CC BY
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BIOTECHNOLOGIA ACTA, V. 8, No 6, 2015
UDC 612.015:616.153.96:616.894
doi: 10.15407/biotech8.06.048
EFFECT OF CURCUMIN LIPOSOMAL FORM ON ANGIOTENSIN CONVERTING ACTIVITY, CYTOKINES AND COGNITIVE CHARACTERISTICS OF THE RATS WITH ALZHEIMER’S DISEASE MODEL
V. V. Sokolik1 1State Enterprise “Institute for Neurology, Psychiatry
S. М. Shulga2 and Narcology of the National Academy of Medical Sciences
of Ukraine”, Kharkiv
2State Enterprise “Institute for Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine”, Kyiv
E-mail: [email protected]
Recieved 12.10.2015
The purpose of the study was the investigation of curcumin liposome form effect on angiotensinconverting enzyme activity, cytokines and mnestic features of rats with experimental model of Alzheimer’s disease. In the animals with intrahippocampal injection of AP42_Human, nasal therapy with curcumin liposome form was used. Cytokine concentration and angiotensin converting enzyme activity in brain regions (cerebral cortex and hippocampus) and in blood serum as well as indicators of conditioned avoidance response were registered. It was found that as a result of curcumin therapy the rats with Alzheimer’s disease had suppressed cytokine and angiotensin converting enzyme activities and recovered mnestic indices. Nasal therapy with curcumin liposome form gave reduction of angiotensin-converting enzyme activity and anti-cytokine effect in the target regions of the brain (cerebral cortex and hippocampus), which helped the rats mnestic features and memory recovery.
Key words: curcumin, liposomes, P-amyloid peptide, cytokines, angiotensin converting enzyme.
The hitherto used approaches to treatment of dementia and amyloidosis in the case of Alzheimer’s disease (AD) concentrated on suppression of P-amyloid peptide (AP) production and aggregation or on symptomatic therapy are ineffective [1-5], therefore correction of chronic inflammation provoked by amyloidosis will have positive effect. The mechanism by which AP causes the damage and death of neurons is generation of oxygen active forms in the course of own aggregation. At the same time neuron membranes lipids peroxidation is activated and ATPases function is deteriorated. As a result AP conduces to depolarization of synaptic membranes, excessive ingress of Ca2+ and mitochondrial insufficiency [6-8]. All these processes are concurrent with non-specific inflammatory reaction which is transformed into chronic form and induces synthesis of AP protein precursor (АРРР) and its processing pursuant to amyloidogenic scenario [9-11]. It is shown that the inflammatory process in case
of AD is characterized by increased peripheral concentrations of anti-inflammatory cytokines and higher TGF-P levels in spinal cord liquid [12]. On the other side, cytokines, similar to AP, are mediators of inborn immunity [13, 14]. Their effect comes up through receptor activation of intracellular signals, which results in translocation of nuclear factor (NFkB) towards nucleus and activation of protein synthesis de novo [12]. In the end, the existing anti-cytokine therapy poorly represented itself for amyloidosis, except for anti-inflammatory effect of IL-10 [15]; although AD risk is lower in patients who take non-steroid anti-inflammatory preparations [16, 17]. Therefore we assumed that curcumin (CUA) with its anti-inflammatory properties may have essential therapeutic effect against AP-induced neurotoxicity and cognitive deficiency.
It is found that natural polyphenol CUA regulates NFkB, АР-1 transcription factors; suppresses expression of cyclooxygenase-2,
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lipoxygenase, NO-synthase, matrix metalloproteinase-9, urokinase of plasminogen activator type, TNF, chemokines, cellular adhesion molecules and D1 cycline, inhibits expression of growth factor receptors and activity of JNK, protein tyrosine kinases as well as some other protein serine/threonine kinases [18, 19]. Curcumin also acts as inhibitor of DNA-methyltransferase therefore it is regarded as DNA hypomethylating agent. It establishes equilibrium between histone acetyltransferase and histone acetylase enzymes activity thus modulates expression of certain genes. At last CUA modulates activity of microRNA and their numerous target genes [20, 21]. Above-mentioned CUA effects are exhibited in its antioxidant, anti-inflammatory, anti-tumor and even anti-amyloidogenic properties [22-27]. The problem with curcumin usage, like with the other hydrophilic anions, lies only in the fact that it cannot enter the cell through plasmatic membrane on its own. Therefore it is reasonable to use nanocarriers, in particular, liposomes, as CUA carriers. Liposome advantages are obvious: prepared from natural phospholipids, they compared to other polymeric delivery systems, completely biodegrade in the body and are biocompatible [28].
