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ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY Оригинальная статья / Original article УДК 579.6; 577.29
DOI: http://dx.doi.org/10.21285/2227-2925-2019-9-1 -44-52
Analysis of microbial phospholipids in processes of biomonitoring of soil condition
© Aida R. Khabibullina*, Tatyana V. Vdovina*, Alexander S. Sirotkin*, Josef Trogl**, Tat'jana Brovdyova**, Pavel Kuran**
* Kazan National Research Technological University, Kazan, Republic of Tatarstan, Russian Federation ** University of Jan Evangelist Purkyne, Usti nad Labem, Czech Republic
Abstract: The purpose of this work was to assess the possibility of applying the method of phospholipid fatty acid analysis in the composition of lipids of cell membranes of soil microorganisms for monitoring the state of the soil in the process of recovery. The experimental part consisted in a determination of phospholipid fatty acids (FFA), including extraction of phospholipids with organic solvents, extraction of the lipid fraction using polar silicate columns, preparation of methyl esters of fatty acids by alkaline meth-anolysis and analysis of the content of FFA with gas chromatography. The identification of microorganisms was carried out on the basis of an evaluation of the experimental results obtained, which are represented by a combination of chromatogram peaks. The impact of stress factors on microorganisms is determined by the presence of cis-trans-isomerism or cycles of three carbon atoms at the ends of phospho-lipid fatty acid radicals. In order to verify the objectivity of the results obtained using the pro posed method for analysing microorganisms, this work also used the traditional method of controlling soil microorganisms based on the activity of their enzymes - glucosidases, proteases, arylsulphatases and phosphatas-es. The data obtained indicate the presence of viable soil microorganisms; however, their number does not match the control samples, indicating a failure of the recovery process. The identification results demonstrate the dominance of Gram-negative (Gr-) bacteria in the control samples in comparison with the samples in conditions of natural recovery. At the same time, in the process of natural restoration, a greater quantity of fungi was found in the soil. According to the data obtained, the largest amounts of cis - isomers and the value of cyclisation of phospholipid fatty acids are characteristic of microorganisms in the control samples, which may be due to the depletion of the substrate when using the soil for agricultural purposes. The experimental data obtained in the course of this work allow us to propose the procedure as an independent method for conducting biological monitoring of ecosystems when analysing phospholipid fatty acids in the composition of cell membrane lipids. Keywords: soil, microorganisms of soil, fermentative activity, phospholipids
Information about the article: Received June 8, 2018; accepted for publication March 4, 2019; available online March 29, 2019.
For citation: Khabibullina A.R., Vdovina T.V., Sirotkin A.S., Trogl J., Brovdyova T., Kuran P. Analysis of microbial phospholipids in processes of biomonitoring of soil condition. Izvestiya Vuzov. Prikladnaya Khimiya i Biotekhnologiya [Proceedings of Universities. Applied Chemistry and Biotechnology]. 2018, vol. 9, no. 1, pp. 44-52. (in Russian). DOI: 10.21285/2227-2925-2019-9-1-44-52.
Анализ микробных фосфолипидов в процессах биомониторинга состояния почв
© А.Р. Хабибуллина*, Т.В. Вдовина*, А.С. Сироткин*, Й. Трёгл**, Т. Бровдыова**, П. Куран**
* Казанский национальный исследовательский технологический университет, г. Казань, Республика Татарстан, Российская Федерация
** Университет Яна Евангелиста Пуркине, г. Усти над Лабем, Чехия
Резюме: Целью данной работы являлась оценка возможности применения метода анализа фосфо-липидных жирных кислот в составе липидов клеточных мембран почвенных микроорганизмов для мо-
ниторинга состояния почв в процессе восстановления. Экспериментальная часть заключалась в определении фосфолипидных жирных кислот (ФЛЖК), включающая экстракцию фосфолипидов органическими растворителями, выделение липидной фракции с помощью полярных силикатных колонок, получение метиловых эфиров жирных кислот путем щелочного метанолиза и анализа содержания ФЛЖК методом газовой хроматографии. Идентификация микроорганизмов осуществлялась на основании оценки полученных экспериментальных результатов, представляющих собой совокупность пиков хроматограммы. Воздействие стрессовых факторов на микроорганизмы определяют по наличию цис-транс-изомерии или циклов из трех атомов углерода на концах радикалов фосфолипидных жирных кислот. С целью проверки объективности результатов, полученных с помощью предлагаемого метода анализа микроорганизмов, в данной работе использовался также традиционный метод контроля почвенных микроорганизмов на основании активности их ферментов - глюкозидаз, протеаз, арилсульфатаз, фосфатаз. Полученные данные свидетельствуют о наличии жизнеспособных почвенных микроорганизмов. Однако их количество не соответствует контрольным пробам, что говорит о незавершении процессов восстановления. Результаты идентификации демонстрируют доминирование грамотрицательных (Го) бактерий в контрольных пробах в сравнении с пробами в условиях естественного восстановления. При этом в процессе естественного восстановления в почве обнаружено большее количество грибов. Согласно полученным данным, наибольшее количество цис-изомеров и значение циклизации фосфолипидных жирных кислот характерны для микроорганизмов в контрольных пробах, что может быть обусловлено истощением субстрата при использовании почвы в сельскохозяйственных целях. Экспериментальные данные, полученные в ходе данной работы, позволяют предложить использование процедуры анализа фосфолипидных жирных кислот в составе липидов клеточных мембран как самостоятельный метод для проведения биологического мониторинга экосистем.
