|Year : 2017 | Volume
| Issue : 2 | Page : 106-114
Analysis of chemical signatures of alkaliphiles using fatty acid methyl ester analysis
Basha Sreenivasulu1, Chinthala Paramageetham1, Dasari Sreenivasulu2, Bukke Suman2, Katike Umamahesh2, Gundala Prasada Babu1
1 Department of Microbiology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
2 Department of Biochemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
|Date of Web Publication||23-Jun-2017|
Department of Microbiology, Sri Venkateswara University, Tirupati - 517 502, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Fatty acids occur in nearly all living organisms as the important predominant constituents of lipids. While all fatty acids have essentially the same chemical nature, they are an extremely diverse group of compounds. Materials and Methods: To test the hypothesis, fatty acids of alkaliphiles isolates, Bacillus subtilis SVUNM4, Bacillus licheniformis SVUNM8, Bacillus methylotrohicus SVUNM9, and Paenibacillus dendritiformis SVUNM11, were characterized compared using gas chromatography-mass spectrometry (GC-MS) analysis. Results: The content of investigated ten fatty acids, 1, 2-benzenedicarboxylic acid butyl 2-methylpropyl ester, phthalic acid, isobutyl 2-pentyl ester, dibutyl phthalate, cyclotrisiloxane, hexamethyl, cyclotetrasiloxane, octamethyl, dodecamethyl, heptasiloxane 1,1,3,3,5,5,7,7,9,9,11,11,13,13-etradecamethyl, 7,15-dihydroxydehydroabietic acid, methyl ester, di (trimethylsilyl) ether, hentriacontane, 2-thiopheneacetic acid, undec-2-enyl ester, obviously varied among four species, suggesting each species has its own fatty acid pattern. Conclusions: These findings demonstrated that GC-MS-based fatty acid profiling analysis provides the reliable platform to classify these four species, which is helpful for ensuring their biotechnological interest and novel chemotaxonomic.
Keywords: Alkaliphiles, fatty acid methyl ester, gas chromatography analysis, muscovite mines
|How to cite this article:|
Sreenivasulu B, Paramageetham C, Sreenivasulu D, Suman B, Umamahesh K, Babu GP. Analysis of chemical signatures of alkaliphiles using fatty acid methyl ester analysis. J Pharm Bioall Sci 2017;9:106-14
|How to cite this URL:|
Sreenivasulu B, Paramageetham C, Sreenivasulu D, Suman B, Umamahesh K, Babu GP. Analysis of chemical signatures of alkaliphiles using fatty acid methyl ester analysis. J Pharm Bioall Sci [serial online] 2017 [cited 2019 Oct 23];9:106-14. Available from: http://www.jpbsonline.org/text.asp?2017/9/2/106/208900
| Introduction|| |
The biological diversity of the Indian subcontinent is one of the richest in the world owing to its vast geographic area, varied topography and climate, and the juxtaposition of several biogeographical regions. Various types of diverse microenvironments and unique ecosystems such as boiling waters, deep sea vents, salts pans, acid mine drainages, cold environment, and deep subsurface environments are present in India that are home to diverse populations of microorganisms.
Muscovite ore granitic pegmatites are the source of muscovite sheet in sedimentary rocks. Formation of sedimentary rocks is one of the important parts of the rock cycle. These ultramafic rocks result in challenging environments for life in continental sites due to the combination of extremely high pH, low salinity, and lack of obvious electron acceptors and carbon sources. In sedimentary rocks, a variety of alkaliphilic microorganisms survives and grows. In these alkaline environments, pH increase is due to microbial ammonification and sulfate reduction and by water derived from leached silicates minerals. Recently, many bacteria capable of growing at a pH as high as 10.5 have been isolated for physiological interest as well as industrial applications.
Fatty acids are organic compounds commonly found in living organisms. They are abundant in the phospholipid bilayer of bacterial membranes. Their diverse chemical and physical properties determine the variety of their biochemical functions. This diversity, which is found in unique combinations in various bacterial species, makes fatty acid profiling a useful identification tool.
