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Detection and analysis of attached microorganisms on the mineral surface during bioleaching of pure chalcopyrite with moderate thermophiles
Hydrometallurgy 106 (2011) 46–50
Contents lists available at ScienceDirect
Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t
Detection and analysis of attached microorganisms on the mineral surface during bioleaching of pure chalcopyrite with moderate thermophiles Weimin Zeng a,b,c, Sunee Tan c, Miao Chen c, Guanzhou Qiu a,b,⁎ a b c
School of Minerals Processing and Bioengineering, Central South University, Changsha, China Key Laboratory of Biometallurgy, Ministry of Education, Changsha, 410083, China CSIRO Process Science and Engineering, Clayton, Victoria, 3168, Australia
a r t i c l e
i n f o
Article history: Received 23 October 2010 Received in revised form 23 November 2010 Accepted 24 November 2010 Available online 1 December 2010 Keywords: Attached microorganisms Chalcopyrite Bioleaching Atomic force microscope Extracellular polymeric substances
a b s t r a c t Moderate thermophiles were used to bioleach pure chalcopyrite and showed good performance of copper extraction in the stirred tank reactor. The attached microorganisms on the mineral surface were detected by atomic force microscope (AFM). The result showed that the attached cells could produce extracellular polymeric substances (EPS) rapidly. However, it was difficult to reduce or eliminate the EPS once it was produced, and thus the large amount of EPS at the later stage of bioleaching would mediate the formation of jarosite and block the continuous copper extraction. Furthermore, the number of attached cells on the mineral surface was enumerated as a function of time and the variation was rather different from that of free cell density in the bioleaching solution. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Chalcopyrite is the most abundant and refractory copper sulphide mineral which is traditionally floated, concentrated and treated by pyrometallurgical methods to obtain copper. Bioleaching of low-grade chalcopyrite ores and tailings is a copper extraction process with low capital and operating cost, and environmental benefits (Brierley 1990; Rohwerder et al., 2003). At present, researchers have paid more attention to bioleach chalcopyrite with moderately thermophilic microorganisms rather than mesophiles as they can greatly improve the leaching reaction kinetics, avoid excessive chalcopyrite passivation and overall improve the total copper extraction (Wu et al., 2007; Zhou et al., 2009). It is reported that the attached microorganisms on the mineral surface played a pivotal role during bioleaching (Beech and Sunner, 2004; Kinzler et al., 2003). The bioleaching microorganisms prefer to attach onto the mineral surface after inoculation and produce extracellular polymeric substances (EPS). EPS could concentrate ferric ions and form a special layer to oxidize chalcopyrite whilst the dissolution of chalcopyrite releases energy sources such as sulphur and ferrous ions for the bioleaching microorganisms. However, the growth trend of attached microorganisms on the mineral surface was not clear. Especially at the later stage of bioleaching, chalcopyrite
⁎ Corresponding author. Key Laboratory of Biometallurgy, Ministry of Education, Changsha, Hunan 410083, China. Tel.: + 86 731 8879212. E-mail address: [email protected] (G. Qiu). 0304-386X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2010.11.014
surface would be covered with a passivation layer to inhibit the flow of microorganisms, nutrients and reaction products to and from the mineral surface (Hackl et al., 1995; Zeng et al., 2010). Then the growth behaviour of attached microorganisms should be different from the free cells in the bioleaching solution. In other research fields, such as soil and waste water treatment, the detection and analysis of attached microorganisms on solid surface had been reported by some authors and several processing methods for extracting attached microorganisms were described. Pedersen et al. (1996) used PCR amplification, cloning and sequencing of 16S rRNA gene methods to investigate the diversity of attached bacteria in soil samples and about 10 species were found, but the amount of attached cells was not enumerated. Yu et al. (1993) adopted a direct viable count way to enumerate the amount of attached microorganisms (Klebsiella pneumonia Kp1) on stainless steel coupons. The attached cells were first scraped from the coupons and moved into the solution, and then the suspension could be observed and counted under the microscope. In the experiments of Delatolla et al. (2008), the nitrifying bacteria microorganism attached on glass beads and produced biofilm. For enumerating the attached cells, the cells were detached by vortexing the beads in a centrifuge tube. Therefore the ways of enumerating the attached microorganisms vary according to the different types of microorganisms, support surfaces and aims of experiments. In this study, bioleaching of pure chalcopyrite was performed by moderate thermophiles in a stirred tank reactor. The attached microorganisms on the mineral surface were detected by atomic force microscope (AFM). In addition, the attached microorganisms
W. Zeng et al. / Hydrometallurgy 106 (2011) 46–50
were removed from the mineral surface and enumerated with a counting chamber under the microscope.
2. Materials and methods 2.1. Microorganisms enrichment Acid Mine Drainage (AMD) samples from several chalcopyrite mines in China were collected. The samples were mixed and then inoculated into the culture medium for enrichment of moderate thermophiles. This medium was modified at 9 K and consisted of the following compounds: (NH4)2SO4 3.0 g/L, Na2SO4 2.1 g/L, MgSO4·7H2O 0.5 g/L, K2HPO4 0.05 g/L, KCl 0.1 g/L, Ca(NO3)2 0.01 g/L. The moderate thermophiles were enriched at 48 °C and initial pH of 2.0 in a stirred tank reactor (see Section 2.3).
