CC BY
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BIOTECHNOLOGIA ACTA, V. 8, No 5, 2015
UDC 579:662.7
doi: 10.15407/biotech8.05.086
SPECIFIC FEATURES OF NATIVE CHEMOLITHOTROPHIC MICROBIOTA WASTES PRODUCED BY THE BIOENERGY INDUSTRY
I. A. Blayda
T. V. Vasilyeva Odesa Mechnikov National University, Ukraine
L. I. Slyusarenko V. F. Khytrych
E-mail: iblayda@ukr.net
Received 08.07.2015
The study’s aims were to isolate and establish pure cultures of prevailing microorganisms from the aboriginal consortium in coal fly ash, describe their physiology, biochemistry and practically-useful properties, and compare the efficiency of bioleaching metals from fly ash using pure cultures and the consortium. Through enrichment cultures on standard media we isolated pure cultures of the microorganisms which were then preliminarily identified using standard techniques. This allowed us to isolate from fly coal ash pure cultures of three prevailing strains of mesophilic and moderately thermophilic coal fly ash acidophilic chemolithotrophic bacteriae, belonging to Acidithiobacillus, such as Acidithiobacillus ferrooxidans, and Sulfobacillus. The strains exhibited high oxidative activity in leaching the rare metals Gallium and Germanium, as well as some heavy metals, from fly ash substrate. A comparison of oxidative activity of the isolated strains and the aboriginal consortium under mesophilic conditions led to the conclusion about advantage of consortium, because it had arisen from syntrophy of microbes in the community. This should be taken into account at the developing of bacterial preparations that are optimal for the technogenic substrate.
Key words: fly ash, aboriginal microbial consortia, acidophilic chemolithotrophic bacteriae, leaching activity, Germanium.
Exhaustion of the world’s worth of mineral resources and growing volumes of technogenic wastes with complex, multicomponent contents demand alternative technological solutions for recycling. Energy industry wastes, such as fly ash and slag from burning coals, are at the same time dangerous to environment and industrially significant, since they contain rare metals. The most profitable and ecologically sound method of their recycling is bio hydrometallurgical. It uses oxidative activity of microbes of the aboriginal consortium shaped by the wastes’ deposition and storage history [1-3]. Microbial biotechnology should be based on research encompassing biological and physicochemical properties of the initial hard substrate and its aboriginal microbial consortium, with subsequent isolation, identification and selection of the most promising highly active strains to create an efficient bacterial preparation. We established that during the process of ash formation, impoundment and storage under the influence of certain technogenic and
native factors, a highly efficient and specific consortium is formed, mostly by heterotrophic and acidophilic chemolithotrophic bacteriae. The highest leaching activity was shown when rare metals from initial technogenic substrates were leached by species of Acidithiobacillus and Sulfobacillus [4-6].
The study’s goal was to isolate the prevailing microorganisms from the coal fly ash (FAAC) of industrial wastes into pure cultures, determine their properties, and to compare the efficiency of bioleaching metals from wastes using pure isolates and the aboriginal consortium. It can be considered as a continuation of our earlier work concerning evaluating quantitative and qualitative structure of microbiocenoses of energy industry wastes [7].
Materials and Methods
The object of our research was fly ash obtained by burning a mixture of native fossil coals on DTEK thermal power station in Ladyzhyn (Vinnytsia region, Ukraine). It
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contained (%): Fe — 5.93; Al — 3.89; Si — 12.10; Ti — 4.16; Ca — 0.20; Cu — 6.82G0-3; Zn — 3.27 .10-2; Mn — 5.73 .10-2; Pb — 1.09 .10-2; Ni — 1.77 G0-2; Cd — 5.3P10-4; Sn — 2.07G0-2; Cr — 2.18G0-2; V — 2.15G0-2; Co — 3.05G0-2; Sr — 6.56G0-2; Ba — 6.34G-2; Zr — 2.37.10-2; Rb — 1.16.10-2; Nb — 1.90G0-3; La — 4.20.10-3; Ce — 7.40.10-3; Ga — 1.02.10-3; Ge — 2.80.10-3; S — 1.24; C (underburning) — 9.98.