The cellular fatty acid analysis for bacterial identification is based on the specific fatty acid composition of the cell wall. The fatty acids are extracted from cultured samples and are separated using gas chromatography (GC). A computer-generated unique profile pattern of the extracted fatty acids is compared through pattern recognition programs, to the existing microbial databases. These databases include fatty acid profiles coupled with an assigned statistical probability values indicating the confidence level of the match. This has become very common in biotechnology. Fatty acid methyl esters (FAMEs) have long been recognized as useful biochemical markers for bacterial classification and characterization.,, The types and relative abundances of fatty acids produced within a cell are largely determined by an organism's genotype and can be used for identification of different species  and strains , and for discriminating between free spores and vegetative cells., Commercial systems that streamline fatty acid extraction and detection procedures  have facilitated the extensive use of fatty acid profiling to identify bacteria in clinical, agricultural, and biodefense settings.,,
FAME analysis has been used to characterize microbial communities in aquifer environments and sediments in some studies.,, Consequently, phospholipid fatty acid analysis is generally preferred for microbiological studies of such environments.,,, In contaminated soils that fatty acids are evaluated to estimate the structure of microbial community and metabolic activity,,, to identify the contamination of surface water polluted by soil which nearby agricultural fields and wooded riparian zones, to evaluate the application of microorganisms to a particular condition,, to found the microbial community distribution in terms of structure,, to determine the relative changes in abundance of microorganisms like bacteria and fungi. The Bacillus sp. strain C6 is characterized by a high content of ramified (iso- and anteiso-C15:0 and C17:0) fatty acids that compose approximately 85% of the total fatty acid pool. The use of fatty acids as biomarkers to analyze the microbial community in air biofilters and polluted soils has also been proposed.,,, This paper describes the fatty acid composition of alkaliphiles in the presence of various branched-chain organic acids.
| Materials and Methods|| |
Four bacteria were chosen for this study: Bacillus subtilis SVUNM4, Bacillus licheniformis SVUNM8, Bacillus methylotrohicus SVUNM9, and Paenibacillus dendritiformis SVUNM11 were isolated from the subsurface environments and metamorphic igneous rocks, gudur division. Muscovite samples (1.0 g) were powdered and mixed with 10 ml of saline solution (0.8% NaCl) in a conical flask and were incubated on the shaker for 30 min at room temperature (30°C ± 2°C). The culture flasks were then allowed to stand for 15 min for the sediment to settle before serial dilution. The slightly turbid supernatant (1.0 ml) and water samples (1.0 ml) were serially diluted (10-fold serial dilution) with normal saline. A 0.1 ml of appropriate dilutions was spreaded onto the plates containing polypeptone-yeast extract-glucose (PPYG) agar (pH 10.5) for selective alkaliphilic bacteria isolation.
Categorization of alkaliphiles based on pH preference
Purified predominant isolates from PPYG agar (pH 10.5) were inoculated into four sets of PPYG broth tubes of pH 7.0, 9.0, 10.0, 11.0, and 12.0 to obtain obligate alkaliphiles and incubated for 48 h at room temperature and the optical density values were recorded on a spectrophotometer at 630 nm. The isolates showing optimum growth at pH 10–12 were selected and considered as true alkaliphiles.
Fatty acid methyl ester analysis
Bacterial isolates were grown on trypticase soy agar at their optimum growth conditions. Whole cell fatty acids were extracted from cell material according to the Musical Instrument Digital Interface (MIDI) protocol. Overnight grown bacterial culture was taken (approximate 40 mg pellet) in a clean screw capped glass tube and 1 ml of Reagent I (45 g NaOH + 150 ml CH3 OH + 150 ml DW) was added to it. The tube was sealed with Teflon-lined screw caps, vortexed briefly, and heated in a boiling water bath for 5 min. The tube was vigorously vortexed for 5–10 s and returned to the water bath (100°C) to complete the 30-min heating (saponification step). The tube was cooled uncapped and 2 ml of Reagent II (325 ml of 6 N HCl + 275 ml CH3 OH) was added. The tube was capped again and briefly vortexed. After vortexing, the tube was heated for 10 min at 80°C (this methylation step is critical with time and temperature). Addition of 1.25 ml of Reagent III (200 ml hexane + 200 ml methyl tetra butyl ether) to the cooled tube was followed by recapping and gentle tumbling on a clinical rotator for about 10 min. The tube was uncapped again and the aqueous (lower) phase was pipetted out and discarded (extraction step). About 3 ml of Reagent IV (10.8 g NaOH + 900 ml D/W) was added to the organic phase remaining in the tube; after that, the tube was recapped and tumbled for 5 min. Following uncapping, about 2/3 of the organic phase was pipetted into a GC vial which was capped and ready for analysis. Gas chromatographic analysis was performed on a GC Sherlock fatty acid identification system (New York, USA) fitted with cross-linked methyl silicon fused capillary column (25 m, 0.2 mm i.d.), flame ionization detector, and a sampler. Helium was used as carrier gas. The sample was injected at oven temperature of 50°C. After 1 min, the oven temperature was raised to 170°C at the rate of 30°C/min and then to 270°C at the rate of 2°C/min and finally to 300°C at 5°C/min.