Ключевые слова: почва, почвенные микроорганизмы, ферментативная активность, фосфолипиды
Информация о статье: Дата поступления 8 июня 2018 г.; дата принятия к печати 4 марта 2019 г.; дата онлайн-размещения 29 марта 2019 г.
Для цитирования: Хабибуллина А.Р., Вдовина Т.В., Сироткин А.С., Трёгл Й., Бровдыова Т., Куран П. Анализ микробных фосфолипидов в процессах биомониторинга состояния почв // Известия вузов. Прикладная химия и биотехнология. 2019. Т. 9, N 1. С. 44-52. РО!: 10.21285/2227-2925-2019-9-1-44-52.
INTRODUCTION
While the environmental effects of soil pollution manifest themselves later than the results of atmospheric and hydrospheric pollution, the latter can be more persistent and long-lasting1. Therefore, the protection of the soil of the biosphere as a whole is possible only on the basis of monitoring the soil environment. Soil microorganisms, which play a key role in soil formation and the maintenance of soil fertility, effecting cycles of all necessary nutrients, also act as decomposers in ecological systems [1].
In order to record the response during biological monitoring at the cellular level, special bi-omarker molecules - indicators of the state of the organism under study - are used. At the present time, special attention is paid to the use of phos-pholipid fatty acids in the composition of cell membrane lipids as biomarkers for identifying microorganisms and assessing the influence of stress-factors on them [2].
Phospholipids, which consist of polyols, fatty acid residues and phosphoric acid, are essentially
Golovanov A.I., Zimin F.M., Smetanin V.I. Re-kul'tivatsiya narushennykh zemel' [Restoration of disturbed lands]. St. Petersburg: Lan' Publ., 2015, 336 p. / Голованов А.И., Зимин Ф.М., Смета-нин В.И. Рекультивация нарушенных земель: учебник для вузов; 2-е изд., испр. и доп. СПб.: Лань, 2015. 336 с.
components of living organisms. In the body, phospholipids act as the main component of cell membranes, participating in the processes of nutrition and adaptation. Phospholipids have the following properties, allowing them to be used as biomarkers:
- are not part of the reserve material of the cell;
- form part of the cell membrane, as a result of which they can signal changes in environmental conditions, as well as changes inside the cell;
- high specificity: each group of microorganisms corresponds to a particular fatty acid radical;
- are only contained in the membranes of living organisms; following cell death, phospholipid fatty acids immediately become dephosphorylated2.
The aim of the research consisted in assessing the possibility of applying the phospholipid fatty acid analysis method in the composition of lipids in the cell membranes of soil microorganisms in order to monitor the state of the soil undergoing processes of recovery from contamination.
2
Zhukova N.V. Zhirnye kisloty morskikh organizmov: taksonometricheskie i troficheskie markery. Avtoref. diss. dokt. biol. Nauk. [Fatty acids of marine organisms: taxonomic and trophic markers: Author's abstract of Dr. Sci. Thesis]. Vladivostok, 2009, 267 p. / Жукова Н.В. Жирные кислоты морских организмов: таксономические и трофические маркеры: Автореферат дис. ... д-ра биолог. наук: 03.00.04. Владивосток, 2009. 267 с.
EXPERIMENTAL PART
The object of research in this work consisted of phospholipid fatty acids in the lipid composition of cell membranes of soil microorganisms.