To obtain pure cultures of acidophilic chemolithotrophic bacteriae we used enrichment cultures on standard media (Table 1), based on literature as well as our own research which showed that in mineral substrates of both geogenic and technogenic origin, there are mesophilic and thermophilic chemolithotrophic bacteriae and Sulfobacillus sp. [3, 8-10]. As energy source, we amended the mineral background with elemental sulfur, its inorganic (sodium thiosulfate Na2S2O3) and organic (thio urea CS(NH2)2) compounds, or bivalent iron (FeSO4.7H2O) [7, 9, 11, 12]. To a 50.0 cm3 Erlenmeyer flask we put 2.0 g of fly ash and 20.0 cm3 of a medium, ratio 1:10 (solid:liquid). The enrichment cultures of mesophilic acidophilic chemolithotrophic bacteriae were kept at 35.0±2.0 ° С, moderately thermophilic — at 55.0±2.0 °С and Sulfobacillus sp. — at 45.0±2.0 °С for 7 days. The microbes’ development was evaluated by the change in the medium’s pH, appearance of slight suspension and a film on the culture’s surface.
The liquid, enriched with bacteriae, was sown on agarized medium of the similar composition, containing sulfur, thiosulfate, thiourea or bivalent iron. The enrichment cultures of Sulfobacilla sp. before resowing were kept for ten minutes at 80.0±2.0 °С. The process allowed to purify initial isolates and to obtain microscopically and colonially homogeneous cultures. Pure cultures on the surface of selective agarized media established colonies which could be readily analyzed. Cultures were concluded to be free on the basis of staining and microscopy. Microscopically and morphologically pure cultures were stained after Gram using standard dyes (solutions of gentian violet, Lugol’s iodine, and fuchsin), and also determined the range and optimal values of temperature and pH, ability to autotrophic and mixotrophic growth, oxidation of different compounds of sulfur and bivalent iron, reaction to organic matters, and ability to bioleach metals from raw mineral materials.
For the measure of biogeochemical activity of isolated strains and FAAC we took the concentration of metals which went into the culture medium from the solid phase. The degree of metal extraction calculated as the ratio (%) of the amount of metal which entered the solution as a result of the medium’s contact with the substrate in the presence of microorganisms, to the amount of the metal in the initial solid substrate (100% stands for all metal being dissolved). As the control, it
Table 1. Medium composition for acidophilic chemolithotrophic bacteriae
Minerals 3 Concentration, g/dm
9K for Acidithiobacillus spp. Modified 9K* for Sulfobacillus spp.
NH4(SO4)2 3.0 0.45
KCl 0.1 0.05
K2HPO4 0.5 -
KH2PO4 - 0.05
MgSO4 0.5 0.5
Ca(NO3)2 0.01 0.014
Na2SO4 - 0.15
Yeast extract - 0.20
Energy source
Na2S2O3 5.0 -
SO 2.0 -
CS(NH2)2 4.8 -
FeSO4-7H2O 44.5 30.0
Note. Here and hereafter 9K* stands for modified 9K medium for Sulfobacillus spp.
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was used the data obtained in sterile leaching solutions of the media on autoclaved fly ash.
Morphology of cells and the colonies they form were studied with light microscopy (Primo Star PC, Germany) and electron microscopy (PЕМ100-01, Ukraine). Biomass growth was determined on a spectrophotometer DR 3900 (Germany) at 540 nm wavelength. The concentration of thiosulfate and the presence of intermediate products of its oxidation were measured by a standard iodometry technique [13]. The concentration of metals in solutions was determined by atomic absorption spectroscopy (AAC-1, Germany and C-115PC Selmi, Ukraine) [14]. The significance of the results was evaluated using Student’s criterion with P < 0.05.
Results and Discussion
As one culture can grow on media differing in both contents and concentrations, primary (initial) isolation of microorganisms using various media can result only in groups of bacteriae, not in pure species [10-12]. In our experiments the development of enrichment culture of mesophilic, moderately thermophilic thionic bacteriae and sulfobacteriae was accompanied by pH increasing (from 1.7 in
the beginning to 3.0-4.5) and a change in the culture medium’s appearance.
