Reduction of arsenic content in a complex galena concentrate by Acidithiobacillus ferrooxidans

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Open AcceResearch articleReduction of arsenic content in a complex galena concentrate by Acidithiobacillus ferrooxidansMario Makita*1,2, Margarita Esperón2, Benito Pereyra3, Alejandro López1 and Erasmo Orrantia*1
Address: 1Centro de Investigación en Materiales Avanzados, Miguel de Cervantes 120, 31109 Chihuahua, México, 2Instituto Tecnológico de Chihuahua II, Chihuahua, México and 3Universidad Autónoma de Nuevo León, Monterrey, México

Email: Mario Makita* - [email protected]; Margarita Esperón - [email protected]; Benito Pereyra - [email protected]; Alejandro López - [email protected]; Erasmo Orrantia* - [email protected]

* Corresponding authors

AbstractBackground: Bioleaching is a process that has been used in the past in mineral pretreatment of refractory sulfides,mainly in the gold, copper and uranium benefit. This technology has been proved to be cheaper, more efficient andenvironmentally friendly than roasting and high pressure moisture heating processes. So far the most studiedmicroorganism in bioleaching is Acidithiobacillus ferrooxidans. There are a few studies about the benefit of metals of lowvalue through bioleaching. From all of these, there are almost no studies dealing with complex minerals containingarsenopyrite (FeAsS). Reduction and/or elimination of arsenic in these ores increase their value and allows theexploitation of a vast variety of minerals that today are being underexploited.

Results: Arsenopyrite was totally oxidized. The sum of arsenic remaining in solution and removed by samplingrepresents from 22 to 33% in weight (yield) of the original content in the mineral. The rest of the biooxidized arsenicform amorphous compounds that precipitate. Galena (PbS) was totally oxidized too, anglesite (PbSO4) formed is virtuallyinsoluble and remains in the solids. The influence of seven factors in a batch process was studied. The maximum rate ofarsenic dissolution in the concentrate was found using the following levels of factors: small surface area of particleexposure, low pulp density, injecting air and adding 9 K medium to the system. It was also found that ferric chloride andcarbon dioxide decreased the arsenic dissolution rate. Bioleaching kinetic data of arsenic solubilization were used toestimate the dilution rate for a continuous culture. Calculated dilution rates were relatively small (0.088–0.103 day-1).

Conclusion: Proper conditions of solubilization of arsenic during bioleaching are key features to improve the percentage(22 to 33% in weight) of arsenic removal. Further studies are needed to determine other factors that influence specificallythe solubilization of arsenic in the bioleaching system such as: pH, dissolved oxygen concentration, redox potentials,nature of concentrate and temperature among others. At. ferrooxidans was able to completely oxidize the mineralspresent during the arsenic bioleaching. Other elements present originally in the concentrate such as Zn, Sb, and Cu werealso solubilized. The process of bioleaching is expected to be influenced by mechanisms that still need to be establisheddue to the diversity of the minerals involved and by the presence of traces of metals in the concentrate. The increase inpulp density generates a decrease in the dissolved arsenic concentration. This decrease is greater in runs where air wasnot injected to the system. The maximum rate of arsenic dissolution in the concentrate was found using; small surfacearea of particle exposure, low pulp density, injecting air and adding 9 K medium to the system. The effect of addition offerric chloride during the arsenic bioleaching resulted in a decrease of the solubilized arsenic in the system. The presenceof CO2 is associated to the decrease in arsenic dissolution.

Published: 13 October 2004

BMC Biotechnology 2004, 4:22 doi:10.1186/1472-6750-4-22

Received: 02 March 2004Accepted: 13 October 2004

This article is available from: http://www.biomedcentral.com/1472-6750/4/22

© 2004 Makita et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundThe term bioleaching refers to the bacterial conversion ofan insoluble metal (usually a metal sulfide, e.g., CuS, NiS,ZnS) into a soluble form (usually the metal sulfate e.g.,CuSO4, NiSO4, ZnSO4). When this happens, the metal isextracted into water [1,2]. The first bacterium discoveredthat was able to oxidize minerals was Acidithiobacillus fer-rooxidans (At. ferrooxidans, previously Thiobacillus ferrooxi-dans), a gram-negative, acidophilic,chemolithoautotrophic, non-spore forming rod. Nutri-tionally, typical At. ferrooxidans isolates are consideredobligate autotrophs. At. ferrooxidans is able to use eitherferrous iron or a wide variety of reduced inorganic sulfurspecies as an electron donor compounds. It is also able togrow using ferric iron as an electron acceptor, providedthat an electron donor, such as reduced inorganic sulfurcompound is present. Energy is derived from the oxida-tion of reduced iron and sulfur compounds, including fer-rous ion, sulfide, elemental sulfur and thiosulfate, withfinal oxidation products being ferric ion and sulfate [3,4].Other microorganisms considered important in commer-cial mineral biooxidation processes are: Acidithiobacillusthiooxidans, Acidithiobacillus caldus, Leptospirillumferrooxidans, and Acidiphilium acidophilum [3].

Bioleaching has emerged as a simpler, safer, and lessexpensive process than other alternatives for most lime-stone, granitic, or other host rocks that have secondaryreplacement of pyritic minerals containing metal values.In recent years, biooxidation has shown itself to requireless capital, reduced operating cost, and less skilled oper-ating and maintenance personnel than the traditionalpressure oxidation or roasting techniques [5].