P-Amyloid peptide aggregation process results in imbalance between its production and degradation. One of the systems, which maintain low AP level in tissues, are zinc metalloproteinases [29]. Angiotensinconverting enzyme (ACE) [EC 3.4.15.1], which is involved in regulation of arterial blood pressure, neuropeptide exchange, immune responses of the body is also among them [30]. This enzyme (chiefly its С-domain) separates C-terminal dipeptides from oligopeptides of various structures which have a free carboxyl group. But ACE interacts with AP exclusively by N-domain and decomposes Arg5-His6 or Asp7-Ser8 peptide bonds [30]. ACE is an integral membrane glycoprotein of the1st type which is released to blood circulation by zinc metalloesterase at the rate of 2% per hour therefore this enzyme functions both in bonded and dissolved forms. According to the conclusions of ACE1 gene polymorphism and enzyme inhibitors studies it was found that ACE activity reduction is associated with AD risk and AP accumulation [31].
The purpose of the study was investigation of curcumin liposome form effect on ACE activity, cytokines and mnestic features of rats with experimental model of Alzheimer’s disease.
Materials and Methods
The study involved 30 male rats of sexually mature age, with 200 to 250 g weight. All the animals were kept under controlled 12-hour light-darkness cycle with standard fodder for rodents and tap water. Experimental protocols were conducted in accordance with the rules of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986).
The rats were randomly distributed into 5 groups (6 animals in each). The reference group included intact animals; group 1 — the rats 1 month after intrahippocampal injection of Ap42_Human (experimental model of AD); group 2 — sham operated animals; group 3 — the rats with AD experimental model which received daily nasal therapy with curcumin liposome form for 1 month, and group 4 — animals with AD experimental model which received daily nasal therapy with empty liposomes also for 1 month.
Beforehand, during 20 days conditioned avoidance response was formed in the rats on the basis of unconditioned reflex [32]. Infallible conditioned responses to metronome sound were considered as positive result. Apart from positive response portion (in %), the study registered duration of latent period of conditioned avoidance response in seconds. Animals from all groups were tested in the conditioned avoidance response parameters after AD experimental model formation in them and nasal therapy with curcumin liposome form or empty liposomes.
Ap42_Human (Human Amyloid P Protein Fragment 1-42, Sigma-Aldrich), dissolved in bidistilled water was aggregated for 24 hours at 37 °C. AP42_Human large size rough conglomerates were dispersed by ultrasound and sterilized immediately before injection. The effect of 42_Human P-amyloid peptide was studied 1 month after its single injection in the dose of 15 nM AP42_Human to the hippocampus of the rats’ brain. Solution volume was 10 mcl per animal. Stereotaxic coordinates of the left hippocampus were determined using the brain map of the rats [33], which corresponds to the distance from the intersection point of sagittal seam and bregma (zero point): distally — 2 mm, laterally — 2 mm and in depth — 3.5 mm. The stereotaxic operations in experimental animals were made under general anesthesia using intra-abdominal injections of thiopental (50 mg/kg of body weight).
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To prepare liposomes with curcumin, lecithin/cholesterol was dissolved in the round-bottom flask at ratio 18:1 in 50% ethanol. After the lipid film was formed as a result of the solvent evaporation, 28.85 mМ CUA in 5 ml of PBS buffer (10 mM Na2HPO4, 1.76 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, рН 7.4) was added and thoroughly mixed for suspension of liposomes with curcumin formation. The suspension of empty liposomes was prepared using the similar protocol but at the final stage PBS buffer without CUA was added. Both liposome suspensions were dissolved with PbS buffer to 0.7 g/l CUA immediately before nasal administration to the rats in the dose of 3.5 pg/animal. Daily nasal therapy of the rats with AD experimental model lasted for 1 month. Administration of liposome form curcumin by nasal method is determined by the fact that, unlike peripheral blood circulation, this is the shortest way to the target regions of the rat neocortex. It is known that after entering the body the dissolved curcumin is nearly unable to overcome the hematoencephalic barrier whereas its liposome form is actively and non-specifically entrapped by the formed elements of blood, which requires big doses of the preparation.