As a result, we isolated from fly ash into pure culture 25 strains of acidophilic chemolithotrophic thionic bacteriae. Three of them were selected for further work, isolated on culture media 9K and 9K* with FeSO47H2O as an energy source. They were numbered according to the conditions of isolation: mesophilic — MesPhLad27, moderately thermophilic — MThPhLad25, sulfobacteriae — SBLad29. The properties of the strains are listed in Table 2, their pictures are given at Fig. 1 and 2.
The properties of strains MesPhLad27 and MThPhLad25 revealed in them representatives of Acidithiobacillus, while their ability to oxidize bivalent iron, as long as other energy sources, allowed us to tentatively identify them as Acidithiobacillus ferrooxidans. The moderately thermophilic strain SBLad29, isolated and studied on 9К* medium at 45.0±2.0 °С, was similarly classified as Sulfo-bacillus [15]. A definite conclusion about their taxonomy would require a molecular-genetical study.
Next, we compared the ability of the isolated strains of acidophilic chemolitho-trophic bacteriae and the aboriginal consortium
Fig. 1. Stained microscopic preparations of strains: MesPhLad27 (a); MThPhLad25 (b); SBLad29 (c). xi 000
Fig. 2. Electron microphotographs: MesPhLad27 (а); MThPhLad25 (b); SBLad29 (c). x11 000
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Table 2. Morphology of cells and cultures of bacterial strains isolated from fly ash
Properties Isolated strains
MesPhLad27 MThPhLad25 SBLad29
Morphology, shape of cells Small thin Small thin Small coccobacilli
Gram stainin Gram-negative Gram-negative Gram-negative
Sporulation Don’t sporulate Don’t sporulate Don’t sporulate
Morphology, shape of colonies Tear-shaped, rounded, convex, grainy, bright-yellow Tear-shaped, rounded, convex, grainy, bright-yellow Tear-shaped, rounded, convex, grainy, bright-yellow
РН value Range 2.0-5.0 2.0-5.0 2.0-5.0
Optimal < 2.0 < 2.0 3.0
Temperature, °С Range 4.0-37.0 10.0-65.0 10.0-60.0
Optimal value 35.0±2.0 55.0±2.0 45.0±2.0
Energy source SO + + +
Na2S2O3 + + +
CS(NH2)2 + + +
FeSO4-7H2O + + +
Degree of oxidation of Na2S2O3,% 47.0 86.0 88.0
Growth under mixotrophic conditions
In presence of sugars (0.02%) Glucose + + +
Sucrose + + +
Molasses + + +
In presence of yeast extract (0.02%) + + +
Growth on meat peptone agar - - -
Extraction of metals from fly ash + + +
to bioleach metals. The results are shown on Fig. 3. The control for metal extraction by strains MТhPhLad25 and МesPhLad27 was sterile medium 9К with bivalent iron as energy substrate at 55.0±2.0 °С (Control 1) and 35.0±2.0 °С (Control 2), respectively, for SBLad29 — sterile 9К* medium with bivalent iron as energy source at 45.0±2.0 °С (Control 3).
The data suggest a relatively high activity of the isolated strains towards extraction of Gallium (67.0-88.2% of extraction), Germanium (75.0-98.8%), Cadmium (53.5-72.1%), copper (87.7-98.8%), nickel (57.0-67.7%), and to a lesser degree — zinc, lead and manganese. Strain MesPhLad27’s bioleaching abilities towards the metals were the least among the researched bacteriae.
Table 3 contains data on metal extraction from the same substrate of fly ash by the FAAC.
The results suggest that as an association, the mesophilic consortium of acidophilic chemolithotrophic bacteria is more efficient than just the isolated strain MesPhLad27.
This might be a result of common for microorganisms syntrophic relationships, e.g., the substrates might be destroyed by certain bacteriae, without which other bacteriae’s action upon them is impossible or substantially slower. Thus, the authors of [8] suppose that Acidithiobacillus thiooxidans, which is often present together with Acidithiobacillus ferrooxidans, due to a more rapid oxidation of sulfur under a non-direct mechanism of bioleaching create favorable conditions for the growth of iron-oxidizing bacteriae. Therefore, the cycle of substrate oxidation runs significantly faster and more efficient: MeS + Fe2(SO4)3 ^ MeSO4 + 2FeSO4 + S0 2S0 + 3O2 + 2H2O ^ (bacteriae) ^ H2SO4.