2.2. Mineral components The chalcopyrite used in the bioleaching experiments was obtained from Yushui copper sulphide mine in Meizhou, Guangdong, China. The mineralogical composition was 98% chalcopyrite and 2% silicate, which mainly includes CaSiO3 and MgSiO3. The pure chalcopyrite was ground and the diameter of the particles was b75 μm. 2.3. Bioleaching experiments Bioleaching experiments were carried out in a 3 L glass cylindrical reactor with a mechanical stirrer operating at 500 r/min. About 1950 mL medium was added into the reactor; then 50 mL seed culture (no soluble copper) was inoculated to get a cell density of 106 cells/ mL. The reactor was placed in a thermostatic bath to keep the temperature constant at 48 ± 0.2 °C. Sterile air was introduced into the base of the reactor at an approximate rate of 360 mL/min. The experiments were performed at an initial pH 2.0 and the pulp density was 6%. The acid consumption was compensated by addition of 10 mol/L sulphuric acid to keep pH value around 2.0 in the beginning of bioleaching. Distilled water was added to the reactor through a peristaltic pump in order to compensate for evaporation losses. Redox potential, the concentration of Cu2+, Pb2+, Fe2+ and total iron in solution were analysed every two days, while pH value and cell density in the solution were analysed every day. The components of the mineral sample and ore residue were analysed by X-ray diffraction (XRD). Copper, lead, and total iron concentrations in solution were determined by ICP-AES. Ferrous ion concentration in solution was assayed by titration with potassium dichromate. Ferric ion concentration is the concentration of total iron minus the concentration of ferrous ions. The pH value was measured with pH S-3 C acid meter. The redox potential was measured using a platinum electrode with an Ag/AgCl reference electrode. Free cells in solution were observed and counted with counting chamber under an optical microscope; the attached cells on the mineral surface were determined with the method shown in Section 2.5.
2.4. Atomic force microscopy view For observing the attached microorganism on the pure chalcopyrite surface, AFM work was performed. Three polished slices of pure chalcopyrite (A, B and C) were put into the bioleaching solution for 1, 4 and 10 days, respectively. The slices after bioleaching for different days were taken out and used for AFM scanning. A Agilent Technology 5500 Scanning Probe Microscope operating in a tapping mode in air was used to image cells.
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2.5. Collection and enumeration of attached microorganisms from the mineral surface The leaching solution was allowed to settle for 1.5 h and the supernatant decanted. Then the thickened sludge was centrifuged at 2000 g for 4 min at 4 °C and 4 g of the pellets were re-suspended with 10 mL Milli Q water in a 50 mL centrifuge tube. After this, one gram of glass beads with a diameter of 0.2 mm was added into the tube. The tube was pressed forcedly on a vortexer and underwent 8 min of vigorous vortexing. Then about 36 mL Milli Q water was added into the tube and the vortexing continued for 2 min. After this, the 50 mL mixture was centrifuged at 2000 g for 2 min to separate the ore residue and solution. 0.1 mL of the supernatant was used to count the cell number under the optical microscope and the rest was transferred to a 150 mL flask. The ore residue was used again, underwent vigorous vortexing for 10 min and centrifugation for 2 min, followed by cell collection and counting. After this, the remaining ore residue was used for the 3rd time and underwent vigorous vortexing for only 5 min and the cells in the supernatant was counted and collected. After three times of vortexing and separation, a total of about 150 mL solution containing the attached cells was collected. The total amount of the cells was analysed by observation under an optical microscope. In addition, the concentration of 2-keto-3-deoxyoctonate (KDO) and DNA in the solution were measured. The measurement of KDO concentration was described as Karkhanis et al. (1978). The DNA concentration was measured spectrophotometrically using a NanoDrop®ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). KDO is part of the cell membrane of microorganisms and therefore can be used as a marker for membrane compound contamination. With low contents of the DNA and KDO indicates that the attached microorganism was not broken during the vortexing of residue (Karkhanis et al., 1978). 2.6. Extraction of extracellular polymeric substances from the mineral surface The detailed processes for extraction and chemical analysis of extracellular polymeric substances from the mineral surface were described in our previous published paper (Zeng et al., 2010). The extracted EPS were analysed for nitrogen (Velghe and Claeys, 1985), uronic acids (Blumenkr and Asboehan, 1973) and protein (Bradford, 1976) spectrophotometrically. Qualitative and quantitative estimations of fatty acids and sugar monomers were achieved by gas–liquid chromatography. Experimental procedures have been described previously (Gehrke et al., 1998). Lastly, the concentration of total iron and ferrous ion in the EPS solution was analysed by ICP-AES. 3. Results and discussion 3.1. Bioleaching of pure chalcopyrite with moderate thermophiles Bioleaching of pure chalcopyrite by moderate thermophiles was performed at 6% pulp density for 40 days in a stirred tank reactor. Fig. 1 shows the copper concentrations as a function of time. Copper extraction increased slowly from the beginning to the 12th day, when the copper concentration was 2.16 g/L. Thereafter, the copper extraction rate increased rapidly till bioleaching performed to the 36th day and then the copper concentration was 12.2 g/L. After that, the chalcopyrite concentrate dissolution rate became slow. When the bioleaching experiment finished (at the 40th day), the copper concentration was 12.6 g/L and the percentage copper extraction achieved was 62%. Fig. 2 shows the variation of iron concentrations during bioleaching of chalcopyrite as a function of time. Iron in solution at the beginning and middle stage was mainly ferrous ion and the concentration of ferric ion was more than ferrous ion only at the
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14
3.2. Production of extracellular polymeric substances by attached microorganisms on the mineral
12
copper Eh
600
10 8
550
6
500
4
450
2
Redox potential / mV
Copper concentration / (g/L)
650
400
0 350 0
5
10
15
20
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35
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Bioleaching / days Fig. 1. The concentration of copper ion and redox potential in the solution during bioleaching of pure chalcopyrite with a mixed culture of moderately thermophilic microorganisms at 6% pulp density and initial pH 2.0.
end stage. After the 26th day, the concentration of ferrous ion began to decrease and that of ferric ion began to increase, which corresponded to an increase of redox potential in the solution over 500 mV. At the 40th day, the concentrations of ferric ion and ferrous ion were 3.12 g/L and 1.35 g/L, respectively. The redox potential value showed an increase from 385 to 634 mV from the beginning to the end of bioleaching process. This increase of redox potential was accompanied with an increase of ferric ion concentration (Fig. 2). Additionally, the oxidation of sulphur compound produced enough sulphuric acid to decrease of the pH value, which is another reason for the increase of redox potential. In the beginning of bioleaching experiment, sulphuric acid was added to the reactor to keep the pH value around 2.0 for acid consumption of chalcopyrite (Mousavi et al., 2006). After that, the pH decreased to a final value of 1.42 since acid was continuously produced during the oxidation of sulphur and the hydrolysis of ferric iron to ferric species such as Fe(OH)2+ and Fe(OH)+ 2 (Sandstorm and Petersson 1997; Gomez et al., 1997). In the first six days, about 7.8 g/L of sulphuric acid was added into the bioleaching system to keep the pH value about 2.0 due to the acid consumption. After this period, the pH value began to decrease and acid addition was stopped, which indicates that the amount of acid-production was more than that of acid consumption.