| Results|| |
Isolation of alkaliphiles from muscovite ore was carried out using PPYG medium with a pH of 10.5. Single, discrete colonies were picked up and purified by repeated streaking. A total of four predominant isolates were selected and named as SVUNM4, SVUNM8, SVUNM9, and SVUNM11 and purified by repeated streaking. Their cultural characteristics such as form, elevation, margin, color, and size were determined. The isolates' size ranges from pinhead to large, without any extracellular or intracellular pigmentation. The colony form of the isolates ranges from circular to rhizoid; the colony margin ranges from entire, filamentous to lobate; the elevation of the isolates ranges from unrex, raised to flat. The color of the colonies ranges from white, yellow, light brown to purple.
The isolate SVUNM4 was in pin head, medium to large, purple, circular with entire margin and unrex elevation. The isolate SVUNM8 was medium to large, yellow, circular, with filamentous margin with raised elevation. The cultural characteristics of the isolate SVUNM9 include small in size, white, irregular, with entire margin and raised elevation. The isolate SVUNM11 exhibited the following structural characteristics. The size of the colonies was large, cream-colored, irregular, with filamentous margin and flat elevation.
Categorization of alkaliphiles for pH preferences
Alkaline-adapted microorganisms can be classified into two main groups; they are alkaliphilic and alkali tolerant according to Krulwich. Alkaline tolerants show optimal growth at the pH 7.0–9.5, but in capable of survival at pH above 9.5, whereas alkaliphiles show optimal growth at the pH range 10.0–12.0. In our study, the isolates were exposed to pH 7, 9, 10, and 12 to categorize them. Based on pH preference, the three isolates, namely, SVUNM4, SVUNM8, and SVUNM9 were alkaliphilic and one isolate, namely, SVUNM11 was alkali tolerant.
Analysis of chemical signatures of alkaliphiles using fatty acid methyl esters analysis
FAMEs were isolated from alkaliphiles B. subtilis SVUNM4 and were characterized by GC-mass spectrometry (MS) retention times and equivalent chain length (ECL) values. These isolates produced major fatty acids. The chromatogram analysis showed that they contained 1, 2-benzenedicarboxylic acid butyl 2-methylpropyl ester (42.743%), phthalic acid, isobutyl 2-pentyl ester (21.753%), dibutyl phthalate (35.684%) as major abundant fatty acid [Figure 1]. The abundance of each fatty acid is presented in [Table 1]. The fatty acid profiles agree with that of other members of species B. subtilis.
|Figure 1: Gas chromatography-mass spectrometry spectra of the fatty acid methyl esters of Bacillus subtilis sp. SVUNM4|
Click here to view
|Table 1: Gas chromatography-mass spectrometry spectra of the fatty acids extracted from Bacillus subtilis sp. SVUNM4|
Click here to view
FAMEs were isolated from B. licheniformis SVUNM8 were characterized by GC-MS retention times and ECL values. These isolates produced major fatty acids. The chromatogram analysis showed that it contained cyclotrisiloxane, hexamethyl (50.089%), octamethyl (27.771%), dodecamethyl (15.794%), heptasiloxane 1, 1, 3, 3, 5, 5, 7, 7, 9, 9, 11, 11, 13, 13-tetradecamethyl (2.607%), 7, 15-dihydroxydehydroabietic acid, methyl ester, di (trimethylsilyl) ether (3.74%) as major abundant fatty acid [Figure 2]. The abundance of each fatty acid is presented in [Table 2]. The fatty acid profiles agree with that of other members of species B. licheniformis.
