with bioacids from fungal metabolism ‘The molasses medium used in these tests was pretreated, with 1.5 g/L potassium ferrocyanide solution (K [Fe (CN)l) 10 remove toxic trace metals (and possibly the ash), which increased the mediums ability to produce citric acid by fermentation. Following the pretreatment, a two-phase pro- cedure, in which laterite ore was leached at 95°C with acid- containing lixiviant produced asaresult of fungus cultivation, under controlled conditions, was performed. Leaching with logically-produced acid metabolites was conducted at a low pH by acidification with sulfuric acid to a free-acid concentration of 0.5¢/L. Cultivation for ligand production ‘was carried out in laboratory air-lift fermenters for eight days. Leaching at 95°C was carried out ina spherical stirred- glass reactor, heated by a thermal mantle that was thermo- statically controlled by the external use of a Fi-monitor. Citric and oxalic acids produced by fungal metabolism were determined using biochemical analytical methods (Boehringer ‘manual, 1989). Results and discussion Nickel tolerance on agar plates Prior to leaching, all strains were adapted to nickel con- centrations of up 10 4000 ppm by increasing, in steps, the nickel content of the growth medium used. All strains tested, with the exception of Fl, when acclimatized, exhibited a much improved tolerance (Fig.1, Fig.2). Ferrocyanide treatment and bioacid production The maximum concentration of citric acid obtained by fermentation of molasses without pretreatment was dramati- cally low (1 t02 g/L). However, all ofthe strains were found MINERALS ANO METALLURGICAL PROCESSINGay ot eames ra h2 yest sans eee ce tran oe oanre a a anee eo sranpat aan wank any sean kz ens wan? sranP2 eran Ps wanPuu ‘san P24 eanFt ores 4567 8 Tine cays) oe ne Fig. 2— Growth of various fungi strains as a function of ime in 18% molasses medium after ‘gradual acclimatization at 4006 ppm Ni Table 1 — Elfect of ferrocyanide troatment on the precipitation ‘of metals and bioacid production Potassium ferrocyanide level (g/L) os 1 18 3 5 Trace metals (% precipitated) Fo 98 95857 cu 7 88 = 8 BBB. ca 2% 2 2 3 8 8 Mn (bal te 7S eels ee Oe ce ote te 08) Bioacid production — Strain F1; fermentation conditions: 15% molasses medium, temperature: 30°C, aoration rato: 150 om3min, time: & days, initial pH: 6.0 Civic acid (Qh) «13S 14981587 B41 52 Osaicacid(g) 38 624 702 42 25 0 Final pH 41341 345 36 38 4a Bioacid production — Strain P2; fermentation conditions: 15% molasses medium, mmperature: 30°C, aeration rate: 150 em%min, imo: 8 days, initial pH: 6.0 Ginie acid (gh) = «78S 02S tz Onaicacid(gh) 41 42 8624021 Final pH 38 354 34 39 422 384 to produce higher concentrations of oxalic acid (3 10 5 g/L), possibly because of the relatively high amount of iron con- tained in the as-received domestic molasses. This is an undesirable characteristic for nickel bioleaching since oxalic, acid is known to be extremely effective at leaching iron but not nickel, which is possibly precipitated in pregnant solu- tions as nickel oxalate (Tzeferis and Agatzini, 1993). MINERALS AND METALLURGICAL PROCESSNG codissolution obtai and 15-16% (Fig. 5), respectively. Results of studies on the effect of the ferrocyanide treatment on mash composi- tion and on bioacid (citric and oxalic acid) production by strains Fl and P2 are pre- sentedin Table 1. Ascan be assumed from Table 1, potassium ferrocyanide makes crude molasses substrate fermentable by effecting changes in the mineral compo- nent of the mash. Heavy metals such as iron, manganese, calcium and copper, which are known to interfere with citric acid formation in synthetic media, are ef- fectively removed as complexes (€.g., Fea{FeCNe]s, FesFeCNeana CU2FCCNg) at ‘most of the potassium ferrocyanide con- centrations added. However, 1.5 g/L was found to be the most effective concentra- tion of potassium ferrocyanide for citric acid production of both strains, even if this concentration was not the best for metals removal. The beneficial effect on bioaci production stimulation could, therefore, result not only from the removal of metals from mash but also from other functions of ferrocyanide on molasses substrate. A subsequent reduction in the ash content by the treatment