The method for determining phospholipid fatty acids (PFLA) comprises four stages [3, 4]:
1. Extraction of lipid fraction with organic solvents. 15 ml of phosphate buffer solution (0,05 mol/l, pH = 7,4), 37,5 ml of methanol and 18,8 ml of chloroform were added to 10 g of sample. Next, the samples were subjected to ultrasonic treatment (15 min) and infused in a rotary mixer (2 h at 10 °C). Following centrifugation (2880 g, 30 min), the supernatant liquid was collected in a separatory funnel. The precipitate was resuspended in 10 ml of the washing mixture (10 ml of chloroform, 8 ml of phosphate buffer, 20 ml of methanol) and centrifuged again (10 min). To the resulting supernatant, which was collected in a separatory funnel, chloroform (18,8 ml) and deionised water (18,8 ml) were added. Following vigorously stirring, the mixture was left to undergo phase separation for 10 hours at ~10 °C.
2. The isolation of phospholipids from the lipid fraction was performed using polar silicate columns (Supelclean™ LC-Si, Sigma-Aldrich) under moderate vacuum (elution rate ~1 ml/min). Each column was pretreated with chloroform (3,5 ml) and methanol (3,5 ml) to ensure the purity of the analysis. The sample was concentrated to ~1 ml using a rotary vacuum evaporator and introduced into the column. Undesirable components of the lipid fraction - in particular, triacylglycerides and glycolipids - were separated out using a mixture of chloroform (7 ml) and acetone (28 ml). Polar lipids were eluted with methanol (7 ml), evaporated under nitrogen atmosphere to ~0,5 ml and stored at -30 °C.
3. Preparation of fatty acid methyl esters was carried out by means of mild alkaline methanolysis. To the polar lipid solution was added 0,5 ml of a mixture of methanol/toluene (1:1) and, following stirring, 0,5 ml of a freshly-prepared solution of potassium hydroxide (KOH) in methanol (1,122 g of KOH in 10 ml of methanol). The mixture was then heated to 37 °C for 30 min. After cooling, 0,5 ml of
acetic acid (0,2 mol/l) was added to the sample. Following shaking, 2 ml of a mixture of hexane: chloroform (4:1) and 2 ml of deionised water were added. Following additional agitation, the mixture was then and centrifuged (2880 g, 10 min). The upper phase was transferred to a clean test tube. The extraction-centrifugation procedure was performed three times. Samples were evaporated in a nitrogen atmosphere to 0,5-1 ml and stored at -30 °C.
4. Qualitative and quantitative analysis of the fatty acid composition of phospholipids was performed by means of gas chromatography using a Varian GC 3800 chromatograph with a VF-5 fused silica capillary (20 m * 0,25 mm * 0,25 ^m) and helium as a carrier gas (1 ml/min). The sample was introduced into the evaporator by means of splitless injection (1 ^l, 0,2 min, 300 °C). The temperature programme was 50 °C for 1 min; 25 °C/min up to 150 °C, 4 °C/min up to 250 °C, 5 min. A Varian 4000 detector with an ion-cyclotron trap was used (ion transport took place at 280 °C, the collector temperature was 45 °C. The mass limits of the compounds to be determined were in the range of 45-450 amu). The aggregate PFLA quantity was determined as the sum of the peaks in the total ion chromatogram with elution in the retention window from a standard mixture of 26 FAME (Sigma-Aldrich). Quantitative determination was performed using the FAME 11 calibration curves (C10-C20) and the internal standard (methyl nonadecanoic acid, 19:0) [5].
The identification of microorganisms was carried out on the basis of an evaluation of the experimental results obtained, consisting in a combination of chromatogram peaks. To ensure the reliability of identification of PFLA groups, reference samples were used - standard solution 26 FAME (Sigma-Aldrich, USA) with commercial FAME standards manufactured by Sigma-Aldrich and Matreya LLC (USA); nonadecanoic acid methyl ester, 19:0. In the framework of this work, it was necessary to confirm the presence of PFLA radicals, characteristic of certain subgroups of microorganisms (Table 1) [6, 7].