After the processing was finished the animals were decapitated. The assays of cerebral cortex and hippocampus were frozen and stored for further measurements. Blood was collected and centrifuged at 1000 g for 20 min. Serum was collected, frozen and stored. The tissues of hippocampus and fronto-coronal cortex were homogenized in Tris-buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5), centrifuged at 14000 g for 5 min producing supernatant.
In the supernatant assays of hippocampus, cerebral cortex and blood serum cytokines were identified using ELISA method in accordance with the protocol Rat ELISA Kit Invitrogen BCM DIAGNOSTICS, USA for interleukine-6 (IL-6), interleukine-10 (IL-10) and tumor necrosis factor a (TNFa). Assay absorption was read out using GBG Stat FAX 2100 (USA) microplate analyzer at 450 nm with wavelength correction at 630 nm. The ELISA data were recalculated into general protein for nerve tissue or expressed in ng/l for blood serum. Concentration of general protein was quantitated by Lowry method [34].
ACE activity was determined by kinetic method [35]. As a substrate the short FAPGG peptide was used, from which by ACE action GG dipeptide was separated and transformed into hippuric acid. Reduction in assay
absorption during 10 minute incubation at 37 °C was measured at 340 nm wavelength. Calculation was made using formula:
EACE = (AAassay / АА]
^■hippuric acid calibrator.
) • Е
calibrator,
where АА — reduction of absorption during 10 min of incubation at 37 °С; Ecalibrator = 82.1 (protocol B HLMANN ACE colometric kit, Switzerland). ACE activity was expressed in U/l, which corresponds to ACE enzyme quantity, which separates 1 pM hippuric acid at 37 °С per minute per liter for blood serum and per mg of protein for areas of the brain (fronto-coronal cortex and hippocampus).
The obtained results were statistically processed, average values and standard deviations were calculated. Statistical analysis of differences was made using f-test. Values at Р < 0.05 were considered significant.
Results and Discussion
Fig. 1 presents data about increase in ACE activity in hippocampus (direct spot of A^42_Human injection) and blood serum and absence of reliable changes in this parameter in cerebral cortex of the rats with AD experimental model. It is first of all due to ACE synthesis induction by one of the substrates local excess, namely АР [36]. Nasal therapy with CUA liposome form significantly reduced ACE activity compared to the effect of empty liposomes (Fig. 1) in both investigated brain sections of the rats and in blood serum.
On physiological level, the intrahippocampal injection of A^42_Human brought suppression of conditioned avoidance response in the rats of group 1 (Fig. 2). Study of mnestic features and memory showed reduction in the share of positive responses and increase of latent period in these animals compared to the reference group. The share of positive responses in the rats with AD experimental model was not different from that of in the sham operated animals, which is a consequence of intracranial intervention. Curcumin in liposomes was responsible for recovery of mnestic features and memory parameters in the rats with AD experimental model, which was not the case when empty liposomes were used (Fig. 2).
The most substantial cytokine activation was registered in hippocampus of the animals with AD experimental model (Table 1): Concentration of TNFa increased by 26%, IL-6 — by 27% and IL-10 — by 95%, respectively. These data show that А^42_
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□ Reference ■ Group 1 Ш Group 2 И Group 3 □ Group 4
Cerebral cortex Hippocampus Blood serum
Fig. 1. Effect of 42_Human P-amyloid peptide and curcumin liposome form on angiotensin-converting activity in brain sections (cerebral cortex and hippocampus) and blood serum of the rats
Hereinafter: * — Р < 0.05 compared to control (intact animals, n = 6); # — Р < 0.05 when comparing the groups 1 (model of Alzheimer’s disease, n = 6); 2 (sham operated animals, n = 6); 3 (nasal therapy of the AD model animals with liposome curcumin, n = 6); 4 (nasal therapy of AD model animals with empty liposomes, n = 6), respectively; & — Р <0.05 compared to the group 1 (Alzheimer’s disease model, n = 6).