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Fig. 3. Degree of extraction (%) of metals from fly ash by strains, isolated from aboriginal association
* — P < 0.05 compared to the control
Table 3. Degree of metal extraction from fly ash by the aboriginal consortium of chemolithotrophic bacteriae (%)
Metals Mesophilic consortium of acidophilic chemolithotrophic bacteriae Moderately thermophilic consorrtium of acidophilic chemolithotrophic bacteriae
9К medium 9К medium 9К* medium
Ge 99.76 99.98 99.82
Ga 94.92 82.75 95.75
Ni 76.64 84.94 87.63
Cd 99.99 69.95 92.35
Cu 89.54 69.37 16.54
Zn 20.22 20.82 5.92
Mn 37.75 47.85 98.52
Pb 35.67 19.34 22.46
When we compared the bioleaching activity of moderately thermophilic consortium of acidophilic chemolithotrophic bacteriae and of the isolated strains of MThPhLad25 and SBLad29 we didn’t observe a general tendency in favor of syntrophic relationships of microorganisms in association.
On the whole, our work on pure cultures of microorganisms from FAAC allowed us to isolate three prevailing strains of mesophilic and moderately thermophilic acidophilic chemolithotrophic bacteriae which we identified as belonging to the genus Acidithiobacillus, in particular
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Acidithiobacillus ferrooxidans, and Sulfobacillus. All isolated strains were characterized by high bioleaching activity towards rare metals Gallium and Germanium, as well as some heavy metals from the studied technogenic substrate. A comparison of activity of the isolated strains and the FAAC under mesophilic conditions provides support for the consortium being a result of syntrophy between microbes in the community.
REFERENCES
1. Karavayko G. I., Kuznetsov S. I., Golomzik Ye. I. The Role of Microorganisms in the leaching of metals from ores. Moskva: Nauka. 1972, 248 p. (In Russian).
2. Brierley J. A. Expanding role of microbiology in metallurgical processes. Mining Engin. 2000, 52 (11), 49-53.
3. Vasil’eva T. V., Blayda I. A., Ivanitsa V. A. The
main groups of microorganisms involved in the biohydrometallurgical process. Problemy ekolohichnoy biotekhnologii. 2013, 1. Available at: http: / /jrnl.nau.edu.ua/index.php /
ecobiotech/article/view/4678. (In Russian).
4. Ivanov M. V., Karavayko G. I. Geological microbiology. Mikrobiologiya. 2004, 5 (73), 581-597. (In Russian).
5. Norris P. R., Burton N. P., Foulis N. A. M. Acidophiles in bioreactor mineral processing. Extremophiles. 2000, V. 4, P. 71-76.
6. Blayda I. A. Extraction of valuable metals from industrial waste biotechnological methods (Review). Energotekhnologii i resursosberezhenie. 2010, V. 6, P. 39-45. (In Russian).
7. Blayda I. A., Vasileva T. V., Slyusarenko L. I., Barba I. N., Ivanitsa V. A. Composition and leaching activity of energy industrial waste microbiocenosis. Problemy ekologichnoy biotekhnologii. 2013, 1. Available at: http:// jrnl.nau.edu.ua/index.php/ ecobiotech/ article/view/4592. (In Russian).
8. Kuzyakina T. I., Haynasova T. S., Levenets O. O. Biotechnology extraction of metals from sulfide ores. Vestnik nauk o Zemle. 2008, 60 (12), 76-85. (In Russian).
The results can be used to develop a highly efficient, stable, optimal for a given substrate type bacterial formula, able to leach both rare and heavy toxic metals. However, this would need a study of the properties of isolated strains’ growth in autotrophic and mixotrophic conditions. It is also necessary to carry out optimization of medium’s composition for the maximal growth rates.
9. Blayda I. A., Vasileva T. V., Slyusarenko L. I., Khitrich V. F., Ivanitsa V. A. Extraction of rare and nonferrous metals by microbial communities of the ash from burning Pavlograd s coal. Microbiology & Biotechnology. 2012, V. 3, P. 91-101. (In Russian).