Table 1
Groups of microorganisms and their typical fatty acid radicals [6, 7]
Таблица 1
Группы микроорганизмов и характерные для них радикалы жирных кислот [6, 7]
Group Subgroup Fatty acid biomarker
Bacteria Gr+ Gr Other i14:0, il5:0, a15:0, il7:0, a17:0 cy17:0, cy19:0 16:1 o7t, 16:1Q7, 16:1Q9
Fungi - 18:2Q6,9
Note. Hereinafter: i - isoacid; a - anteisoacid; cy - cyclic form of the acid; ш - double bond. In the above notation of acids, the first digit indicates the number of carbon atoms, while the second indicates the degree of unsaturation of the radical; the number after ® is the position of the double bond from the methyl end. / Примечание. Здесь и далее: i - изокисло-та; а - антеизокислота; су - циклическая форма кислоты; ш - двойная связь. В приведенных обозначениях кислот первая цифра указывает число атомов углерода, вторая - степень ненасыщенности радикала; цифра после ® - положение двойной связи с метильного конца.
The calculation of the values of equivalent chain lengths (ECL) was carried out on the basis of the work of S.A. Mj0s [8], T.K. Miwa et al. [9]. Thus, the presence of anteiso- or isoacids in the final concentrate indicates the presence of Grampositive (Gr+) bacteria, while cyclic acid forms are characteristic of Gram-negative (Gr-) bacteria; fungi in the composition of membrane lipids have two double bonds.
The impact of stress factors on microorganisms is determined by the presence of cis-trans-isomerism or cycles of three carbon atoms at the ends of phospholipid fatty acid radicals.
In order to verify the objectivity of the results obtained using the proposed method for analysing microorganisms, the present work also used the traditional method for monitoring soil microorganisms based on the activity of their enzymes -glucosidases, proteases, arylsulfatases, phosphatases. [10, 11]. The analysed enzymes are of interest, since they determine the circulation of the most essential nutrients for plants, such as carbon, nitrogen, sulphur and phosphorus.
The subject of the research comprised soil samples selected from the Radovesice district near the city of Usti nad Labem (Czech Republic). This area had previously been used for coal mining and was then backfilled with soil.
Soil sampling was carried out in 4 replications from an average depth of 5 cm (since it is at this depth to which the roots of most plants extend and where the majority of soil microorganisms actively develop) [12].
In order to establish the possibility of applying the analysis of phospholipid fatty acids to assess the state of soil microorganisms, samples were taken from various locations:
- undergoing reclamation process including artificial tree planting;
- undergoing natural recovery, associated with the process of succession under the influence of natural factors only and in the absence of any particular interventions.
Soil from agricultural land acted as a control (following the completion of restoration processes, it was planned to use the soil in agriculture).
In order to ensure statistically reliable results, multiple sampling was carried out from each site of the surveyed territory. Statistical processing was performed using standard procedures of the Microsoft Excel application package. In this way, variance analysis methods were used together with determinations of the average value, standard deviation, confidence interval and relative error.
RESULTS AND DISCUSSION
As a result of the studies carried out using the above methods, phospholipid fatty acids and related substances were identified in the samples (Table 2).
Table 3 shows the quantitative (in mg) content of fatty acids, which are biomarkers in terms of absolutely dry matter (ADM) of the studied samples, as well as the limits of errors in their determination.
Table 2
Identified PLFA in the studied samples with retention time and equivalent chain length (ECL)
Таблица 2
Идентифицированные ФЛЖК в исследованных пробах с указанием времени удерживания и эквивалентных длин цепей (ECL)
PLFA Time holding, min ECL PLFA Time holding, min ECL
10:0 5,716 9,9263 16:0 15,543 16,0014
12:0 7,281 11,9963 i-17:0 16,891 16,6837
12:1 7,681 11,8350 a-17:0 17,086 16,7904
13:0 9,529 13,0043 cy17:0 17,321 16,9024
13:1 9,546 12,8460 17:0 17,607 17,0324
14:0 11,521 13,9946 2-OH 16:0 18,233 17,3134
i-15:0 12,261 14,6309 18:2Q6,9 19,089 17,7390
a-15:0 12,881 14,1706 18:1 q9 19,215 17,7624
15:1 13,045 14,7786 18:1Q7 19,342 17,8661
15:0 13,486 14,9979 18:0 19,793 18,0021
2-OH 14:0 14,248 15,1719 10-Ме 18:0 20,605 18,5201
3-OH 14:0 14,753 15,6420 19:1 21,313 18,8269
i-16:0 15,086 15,7950 cy19:0 21,566 18,8304
16:1o7t 15,149 15,8315 19:0 21,887 19,0041
16:1Q9 15,233 15,8587 20:0 23,363 20,0073
16:1Q7 15,311 15,8622 - - -
Biomarkers are in bold, PLFA 10:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0 are standards (see «Objects and methods of research») / Биомаркеры выделены жирным шрифтом, ФЛЖК состава 10:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0 являются стандартами (см. «Объекты и методы исследований»)
In accordance with the data given in Table 1, the content of PLFA in the composition of the cell membranes of microorganisms per ADM of the studied samples was determined (Table 4).