□ Reference ■ Group 1 D Group 2 ■ Group 3 □ Group 4
%
100 -I
75 ■
SO ■
25 ■
0 -
Share of positive responses Latent period
Fig. 2. Dynamics of mnestic ability and memory parameters (share of positive responses and latent period) in effect of 42_Human P-amyloid peptide and curcumin liposome form therapy in the rats
Human in hippocampus of the experimental rats causes neuroinflammation specifically and mainly in the spot of injection. But in the brain cerebral cortex the activation of neuroinflammation was also shown, although to lower extent (Table 2). Namely, only the IL-6 level reliably increased by nearly 54%. Specificity of Ap42_Human inflammatory effect in the brain of the rats was also proved by difference in the levels of the studied cytokines between groups 1 and 2 (Tables 1 and 2).
Effect of liposome curcumin on cytokine levels in hippocampus of the animals was marked by essential suppression of inflammation (Table 1): TNFa level decreased by 56%, IL-6 — by 39% and IL-10 — by 52%, respectively. But not a single cytokine normalized its concentration, unlike in the case with the effect of empty liposomes (Table 1). Effect of CUA as component of liposomes in the cerebral cortex of the
rats with AD gave similar suppression of cytokine response (Table 2): TNFa level decreased by 71%, IL-6 — by 67% and IL-10 — by 41%, respectively. The obtained results are in agreement with our previous data for curcumin water solution only for the brain cerebral cortex of the animals with intrahippocampal injection of Ap42_Human [37]. In hippocampus of the rats with AD model the CUA liposome form showed more intensive suppression of cytokine chain of neuroinflammation compared to its water solution.
The level of peripheral cytokines (TNFa, IL-6 and IL-10 in blood serum) did not reflect neither specific neuroinflammatory effect of 42_Human P-amyloid peptide in the brain of the rats, nor suppression effect of liposome CUA (Table 3). 20-50% rise in TNFa concentration, 54-67% decrease in IL-10 concentration and absence of changes in IL-6 content in blood serum of the rats of all the
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BIOTECHNOLOGIA ACTA, V. 8, No 6, 2015
Table 1. Effect of curcumin liposome form on TNFa, IL-6 and IL-10 in hippocampus of the rats
with the model of Alzheimer’s disease
Cytokine Reference Group 1 Group 2 Group 3 Group 4
n = 6 n = 6 n = 6 n = 6 n = 6
TNFa 50.7±2.1 63.8±3.5*# 46.8±1.9& 28.3±1.7*#& 46.3±2.3&
IL-6 57.3±8.3 72.8±6.8# 98.3±6.8*& 44.7±5.9*& 58.5±8.2
IL-10 130.4±11.0 254.3±16.7*# 152.8±12.9& 122.4±13.4*#& 151.0±12.4&
Note. Hereinafter: the results are represented as M ± m, ng/g of protein.
Table 2. Effect of curcumin liposome form on TNFa, IL-6 and IL-10 in cerebral cerebral cortex of the rats with the model of Alzheimer’s disease
Cytokine Reference n = 6 Group 1 n = 6 Group 2 n = 6 Group 3 n = 6 Group 4 n = 6
TNFa 50.8±2.5 46.2±2.4# 40.6±2.9*& 13.2±0.9*#& 47.1±2.8
IL-6 52.5±4.2 80.8±7.4* 68.5±5.8 26.8±2.0*#& 49.3±3.9&
IL-10 179.5±13.0 150.8±10.6# 206.4±24.2& 89.4±7.6*#& 177.0±12.8&
Table 3. Effect of curcumin liposome form on TNFa, IL-6 and IL-10 in blood serum of the rats
with the model of Alzheimer’s disease
Cytokine Reference n = 6 Group 1 n = 6 Group 2 n = 6 Group 3 n = 6 Group 4 n = 6
TNFa 7.9±0.8 9.5±0.6 * 10.7±1.0 * 10.4±0.4 *# 11.9±0.9 *&
IL-6 48.3±10.4 37.5±6.9 53.2±11.3 44.8±7.7 47.0±14.1
IL-10 19.5±2.4 9.8±1.2 *# 6.5±0.9 * 7.0±1.8 *#& 9.3±2.5 *
experimental groups compared to the reference were marked.