could be one of these pos- sible functions. Another explanation is provided by the postulation of a direct effect of ferrocyanide on the mold (by controlling growthof the organisms), which is incompatible with acid production, Martin(1955) concluded that ferrocyanide ion directly inhibited mold growth as well as removing unwanted heavy metals from molasses. This can also be supported by the observation that in molasses a moder- ately high residual ferrocyanide level is required for satisfactory fermentations (Heyer and Schwartz, 1964) Apart from FI and P2, all other strains used for citric acid production, with or without pretreatment, were excluded from any further study because, in most cases, theacid concentrations produced were near the minimum limit of detection by the methods used. Leaching with bioacids from fungal ‘metabolism Leaching of the ore at 95°C by meta- bolic products of strains P2 and F1 in 15% pretreated molasses medium after acidi ‘cation ofthe fermented liquid to [H*] =0. g/L by means of sulfuric acid gave nickel lets of $49 and 62% respectively Maximum Co recovery and Fe by both strains were 44-49% (Fig. 4)‘A comparison of the nickel recoveries obtained in molasses media (up to 62%), pare sucrose media (up to 72%) (Tzeferis, et.al, 1991) and from chemical leaching with sulfuric acid (45.5%) (Fig. 3), under the same leaching conditions, indicated that leaching with bioacids from the fungal ‘metabolism of molasses could be a prom- ising altemative despite the cost of the necessary pretreatment. The price of un- treated molasses in the Greek industrial market is estimated at 13-14 drs/kg (=0.05 2z BB HRSO4,(He}easot ED lcting wih arin 2. HeOSo4 a 94) | Geen an at So Zh. Ey g Leaching conditions: y Z| ote tents Z 7 ‘$/kg), considerably lower than the price of industrially produced sulfuric acid and at Teast twenty times less than the price of food-grade sucrose. Moreover, ferrocya- nide treatment costs might be covered by Fig. 9— Re oftirains P2 and Tet) of nickel aftr aching ofthe ore sulluric acid and metabolic products altor cultvation for 8 days in 15 pretreated molasses medium. the commercial price of the produced bio- mass, which could be used as a protein source for animal consumption, 60 ‘0 Conclusions and prospects + Thebioleaching effectivenessof beet ‘molasses medium was found to depend on its ability to produce hydroxycarboxylicacids, especially cit- ric acid, and also other metabolites. + Thebioacid yields by all strains from 1B 2504, PeI050H 2 leeoing wm aan P2, M0 08 1 leching wh ara, He0 od untreated solutions of molasses were Jow in comparison to those obtained from ferrocyanide treated molasses media. + Potassium ferrocyanide treatment, at ° Fig. 4— of strains P2 a Recove nd eat Leaching conditions: y a Y te 4g snc g A 2 Tene (he) eee octagon ey sta ook 1.5 g/L, increased citric acid production by removing the trace metals that have a detrimental affect on citric acid fer- ‘mentation and/or possibly by exerting a direct toxic action on the strains which facilitate citric acid production. + More than 60% of the nickel was bioleached with molasses pretreated media following the two-phase process in which acid metabolites were pro- duced after cultivation of strains under controlled conditions and then used for HeS04. eho sor 1 lecring wi ran P20 08 (Gsching wits ara, so Soh 150 aaching condone In oe aid concentratond ine Shr ” Tempete ssc laterite leaching at strong acid pH, [1"1 = 0.5 g/L. This process is almost as effective as comparable processes us- ing pure carbohydrate media, and, more- ‘over, itappearsto have the advantage of a lower overall cost, despite the cost of the necessary pretreatment. ° Fig. 5 ~ Iron codiss trains B2 and F ‘The objectives of any further investigations should be to ‘optimize the nutrient composition of molasses media for citric acid production and to determine the mode and exact ‘mechanism of ferrocyanide action on the media used. An- other area of considerable importance isto find inexpensive alternative carbon sources (¢.g.,lignocellulose sugars) or organic-waste products (cg., from sewage, paper, agricul- tural, food and beverage industries) that could be beneficial for bioleaching. A cost-benefit analysis would indicate a sizong