According to the results of PLFA determination,
it should be noted that the number of microorganisms in the biocenosis of the studied soils differs from their number in the control sample, indicating the incompleteness of the processes of soil restoration (Table 5, Fig. 1).
Table 3
Quantitative content and error limits in the determination of fatty acids acting as markers
Таблица 3
Количественное содержание и пределы погрешностей при определении жирных кислот, выступающих в роли маркеров
Marker Quantity of PLFA, mg/g ADM
Control Reclamation Natural recovery
i-15:0 3,01 ± 0,15 2,03 ± 0,09 1,12 ± 0,02
a-15:0 2,58 ± 0,11 0,96 ± 0,01 0,61 ± 0,01
16:1 o7t not open not open not open
16:1 Q9 0,97 ± 0,05 not open not open
16:1 Q7 1,83 ± 0,08 1,62 ± 0,01 0,73 ±0,03
i-17:0 0,58 ± 0,03 0,38 ± 0,01 0,21 ± 0,01
a-17:0 0,35 ± 0,01 0,26 ± 0,01 0,19 ± 0,01
cy17:0 2,71 ± 0,01 1,28 ± 0,01 0,44 ± 0,01
18:2Q6,9 5,22 ± 0,52 1,17 ± 0,26 0,42 ± 0,16
cy19:0 4,69 ± 0,22 2,71 ± 0,001 1,51 ± 0,002
Table 4
Phospholipid fatty acid (PLFA) content in the studied samples
Таблица 4
Содержание фосфолипидных жирных кислот (ФЛЖК) в исследованных образцах
Sample Quantity of PLFA, mg/g ADM
General Bacteria Fungi Other
Gr+ Gr-
Control 22,02 ± 5,73 6,02 ± 2,03 7,96 ± 3,52 5,23 ± 0,52 2,81 ± 0,81
Reclamation 12,05 ± 2,46 3,75 ± 0,83 3,99 ± 1,08 1,19 ± 0,26 1,64 ± 0,38
Natural regeneration 6,01 ± 2,39 2,19 ± 0,64 1,97 ± 0,71 0,41 ± 0,16 0,78 ± 0,32
Table 5
Number of isomerised phospholipid radicals
Таблица 5
Количество изомеризованных фосфолипидных радикалов
Sample Number of isomers, mg/g ADM
Cis-isomers Trans-isomers Cyclic forms Precursors of cyclic forms
Control 6,94 ± 0,35 not open 2,78 ± 0,34 6,93 ± 0,54
Reclamation 2,37 ± 0,22 not open 0.57 ± 0,03 2,38 ± 0,12
Natural regeneration 1,38 ± 0,07 not open 0,26 ± 0,08 1,37 ± 0,17
D
A g
P g
m
30 25 20 15 10 5 0
Control Контроль
Restoration Рекультивация
Natural regeneration
Естественное
восстановление
Samples
Fig. 1. Total phospholipid fatty acid content in soil samples Рис. 1. Общее содержание фосфолипидных жирных кислот в почвенных образцах
The estimation of the microorganism content according to the results of the identification of characteristic groups of PLFAs (Fig. 2, see Table 2) demonstrates the dominance of Gr- bacteria in the control sample in comparison with samples under conditions of natural recovery. At the same time, during the process of natural restoration, a greater quantity of fungi was found in the soil. Minor discrepancies in the results of the control and recultivated systems are due to the fact that the recultivation involves the rapid restoration of the soil to a condition suitable for use in agriculture.
However, it should be noted that the process of natural restoration implies succession, i.e. a consistent replacement of some biological communities by others. The obtained results confirm the longer duration of the natural restoration of soil fertility.
The influence of stress factors on soil microorganisms leads to the formation of cis-trans-isomers or cycles of three carbon atoms at the ends of fatty acid radicals (see Table 5). For bacteria that already have cyclic fatty acid radicals in cellular membranes (Gr- i.e. cycles of 17 and 19 carbon atoms), the number of cyclic forms precursors (radicals with double bonds of 16 and 18 carbon atoms) is analysed.