This technology has been used for treating specific min-eral ores, mainly copper and gold bearing ores [6-8].Moreover, bacterial leaching in acid medium has beensuccessfully applied in: uranium metallurgy [9]; silver,gold and lead recovery [10]; zinc [11]; and new processeshave been developed for cobalt recovery [12,13].

A complex sulfide ore is an association of galena (PbS),sphaelerite (ZnS) and chalcopyrite (CuFeS2), dissemi-nated in a pyritic matrix. Besides of lead, zinc and copperas valuable metals, such deposits may contain significantquantities of silver, gold, arsenic, antimony, bismuth andmercury. Numerous economically important deposits ofthese ores exist in the world [14].

Complex ores are often characterized by particularly fineintergrowth of the mineral values. Due to these specificmineralogical characteristics, it is necessary to finely grindand concentrate the ore prior to the solubilization of thevaluable metals. To obtain separate concentrates by selec-

tive flotation involves high unit-cost, poor quality of theconcentrates and relatively low overall recoveries [15].

Arsenic is a major impurity present in numerous sulfidedeposits. The presence of arsenic in mineral concentratesdrastically diminishes their value, and results in two typesof problems. On one hand, arsenic produces metallurgicalproblems, making difficult the metal extraction and therecovery of a final product of high purity. On the otherhand, arsenic is regarded as a highly toxic contaminantresulting in environmental problems due to its atmos-pheric release and possible water contamination associ-ated to the processing of arsenic bearing ores andconcentrates [16].

Furthermore, this element is generally toxic for microor-ganisms and its dissolution could inhibit bacterial activityin the bioleaching process. It has been shown that highconcentrations of arsenic in solution inhibit bacterialgrowth, with As(III) reported to inhibit bacteria to agreater degree than As(V) [17].

The processing of complex arsenic bearing ores and con-centrates requires a good understanding of the mecha-nisms involved previous to the design and developmentof an adapted sequence of process units. In particular, acomplete chemical and mineralogical characterization ofthe ore is essential from both biological and metallurgicalpoints of view in order to determine the possible inhibi-tion problems and the requirement for effluent treatmentprocesses [12].

The interest in the study of bioleaching of arsenic-contain-ing minerals, started two decades ago with a publicationdealing with the degradation of arsenopyrite (FeAsS)through At. ferrooxidans in gold-arsenic concentrates. Thisstudy established the economic feasibility of this technol-ogy and its potential for alleviating some environmentrelated problems [18]. Later, other researchers [19] stud-ied the physical changes occurring in the mineral-bacteriainterphase in pure crystals of arsenopyrite. They foundthat bacterial oxidation is characterized by three stagesthat coincide with the phases of the general bacterialgrowth and that the amorphous arsenates produced aredeposited over the crystal surface, thus interrupting thebioleaching process.

There have been other studies aimed to determine theinfluence of several factors over the arsenic bioleaching ingold minerals and concentrates. These studies have beenmainly focused to maximize gold extraction and consid-ered bioleaching as a pretreatment in the cyanidationprocess, consequently leaving the importance of arsenicdissolution and extraction in a second term.



Ubaldini et al [20] studied the arsenopyrite bioleachingover a refractory mineral using a mixture of At. ferrooxidansand At. thiooxidans. They achieved an increase in goldextraction from 55.3% to 96.8% using the following con-ditions; pulp density 20%, pH 2, stirring conditions 200rpm, temperature 30°C, time 7 days. Another researchgroup used a domestic strain of At. ferrooxidans and a goldrefractory mineral to study the influence of a magneticfield, surfactant addition and the presence of Ag+, Bi3+,Co2+ y Hg2+ ions over the bioleaching process [21]. Theyfound a reduction to 120 hours to leachate 60% of arsenicusing magnetized water and the addition of tween-80 sur-factant and Ag+ ion [21].

As indicated above, previous studies are focused in usingbioleaching as a technique to eliminate arsenic in high-value minerals such as gold concentrates. However, thereis no reported work on arsenic elimination or reduction inminerals of low value such as lead.

This work is aimed to reduce the arsenic content in com-plex concentrates of galena and to generate preliminarydata to allow us to conduct further studies to understandthe complex behavior of the bioleaching process. This pre-liminary study deals with the influence of main factors inbatch bioleaching over the arsenic solubilization from acomplex lead concentrate.

The main factors influencing the biooxidative treatmentwere tested using a two level fractional factorial plan ofexperiments (27–4), and they were: surface area, pulp den-sity, carbon dioxide bubbling, air bubbling, 9 K mediumaddition, FeCl3 addition, and two different At. ferrooxidansstrains.

Results and discussionChemical and mineralogical characteristics of the concentrateThe chemical composition of the concentrate, determinedby atomic absorption spectrometry (AAS) and AASHydride System for arsenic was: 3.85% As, 14.75% Fe,23.53% S, 49.76% Pb, 2.48% Zn, 0.51% Sb, 0.29% Cu,0.28% Bi. The remaining 4.56% being other elementssuch as: Cd, Ca, Ag, K, Mn, Na, Ni, Ba, Mo, Sn, Si, O.

Table 1 shows major phases in the concentrate deter-mined by X-ray diffraction (XRD).

Photographs of prepared mounts (Figures 1, 2 and 3)show several mineral species and some mineral associa-tions found in the concentrate.

Studies dealing with bioleaching through At. ferrooxidanshave provided great amount of basic knowledge aboutthis process and have been useful in the understanding of

the physicochemical and microbiological aspects of thisphenomenon [3,22].