|Figure 2: Gas chromatography-mass spectrometry spectra of the fatty acid methyl esters of Bacillus licheniformis sp. SVUNM8|
Click here to view
|Table 2: Gas chromatography-mass spectrometry spectra of the fatty acids extracted from Bacillus licheniformis sp. SVUNM8|
Click here to view
FAMEs were isolated from B. methylotrohicus SVUNM9 were characterized by GC-MS retention times and ECL values. These isolates produced major fatty acids. The chromatogram analysis showed that it contained hentriacontane (74.593%), 1, 2 benzenedi carboxylic acid, mono (2-ethylhexyl) ester (25.388%) as major abundant fatty acid [Figure 3]. The abundance of each fatty acid is presented in [Table 3]. The fatty acid profiles agree with that of other members of B. methylotrophicus.
|Figure 3: Gas chromatography-mass spectrometry spectra of the fatty acids methyl ester of Bacillus methylotrophicus sp. SVUNM9|
Click here to view
|Table 3: Gas chromatography-mass spectrometry spectra of the fatty acids extracted from Bacillus methylotrophicus sp. SVUNM9|
Click here to view
FAMEs were isolated from P. dendritiformis SVUNM11 were characterized by GC-MS retention times and ECL values. These isolates produced major fatty acids. The chromatogram analysis showed that it contained 2-thiopheneacetic acid, undec-2-enyl ester (11.477%), hentriacontane, 1,2 benzene dicarboxylic acid, mono (2-ethylhexyl) ester (23.072%) as major abundant fatty acid [Figure 4]. The abundance of each fatty acid is presented in [Table 4]. The fatty acid profiles agree with that of other members of P. dendritiformis.
|Figure 4: Gas chromatography-mass spectrometry spectra of the fatty acids esters of Paenibacillus dentritiformis sp. SVUNM11|
Click here to view
|Table 4: Gas chromatography-mass spectrometry of the fatty acids extracted from Paenibacillus dendritiformis sp. SVUNM11|
Click here to view
| Discussion|| |
The microbial studies pertaining to deep surface often contain unique assets, and they are ideal for several biotechnological and environmental applications. Further, the microbial studies pertaining to muscovite ore are very scarce. The choice of indigenous microbes for this investigation is essentially due to possibility of their better acclimatization to the biobeneficiation environments. Alkaliphilic prokaryotes, in their rich phylogenetic diversity and metabolic versatility, are central participants in useful bioprocessing settings. A range of pH values are used by different investigation to define extreme alkaliphiles, alkaliphiles, and alkaline tolerant bacteria. Extremely alkaliphilic bacteria are generally defined as those that grow at an external pH ≥10 (the more extreme strains growing at pH ≥12), moderate alkaliphiles as those that can grow in the pH 9–10 range, the alkaline tolerant bacteria as those that can survive and grow suboptimally at ~pH 9. It has always been a very interesting and challenging area to explore microbes that reside in extreme environment. Alkaliphiles were isolated using culture-dependent analysis. Microbial communities in subsurface muscovite mine have escaped our attention so far. These mines harbor unique extreme environments for microorganisms, both natural and anthropogenic, including extreme temperature pressure, low oxygen concentration toxic heavy metals oligotrophic conditions, low water availability, and pH. The physical and chemical characterization of the sample also revealed its nature. The chemical signatures of the four alkaliphilic isolates were analyzed using FAME analysis. A total of ten fatty acids were identified in all the four isolates. The predominant fatty acid was 1, 2-benezendicarboxylic acid butyl 2-methylpropyl ester and hentricantane, phthalic acid, isobutyl 2-pentyl ester, dibutyl phthalate. The abundance of each fatty acid is presented in [Table 5]. Pujari  reported 12 fatty acids from marine bacteria. Cooney  reported 13 fatty acids from marine Fungi. Devi  reported ten fatty acids from marine Fungi. Thompson  due to the estimation of community diversity by FAME content of bacterial isolates exposed that the genetically modified Pseudomonas fluorescens had less impact on the bacterial community than the wild type. Haack  applied principal components analysis of MIDI-FAMEs profiles in a clear separation of two different communities. They found comparative similarities