According to the data obtained, the largest quantity of cis-isomers and the highest cyclisation value of FLFA are noted for microorganisms in the control system, which may be due to the effect of a combination of adverse factors in agricultural production (use of chemical plant protection products, fertilisers, etc.). The smallest amount of cis-isomers is characterised by the soil in the process of natural restoration, since this process involves a flow of successive processes under natural conditions.
Table 6 shows the activity values of four groups of microbial enzymes for the studied soil samples, confirming the data on the PLFA content in the composition of their cell membranes.
It is noted that the activity of the studied microbial enzymes under the conditions of the natural restoration of the soil is lower than in the samples of the control and recultivated soil. In this case, the values of the enzymatic activity of microorganisms in the reclaimed soils are close to those of control samples. An exception to this is seen in the proteolytic activity of microorganisms (see Table 6).
Higher values of protease activity in soil samples taken from the recultivated areas compared to the control sample may be due to the uneven distribution of microorganisms in the soil.
Fig. 2. Ratio of microbial groups in soil samples, from left to right: control sample, samples from the restoration areas and from the areas of natural regeneration
Рис. 2. Соотношение микробных групп в почвенных образцах, слева направо: контрольная проба, пробы с территорий рекультивации и с территорий естественного восстановления
Enzymatic activity values of soil microorganisms
Table 6
Значения ферментативной активности почвенных микроорганизмов
Таблица 6
Enzyme Activity value, nmol/min /g ADM
Control Reclamation Natural recovery
Glucosidase Protease Arylsulfatase Phosphatase 0,0057 ± 0,0024 0,0205 ± 0,0071 0,0159 ± 0,0066 0,1317 ± 0,0347 0,0050 ± 0,0017 0,0362 ± 0,0029 0,0040 ± 0,0007 0,0742 ± 0,0280 0,0035 ± 0,0010 0,0125 ± 0,0040 0,0021 ± 0,0008 0,0313 ± 0,0180
CONCLUSION
In the course of the study, results of the analysis of phospholipid fatty acids in the lipid composition of cell membranes of microorganisms of soil ecosystems, which correlate with indicators of the enzymatic activity of microorganisms, were obtained.
The total PLFA and enzymatic activity values obtained indicate the presence of viable cells with a full cell wall and a set of active enzymes in all soil samples in the microbial communities; a significant stress effect on the microbial ecosystems in the case of long-term production processes in a given territory is also indicated. It was empirically demonstrated that the minimum amount of phospholipid fatty acids in the composition of lipids of cell membranes is typical for soil samples under processes of natural restoration and recultivation, i.e. 6 and 3 times smaller compared to control samples respectively. A similar dependence is noted for the activity values of the studied enzyme groups. Based on the data obtained, it is concluded that the recovery pro-
cesses are not complete.
In addition, the high content of isomerised phospholipid radicals (cis-isomers and cyclic forms) for control soil samples indicates a significant stress effect on microbial communities in the development of natural areas for agricultural production.
Despite the labour-intensive nature of the analysis of phospholipids, this complex method allows detailed information to be obtained about the systems under study, including: determining the number of living microorganisms, identifying groups of microorganisms in the microbial communities and evaluating the influence of external factors on the microbiocenosis. The experimental data obtained in the course of this work allow us to propose the use of analysis procedures of phos-pholipid fatty acids forming part of cell membrane lipids for the development of theory and to support environmental monitoring in order to preserve and restore soil fertility on agricultural lands and as part of rural landscapes.
REFERENCES
1. Shlegel' G. Obshchaya mikrobiologiya [General Microbiology]. Moscow: Mir Publ., 1987, 567 p.
2. Akhmetshina E.A., Sirotkin A.S. Analysis of phospholipid fatty acids of microorganisms as environmental biomarkers. Vestnik Kazanskogo tekhnologicheskogo universiteta. 2014, vol. 17, no. 19, pp. 233-236. (In Russian)
3. Kurän P., Trögl J., Noväkovä J., Pilarovä V., Dänovä P., Pavlorkovä J., Kozler J., Noväk F., Popelka J. Biodegradation of spilled diesel fuel in agricultural soil: Effect of humates, zeolite, and bioaugmentation. The Scientific World Journal. 2014, vol. 2014, 8 p. http://dx.doi.org/10.1155/2014/642427
4. Trögl J., Jirkovä I., Zemänkovä P. et al. Estimation of the quantity of bacteria encapsulated in Lentikats Biocatalyst via phospholipid fatty acids content: a preliminary study. Folia Microbiol. 2013, vol. 58, pp. 135-140.