Biooxidative dissolution of arsenopyrite [19,23-29] andthe parameters for adapting the bacteria to arsenic havealso been studied [17,30]. However, a major disadvantageof these studies is the fact that they were performed usingchemicals reagents or pure crystals of pyrite and/or arse-nopyrite, synthetic or manually sorted with the aid of amicroscope and therefore, they do not represent the con-ditions and complexity involved in the bioleaching treat-ment of concentrates.

There are only a few of studies dealing with arsenic-bear-ing ores and concentrates of natural sources[12,14,31,32]. It is desirable that the trend of today's stud-ies of bioleaching of natural ores be aimed to understandthe factors influencing these phenomena if this technol-ogy is expected to reach industrial importance. However,it is clear that the complexity of some sulfide minerals aredue to the association among species and to the coexist-ence of inclusion forms of many of these sulfide materials.These two issues (complexity and inclusions) are impor-tant factors that may inhibit the process and produce non-successful intrinsic behavior of the bioleaching mineralsystems [33].

The concentrated used in this study was highly complex ascan be seen in Figures 1, 2 and 3. Also in these Figures therelative composition of the mineral species and their asso-ciations described in Table 1 are shown. The complexarray of compositions showed in these Figures means thatthe process of bioleaching is expected to be influenced bymechanisms that still need to be established by the diver-sity of the mineral species involved and by the presence oftraces of metals in the concentrate.

Table 1: Mineral species and associations

Mineral species and associations Relative proportion

Galena (free) 50.41Galena + Arsenopyrite 13.95Pyrite (free) 5.42Galena + Sphaelerite 4.60Arsenopyrite (free) 3.87Pyrrotite (free) 3.09Sphaelerite 3.09Galena + Pyrite 2.33Galena + Sphaelerite + Arsenopyrite 1.55Galena + Arsenopyrite with inclusions of Galena

1.55

Other complex associations 10.14



Mineral species oxidizedXRD analysis of the residual solid from bioleaching (Fig-ure 4) shows only the presence of crystalline phase angles-ite (PbSO4). Chemical analysis performed through AASresulted in the presence of residual arsenic. This indicatesthat all crystalline species present in the concentrate werecompletely oxidized through the bioleaching process.This means that all arsenic present in arsenopyrite waseither solubilized (22 to 33%) or precipitated in amor-phous compounds (67 to 78%). Also, galena (PbS) wascompletely oxidized to anglesite. Only a very small por-tion of anglesite remained in solution (25 mg/L, seeFigure 5) while, the main phase of this appeared as a solidprecipitate. Other elements present originally in the con-centrate such as Zn, Sb, and Cu were also solubilized. Thisfact opens a window of a great potential for separatinglead from other elements that often are present along inthe mineral concentrates [2].

Formation of precipitates during biooxidationFormation of precipitates of arsenic and iron has beencommonly observed with the use of At. ferrooxidans [34].Several other compounds have also been observed insolid state during the bioleaching of arsenopyrite, amongthese are; ferric arsenate, elemental sulfur, amorphous fer-ric arsenate FeAsO4

·xH2O, jarosite KFe3(SO4)2(OH)6 andscorodite FeAsO4

·2H2O [19,25-27].

It has been reported that the elemental sulfur and ferricarsenate are accumulated in the surface of the grains form-ing a coating barrier that avoids the contact between themineral and the Fe+3 ions in the indirect oxidation, inhib-iting the leaching of the mineral species [19,27]. The for-mation of precipitates of the jarosite-type are prevented bya pH control to levels lower than 1 [33], while the forma-tion of S0 can be avoided by the use of bacterial consortiain which sulfur-oxidant bacteria such as At. caldus [28] orAt. thiooxidans are included [3].

Photography of a site in surface of a mountFigure 1Photography of a site in surface of a mount G: Galena; S: Sphaelerite; A: Arsenopyrite; P: Pyrite



In this study the precipitation of amorphous arsenic com-pounds was important during the bioleaching process. Insamples taken at the end of the experiment there was a dif-ference in the arsenic content in the digested mineral res-idue, which was treated by a hydrochloric acid digestion,to the non-digested residue (see Table 2). This last, sug-gests the presence of two types of arsenic compounds inthe residue, probably amorphous ferric arsenates andjarosite-type precipitates due to the acidic conditions usedin the experiment (pH ≥ 2). However, other studies areneeded using this kind of concentrates in order to deter-mine the type and amount of precipitates formed and thekinetics involved in this process.

Significant factors in arsenic dissolutionArsenic bioleaching results for the eight experimental runs(Table 3) are shown in Figure 6. In this plot a great deal ofscattered data among experimental runs is observed, thismeans that still a great deal of variability exists among theeffect of the factors considered in this study.

Table 4 shows the results of the least-squares-multiple-regression model fitting to arsenic dissolution data usingtime and factors as explanatory variables. With the excep-tion of At. ferrooxidans strains, all of the other factorsresulted to be highly significant in its influence to thearsenic dissolution. The resulting model was the follow-ing (Minitab 13.0):

Arsenic = 52.0 - 66.3 Pulp Density - 32.6 Surface Area -21.1 Ferric chloride - 12.5 Carbon dioxide + 29.3 Air +29.3 9 K Medium + 3.00 Strain + 3.94 Days. (1)

With a determination coefficient of R2 = 0.834

The negative sign in some of the variables of the modelindicates that in order to maximize bioleaching of arsenic,these factors must be kept in low levels. These weredenoted with a zero value in the model. Thus, the levels offactors that should be considered in the model are the fol-

Photography of a site in surface of a mountFigure 2Photography of a site in surface of a mount G: Galena; S: Sphalerite; A: Arsenopyrite; H: Hematite; Q: Quartz



lowing: pulp density at 10%, low surface area, no FeCl3and CO2 addition, air and 9 K Medium addition and anystrain present.