10. Kondrateva T. F., Pivovarova T. A., Tsaplina I. A. A variety of community chemolithotrophic acidophilus microorganisms in natural and man-made ecosystems. Mikrobiologiya. 2012, 1 (81), 3-27. (In Russian).
11. Karavayko G. I. Practical Guide to biogeotechnology metals. Moskva: AN SSSR. 1989, 371 p. (In Russian).
12. Methods for General Bacteriology. V. 2. Moskva: Mir. 1984, 265 p. (In Russian).
13. Chernyak S. M., Kolobova T. P., Pershina I. V. Methods of hydro-chemical analysis of objects of the marine environment. In: Methodical bases of an integrated environmental monitoring of ocean. Moskva: Gidrometeoizdat. 1988, P. 23-25. (In Russian).
14. Khavezov I., Tsalev D. Atomic absorption analysis. Leningrad: Khimiya. 1983, 144 p. (In Russian).
15. Kelly D. P., Wood A. P. Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Hallothiobacillus gen. nov. and Thermithiobacillus gen. nov. Int. J. Syst. Evolution. Microbiol. 2000, V. 50, P. 512-516.
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ОСОБЛИВОСТІ
АБОРИГЕННОГО УГРУПОВАННЯ ХЕМОЛІТОТРОФНИХ БАКТЕРІЙ ІЗ ВІДХОДІВ БІОЕНЕРГЕТИКИ
І. А. Блайда Т. В. Васильєва Л. І. Слюсаренко В. Ф. Хитрич
Одеський національний університет імені І. І. Мечникова, Україна
Е-mail: iblayda@ukr.net
Метою роботи було виділення домінуючих чистих культур мікроорганізмів з аборигенного угруповання золи-виносу від спалювання вугілля, встановлення їхніх властивостей, порівняння ефективності біовилуговуван-ня металів із золи-виносу з використанням чистих культур та аборигенного угруповання. Методом накопичувальних культур із застосуванням стандартних живильних середовищ виділено чисті культури мікроорганізмів, а стандартними мікробіологічними методами встановлено їх передбачувану таксономічну належність. У результаті з аборигенного угруповання мікроорганізмів золи-виносу в чисті культури виділено три домінуючих штами мезофільних і помірно термофільних ацидофільних хемолітотроф-них бактерій і встановлено їх належність до представників роду Acidithiobacillus, зокрема Acidithiobacillus ferrooxidans, а також Sulfobacillus. Ці штами відзначалися високою окиснювальною активністю стосовно вилучення рідкісних металів галію і германію, а також деяких важких металів із субстрату золи-виносу. Порівняння окиснювальної активності виділених штамів і аборигенного угруповання в мезофільних умовах свідчить на користь консорціуму як результату син-трофних відносин мікроорганізмів в угрупованні. Це слід враховувати при створенні оптимального для даного техногенного субстрату ефективного бактеріального препарату.
Ключові слова: зола-винос, аборигенне угруповання, ацидофільні хемолітотрофні бактерії, активність вилуговування, германій.
ОСОБЕННОСТИ
АБОРИГЕННОГО СООБЩЕСТВА ХЕМОЛИТОТРОФНЫХ БАКТЕРИЙ ИЗ ОТХОДОВ БИОЭНЕРГЕТИКИ
И. А. Блайда Т. В. Васильева Л. И. Слюсаренко В. Ф. Хитрич
Одесский национальный университет имени И. И. Мечникова, Украина
Е-mail: iblayda@ukr.net
Целью работы было выделение доминирующих чистых культур микроорганизмов из аборигенного сообщества золы-уноса от сжигания угля, установление их свойств, сравнение эффективности биовыщелачивания металлов из золы-уноса при использовании чистых культур и аборигенного сообщества. Методом накопительных культур с использованием стандартных питательных сред были выделены чистые культуры микроорганизмов, а стандартными микробиологическими методами определена их предполагаемая таксономическая принадлежность. В результате из аборигенного сообщества микроорганизмов золы-уноса в чистые культуры выделены три доминирующих штамма мезофильных и умеренно термофильных ацидофильных хемолитотрофных бактерий и установлена их принадлежность к представителям рода Acidithiobacillus, в частности Acidithiobacillus ferrooxidans, а также Sulfobacillus. Эти штаммы отличались высокой окислительной активностью по отношению к извлечению редких металлов галлия и германия, а также некоторых тяжелых металлов из субстрата золы-уноса. Сравнение окислительной активности выделенных штаммов и аборигенного сообщества в мезофильных условиях свидетельствует в пользу консорциума как результата синтрофных отношений микроорганизмов в сообществе. Это следует учитывать при создании оптимального для данного техногенного субстрата эффективного бактериального препарата.