and differences of microbial communities that differed in taxonomic status. Petersen and Klug  observed a major change in the fatty acid profiles in soil at a near-freezing temperature and 25°C, whereas Nazih  did not observe changes in FAMEs profiles at 22°C and 34°C. Kozdrój  used FAME analysis to assess microbial community structure in technogenous wastes such as coal mine spoil, nonferrous metallurgical slag, and coal fly ash. He noticed a high content of 18:2 ω6,9 in the metallurgical slag, indicating the domination of Fungi in this waste. In difference, representatives of the Cytophaga-Flavobacterium group, for which 16:1 ω5c fatty acid was used as a marker, dominated in the coal fly ash. Bossio and Scow  concluded that fatty acid profiles were sensitive indicators of changes occurring in the structure of soil microbial communities due to agricultural management. In previous cases, information obtained from lipid analysis provides insight into the community composition as well. It has been proposed that particular groups of microorganisms contain characteristic fatty acid profiles that can be used as biomarkers. Mummey  applied FAME biomarkers to monitor the recovery of ecosystems following surface mine reclamation. In this study, it was found that the percentage of FAME bacterial to fungal biomarkers reflected changes in other indicators of soil health signifying that this ratio is a useful indicator of reclamation progress. Fatty acids from whole cells of Chlorobium are within the range of C12–C18, and the main ones are n-tetradecanoic (C14:0), hexadecenoic (C16:1), and n-hexadecanoic (C16:0).,, Cha et al. identified signature fatty acids of Nocardia amarae : 1o8, 16: 1o6c, i15: 0 2OH) and used their relative abundance to reveal their potential to quantitatively monitor the abundance of Nocardia in mixed liquor samples of activated sludge. The use of fatty acid patterns has also been applied to full-scale biological wastewater treatment plants to estimate activated sludge microbial communities, demonstrating that FAME profiles could be a valuable technique in evaluating the alters in bacterial communities when a wastewater treatment system is operated in a particular way.
|Table 5: Fatty acid methyl esters profiles of the SVUNM4, SVUNM8, SVUNM9 and SVUNM11|
Click here to view
| Conclusions|| |
Although advances in the technologies for fatty acid analysis have been reported, the fatty acid analysis of alkaliphiles prokaryotes remains scarce, and it is very important to encourage the investigation of these microorganisms. The chemical signature of the four alkaliphilic isolates was analyzed using FAME analysis. A total of ten fatty acids were identified in all the four isolates. The predominant fatty acid was 1,2-benezendicarboxylic acid butyl 2-methylpropyl ester and hentricantane, phthalic acid, isobutyl 2-pentyl ester, dibutyl phthalate. With new sources of genetic materials from high pH bacteria and the application of new technologies – for example, lipidomics – which are more precise, it will be possible to discover new fatty acid structures and to evaluate the sources of these compounds of biotechnological interest and novel chemotaxonomic biomarkers.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ratledge C, Wilkinson SG. Microbial lipids. Vol. 1. New York: Academic Press Inc.; 1988.
Welch DF. Applications of cellular fatty acid analysis. Clin Microbiol Rev 1991;4:422-38.
Weyent RS, Moss CW, Weaver RE, Hollis DG, Jordan JG, Cook EC, et al.
Bacterial identification by cellular fatty acid analysis: Identification of unusual Gram-negative aerobic and facultatively anaerobic bacteria. 2nd
ed. Baltimore, MD: Williams & Wilkins; 1996. p. 565-721.
Vandamme P, Pot B, Gillis M, de Vos P, Kersters K, Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996;60:407-38.
Leonard RB, Mayer J, Sasser M, Woods ML, Mooney BR, Brinton BG, et al.
Comparison of MIDI Sherlock system and pulsed-field gel electrophoresis in characterizing strains of methicillin-resistant Staphylococcus aureus
from a recent hospital outbreak. J Clin Microbiol 1995;33:2723-7.
Von Wintzingerode F, Rainey FA, Kroppenstedt RM, Stackebrandt E. Identification of environmental strains of Bacillus mycoides
by fatty acid analysis and species-specific rDNA oligonucleotide probe. FEMS Microbiol Ecol 1997;24:201-9.