Best runOne of the main disadvantages for the commercial use ofbioleaching is the slow nature of this process, which is duemainly to the relative slow growth rate of the bacteria[5,35], to the long period of time needed for the bacteriato adapt to the mineral environment and particularly tomineral complexes [31] and to inhibition problems dueto the products of the bioleaching [36].

In Figure 6, Run 1 presents the highest arsenic concentra-tion in the leachate (approximately 200 mg/L). The com-bination of the previously established levels of factors forthis run are shown in Table 3 and coincides with therequired combination of levels to maximize the arsenic

dissolution as stated in the model of the above Equation(1).

The graphical pattern of the arsenic bioleaching for Run 1(Figure 7) shows some features that are potentially ade-quate for the use of this combination in reduction ofarsenic content in future studies. Some of these featuresare: the lack of an adaptation period (lag-time), a verypronounced slope and that the stability of the system isreached in a relatively short time (four days) compared tothe other experimental runs.

The data for this run (Run 1) were fitted through a thirdorder polynomial linear regression model and statisticallyanalyzed over a 95% confidence interval (see Figure 7).The model of arsenic present in solution in Run 1 is thefollowing (Minitab 13.0):

Photography of a site in surface of a mountFigure 3Photography of a site in surface of a mount S: Sphaelerite; A: Arsenopyrite; P: Pyrite; Pr: Pyrrotite



Arsenic = 100.16 + 11.487 Days - 0.4705 Days2 + 0.006Days3 (2)

With a determination coefficient of R2 = 0.6585

Besides of all these convenient features in Run 1, the per-centage of bioleached arsenic is still low. Proper condi-tions of solubilization of arsenic during bioleaching arekey feature to improve the percentage (yield) of arsenicremoval. Therefore, further studies are needed in order todetermine the other factors (not considered in this work)that influence specifically the solubilization of arsenic inthe bioleaching system such as: pH, dissolved oxygen con-centration, redox potentials, nature of concentrate andtemperature among others.

General evolution of arsenic in the leachateWhen time (days) was used as the only independent vari-able, data collected in all runs were adjusted to a thirdorder polynomial regression model and the analysis isshown in Figure 8. The resulted general equation of thismodel is the following (Minitab 13.0):

Arsenic = 23.888 - 1.0597 Days + 0.5161 Days2 - 0.0131Days3 (3)

The determination coefficient of this model was R2 =0.3237, which represents a value relatively low due to theabsence of the other significant factors (pulp density,CO2, 9 K medium, particle size, air injection and ferricchloride added).

It is worth to note that this model resembles the wellknown general three-phase bacterial growth model.

Effect of pulp densityThe pulp density effect is shown in Figure 9. Here a greaterarsenic dissolution is achieved with a low level of 10% ofsolids. This result is in agreement with results reported byother researchers working with complex concentrates ofcopper and zinc [14], complex sulfides of copper, leadand zinc [37] and copper concentrates [38,39]. In all ofthese studies greater levels of leaching were achieved withlow pulp densities. The reduction in the bioleaching ratecan be due to the fact that at higher concentrations ofsolids causes an increase in the friction between particles,and probably avoiding the adhesion between the particleand bacteria [40]. This friction may consequently causesome mechanical damage to the cell [41].

XRD of the residual solid from bioleachingFigure 4XRD of the residual solid from bioleaching



In the case of arsenic, it is possible that a high pulp densitymay cause a greater interaction between the dissolvedarsenic and other products of biooxidation. Thus,increasing the rate of formation of precipitates and conse-quently decreasing the concentration of arsenic insolution.

In our study it was found a strong interaction between thepulp density and the air injection with respect to thearsenic removal. The ANOVA analysis of this interaction isshown in Table 5, and the plot in Figure 10. The increasein pulp density generates a decrease in the dissolved

Lead SolubilizedFigure 5Lead Solubilized

Table 2: Arsenic content in mineral residue before and after digestion with hydrochloric acid

Run Residue before digestion % arsenic Residue after digestion % arsenic Difference % arsenic

1 3.5208 2.6332 0.88762 3.5751 2.7105 0.86463 3.6283 3.0034 0.62494 3.6452 2.9911 0.65415 3.5291 2.5907 0.93846 3.5325 2.5821 0.95047 3.6334 2.9436 0.68988 3.6021 2.9784 0.6237

-1

4

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Bioleaching time, Days

Lea

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hat

e,m

g/l

Run 1

Run 2

Run 3

Run 4

Run 5

Run 6

Run 7

Run 8



arsenic concentration in all runs. However, this decrease isgreater in runs where air was not injected to the system.

Effect of surface areaThe effect of surface area (particle size) can be observed inFigure 11. Here a greater arsenic dissolution is presentedwhen the particle surface area is small, in other words,where bigger particles are present in the system. One

would expect that a greater exposed surface area to themicrobial attack would reflect an increase of the arsenicdissolution. However, this unexpected result is in agree-ment with other studies reported in the literature [42,43].Explanations for this phenomenon are in the sense that itis possible for the bacteria to preferentially attack somesites formed during the solids grinding process [42].Other study of the leaching of chalcopyrite reported that

Table 3: The 27–4 design used.