Ключевые слова: зола-унос, аборигенное сообщество, ацидофильные хемолитотрофные бактерии, активность выщелачивания, германий.
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UDK 579.695 doi: 10.15407/biotech8.05.093
COPPER RESISTANT STRAIN Candida tropicalis ROMCU5 INTERACTION WITH SOLUBLE AND INSOLUBLE COPPER COMPOUNDS
Ie. P. Prekrasna Zabolotny Institute of Microbiology and Virology
O. B. Tashyrev of the National Academy of Sciences of Ukraine, Kyiv
E-mail: preckrasna@gmail.com
Received 25.06.2015
The focus of the study was interaction of Candida tropicalis RomCu5 isolated from highland Ecuador ecosystem with soluble and insoluble copper compounds.
Strain C. tropicalis RomCu5 was cultured in a liquid medium of Hiss in the presence of soluble (copper citrate and CuCl2) and insoluble (CuO and CuCO3) copper compounds. The biomass growth was determined by change in optical density of culture liquid, composition of the gas phase was measured on gas chromatograph, redox potential and pH of the culture fluid was defined potentiometrically. The concentration of soluble copper compounds was determined colorimetrically.
Maximal permissible concentration of Cu2+ for C. tropicalis RomCu5 was 30 000 ppm of Cu2+ in form of copper citrate and 500 ppm of Cu2+ in form of CuCl2. C. tropicalis was metabolically active at super high concentrations of Cu2+, despite the inhibitory effect of Cu2+. C. tropicalis immobilized Cu2+ in the form of copper citrate and CuCl2 by its accumulation in the biomass. Due to medium acidification C. tropicalis dissolved CuO and CuCO3. High resistance of C. tropicalis to Cu2+ and ability to interact with soluble and insoluble copper compounds makes it biotechnologically perspective.
Key words: yeast, Candida tropicalis, copper, resistance to copper, inhibition of metabolism, copper immobilization, copper mobilization.
Nowadays anthropogenic pressure on the environment dramatically increases. Various industries produce and discharge wastes into the environment, such as mining, energy and fuel production, electroplating, electrolysis, leatherworking, photography, etc. One of the main components of the various industrial wastes is heavy metals, such as copper, zinc, cobalt, mercury, chromate, etc. So, the problem of environmental metal pollution drastically becomes more acute.
Microorganisms are plentiful in nature and play vital roles in the geochemical cycling of metals by protonation, chelation, redox and chemical transformation, metal accumulation [1]. Mechanisms of microbial interaction with metals are being exploited in various environmental biotechnologies. Microbial biotechnologies are used for both removing of toxic metals from the industrial wastewater and recovery of heavy metals from low grade ores, dumps, soils, sediments, dumps and industrial wastes [2-5].
Microbial technologies of metal removing or bioleaching have advantages over physical and chemical technologies. For example, chemical precipitation and electrochemical treatment are ineffective and produce large quantity of sludge required to treat with great difficulty. Ion exchange, membrane technologies and activated carbon adsorption process are extremely expensive [2]. Contrary microbial biotechnologies have low operating cost, minimal use of chemicals [2]. So, microbial biotechnologies of both metal removing and bioleaching can be assumed as environment-friendly. Accordingly, search for microorganisms promising for metal biotechnologies is actual area of investigation.
Resistance to toxic metals and ability to interact with toxic metals are the main criteria for microorganisms perspective for metal biotechnologies. In our recent researches high resistant to copper strain Candida tropicalis RomCu5 was isolated from highland
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