5. Trögl J., Jirkova I., Kuran P., Achmetshina E., Brovdyova T., Sirotkin A., Kirilina T. Phospholipid Fatty Acids as Physiological Indicators of Paracoccus Denitrificans Encapsulated in Silica Sol-Gel Hydrogels. Sensors. 2015, no. 15, pp. 3426-3434.
6. Federici E., Giubilei M.A., Cajthaml T., Petruccioli M., D'Annibale A. Lentinus (Panus) Tigrinus augmentation of a historically contaminated soil: Matrix Decontamination and Structure and Function of the Resident Bacterial Communi-
ty. Journal Hazard. Mater. 2011, no. 186, pp. 1263-1270.
7. Trogl J., Pavlorkova J., Packova P., Sejak J., Kuran P., Popelka J., Pacina J. Indication of Importance of Including Soil Microbial Characteristics into Biotope Valuation Method. Sustainability. 2016, no. 8, pp. 253-263.
8. Mj0s S.A. Identification of Fatty Acids in Gas Chromatography by Application of Different Temperature and Pressure Programs on a Single Capillary Column. Journal Chromatogr. A. 2003, vol. 1015, pp. 151-161.
9. Miwa T.K., Mikolajczak K.L., Earle F.R., Wolff I.A. Identification of peaks in gas-liquid chromatography. Anal. Chem. 1960, no. 32, pp. 1739-1742.
10. Khaziev F.H. Metody pochvennoi enzim-ologii [Methods of soil enzymology]. Moscow: Nauka Publ., 2005, 252 p.
11. Baldrian P., Trogl J., Frouz J., Snajdr J., Valaskova V., Merhautova V., Cajthaml T., He-rinkova J. Enzyme activities and microbial biomass in top soil layer during spontaneous succession in spoil heaps afterbrown coal mining. Soil. Biol. Bio-chem. 2008, no. 40, pp. 2107-2115.
12. Ivanova T.I., Kuz'mina N.P., Savvinov D.D. The number of microorganisms in the permafrost soils of Alas Muru. Nauka i obrazovanie. 2007, no. 2, pp. 76-82. (In Russian)
БИБЛИОГРАФИЧЕСКИМ СПИСОК
1. Шлегель Г. Общая микробиология / пер. с нем.; 6-е изд., перераб. и доп. М.: Мир, 1987. 567 с.
2. Ахметшина Э.А., Сироткин А.С. Анализ фосфолипидных жирных кислот микроорганизмов как биомаркеров окружающей среды // Вестник Казанского технологического университета. 2014. Т. 17. N19. С. 233-236.
3. Kurän P., Trögl J., Noväkovä J., Pilarovä V., Dänovä P., Pavlorkovä J., Kozler J., Noväk F., Popelka J.
Biodegradation of spilled diesel fuel in agricultural soil: Effect of humates, zeolite and bioaugmentation // The Scientific World Journal. 2014. Vol. 2014. 8 p. http://dx.doi.org/10.1155/2014/642427
4. Trögl J., Jirkovä I., Zemänkovä P. et al. Estimation of the quantity of bacteria encapsulated in Lentikats Biocatalyst via phospholipid fatty acids content: a preliminary study // Folia Microbiol. 2013. Vol. 58. P. 135-140.
5. Trogl J., Jirkova,I., Kuran P., Achmetshina E., Brovdyova T., Sirotkin A., Kirilina T. Phospholipid Fatty Acids as Physiological Indicators of Para-coccus Denitrificans Encapsulated in Silica Sol-Gel Hydrogels // Sensors. 2015. No. 15. P. 3426-3434.
6. Federici E., Giubilei M.A., Cajthaml T., Petruccioli M., D'Annibale A. Lentinus (Panus) Tigri-nus augmentation of a historically contaminated soil: Matrix Decontamination and Structure and Function of the Resident Bacterial Community // Journal Hazard. Mater. 2011. No 186. P. 1263-1270.
7. Trogl J., Pavlorková J., Packová P., Seják J., Kuráñ P., Popelka J., Pacina J. Indication of Importance of Including Soil Microbial Characteristics into Biotope Valuation Method // Sustainability. 2016. No. 8. P. 253-263.