8
iron concentraction / (g/L)
7
Ferrous iron Ferric iron Total iron
6 5 4 3 2 1 0 0
5
10
15
20
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30
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Bioleaching time / days Fig. 2. The variation of the total iron, ferrous and ferric ions concentrations in solution as a function of time during bioleaching of pure chalcopyrite by a mixed culture of moderately thermophilic microorganisms at 6% pulp density and initial pH 2.0.
The attached microorganisms on the polished slices of pure chalcopyrite bioleached after different days were detected by AFM. It can be seen from Fig. 3 that on the first day of bioleaching, microorganisms began to attach onto the mineral surface and produced a small amount of EPS. At the 4th day of bioleaching, the amount of EPS covering on the cells increased significantly and EPS tended to spread onto the mineral surface. At the 10th day of bioleaching, the attached cell and mineral surface were both fully covered by EPS. It is reported (Beech and Sunner, 2004; Kinzler et al., 2003) that the production of EPS benefits the concentration of ferric ions and thus oxidizes the chalcopyrite. It is believed that uronic acid and other unidentified compounds in the EPS complexes with ferric ion. Therefore, the rapid production of EPS on the mineral surface can improve the copper extraction. After the 10th day, it was hard to get a good AFM view of attached cells (data not shown) due to the production of a large amount of EPS and the formation of passivation substances on the mineral surface. The sulphur content in the ore residue was about 2.4% at this time. The EPS on the mineral surface was extracted and characterized from the mineral surface as a function of bioleaching time (Fig. 4). The collected EPS was primarily composed of sugars and fatty acids and the content of protein was relatively low. The variation of total EPS and the EPS fraction contents per gram of ore residue as a function of bioleaching time are shown in Fig. 4. It was found that the amount of total EPS increased rapidly at the beginning of bioleaching and achieved a maximum of 75.6 mg per gram of ore residue at the 10th day. Once the EPS were produced, it was difficult to remove or degrade them from the bioleaching system. As a result, a large amount of EPS would remain on the mineral surface and possibly enhance the passivation layer at the end of bioleaching (Zeng et al., 2010). In this case, the copper extraction rate decreased largely and the attached microorganisms would be difficult to obtain nutrients and oxygen from the bioleaching solution. 3.3. Enumeration of the attached microorganisms on the mineral surface and their function analysis during the bioleaching of chalcopyrite As described previously, the microorganisms attached on the mineral surface were removed by three repeated vortexing with glass beads and separation by centrifugation. The cell density in the supernatant in the 50 mL tube after each separation gradually decreased and was lower than 4 × 106 cells/mL after the 3 rd separation, indicating that the majority of attached cells had detached from the minerals and were recovered from the supernatant. DNA and KDO analysis of the supernatant showed that less than 0.3 mg of DNA and 0.06–0.09 mg of KDO were present in solution originating from one gram of ore residue. Thus the low content of DNA and KDO indicates that the method could separate the attached microorganisms from mineral surface, without breaking the attached cells (Gehrke et al., 1998; Karkhanis et al., 1978). Fig. 5 shows the attached cell amount on the mineral surface and the free cell density in the solution as a function of bioleaching time. It can be seen that the amount of attached cells reached a maximum at the 20th day (1.3 × 1010 cells per gram of ore residue). After that, the amount of attached cell remained stable till the 36th day and then began to decrease. However, the free cell density in the solution increased slowly after a long lag phase and reached the maximum at the 28th day. During the bioleaching of pure chalcopyrite, the microorganisms after being inoculated preferred to attach onto the surface of chalcopyrite (Harneit et al., 2006; Ghauri et al., 2007). At the beginning of attachment, the sessile cells produce EPS to form a special growth environment and multiply continuously to large quantities (Gehrke et al., 1998). Only after the sessile cell amounts
The amount of EPS components / (mg/g)
W. Zeng et al. / Hydrometallurgy 106 (2011) 46–50
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80 70
Protein Sugar Fatty acid Total EPS
60 50 40 30 20 10 0 A
B
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D
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Bioleaching time Fig. 4. EPS components and contents in ore residue at the 1st (A), 4th (B), 10th (C), 20th (D) and 40th (E) day during bioleaching of pure chalcopyrite by a mixed culture of moderately thermophilic microorganisms.
26 24 22 20 18 16 14 12 10 8 6 4 2 0 -2
20 18 Free cell Attached cell
14 12 10 8 6 4 2 0 0
Fig. 3. AFM images of attached microorganisms on the surface of slices of pure chalcopyrite bioleached by moderate thermophiles at 48 °C. A: bioleached for 1 day; B: bioleached for 4 days; C: bioleached for 10 days.