Pattern Run Pulp Density % Surface Area Ferric Chloride Carbon Dioxide Air 9 K Medium Strain

----+++ 1 10 Low No added No added Added Added T18--++--+ 2 10 Low Added Added No added No added T18-+-+-+- 3 10 High No added Added No added Added T1-++-+-- 4 10 High Added No added Added No added T1+--++-- 5 20 Low No added Added Added No added T1+-+--+- 6 20 Low Added No added No added Added T1++----+ 7 20 High No added No added No added No added T18+++++++ 8 20 High Added Added Added Added T18

Arsenic SolubilizedFigure 6Arsenic Solubilized

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the rate of leaching increased with the use of largerparticles sizes [42]. They suggested that this was due to agreater efficiency of bacteria attachment to the particles.Other explanations are focused on disregarding the asso-ciated physical factors such as surface area and stressingthe specific and non specific interactions. Among thespecific are the ionic and hydrogen bonds, and chemicaland protein interactions. Non-specific are hydrophobic

interactions such as surface free energy and electrostatic[44].

A possible explanation for the behavior observed in thisstudy is based in the great complexity of the mineral con-centrate that may enhance the specific interactions. Theseinteractions are affected by the particles of smaller sizeswhere the greater surface area leaves each particle exposedto a greater amount of different mineral species. These

Table 4: Regression analysis results

Predictor Coefficient T value Probability

Constant 52.0270 6.76 0.000Pulp density % -66.3470 -14.05 0.000Surface area -32.5570 -6.89 0.000Ferric chloride -21.0730 -4.46 0.000Carbon dioxide -12.5350 -2.65 0.009Air 29.3000 6.20 0.0009 K medium 29.3010 6.20 0.000Strain 3.0020 0.64 0.526Days 3.9396 14.41 0.000

Third order model fit and confidence interval for Run 1Figure 7Third order model fit and confidence interval for Run 1

0

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0 5 10 15 20 25 30


Ars

enic

in

lea

chat

e, m

g/l Run 1

Run 2Run 3Run 4Run 5Run 6Run 7Run 8UCLLCLPolynomic Run 1



species increase the complexity of the medium thus affect-ing directly the solubilization of arsenic during thebioleaching. The complexity of the medium due to smallsize particles can be further increased when a high pulpdensity is used as pointed out in the previous section.These two issues, complexity and pulp density, combinedin the conditions above mentioned may produce a nega-tive effect over the arsenic dissolution.

Effect of ferric chlorideIn the present study the effect of addition of ferric chlorideduring the arsenic bioleaching resulted in a decrease of thesolubilized arsenic in the system as observed in Figure 12.Information on this effect is limited in the literature withonly some studies reporting the influence of the ionchloride on the sulfur and iron oxidation during bioleach-ing of some chemical species using At. ferrooxidans and /orAt. thiooxidans [45-47].

Effect of carbon dioxideIt is well known that autotrophic organisms such as At.ferrooxidans require CO2 for its growth. This is due to thefact that these bacteria are able to use CO2 as its onlysource of carbon for their biosynthetic reactions. CO2 fix-ation is usually achieved through the so-called Calvin-Benson-Bassham cycle [48]. In Figure 13 it can be seenthat the arsenic dissolution is reduced when CO2 isbubbled into the system. This behavior is in agreementwith studies reported in literature that established that theconsumption of carbon by the bacteria decreases at highCO2 concentrations implying the formation of intracellu-lar bicarbonates [49]. Other authors coincide in that theamount of CO2 present in the air is enough to withstandthe bacterial growth [14,25].

In our study, it was found a strong interaction between thetwo levels of CO2 and air into the system. The ANOVAanalysis of this interaction is also shown in Table 6 andFigure 14. However, in our study the presence of CO2according to the results shown in Figure 13 is associated

Third order model fit for all dataFigure 8Third order model fit for all data

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to the decrease in arsenic dissolution. At this time, it is dif-ficult to establish the influence of the presence and con-centration of CO2 on the dissolution of arsenic. Therefore,further studies oriented to establish the relationshipbetween the dissolved oxygen and the concentration of

CO2 in the system are needed in order to elucidate thiseffect.

Effect of Pulp DensityFigure 9Effect of Pulp Density

Table 5: Analysis of variance for interaction Pulp density – Air

Source DF SS MS F P

Air 1 4714 4714 1.92 0.168Pulp Density 1 132058 132058 53.84 0.000Interaction 1 25775 25775 10.50 0.002Error 116 284501 2453Total 119 447028

DF: Degrees of Freedom; SS: Sum of Squares; MS: Mean Square; F: Fisher Statistic; P: Probability

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10% Solids 20% Solids Polynomial 10% Polynomial 20%



Effect of airAcidithiobacillus ferrooxidans is an aerobic microorganismthat uses oxygen as a final electron acceptor during theoxidation process. However, in absence of oxygen it is stillable to grow in the presence of inorganic reduced sulfurcompounds by using the ferric ion as an alternativeelectron acceptor [50]. In these conditions, however, itsgrowth is slower [48]. In the present study, it is notsurprising that the arsenic dissolution was increased withthe aeration of the system as can be observed in Figure 15.As it was shown previously, the availability of oxygen inthe medium decreases when the pulp density increasesand also when CO2 is admitted into the system. Again amore definite study targeted to measure dissolved oxygenin the system may bring light into the effect of air duringthe arsenic bioleaching.