Scandella CJ, Kornberg A. Biochemical studies of bacterial sporulation and germination. XV. Fatty acids in growth, sporulation, and germination of Bacillus megaterium
. J Bacteriol 1969;98:82-6.
Song Y, Yang R, Guo Z, Zhang M, Wang X, Zhou F. Distinctness of spore and vegetative cellular fatty acid profiles of some aerobic endospore-forming bacilli. J Microbiol Methods 2000;39:225-41.
Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI technical note. Newark, DE: MIDI; 1990.
Kim WY, Song TW, Song MO, Nam JY, Park CM, Kim KJ, et al
. Analysis of cellular fatty acid methyl esters (FAMEs) for the identification of Bacillus anthracis
. J Korean Soc Microbiol 2001;35:31-40.
White DC, Lytle CA, Gan YD, Piceno YM, Wimpee MH, Peacock AD, et al.
Flash detection/identification of pathogens, bacterial spores and bioterrorism agent biomarkers from clinical and environmental matrices. J Microbiol Methods 2002;48:139-47.
Elvert M, Boetius A, Knittel K, Jørgensen BB. Characterization of specific membrane fatty acids as chemotaxonomic markers for sulfate-reducing bacteria involved in anaerobic oxidation of methane. Geomicrobiol J 2003;20:403-19.
Knief C, Altendorf K, Lipski A. Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of 13C-labelled fatty acids. Environ Microbiol 2003;5:1155-67.
Dias AC, Andreote FD, Andreote FD, Lacava PT, Sa' AL, Melo IS, et al.
Diversity and biotechnological potential of culturable bacteria from Brazilian mangrove sediment. World J Microbiol Biotechnol 2009;25:1305-11.
Salomonova S, Lamacova J, Rulik M, Rolcik J, Cap L, Bednar P, et al
. Determination of phospholipid fatty acids in sediments. Chemica 2003;42:39-49.
Harji RR, Bhosie NB, Garg A, Sawant SS, Venkat K. Sources of organic matter and microbial community structure in the sediments of the Visakhapatnam harbor, East coast of India. Chem Geol 2010;276:309-17.
Pratt B, Riesen R, Johnston CG. PLFA analyses of microbial communities associated with PAH-contaminated riverbank sediment. Microb Ecol 2012;64:680-91.
Zelles L. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterization of microbial communities in soil: A review. Biol Fertil Soils 1999;219:111-29.
Kozdroj J, van Elsas JD. Structural diversity of microbial communities in arable soils of a heavily industrialised area determined by PCR-DGGE fingerprinting and FAME profiling. Appl Soil Ecol 2001;17:31-42.
Pinkart HC, Ringelberg DB, Piceno YM, Macnaughton SJ, White DC. Biochemical approaches to biomass measurements and community structure analysis. In: Hurst CJ, Crawford RL, Knudsen GR, McInerney MJ, Stentzenbach LD, editors. Manual of Environmental Microbiology. Washington, DC: ASM Press; 2002. p. 101-13.
Banowetz GM, Whittaker GW, Dierksen KP, Azevedo MD, Kennedy AC, Griffith SM, et al.
Fatty acid methyl ester analysis to identify sources of soil in surface water. J Environ Qual 2006;35:133-40.
Werker AG, Hall ER. Using microbial fatty acids to quantify, characterize and compare biofilm and suspended microbial population in wastewater treatment systems. Water Sci Technol 1998;38:273-80.
Quivey RG Jr., Faustoferri R, Monahan K, Marquis R. Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutans. FEMS Microbiol Lett 2000;189:89-92.
Sundh I, Nilsson M, Borga P. Variation in microbial community structure in two boreal peatlands as determined by analysis of phospholipid fatty acid profiles. Appl Environ Microbiol 1997;63:1476-82.
Zeller V, Bardgett RD, Tappeiner U. Site and management effects on soil microbial properties of subalpine meadows: A study of land abandonment along a North-South gradient in the European Alps. Soil Biol Biochem 2001;33:639-49.