8. Mj0s S.A. Identification of Fatty Acids in Gas Chromatography by Application of Different Tempe-
Contribution
Aida R. Khabibullina, Tatyana V. Vdovina, Alexander S. Sirotkin, Josef Trogl, Tafjána Brovdyová, Pavel Kuráñ carried out the experimental work, on the basis of the results summarized the material and wrote the manuscript. Aida R. Khabibullina, Tatyana V. Vdovina, Alexander S. Sirotkin, Josef Trogl, Tafjána Brovdyová, Pavel Kuráñ have equal author's rights and bear equal responsibility for plagiarism.
Conflict of interests
The authors declare no conflict of interests regarding the publication of this article.
AUTHORS' INDEX
Aida R. Khabibullina El
Postgraduate Student Kazan National Research Technological University e-mail: aida_khabibullin@mail.ru
Tatyana V. Vdovina
Ph.D. (Engineering), Associate Professor Department of Industrial Biotechnology Kazan National Research Technological University e-mail: tvkirilina@gmail.com
Alexander S. Sirotkin
Dr. Sci. (Engineering), Professor, Head of the Department of Industrial Biotechnology Kazan National Research Technological University e-mail: asirotkin66@gmail.com
Josef Trogl
Ph.D. (Engineering), Head of the Department of Technical Science University of Jan Evangelista Purkine e-mail: Josef.Trogl@ujep.cz
rature and Pressure Programs on a Single Capillary Column // Journal Chromatogr. A 2003. Vol. 1015. P. 151-161.
9. Miwa T.K., Mikolajczak K.L., Earle F.R., Wolff I.A. Identification of peaks in gas-liquid chromatography // Anal. Chem. 1960. No. 32. P. 1739-1742.
10. Хазиев Ф.Х. Методы почвенной энзи-мологии. М.: Наука, 2005. 252 с.
11. Baldrian P., Trogl J., Frouz J., Snajdr J., Valaskova V., Merhautova V., Cajthaml T., He-rinkova J. Enzyme activities and microbial biomass in top soil layer during spontaneous succession in spoil heaps afterbrown coal mining // Soil. Biol. Biochem. 2008. No. 40. P. 2107-2115.
12. Иванова Т.И., Кузьмина Н.П., Савви-нов Д.Д. Численность микроорганизмов в мерзлотных почвах аласа Мюрю // Наука и образование. 2007. N 2. С. 76-82.
Критерии авторства
Хабибуллина А.Р., Вдовина Т.В., Сироткин А.С., Трёгл Й., Бровдыова Т., Куран П. выполнили экспериментальную работу, на основании полученных результатов провели обобщение и написали рукопись. Хабибуллина А.Р., Вдовина Т.В., Сироткин А.С., Трёгл Й., Бровдыова Т., Куран П. имеют на статью равные авторские права и несут равную ответственность за плагиат.
Конфликт интересов
Авторы заявляют об отсутствии конфликта интересов.
СВЕДЕНИЯ ОБ АВТОРАХ
Хабибуллина Аида Рамилевна СЕН ,
аспирант
Казанский национальный исследовательский технологический университет e-mail: aida_khabibullin@maul.ru
Вдовина Татьяна Владимировна,
к.т.н., доцент кафедры
промышленной биотехнологии
Казанский национальный исследовательский
технологический университет
e-mail: tvkirilina@gmail.com
Сироткин Александр Семенович,
д.т.н., профессор, заведующий кафедрой промышленной биотехнологии Казанский национальный исследовательский технологический университет e-mail: asirotkin66@gmail.com
Трёгл Йозеф,
к.т.н., заведующий кафедрой технических наук Университет Яна Евангелиста Пуркине e-mail: Josef.Trogl@ujep.cz
Tat'jána Brovdyová
Ph.D. (Chemistry), Assistant Professor Department of Technology and Material Engineering University of Jan Evangelista Purkine e-mail: tatjana.brovdyova@ujep.cz
Pavel Kuráñ
Vice-Dean of Science
University of Jan Evangelista Purkine
e-mail: P.Kuran@gmx.de
Бровдыова Татьяна,
к.х.н., доцент кафедры технологии и материаловедения
Университет Яна Евангелиста Пуркине e-mail: tatjana.brovdyova@ujep.cz
Куран Павел,
вице-декан по науке
Университет Яна Евангелиста Пуркине
e-mail: P.Kuran@gmx.de