Effect of 9 K mediumThe 9 K medium is the source of nitrogen and phospho-rous [51] needed for bacterial growth. These two elementsare not available in the sulfide minerals. Therefore, theyneed to be provided during the bioleaching process. Fig-

ure 16 shows the effect of the addition of 9 K medium. Asexpected, the arsenic bioleaching is increased when 9 Kmedium is used.

Effect of strainIn Figure 17 differences of two At. ferrooxidans strains usedin this study are shown. Strain T1 presents the commonbacterial model of growth in three stages, while strain T18presents a straight model that requires less adaptationtime. This can be due to the fact that T18 was previouslygrown in systematically increased arsenic concentrationsas discussed in the material and methods section of thispaper.

Continuous culture dilution rate calculusData generated from the batch arsenic bioleaching wereused as a first approximation for the calculation of thedilution rate D for a future design of a continuousbioleaching system. The procedure used consisted in toderive the third order polynomial equations (2) and (3)with respect to time and plot each equation versus thearsenic concentration in the system. Then to draw a

Interaction plot Pulp density – AirFigure 10Interaction plot Pulp density – Air

AddedNo added

2010

130

80

30

Pulp Density

Air

Mea

nof

Ars

enic

inle

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tem

g/l Added

No added

2010

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Pulp Density

Air

Mea

nof

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straight line from the origin to the maximum point ineach curve (Figure 18). The slope of this line was thendefined as dilution rate in agreement with the equation ofproduct balance in a continuous culture vessel in steadystate [52,53] as shown in equation (4):

where

F = Rate of medium flow through the vessel (volume/time)

V = Volume of the vessel (volume)

D = F/V Dilution rate (time-1)

Pn-1 = Inner product concentration

Pn = Outer product concentration

dPn/dt = Total variation of the product concentration inthe vessel

(dPn/dt)production= Product concentration variation due tothe production in the vessel

Rearranging Eq. (4):

In steady state, dPn/dt = 0 and assuming that Pn-1 = 0 in asingle vessel,

From which

Effect of Surface AreaFigure 11Effect of Surface Area

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ate,

mg

/l

Low High Polynomial Low Polynomial High

VdP

dtFP FP V

dP

dtn

n nn

production= − +

−1 4( )

dP

dt

F

VP P

dP

dtn

n nn

production= − +

−( ) ( )1 5

dP

dt

dP

dtDP

Total production

= =

−0 6( )



Equation (7) represents a straight line through the originhaving a slope of D (dilution rate) and corresponds to thestraight line plotted in Figure 18.

Using this method, the calculated dilution rate for thegeneral model of equation (3) is 0.088 days-1, while forrun 1 in equation (2) is of 0.103 days-1 (not shown in theplot). However, these dilution rates are still relativelysmall compared to the dilution rates used in industrialscale processes (0.05–0.6 h-1) [53].

ConclusionsSince the bioleaching rate and the levels of leached arsenicare limited (22–33% of the originally present in the con-centrate), proper conditions of solubilization of arsenicduring bioleaching is the key feature to improve the per-

centage (yield) of arsenic removal. Therefore, furtherstudies are needed in order to determine the other factors(not considered in this work) that influence specificallythe solubilization of arsenic in the bioleached system suchas: pH, dissolved oxygen concentration, redox potentials,nature of concentrate and temperature among others.

The performance of the bacteria used in this study (At. fer-rooxidans) was able to completely oxidize the mineralspresent during the arsenic bioleaching. Since, galena(PbS) was completely oxidized to anglesite (PbSO4) withonly a very small portion of anglesite remaining in solu-tion, while the main phase of this appeared as a solid pre-cipitate. Other elements present originally in theconcentrate such as Zn, Sb, and Cu were also solubilized.

The complex array of compositions contained in the con-centrate, employed in this study, means that the process ofbioleaching is expected to be influenced by mechanisms

Effect of Ferric ChlorideFigure 12Effect of Ferric Chloride

0

50

100

150

200

250

0 5 10 15 20 25 30


Ars

enic

in le

ach

ate,

mg

/l

Ferric Chloride Added No Ferric Chloride added

Polynomic (No Fe chloride added) Polinómica (Ferric Chloride Added)

dP

dtDP

production

= ( )7



that still need to be established due to the diversity of themineral species involved and by the presence of traces ofmetals in such concentrate.

The precipitation of amorphous arsenic compounds wasimportant during the bioleaching process. Results suggestthe presence of two types of arsenic compounds con-tained in the residue, probably amorphous ferric arsenates

and jarosite-type precipitates due to the acidic conditionsused in the experiment (pH ≥ 2).

The increase in pulp density generates a decrease in thedissolved arsenic concentration. However, this decrease isgreater in runs where air was not injected to the system.

Effect of Carbon DioxideFigure 13Effect of Carbon Dioxide

Table 6: Analysis of variance for interaction Carbon dioxide – Air

Source DF SS MS F P

Air 1 4714 4714 1.92 0.168Carbon dioxide 1 25755 25775 10.50 0.002Interaction 1 132058 132058 53.84 0.000Error 116 284501 2453Total 119 447028

DF: Degrees of Freedom; SS: Sum of Squares; MS: Mean Square; F: Fisher Statistic; P: Probability

0

50

100

150

200

250

0 5 10 15 20 25 30


Ars

enic

in

leac

hat

e, m

g/l

No CO2 B ubbling CO2 B ubbling

P o lynomic No CO2 B ubbling P o lyno mic CO2 B ubbling



The maximum rate of arsenic dissolution in the concen-trate was found using small surface area of particle expo-sure, low pulp density, injecting air and adding 9 Kmedium to the system.