The obtained results showed activation of cytokine system in the brain of the rats with AD experimental model (Tables 1, 2). These data are in agreement with other studies on activation of neuroinflammation by Ap aggregates [38-41]. Ap deposits are responsible for activation of microglia [38]. Ap conduces to higher inflammatory response to NFkB stimulation, the nuclear factor which is involved in regulation of ERK (extracellular signal-regulated kinases) and МАРК (mitogen-activated protein kinases) routes which lead to cytokines and chemokines production [39]. Modification of the inflammatory condition of microglia/macrophages plays prominent role in the course of amyloidosis [40].
Nasal therapy of AD model rats with curcumin liposome form was responsible for suppression of ACE activity and cytokine chain of neuroinflammation. Revealed anti-
inflammatory activity of CUA resulted in recovery of memory parameters and mnestic functions in the animals. Therefore, the previous assumption that its liposome form may be an efficient anti-inflammatory factor in the effect of exogenous p-amyloid peptide was confirmed by experimental data. This natural polyphenol prevents activation of NFkB transcription nuclear factor suppressing phosphorylation and degradation of 1кВа (NFkB inhibitor). Since curcumin effect lies in inhibition of IB kinase (IKK) activation, needed for NFkB activation [42-44], it is just this fact that explains the revealed anti-cytokine effect of curcumin in the experimental animals. The mechanism of curcumin effect on ACE activity is substantiated by the proved suppressor effect of this polyphenol on expression of enzyme gene [45]. Above-mentioned data show high anti-cytokine potential of especially the liposome form of curcumin.
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ВПЛИВ ЛШОСОМНО1 ФОРМИ КУРКУМ1НУ НА АНГ1ОТЕНЗИН-ПЕРЕТВОРЮВАЛЬНУ АКТИВН1СТЬ, ЦИТОК1НИ I КОГН1ТИВН1 ВЛАСТИВОСТ1 ЩУР1В З МОДЕЛЛЮ ХВОРОБИ АЛЬЦГЕЙМЕРА
B. В. Сокол1к1
C. М. Шульга2
1ДУ «1нститут неврологи, псих1атрИ i наркологи НАМН Укра!ни», Харшв 2ДУ «Институт харчово! бмтехнологи i геномiки НАН Украши», Ки!в
Е-mail: [email protected]
Метою дослщження було вивчення впли-ву лшосомно! форми куркумiну на актившсть ангiотензинперетворювального ензиму, цито-кiни i мнестичнi властивостi щурiв з експери-ментальною моделлю хвороби Альцгеймера. У тварин з штрагшокампальним уведенням АР42_Нитап застосовували назальну тера-пiю лшосомною формою куркумiну. Реестру-вали концентращю цитокiнiв i активнiсть ангiотензинперетворювального ензиму у вщд^ах головного мозку (лобно-фронтальна кора i гiпокамп) та сироватщ кровi, а та-кож показники умовно-рефлекторно! реакци уникнення. У результат терапи куркумiном встановлено пригшчення активностi цитокь нiв, ангiотензинперетворювального ензиму та вщновлення мнестичних показникiв у щурiв iз хворобою Альцгеймера. Назальна терашя лшосомно! форми куркумiну мала наслщ-ком зменшення активностi ангштензинпе-ретворювального ензиму та антицитокшо-вого ефекту в щльових вiддiлах головного мозку (лобно-фронтальна кора i гшокамп), що сприяло вiдновленню мнестичних власти-востей i пам’ятi щурiв.
Ключовi слова: куркумш, лiпосоми, Р-амшо-!дний пептид, цитокiни, ангiотензинперетво-рювальний ензим.
ВЛИЯНИЕ ЛИПОСОМНОЙ ФОРМЫ КУРКУМИНА НА АНГИОТЕНЗИН-ПРЕВРАЩАЮЩУЮ АКТИВНОСТЬ, ЦИТОКИНЫ И КОГНТИТИВНЫЕ СВОЙСТВА КРЫС С МОДЕЛЬЮ БОЛЕЗНИ АЛЬЦГЕЙМЕРА
B. В. Соколик1
C. М. Шульга2
1ГУ «Институт неврологии, психиатрии и наркологии НАМН Украины», Харьков 2ГУ «Институт пищевой биотехнологии и геномики НАН Украины», Киев
Е-mail: [email protected]
Целью исследования было изучение влияния липосомной формы куркумина на активность ангиотензин-превращающего энзима, цитокины и мнестические свойства крыс с экспериментальной моделью болезни Альцгеймера. У животных с интрагипокампальным введением АР42_Нитап применяли назальную терапию липосомной формой куркумина. Регистрировали концентрацию цитокинов и активность ангиотензинпревращающего энзима в отделах головного мозга (лобно-фронтальная кора и гиппокамп) и сыворотке крови, а также показатели условно-рефлекторной реакции избегания. В результате терапии курку-мином установлено угнетение активности цитокинов, ангиотензинпревращающего энзима и восстановление мнестических показателей у крыс с болезнью Альцгеймера. Назальная терапия липосомной формой куркумина обусловила уменьшение активности ангиотензин-превращающего энзима и антицитокинового эффекта в целевых отделах головного мозга (лобно-фронтальная кора и гиппокамп), что способствовало восстановлению мнестических свойств и памяти крыс.