Grabova GY, Dragovoz IV, Zelena LB, Ostapchuk AN, Avdeeva LV. Polyphasic taxonomic analysis of Bacillus sp. strain C6 – The antagonist of phytopathogenic microorganisms. Cytol Genet 2016;50: 251-6.
Zelles L, Palojarvi A, Kandeler E, von Lutzow M, Winter K, Bai QY, et al
. Changes in soil microbial properties and phospholipid fatty acid fractions after chloroform fumigation. Soil Biol Biochem 1997;29:1325-36.
Sorokin DY, van den Bosch PL, Abbas B, Janssen AJ, Muyzer G. Microbiological analysis of the population of extremely haloalkaliphilic sulfur-oxidizing bacteria dominating in lab-scale sulfide-removing bioreactors. Appl Microbiol Biotechnol 2008;80:965-75.
Sarethy IP, Saxena Y, Kapoor A, Sharma M, Sharma SK, Gupta V, et al
. Alkaliphilic bacteria: Applications in industrial biotechnology. J Ind Microbiol Biotechnol 2011;38:769-90.
Krulwich TA, Agus R, Schneier M, Guffanti AA. Buffering capacity of bacilli that grow at different pH ranges. J Bacteriol 1985;162:768-72.
Pujari S, Roy R, Bhosle S. Screening of bacteria from sediments of coastal ecosystem, as potential sources of alpha linolenic acid. Indian J Mar Sci 2001;33:243-7.
Cooney JJ, Doolittle MM, Grahl-Nielsen O, Haaland IM, Kirk PW Jr. Comparison of fatty acids of marine Fungi
using multivariate statistical analysis. J Ind Microbiol 1993;12:373-8.
Devi P, Divya Shridhar MP, D'Souza L, Naik CG. Cellular fatty acid composition of marine-derived Fungi
. Indian J Mar Sci 2006;35:359-63.
Thompson IP, Ellis RJ, Bailey MJ. Autecology of a genetically modified fluorescent pseudomonad on a sugar beet. FEMS Microbiol Ecol 1995;17:1.
Haack SK, Garchow H, Odelson DA, Forney LJ, Klug MJ. Accuracy, reproducibility, and interpretation of fatty acid methyl ester profiles of model bacterial communities. Appl Environ Microbiol 1994;60:2483-93.
Petersen SO, Klug MJ. Effects of sieving, storage, and incubation temperature on the phospholipid fatty acid profile of a soil microbial community. Appl Environ Microbiol 1994;60:2421-30.
Nazih N, Finlay-Moore O, Hartel PG, Fuhrmann JJ. Whole soil fatty acid methyl ester (FAME) profiles of early soybean rhizosphere as affected by temperature and matric water potential. Soil Biol Biochem 2001;33:693.
Kozdrój J. Microflora of technogenous wastes characterised by fatty acid profiling. Microbiol Res 2000;155:149-56.
Bossio DA, Scow KM. Impacts of carbon and flooding on soil microbial communities: Phospholipid fatty acid profiles and substrate utilization patterns. Microb Ecol 1998;35:265-78.
Mummey DL, Stahl PD, Buyer JS. Microbial biomarkers as an indicator of ecosystem recovery following surface mine reclamation. Appl Soil Ecol 2002;21:251-9.
Knudsen E, Jantzen E, Bryan K, Ormerod JG, Sirevag R. Quantitative and structural characteristics of lipids in Chlorobium
. Arch Microbiol 1982;132:149-54.
Kenyon CN, Gray AM. Preliminary analysis of lipids and fatty acids of green bacteria and Chloroflexus aurantiacus
. Arch Microbiol 1974;120:131-78.
Núñez-Cardona MT. Fatty acids analysis of photosynthetic sulfur bacteria by gas chromatography. Gas chromatography-biochemicals, narcotics and essential oils. Mexico. InTech Publications; 2012. p. 117-38.
Werker AG, Becker J, Huitema C. Assessment of activated sludge microbial community analisis in full-scale biological wastewater treatment plants using patterns of fatty acid isopropyl esters (FAPEs). Water Res 2003;37:2162-72.
Cha DK, Fuhrmann JJ, Kim DW, Golt CM. Fatty acid methyl ester (FAME) analysis for monitoring Nocardia levels in activated sludge. Water Res 1999;33:1964-6.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]