dependence on the cost of the organic. The use of carob pod, an inexpensive agricultural product growing on 4 August 1904 solution after leaching aftr cultivation for. ‘by sulfuric acid and metabolic products ot "Bays in 15% protroated molasses medium barren soils in mast warm regions of the Mediterranean, is rich in water-soluble sugars (mainly sucrose) and is another possible carbon source (Marakis, 1992). Acknowledgments The financial support of the Commission of European Communities DGXIT (under contract No. MAIM,0017.C (H) is gratefully acknowledged. The helpful collaboration of Dr. S. Agatzini (NTUA, Athens), Dr. E.T.Nerantzis (TEI, Athens), Dr. D.Leak, Dr. K.Alibhai and Dr. A.Dudeney (LCST, London) are also acknowledged. MINERALS AND METALLURGICAL PROCESSINGReferences ‘Bostvinge Mannheim GMBH Users Manu, 1680, ethos of loshercal Anahyee and Food Anais ing Tet-Comnatons, Revo econ po. 142. Boseckar.K 1096, "Lasching fate rckl oan wth heterotrophic microrgaiaa, Proceedings ‘of to 6th intrnaoraleypeakm on Bihysrometalirgy, Vancower, Canada, Augut 21-24 ak, 0 eta 1088, "cect ot potasiomfracyangeon the chemical compoetion ot relasses mash used nthe vie a ermertaion Bltacraclogy and oengineang vol Vik pe 208-278, ‘unger, Wand Crosger A. 1950, Bitectnology:A textbook ol indua mcrobibgy ‘Sinauer Ascites, Inc, Sunderland, USA second elon, pp 2. Dave &.R, Natarajan, KR. and Bat, JV. 198, “Leaching of Copper and Ze rom centaed ofe by ng” Hycrmetargy, VSL 7, pp 238-242 Ete, HL. and Holmes, 2.8, a. 1086, "Worhabop on betecnology fr the ming ‘matal-ering ad fon fue precesing nduetien > Bltchnoogy and Bloangineehg ‘Syrpokm Ne.16. John Whey & Sor, rep. 386 Crovdev, 8. and Gancey, FN. 1978, Bioleaching of bauto by wid and brary ‘bed mite train th Congross,LCS.O.BA, Ahens, 1p. 271-278. Croudey, 8. ot al, 1985, ‘Biogial Removal ron rom quar sande, Kaoine and ‘lays Itraiorliineral Processing Congress, Canes 28 Jane 1986, Haya and Sehwart, 1964, -Wikng von Hexacyanoleate aul Ciroransauroganng von Anigorim Molaasemedum 2 Alp. Mivoblobgy. Vo. 4, No.3, p. 190-24, Karavi, GL, at al, 1968, “logectchnology of Meta,” Marval, UNEP, USSR CCommieion or UNEF, Conte tor ntarational Projects GKNT, Motcow. BE. 12, King, AB, 1986, “Te bedapracaton of naptelne by Aspergilv rigs Ph. Thess, Unvry f London Leak 0. oa, 1081, "Recovery of karo non sutoe sources lng mirorgand ied leaching proces” Final Rept susie to te C.EC, Contact No, MAM, ore INERALS AND METALLURGICAL PROCESSING Mara, 5.6, 1990, “Svcroee sy tom carob Bltchnology Let, Vo. 14 No. ‘pp 1075-1080, Manin SM, 1985, "Efecto onacyande on growth and acid preducon of Aapergioe ‘igen Canaan Joumal ot Miroiogy, VO. 1,644 82, Noble E.G. ta, 1991, ~Bllogialiaching of manganese oes," presenie athe SME ‘Annis Mesng, Denver, Colorado, February 25.28. "Noy. 1860, "Cvc cd production proceasee Noyes Development Cotpeaton Park ge, USA. Panagtopovlos,N. and Konopouoe, A. 1068, “Almogphele pressure auto als leaching ow-grade hema atarhes"Extractve Metagy ot Neel ané Cabal? ‘Tyler and CA Lando edt, The Metalugiea Society, pp. 447-58 Periman.0. oral, W. and JohntonM. 1948, “Elec of tacos nthe proton eric cd by A rige: Archives of Blochematy, Vol. 11 po 11-143, Ross. G. 1978, Potassium recovery through levee blleacing: poets and lerfatons* Metalic! eppleations of Pacer eachiog and related ricobislogcal Phenomena, Mur ol, ee, Acadoricpress LY. SanFaneluco, Landon op.257°319, ‘Shu, P. and Johnton, M.L, 148, "The interdependence ot medium contents nee ‘=x production by suomergediemetaion = Juralot Bactrloy, Vol 8 pp 677-05, Sivaran, M. ard Munoz. E1971 Fungal leaching of ttanlum fom feck Apes Mrosioogy. Val. 22, No pp. 923.824. ‘Smth, RW. and Mla, M, (edt, 1901, Proceeding ofthe conterance “Mineral Blprocesting” Santa Barbara, Calflora, June 1622, 101, Taal, Pe a, 1661, “Blloaching ol Grask nora rck owe ung micorgan- lam assed leaching process” Forum or Appa Botacnotogy 25-27 Soperta, Gort, Beigum| ‘asa, P.., 1961 “Blolaaching of non-autide ihaltarous ore using Hatarotophle ricrorgariams."PR.D. Thal, Naoral Technical Unwery ol Aone, Greece, 0376. ‘Taster 6, and Agazin 5 1989, "Leaching fica andron rom nn-stide Grek cketrous ores by orgric sid, submted Ter pubcaton In Hydomealugy AUGUST 194