16
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Attached cell amount / (109 cells)
Free cell density / (108 cells/mL)
reached a maximum, could the increase of free cell density begin to accelerate (Fig. 5). This may be due to the increase of the concentration of energy source such as ferrous ion in the bioleaching solution (Fig. 2). From the 28th day, the free cells entered into a stationary phase, which was maintained for about 10 days. After this, the total concentration of sulphate ion increased significantly in the solution and the pH value decreased to a low value which would inhibit the growth of some moderate thermophiles such as Acidithiobacillus caldus. Thus the cell density in the solution began to decrease. It can be seen from Fig. 5 that the growth trend between the attached microorganisms on the mineral surface and the free microorganisms in the bioleaching solution was different. Thus their function during bioleaching of chalcopyrite concentrate may also be different. At the beginning of bioleaching, the cell density in the solution was very low and the copper extraction was mainly due to the contribution of the attached cells. After the 18th day, the free cells multiplied quickly and played a key role in oxidizing soluble sulphur compounds and ferrous ion. This was very important to continuously produce enough ferric ion to dissolve mineral. At the end of bioleaching, the formation of a passivation layer like jarosite on the mineral surface inhibits the flow of microorganisms, nutrients and reaction products to and from the mineral surface, thus the copper extraction rate from the chalcopyrite begins to decrease (Hackl et al. 1995; Zeng et al. 2010; Zhou et al. 2009).
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Bioleaching time / days Fig. 5. The variation of the attached cell amount per gram of ore residue and the free cell density in the solution during bioleaching of pure chalcopyrite with a mixed culture of moderately thermophilic microorganisms at 6% pulp density and initial pH 2.0.
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W. Zeng et al. / Hydrometallurgy 106 (2011) 46–50
4. Conclusions The moderate thermophiles showed good performance during bioleaching of pure chalcopyrite in the stirred tank reactor. The attached microorganisms on the mineral surface play a pivotal role for the increase of copper extraction by producing EPS rapidly after attachment which concentrates ferric ions to oxidize chalcopyrite. Furthermore, the growth curve between the attached microorganisms on the mineral surface and the free microorganisms in the bioleaching solution were different. This difference possibly originates from the different growth sites of the microorganisms. Further work is required to understand the binding mechanism of ferric ion on EPS and to investigate the interface between the biofilm and the mineral surface when jarosite is present. In the next planned experiments, the differences between the free and attached microorganism according to metal ion concentration, organic compound composition and electrochemical behaviour will be investigated during bioleaching of chalcopyrite. Acknowledgements This work was supported by the National Nature Science Foundation of China (No. 50321402 and 50621063), the China National Basic Research Program (No. 2004CB619201 and 2010CB630901), Australia CSIRO Science Leader Grant and the Graduate Degree Thesis Innovation Foundation of Central South University (No. 1960-71131100009). References Beech, W.B., Sunner, J., 2004. Biocorrosion: towards understanding interactions between biofilms and metals. Current Opinion in Biotechnology 15 (3), 181–186. Blumenkr, N., Asboehan, G., 1973. Improved method for assay of hydroxylysine. Analytical Biochemistry 56 (1), 10–15. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 (1–2), 248–254. Brierley, J.A., 1990. Acidophilic thermophilic archaebacteria: potential application for metals recovery. FEMS Microbiology 75, 287–292.
Delatolla, R., Berk, D., Tufenkji, N., 2008. Rapid and reliable quantification of biofilm weight and nitrogen content of biofilm attached to polystyrene beads. Water Research 42 (12), 3082–3088. Gehrke, T., Telegdi, J., Thierry, D., Sand, W., 1998. Importance of extracellular polymeric substances from Thiobacillus ferrooxidans for bioleaching. Applied and Environmental Microbiology 64 (7), 2743–2747. Ghauri, M.A., Okibe, N., Johnson, D.B., 2007. Attachment of acidophilic bacteria to solid surface: the significance of species and strain variations. Hydrometallurgy 85 (2–4), 72–80. Gomez, C., Roman, E., Blazquez, M.L., Ballester, A., 1997. SEM and AES studies of chalcopyrite bioleaching in the presence of catalytic ions. Minerals Engineering 10 (8), 825–835. Hackl, R.P., Dreisinger, D.B., Peters, E., 1995. Passivation of chalcopyrite during oxidative leaching in sulfate media. Hydrometallurgy 39 (1), 25–48. Harneit, K., Göksel, A., Kock, D., Klock, J.H., Gehrke, T., Sand, W., 2006. Adhesion to metal sulfide surfaces by cells of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans. Hydrometallurgy 83 (1–4), 245–254. Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J., Carlo, D.J., 1978. A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharides of gramnegative bacteria. Analytical Biochemistry 85, 595–601. Kinzler, K., Gehrke, T., Telegdi, J., Sand, W., 2003. Bioleaching—a result of interfacial processes caused by extracellular polymeric substances (EPS). Hydrometallurgy 71 (1–2), 83–88. Mousavi, S.M., Jafari, A., Yaghmaei, S., Vossoughi, M., 2006. Bioleaching of low-grade sphalerite using a column reactor. Hydrometallurgy 82 (1–2), 75–82. Pedersen, K.A., Johanna, E., Susanne, H.L., 1996. 16S rRNA gene diversity of attached and unattached bacteria in boreholes along the access tunnel to the Äspö hard rock laboratory, Sweden. FEMS Microbiology Ecology 19 (4), 249–262. Rohwerder, T., Gehrke, T., Kinzler, K., Sand, W., 2003. Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Applied Microbiology and Biotechnology 63, 239–248. Sandstorm, A., Petersson, S., 1997. Bioleaching of a complex sulphide ore with moderate thermophilic and extreme thermophilic microorganism. Hydrometallurgy 46 (1– 2), 181–190. Velghe, N., Claeys, A., 1985. Rapid spectrophotometric determination of nitrate in mineral water with resorcinol. The Analyst 110, 313–314. Wu, C.B., Zeng, W.M., Zhou, H.B., Fu, B., Huang, J.F., Qiu, G.Z., Wang, D.Z., 2007. Bioleaching of chalcopyrite by mixed culture of moderately thermophilic microorganisms. Journal of Central South University of Technology 14, 474–478. Yu, F.P., Pyle, B.H., McFeters, G.A., 1993. A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection. Journal of Microbiological Methods 17 (3), 167–180. Zeng, W.M., Qiu, G.Z., Zhou, H.B., Liu, X.D., Chen, M., Chao, W.L., Zhang, C.G., Peng, J.H., 2010. Characterization of extracellular polymeric substances extracted during the bioleaching of chalcopyrite concentrate. Hydrometallurgy 100 (3–4), 177–180. Zhou, H.B., Zeng, W.M., Yang, Z.F., Xie, Y.J., Qiu, G.Z., 2009. Bioleaching of chalcopyrite concentrate by a moderately thermophilic culture in a stirred tank reactor. Bioresource Technology 100, 515–520.