The effect of addition of ferric chloride during the arsenicbioleaching resulted in a decrease of the solubilizedarsenic in the system.

The presence of CO2 according to the results is associatedto the decrease in arsenic dissolution. Further studiesoriented to establish the relationship between the dis-solved oxygen and the concentration of CO2 in the systemare needed in order to elucidate this effect.

Arsenic dissolution was increased with the aeration of thesystem. The availability of oxygen in the mediumdecreases when the pulp density increases and also whenCO2 is admitted into the system. A study using dissolvedoxygen measurements is needed in the future to deter-mine the effect of air during the arsenic bioleaching.

MethodsChemical and mineralogical analysisThe flotation concentrate was obtained from La Soledadmine (Parral, Chihuahua, México). Chemical analysis wascarried out by atomic absorption spectrometry throughAAS (GBC Avante Σ), arsenic was determined by AASHydride System. The major phases in the concentrate weredetermined by X-ray diffraction (Siemens D5000).Mineral samples were mounted in polyester resin blocksusing approximately 0.2 g per mount and surface waspolished. Mounts were examined using a microscope(Olympus AX70) and photographs were taken at varioussites of each sample.

Design of experimentsOne of the most useful types of multifactor experiment isthe 2k factorial series. In this series, there are k factors eachat two levels. Hence there are a total of 2k treatments in thefull factorial set. This series is particularly useful in theexploratory stages of an investigation because it permitsthe examination of a fairly large number of factors andtheir interactions in a trial of reasonable size [54].

Interaction plot Carbon dioxide – AirFigure 14Interaction plot Carbon dioxide – Air

AddedNo added

No addedAdded

130

80

30

Carbon dioxide

Air

Mea

nof

Ars

enic

inle

acha

tem

g/l Added

No added

No addedAdded

130

80

30

Carbon dioxide

Air

Mea

nof

Ars

enic

inle

acha

tem

g/l



In almost all experiments the investigator would like toreduce the number of observations required for a com-plete factorial. If certain assumptions can be met the useof fractional factorials is a most efficient technique toreduce the number of observations and still obtain thedesired information. The usual fractional factorial is stillorthogonal, which means that certain effects are estimatedindependently of one other [55].

A major use of fractional factorial designs is in screeningexperiments. These are experiments in which many factorsare considered with the purpose to identifying only theimportant variables that affect the response and theirinteractions. The factors that are identified as importantare then investigated more thoroughly in subsequentexperiments. A fractional factorial of the 2k design con-taining 2k-p runs is called a 1/2p fraction of the 2k or, moresimply, a 2k-p fractional factorial design. It is possible toconstruct this type of designs for investigating up to k = N-1 factors in only N runs. If k = N-1 the fractional factorial

design is said to be saturated. Of particular importance isa very useful saturated fractional factorial design for stud-ying seven factors in eight runs; that is, the 27–4 design.This design is a one-sixteenth fraction of the 27 [56].

This was the design used in this work to test the factorsinfluencing the biooxidative treatment. The objective wasto maximize the arsenic solubilization. The factors, levelsand runs are presented in Table 3.

Factors and levels in experimental runsPulp densityThe bioleaching experiments were carried out using twosolid concentrations. The low level for pulp density wassettled as 10 % w/v; the high level as 20% w/v.

Surface areaThe lead concentrate was washed with distilled water; themineral suspension was wet sieved using a 75 µm sieve inorder to obtain two fractions. Both of them were dried

Effect of AirFigure 15Effect of Air

0

50

100

150

200

250

0 5 10 15 20 25 30


Ars

enic

in le

acha

te, m

g/l

No Air bubbling Air bubbling

Polynomic No Air bubbling Polynomic A ir bubbling



and analyzed for specific surface area by a laser scatteringparticle sizer (Malvern Master Sizer 2000). The specificsurface area for fractions >75 µm and <75 µm were 0.42and 1.65 m2/g and were established as low and high levelsrespectively.

Ferric chlorideAddition of ferric chloride at concentration of 150 mg perliter of liquid medium was settled as high level, no ferricchloride addition was low level.

Carbon dioxideLow level: no carbon dioxide bubbling; high level: carbondioxide flow from a compressed cylinder, injected into themixture at a rate of 0.2 vol/vol min-1.

AirNo air bubbling was considered as low level; no sterile airbubbles injected into the liquid-concentrate mixture at arate of 0.3 vol/vol min-1 was high level.

9 K MediumPure distilled water as culture medium was established aslow level; the use of 9 K medium [51], which contained(per liter of distilled water) 3.0 g of (NH4)2SO4, 0.5 g ofMgSO4·7H2O, 0.1 g of KCl, 0.5 g of K2PO4, 0.01 g ofCa(NO3)2 was settled as high level. The pH was adjustedto 5.7 with sulfuric acid.

