Analysis of lipid biomarkers in rocks of Archean crystalline basement

Shekhovtsova N.V., Osipov G.A., Verkhovtseva N.V., Pevzner L.A. Analysis of lipid biomarkers in rocks of the Archean crystalline basement //Proceedings of SPIE.- 2003.- Vol.4939.- P. 160 — 168. 

Analysis of lipid biomarkers in rocks of Archean crystalline basement

 Nina V. Shekhovtsova*^a, George A. Osipov^b, Nadejda V. Verkhovtseva^a, Lev A. Pevzner^c

^aChair of Botany and Microbiology, Yaroslavl State Univ./

proezd Matrosova, 9, Yaroslavl, Russia, 150057

^bResearch Group of Academician Yu. Isakov, Russian Academy of Medical Science/Moscow, Russia

^cFSUE SIC «Nedra»/Yaroslavl, Russia

ABSTRACT
The Earst-European platform Archean crystalline basement rocks opened by Vorotilov Deep Well (VDW) were studied within depths 2575 — 2805 m. VDW was drilled through Puchezh-Katunki impact structure and opened some rocks characterized by high magnetic saturation. Micro-dispersed structure of magnetite indicated on a possibility of its biogenic origin. Really some pure cultures of magnet-ordered compound producing bacilli were isolated. Thus, the identification of fatty acids and other lipid components of microbial cells inside rocks was made to establish the iron reduction role in common biochemical activity in deep subsurface.
34 microbial lipid markers were detected by gas chromatography — mass-spectrometry and 22 species were identidied by private database. Bacteria of g. Bacillus reached 6,8 % in subsurface communities. However, representatives of gg. Clostridium (37,1 — 33,2 %) and Rhodococcus (27,6 — 33,7 %) were absolute dominants within studied depth interval.
Geochemical conditions in situ as well as physiological features of these microorganisms allow to constitute a following trophic chain: subsurface  fluid hydrocarbons ® it oxidizing rhodococci ® free aminoacids and biomass proteins (products of rhodococci metabolism) ® it fermenting clostridia. This syntrophic association may be a new basement for subsurface ecosystem and can support the magnet-ordered compounds production.
Keywords: Lipid biomarkers, Archean crystalline basement, impact structure, subsurface microbial communities

1. INTRODUCTION

Studying of microbial complex in situ is a key to receiving notions about real geochemical activity in rocks. Revealing of fossilized microorganisms by electrone microscope nothing says about their viability. Culturing methods very often don’t allow to render subsurface environment conditions as well as to guess a kind of carbon source for isolating bacteria. Molecular probe methods are very usefull to detect indigenous biodiversity. However, the results of very popular 16 S rRNA genes analysis are depend on success of DNA extraction and following PCR amplification. Lipid biomarkers identification is the most express method for studying species composition of microbial communities in situ. Recently it was successfully used for identification of microbiota in kaolin¹ as well as  fine structure of fossilized bacteria in Volyn kerite².
In present research this method has been used for understanding the bacteria role in magnetization of Archean crystalline basement rocks penetrated by Vorotilov Deep Well.^*