Contents lists available at ScienceDirect
Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t
Detection and analysis of attached microorganisms on the mineral surface during bioleaching of pure chalcopyrite with moderate thermophiles Weimin Zeng a,b,c, Sunee Tan c, Miao Chen c, Guanzhou Qiu a,b,⁎ a b c
School of Minerals Processing and Bioengineering, Central South University, Changsha, China Key Laboratory of Biometallurgy, Ministry of Education, Changsha, 410083, China CSIRO Process Science and Engineering, Clayton, Victoria, 3168, Australia
a r t i c l e
i n f o
Article history: Received 23 October 2010 Received in revised form 23 November 2010 Accepted 24 November 2010 Available online 1 December 2010 Keywords: Attached microorganisms Chalcopyrite Bioleaching Atomic force microscope Extracellular polymeric substances
a b s t r a c t Moderate thermophiles were used to bioleach pure chalcopyrite and showed good performance of copper extraction in the stirred tank reactor. The attached microorganisms on the mineral surface were detected by atomic force microscope (AFM). The result showed that the attached cells could produce extracellular polymeric substances (EPS) rapidly. However, it was difficult to reduce or eliminate the EPS once it was produced, and thus the large amount of EPS at the later stage of bioleaching would mediate the formation of jarosite and block the continuous copper extraction. Furthermore, the number of attached cells on the mineral surface was enumerated as a function of time and the variation was rather different from that of free cell density in the bioleaching solution. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Chalcopyrite is the most abundant and refractory copper sulphide mineral which is traditionally floated, concentrated and treated by pyrometallurgical methods to obtain copper. Bioleaching of low-grade chalcopyrite ores and tailings is a copper extraction process with low capital and operating cost, and environmental benefits (Brierley 1990; Rohwerder et al., 2003). At present, researchers have paid more attention to bioleach chalcopyrite with moderately thermophilic microorganisms rather than mesophiles as they can greatly improve the leaching reaction kinetics, avoid excessive chalcopyrite passivation and overall improve the total copper extraction (Wu et al., 2007; Zhou et al., 2009). It is reported that the attached microorganisms on the mineral surface played a pivotal role during bioleaching (Beech and Sunner, 2004; Kinzler et al., 2003). The bioleaching microorganisms prefer to attach onto the mineral surface after inoculation and produce extracellular polymeric substances (EPS). EPS could concentrate ferric ions and form a special layer to oxidize chalcopyrite whilst the dissolution of chalcopyrite releases energy sources such as sulphur and ferrous ions for the bioleaching microorganisms. However, the growth trend of attached microorganisms on the mineral surface was not clear. Especially at the later stage of bioleaching, chalcopyrite
⁎ Corresponding author. Key Laboratory of Biometallurgy, Ministry of Education, Changsha, Hunan 410083, China. Tel.: + 86 731 8879212. E-mail address: [email protected] (G. Qiu). 0304-386X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2010.11.014
surface would be covered with a passivation layer to inhibit the flow of microorganisms, nutrients and reaction products to and from the mineral surface (Hackl et al., 1995; Zeng et al., 2010). Then the growth behaviour of attached microorganisms should be different from the free cells in the bioleaching solution. In other research fields, such as soil and waste water treatment, the detection and analysis of attached microorganisms on solid surface had been reported by some authors and several processing methods for extracting attached microorganisms were described. Pedersen et al. (1996) used PCR amplification, cloning and sequencing of 16S rRNA gene methods to investigate the diversity of attached bacteria in soil samples and about 10 species were found, but the amount of attached cells was not enumerated. Yu et al. (1993) adopted a direct viable count way to enumerate the amount of attached microorganisms (Klebsiella pneumonia Kp1) on stainless steel coupons. The attached cells were first scraped from the coupons and moved into the solution, and then the suspension could be observed and counted under the microscope. In the experiments of Delatolla et al. (2008), the nitrifying bacteria microorganism attached on glass beads and produced biofilm. For enumerating the attached cells, the cells were detached by vortexing the beads in a centrifuge tube. Therefore the ways of enumerating the attached microorganisms vary according to the different types of microorganisms, support surfaces and aims of experiments. In this study, bioleaching of pure chalcopyrite was performed by moderate thermophiles in a stirred tank reactor. The attached microorganisms on the mineral surface were detected by atomic force microscope (AFM). In addition, the attached microorganisms
W. Zeng et al. / Hydrometallurgy 106 (2011) 46–50
were removed from the mineral surface and enumerated with a counting chamber under the microscope.
2. Materials and methods 2.1. Microorganisms enrichment Acid Mine Drainage (AMD) samples from several chalcopyrite mines in China were collected. The samples were mixed and then inoculated into the culture medium for enrichment of moderate thermophiles. This medium was modified at 9 K and consisted of the following compounds: (NH4)2SO4 3.0 g/L, Na2SO4 2.1 g/L, MgSO4·7H2O 0.5 g/L, K2HPO4 0.05 g/L, KCl 0.1 g/L, Ca(NO3)2 0.01 g/L. The moderate thermophiles were enriched at 48 °C and initial pH of 2.0 in a stirred tank reactor (see Section 2.3).