StrainsA native Acidithiobacillus ferrooxidans wild type straintermed T1 [57], isolated from a domestic mining site aciddrainage, was used as a low level; and an arsenic-resistantstrain, called T18, derived from T1 by serial transfers toflasks containing increasing arsenic amounts, was used ashigh level. T18 is able to grow at arsenic concentration ashigh as 1800 mg l-1 [57]. The strains were cultured in arotary shaker incubator (30°C, 175 rpm), in a mediumcontaining 44.22 g FeSO4, 3.0 g (NH4)2SO4, 0.5 gKH2PO4, 0.5 g MgSO4· 7H2O, 0.1 g KCl, 0.01 g Ca(NO3)2per liter, adjusted to pH 2.0 with sulfuric acid. After bacte-

Effect of 9 K mediumFigure 16Effect of 9 K medium

0

50

100

150

200

250

0 5 10 15 20 25 30


Ars

enic

inle

acha

te, m

g/l

9K No added 9K A dded P o lyno mic 9K added P o lyno mic 9K no added



rial growth for ten days, cultures were filtered and the clearliquid was used as inoculum (20% v/v).

Conditions of cultivation and samplingThe eight biooxidation runs (Table 3) were conducted in1000 ml Pyrex culture flasks containing 500 ml of mix-ture, placed in a rotary shaker incubator (30°C, 175 rpm).The pH was maintained to 2.0 with sulfuric acid. Theexperiment was monitored each 48 h. After a short periodfor sedimentation of solid particles, each flask wassampled extracting 2 ml of clear leachate in which totalarsenic, lead and iron concentrations were determined.The liquid extracted was compensated by the addition ofdistilled water or 9 K medium. Little hoses were sub-merged in liquid mixture to inject compressed air, carbondioxide, or both. The experiment was carried out for 28days; this is twice the time needed to reach the stability ofarsenic concentration in a previous laboratory test.

At the end of the experiment the resulting pulp in each runwas filtered and the bioleached mineral was washed with

distilled water and dried in a stove at 40°C. Two grams ofthis material were taken and were submitted to digestionusing 10 ml of HCl 0.6 N during 3 hours at roomtemperature. The digested mineral was filtered, washedwith distilled water and dried at 40°C. In both cases thearsenic content was determined through chemicalanalysis.

Data analysisTable 3 shows the saturated experimental design used tocarry out the experiment, the eight trials provide a total ofseven degrees of freedom for the entire experiment, allo-cated to seven columns of two levels, each column havingone factor assigned (Pulp density, Surface area, Ferricchloride, Carbon dioxide, Air, 9 K medium, Strain). Allcolumns provide four tests under the low level of the fac-tor and four tests under the high level of the factor. This isone of the features that provides the orthogonality amongall the columns (factors) [58]. Orthogonality permits thecomparison between low and high levels for each factor intheir ability to dissolve arsenic. Comparison was per-

Effect of StrainFigure 17Effect of Strain

0

50

100

150

200

250

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30


Ars

enic

inle

acha

te, m

g/l

T1 Strain T18 Strain Polynomic T1 Strain Polynomic T18 Strain



Dilution rate calculusFigure 18Dilution rate calculus

Table 7: Factors coded as dummy variables

Pattern Run Pulp Density

Surface Area

Ferric Chloride

Carbon Dioxide

Air 9 K medium

Strain Results As mg/l

----+++ 1 0 0 0 0 1 1 1 R1--++--+ 2 0 0 1 1 0 0 1 R2-+-+-+- 3 0 1 0 1 0 1 0 R3-++-+-- 4 0 1 1 0 1 0 0 R4+--++-- 5 1 0 0 1 1 0 0 R5+-+--+- 6 1 0 1 0 0 1 0 R6++----+ 7 1 1 0 0 0 0 1 R7+++++++ 8 1 1 1 1 1 1 1 R8

Results of As in leachate (mg/l) were determined each 48 hours, to finally have the results of this table 15 times during the 28 days of the experiment

-4

-3

-2

-1

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80 90 100 110 120 130

P = Mean arsenic concentration mg/l

dP/d

t



formed by fitting a multiple regression model to arsenicdissolution data, to make the model function, all sevenfactors were treated as dummy variables [59] taking thelow level as 0 and high level as 1 (Table 7). Time in dayswas included in the model as the only true quantitativefactor. Model fitted is:

Arsenic = β0 + β1 Pulp Density + β2 Surface Area + β3 Ferricchloride + β4 Carbon dioxide + β5 Air + β6 9 K Medium + β7Strain + β8 Days. (8)

Where

Arsenic Expected value of arsenic concentration in mg l-1 inleachate

β0, β1,... β8 Regression coefficients

As the experiment was monitored each 48 hours, andarsenic concentration was determined in leachate, arsenicdissolution data gathered during the bioleaching experi-ment were analyzed as time series (time as independentvariable) by multiple regression to fit the third order pol-ynomial model:

Arsenic = β0 + β1t + β2t2 + β3t3 (9)

Where

Arsenic Expected value of arsenic concentration in mg l-1 inleachate

t Time in days of bioleaching

β0, β1, β2, β3 Regression coefficients

SoftwareAnalytical procedures and graphing was performed usingMS Excel or Minitab 13.0

Authors' contributionsMM designed and performed the experimental runs andprepared the manuscript. ME performed the statisticalanalysis. BP provided the bacterial strains and helped inthe culture production. AL performed the technical revi-sion of the manuscript. EO got the funds and provided thetutoring during the development of this work. All authorsread and approved the final manuscript.

AcknowledgementsThe authors wish to express sincere gratitude to the Consejo Nacional de Ciencia y Tecnología CONACYT (México) for funding this research through project CONACYT-DAIC 34223-B.

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Reduction of arsenic content in a complex galena concentrate by Acidithiobacillus ferrooxidans

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