Ключевые слова: куркумин, липосомы,
Р-амилоидный пептид, цитокины, ангиотен-зинпревращающий энзим.
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BIOTECHNOLOGIA ACTA, V. 8, No 6, 2015
УДК 579:662.7
doi: 10.15407/biotech8.06.056
PROPERTIES OF CHEMOLITHOTROPHIC BACTERIA
NEW STRAINS ISOLATED FROM INDUSTRIAL SUBSTRATES
I. A. Blayda1 T. V. Vasyleva1 V. I. Baranov2
K. I. Semenov1
L. I. Slysarenko1 I. M. Barba1
1Mechnykov Odesa National University, Ukraine 2Franko Lviv National University, Ukraine
E-mail: [email protected]
Received 23.07.2015
The purpose of the research was determination of strains Acidithiobacillus ferrooxidans MFLv37 and Acidithiobacillus ferrooxidans MFLad27, isolated from aboriginal consortium of coal beneficiation dumps and fly ash from coal combustion, resistance to heavy metals, forming part of these waste, as well as adaption ability of the strains to new substrates. New strains increased resistance to heavy metal ions as compared to A. ferrooxidans standard and collection strains is found; minimal inhibitory concentrations of heavy and toxic metals are determined; a number of metals that have negative impact on growth of isolated cultures are identified. It is shown that the minimal metals concentrations, at which strains growth still happens, are several times higher than their concentrations in technogenic waste. It has been found that isolated strains differed in their ability to adapt, as well as in growth rate and substrates oxidation. This is due to the specific conditions of microbiocenoses formation in making and further storage of rock dumps and fly ash, whereof the appropriate strains are isolated. The investigations indicate the necessity in directional selection of strains that are resistant to the toxic compounds and are able to oxidize various mineral substrates, as well as in their adaptation to new substrates for the extraction of heavy metals.
Key words: fly ash, rock dumps, aboriginal bacterial community, acidophilic chemolithotrophic bacteria, strains, leaching activity, ions of heavy metals.
The members of the genera Acidithiobacillus and Sulfobacillus have the greatest activity regarding leaching of metals from raw natural ores and technogenic substrates [1-3]. In previous investigations it was found that the most active group of microorganisms in the aboriginal consortium of coal beneficiation dumps and fly ash from coal combustion substrates is the group of acidophilic chemolithotrophic microorganisms as small mesophilic and most numerous moderately thermophilic ones — the members of the genera Acidithiobacillus and Sulfobacillus [4, 5]. The study of the properties of the most active pure cultures of the microorganisms isolated from the substrates aboriginal association has allowed to classify them as the representatives of Acidithiobacillus ferrooxidans and assign them the strain numbers MFLv37 and MFLad27 in a view of their habitats — Lviv-Volyn Coal Basin coal
beneficiation dumps and fly ash from the coal combustion at Ladyzhynskaya thermal power station, respectively.
Acidithiobacillus ferrooxidans group is the most studied among acidophilic chemolithotrophic bacteria receiving energy from the oxidation of ferrous iron, elemental sulfur and its reduced compounds and sulfide minerals; it has a high level of resistance to heavy metals. Resistance is an important property of microorganisms of different taxonomic groups, thanks to which resistant forms of microorganisms in the environment with a high content of heavy metals appear. So, in [6] it is shown that the differences between the strains in substrate oxidation activity are caused by environment and conditions of microbiocenosis formation whereof the strains were isolated. A. ferrooxidans strain, isolated from a complex mineralogical composition substrate, is characterized by a higher growth
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