2.2. Mineral components The chalcopyrite used in the bioleaching experiments was obtained from Yushui copper sulphide mine in Meizhou, Guangdong, China. The mineralogical composition was 98% chalcopyrite and 2% silicate, which mainly includes CaSiO3 and MgSiO3. The pure chalcopyrite was ground and the diameter of the particles was b75 μm. 2.3. Bioleaching experiments Bioleaching experiments were carried out in a 3 L glass cylindrical reactor with a mechanical stirrer operating at 500 r/min. About 1950 mL medium was added into the reactor; then 50 mL seed culture (no soluble copper) was inoculated to get a cell density of 106 cells/ mL. The reactor was placed in a thermostatic bath to keep the temperature constant at 48 ± 0.2 °C. Sterile air was introduced into the base of the reactor at an approximate rate of 360 mL/min. The experiments were performed at an initial pH 2.0 and the pulp density was 6%. The acid consumption was compensated by addition of 10 mol/L sulphuric acid to keep pH value around 2.0 in the beginning of bioleaching. Distilled water was added to the reactor through a peristaltic pump in order to compensate for evaporation losses. Redox potential, the concentration of Cu2+, Pb2+, Fe2+ and total iron in solution were analysed every two days, while pH value and cell density in the solution were analysed every day. The components of the mineral sample and ore residue were analysed by X-ray diffraction (XRD). Copper, lead, and total iron concentrations in solution were determined by ICP-AES. Ferrous ion concentration in solution was assayed by titration with potassium dichromate. Ferric ion concentration is the concentration of total iron minus the concentration of ferrous ions. The pH value was measured with pH S-3 C acid meter. The redox potential was measured using a platinum electrode with an Ag/AgCl reference electrode. Free cells in solution were observed and counted with counting chamber under an optical microscope; the attached cells on the mineral surface were determined with the method shown in Section 2.5.
2.4. Atomic force microscopy view For observing the attached microorganism on the pure chalcopyrite surface, AFM work was performed. Three polished slices of pure chalcopyrite (A, B and C) were put into the bioleaching solution for 1, 4 and 10 days, respectively. The slices after bioleaching for different days were taken out and used for AFM scanning. A Agilent Technology 5500 Scanning Probe Microscope operating in a tapping mode in air was used to image cells.
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2.5. Collection and enumeration of attached microorganisms from the mineral surface The leaching solution was allowed to settle for 1.5 h and the supernatant decanted. Then the thickened sludge was centrifuged at 2000 g for 4 min at 4 °C and 4 g of the pellets were re-suspended with 10 mL Milli Q water in a 50 mL centrifuge tube. After this, one gram of glass beads with a diameter of 0.2 mm was added into the tube. The tube was pressed forcedly on a vortexer and underwent 8 min of vigorous vortexing. Then about 36 mL Milli Q water was added into the tube and the vortexing continued for 2 min. After this, the 50 mL mixture was centrifuged at 2000 g for 2 min to separate the ore residue and solution. 0.1 mL of the supernatant was used to count the cell number under the optical microscope and the rest was transferred to a 150 mL flask. The ore residue was used again, underwent vigorous vortexing for 10 min and centrifugation for 2 min, followed by cell collection and counting. After this, the remaining ore residue was used for the 3rd time and underwent vigorous vortexing for only 5 min and the cells in the supernatant was counted and collected. After three times of vortexing and separation, a total of about 150 mL solution containing the attached cells was collected. The total amount of the cells was analysed by observation under an optical microscope. In addition, the concentration of 2-keto-3-deoxyoctonate (KDO) and DNA in the solution were measured. The measurement of KDO concentration was described as Karkhanis et al. (1978). The DNA concentration was measured spectrophotometrically using a NanoDrop®ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). KDO is part of the cell membrane of microorganisms and therefore can be used as a marker for membrane compound contamination. With low contents of the DNA and KDO indicates that the attached microorganism was not broken during the vortexing of residue (Karkhanis et al., 1978). 2.6. Extraction of extracellular polymeric substances from the mineral surface The detailed processes for extraction and chemical analysis of extracellular polymeric substances from the mineral surface were described in our previous published paper (Zeng et al., 2010). The extracted EPS were analysed for nitrogen (Velghe and Claeys, 1985), uronic acids (Blumenkr and Asboehan, 1973) and protein (Bradford, 1976) spectrophotometrically. Qualitative and quantitative estimations of fatty acids and sugar monomers were achieved by gas–liquid chromatography. Experimental procedures have been described previously (Gehrke et al., 1998). Lastly, the concentration of total iron and ferrous ion in the EPS solution was analysed by ICP-AES. 3. Results and discussion 3.1. Bioleaching of pure chalcopyrite with moderate thermophiles Bioleaching of pure chalcopyrite by moderate thermophiles was performed at 6% pulp density for 40 days in a stirred tank reactor. Fig. 1 shows the copper concentrations as a function of time. Copper extraction increased slowly from the beginning to the 12th day, when the copper concentration was 2.16 g/L. Thereafter, the copper extraction rate increased rapidly till bioleaching performed to the 36th day and then the copper concentration was 12.2 g/L. After that, the chalcopyrite concentrate dissolution rate became slow. When the bioleaching experiment finished (at the 40th day), the copper concentration was 12.6 g/L and the percentage copper extraction achieved was 62%. Fig. 2 shows the variation of iron concentrations during bioleaching of chalcopyrite as a function of time. Iron in solution at the beginning and middle stage was mainly ferrous ion and the concentration of ferric ion was more than ferrous ion only at the
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3.2. Production of extracellular polymeric substances by attached microorganisms on the mineral
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end stage. After the 26th day, the concentration of ferrous ion began to decrease and that of ferric ion began to increase, which corresponded to an increase of redox potential in the solution over 500 mV. At the 40th day, the concentrations of ferric ion and ferrous ion were 3.12 g/L and 1.35 g/L, respectively. The redox potential value showed an increase from 385 to 634 mV from the beginning to the end of bioleaching process. This increase of redox potential was accompanied with an increase of ferric ion concentration (Fig. 2). Additionally, the oxidation of sulphur compound produced enough sulphuric acid to decrease of the pH value, which is another reason for the increase of redox potential. In the beginning of bioleaching experiment, sulphuric acid was added to the reactor to keep the pH value around 2.0 for acid consumption of chalcopyrite (Mousavi et al., 2006). After that, the pH decreased to a final value of 1.42 since acid was continuously produced during the oxidation of sulphur and the hydrolysis of ferric iron to ferric species such as Fe(OH)2+ and Fe(OH)+ 2 (Sandstorm and Petersson 1997; Gomez et al., 1997). In the first six days, about 7.8 g/L of sulphuric acid was added into the bioleaching system to keep the pH value about 2.0 due to the acid consumption. After this period, the pH value began to decrease and acid addition was stopped, which indicates that the amount of acid-production was more than that of acid consumption.
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iron concentraction / (g/L)
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Bioleaching time / days Fig. 2. The variation of the total iron, ferrous and ferric ions concentrations in solution as a function of time during bioleaching of pure chalcopyrite by a mixed culture of moderately thermophilic microorganisms at 6% pulp density and initial pH 2.0.
The attached microorganisms on the polished slices of pure chalcopyrite bioleached after different days were detected by AFM. It can be seen from Fig. 3 that on the first day of bioleaching, microorganisms began to attach onto the mineral surface and produced a small amount of EPS. At the 4th day of bioleaching, the amount of EPS covering on the cells increased significantly and EPS tended to spread onto the mineral surface. At the 10th day of bioleaching, the attached cell and mineral surface were both fully covered by EPS. It is reported (Beech and Sunner, 2004; Kinzler et al., 2003) that the production of EPS benefits the concentration of ferric ions and thus oxidizes the chalcopyrite. It is believed that uronic acid and other unidentified compounds in the EPS complexes with ferric ion. Therefore, the rapid production of EPS on the mineral surface can improve the copper extraction. After the 10th day, it was hard to get a good AFM view of attached cells (data not shown) due to the production of a large amount of EPS and the formation of passivation substances on the mineral surface. The sulphur content in the ore residue was about 2.4% at this time. The EPS on the mineral surface was extracted and characterized from the mineral surface as a function of bioleaching time (Fig. 4). The collected EPS was primarily composed of sugars and fatty acids and the content of protein was relatively low. The variation of total EPS and the EPS fraction contents per gram of ore residue as a function of bioleaching time are shown in Fig. 4. It was found that the amount of total EPS increased rapidly at the beginning of bioleaching and achieved a maximum of 75.6 mg per gram of ore residue at the 10th day. Once the EPS were produced, it was difficult to remove or degrade them from the bioleaching system. As a result, a large amount of EPS would remain on the mineral surface and possibly enhance the passivation layer at the end of bioleaching (Zeng et al., 2010). In this case, the copper extraction rate decreased largely and the attached microorganisms would be difficult to obtain nutrients and oxygen from the bioleaching solution. 3.3. Enumeration of the attached microorganisms on the mineral surface and their function analysis during the bioleaching of chalcopyrite As described previously, the microorganisms attached on the mineral surface were removed by three repeated vortexing with glass beads and separation by centrifugation. The cell density in the supernatant in the 50 mL tube after each separation gradually decreased and was lower than 4 × 106 cells/mL after the 3 rd separation, indicating that the majority of attached cells had detached from the minerals and were recovered from the supernatant. DNA and KDO analysis of the supernatant showed that less than 0.3 mg of DNA and 0.06–0.09 mg of KDO were present in solution originating from one gram of ore residue. Thus the low content of DNA and KDO indicates that the method could separate the attached microorganisms from mineral surface, without breaking the attached cells (Gehrke et al., 1998; Karkhanis et al., 1978). Fig. 5 shows the attached cell amount on the mineral surface and the free cell density in the solution as a function of bioleaching time. It can be seen that the amount of attached cells reached a maximum at the 20th day (1.3 × 1010 cells per gram of ore residue). After that, the amount of attached cell remained stable till the 36th day and then began to decrease. However, the free cell density in the solution increased slowly after a long lag phase and reached the maximum at the 28th day. During the bioleaching of pure chalcopyrite, the microorganisms after being inoculated preferred to attach onto the surface of chalcopyrite (Harneit et al., 2006; Ghauri et al., 2007). At the beginning of attachment, the sessile cells produce EPS to form a special growth environment and multiply continuously to large quantities (Gehrke et al., 1998). Only after the sessile cell amounts
The amount of EPS components / (mg/g)
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26 24 22 20 18 16 14 12 10 8 6 4 2 0 -2
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Fig. 3. AFM images of attached microorganisms on the surface of slices of pure chalcopyrite bioleached by moderate thermophiles at 48 °C. A: bioleached for 1 day; B: bioleached for 4 days; C: bioleached for 10 days.
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reached a maximum, could the increase of free cell density begin to accelerate (Fig. 5). This may be due to the increase of the concentration of energy source such as ferrous ion in the bioleaching solution (Fig. 2). From the 28th day, the free cells entered into a stationary phase, which was maintained for about 10 days. After this, the total concentration of sulphate ion increased significantly in the solution and the pH value decreased to a low value which would inhibit the growth of some moderate thermophiles such as Acidithiobacillus caldus. Thus the cell density in the solution began to decrease. It can be seen from Fig. 5 that the growth trend between the attached microorganisms on the mineral surface and the free microorganisms in the bioleaching solution was different. Thus their function during bioleaching of chalcopyrite concentrate may also be different. At the beginning of bioleaching, the cell density in the solution was very low and the copper extraction was mainly due to the contribution of the attached cells. After the 18th day, the free cells multiplied quickly and played a key role in oxidizing soluble sulphur compounds and ferrous ion. This was very important to continuously produce enough ferric ion to dissolve mineral. At the end of bioleaching, the formation of a passivation layer like jarosite on the mineral surface inhibits the flow of microorganisms, nutrients and reaction products to and from the mineral surface, thus the copper extraction rate from the chalcopyrite begins to decrease (Hackl et al. 1995; Zeng et al. 2010; Zhou et al. 2009).
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Bioleaching time / days Fig. 5. The variation of the attached cell amount per gram of ore residue and the free cell density in the solution during bioleaching of pure chalcopyrite with a mixed culture of moderately thermophilic microorganisms at 6% pulp density and initial pH 2.0.
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4. Conclusions The moderate thermophiles showed good performance during bioleaching of pure chalcopyrite in the stirred tank reactor. The attached microorganisms on the mineral surface play a pivotal role for the increase of copper extraction by producing EPS rapidly after attachment which concentrates ferric ions to oxidize chalcopyrite. Furthermore, the growth curve between the attached microorganisms on the mineral surface and the free microorganisms in the bioleaching solution were different. This difference possibly originates from the different growth sites of the microorganisms. Further work is required to understand the binding mechanism of ferric ion on EPS and to investigate the interface between the biofilm and the mineral surface when jarosite is present. In the next planned experiments, the differences between the free and attached microorganism according to metal ion concentration, organic compound composition and electrochemical behaviour will be investigated during bioleaching of chalcopyrite. Acknowledgements This work was supported by the National Nature Science Foundation of China (No. 50321402 and 50621063), the China National Basic Research Program (No. 2004CB619201 and 2010CB630901), Australia CSIRO Science Leader Grant and the Graduate Degree Thesis Innovation Foundation of Central South University (No. 1960-71131100009). References Beech, W.B., Sunner, J., 2004. Biocorrosion: towards understanding interactions between biofilms and metals. Current Opinion in Biotechnology 15 (3), 181–186. Blumenkr, N., Asboehan, G., 1973. Improved method for assay of hydroxylysine. Analytical Biochemistry 56 (1), 10–15. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 (1–2), 248–254. Brierley, J.A., 1990. Acidophilic thermophilic archaebacteria: potential application for metals recovery. FEMS Microbiology 75, 287–292.
Delatolla, R., Berk, D., Tufenkji, N., 2008. Rapid and reliable quantification of biofilm weight and nitrogen content of biofilm attached to polystyrene beads. Water Research 42 (12), 3082–3088. Gehrke, T., Telegdi, J., Thierry, D., Sand, W., 1998. Importance of extracellular polymeric substances from Thiobacillus ferrooxidans for bioleaching. Applied and Environmental Microbiology 64 (7), 2743–2747. Ghauri, M.A., Okibe, N., Johnson, D.B., 2007. Attachment of acidophilic bacteria to solid surface: the significance of species and strain variations. Hydrometallurgy 85 (2–4), 72–80. Gomez, C., Roman, E., Blazquez, M.L., Ballester, A., 1997. SEM and AES studies of chalcopyrite bioleaching in the presence of catalytic ions. Minerals Engineering 10 (8), 825–835. Hackl, R.P., Dreisinger, D.B., Peters, E., 1995. Passivation of chalcopyrite during oxidative leaching in sulfate media. Hydrometallurgy 39 (1), 25–48. Harneit, K., Göksel, A., Kock, D., Klock, J.H., Gehrke, T., Sand, W., 2006. Adhesion to metal sulfide surfaces by cells of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans. Hydrometallurgy 83 (1–4), 245–254. Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J., Carlo, D.J., 1978. A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharides of gramnegative bacteria. Analytical Biochemistry 85, 595–601. Kinzler, K., Gehrke, T., Telegdi, J., Sand, W., 2003. Bioleaching—a result of interfacial processes caused by extracellular polymeric substances (EPS). Hydrometallurgy 71 (1–2), 83–88. Mousavi, S.M., Jafari, A., Yaghmaei, S., Vossoughi, M., 2006. Bioleaching of low-grade sphalerite using a column reactor. Hydrometallurgy 82 (1–2), 75–82. Pedersen, K.A., Johanna, E., Susanne, H.L., 1996. 16S rRNA gene diversity of attached and unattached bacteria in boreholes along the access tunnel to the Äspö hard rock laboratory, Sweden. FEMS Microbiology Ecology 19 (4), 249–262. Rohwerder, T., Gehrke, T., Kinzler, K., Sand, W., 2003. Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Applied Microbiology and Biotechnology 63, 239–248. Sandstorm, A., Petersson, S., 1997. Bioleaching of a complex sulphide ore with moderate thermophilic and extreme thermophilic microorganism. Hydrometallurgy 46 (1– 2), 181–190. Velghe, N., Claeys, A., 1985. Rapid spectrophotometric determination of nitrate in mineral water with resorcinol. The Analyst 110, 313–314. Wu, C.B., Zeng, W.M., Zhou, H.B., Fu, B., Huang, J.F., Qiu, G.Z., Wang, D.Z., 2007. Bioleaching of chalcopyrite by mixed culture of moderately thermophilic microorganisms. Journal of Central South University of Technology 14, 474–478. Yu, F.P., Pyle, B.H., McFeters, G.A., 1993. A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection. Journal of Microbiological Methods 17 (3), 167–180. Zeng, W.M., Qiu, G.Z., Zhou, H.B., Liu, X.D., Chen, M., Chao, W.L., Zhang, C.G., Peng, J.H., 2010. Characterization of extracellular polymeric substances extracted during the bioleaching of chalcopyrite concentrate. Hydrometallurgy 100 (3–4), 177–180. Zhou, H.B., Zeng, W.M., Yang, Z.F., Xie, Y.J., Qiu, G.Z., 2009. Bioleaching of chalcopyrite concentrate by a moderately thermophilic culture in a stirred tank reactor. Bioresource Technology 100, 515–520.