2. OBJECTS AND METHODS

2.1. Rock bedding conditions in  Puchezh-Katunki impact structure and Vorotilov deep well
The Puchezh-Katunki impact structure is situated in central part of the Russian Platform (56^o 58′ N, 43^o 43′ E) to the North of Nizhny Novgorod and it has diameter of about 80 km. The impact structure is not marked in relief. It is almost completely overlaid by the Middle — Upper Jurassic, Cretaceous and Cenozoic sediments with a thickness up to several hundreds of meters. In center of impact structure the Vorotilov deep well (VDW ) was drilled down to 5374 m with continuous coring by FSUE SIC «Nedra» as a certain contribution to the International Scientific Drilling Program (ICDP).
The Archean crystalline basement around the impact structure occurs at a depth about 1.8 — 2.2 km and it is mainly composed by biotite-amphibole gneiss. In a whole the Archean rocks underwent two stages of metamorphism at an amphibolite facies. The first stage had t^o = 700 — 800 ^oC and P = 6 — 8 kbar and the second one — t^o = 600 — 700 ^oC, P = 3.5 – 5.0 kbar. The regional metamorphism occured over 1800 — 1900 mln. years ago and a model age of gneiss substrate is estimated at 2.65 bln. years. But as estimated the impact event happened  about 180 mln. years ago.
Basic elements of the inner structure of Puchezh-Katunki impact crater are a peripheral ring terrace, ring trough and central uplift. The Vorotilov deep well penetrated the crystalline uplift in the central pit and at a depth 546 m it entered brecciated and broken-down crystalline rocks of the basement (authigenic breccia), which had been transformed by shock compression, additional heating and suffered impact melt injections and hydrothermal alterations. In near surface zone the rocks underwent a compression with an amplitude of more than 45 GPa and at a depth of 5 km — above 15 GPa. Core samples from the well clearly indicate attenuation of the indicated processes and the change of physical properties of the rocks: increase of density with depth (from 2.4 to 2.75 g/cm³), decrease of effective porosity (from 12 to 2 %), thermal conductivity etc. In the well there are observed rock blocks with high and low magnetization (interchange). Magnetic susceptibility and residual magnetization tend to go down with depth. The most contrast changes of rock properties are observed to a depth of 3300 m. Borehole geophysics indicated increase of rock resistivity with depth. Max t^o = +98.3 ^oC was measured at depth of 5270 m. A thermal metamorphism is accompanied by formation of magnetite. But preliminary it micro-dispersed structure was explained be two reasons. One is destruction of mineral material under impact. The other may was connected with magnetite biogenic properties.
Two studied core samples being a biotite-amphibole gneiss were isolated from interval 2575 – 2805 m. In situ these rocks had a temperature about 60 ^oC and were included in interval with increased content of gases: CO₂ > H₂ > N₂ > hydrocarbons. A little amount of non carbonate carbon was dispersed within rocks. A hard methane homologies dominated among hydrocarbons. Moreover these samples were taken from interval with  high magnetic susceptibility of rocks where only a micro-dispersed magnetite were found³.
2.2. Preparing rocks for analysis
Before microbiological analysis every core sample was sterilized 30 seconds by fire for killing contaminating undersurface microorganisms. Then purified sample were pounded in sterile conditions by hand into film isolated metal mortar which was previously autoclaved at 120 ^oC during 30 minutes. Pounded rock with particles no more than 1 mm in diameter was used as nature sample for fatty acid analysis as well as inoculate for isolation of iron reducing bacteria.
2.3. Detection of magnet-ordered compounds producing bacteria
Iron-reducing bacteria were isolated from VDW rocks as enrichment cultures with Lovley medium included lactate or acetate as a carbon source and freshly precipitated Fe(III)-hydroxide⁴. Further on from enrichment cultures it was isolated the pure cultures  and it’s ability to produce the magnet-ordered iron compounds was tested.
Magnetic parameters: magnetic susceptibility, c_par and magnetization saturation,s_sat, of rocks and cells were detected with the help of magnetic scales by Faradei method⁵.
2.4. Research of microbial marker content in Vorotilov deep well rocks by chromate-mass spectrometry
A pounded sample (0,6 g) was underwent the acid methanolysis  by 0,6 ml 1M HCL solution in methanol during 3 hours at 80 ^oC. In this stage both fatty acids and aldehydes release from compound lipids of microorganism and other cells within mixture. As the result a fatty acids were received as methyl esters, which were extracted by 200 mkl of hexane, after that were dried and treated by 20 mkl of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to 80 ^oC for 15 minutes to produce a trimethylsilyl esters of oxy-acids, alcohol and sterols. For analysis a reactionary mixture (2 ml) was introduce into injector of chromate-mass-spectrometer (GC-MS). This is a usual procedure for investigation of biologic matter lipid fraction.
GC-MS analysis was provided by chromate-mass-spectrometer GCMS-QP 2000 (Shimadzu, Japan). The quadrupole mass spectrometer has a resolution of 0.5 mass units over the whole mass range of 2 — 1000 amu. Ionization is performed by electrons at 70 eV. The sensibility of GC-MS system is 1 ng of methyl stearate in permanent scanning regime as well as 1 ng in selective ions regime. A fused quartz capillary column HP-5MS with length of 25 meters and internal diameter of 0.25 millimeters was used for chromatographic probe division. Chromatography was provide by programmed temperature regime from 130  to 320 ^oC with rate of 5 grad/min. The injector temperature was 280 as well as interface temperature — 250  ^oC.
Mass-fragmentography method (MF) was used to analysis of minor biogenic chemical components. A special program was design for selective detection with accumulation of specific ion signals from microorganism marker compounds. The program made periodic inquest of intensity among five temporal groups each from which includes up to 8 ions. Groups are ordered into probe chromatography  time from 4 till 45 minutes since moment of probe injection into chromatograph and occupy the range compounds from decane acid to sitosterol. Measurement algorithm allows to detect about 80 microbial marker compounds if 3 — 4 parameters per compound are used: two specific ions, characteristic retention time as well as correlation of chromatographic peak areas.
Calculation the species structures of rock microbial communities is provided on the private data base of fatty acid profiles and markers of microorganisms according earlier described protocol^1,6. According to fatty acid structure the identifying of isolated microbial pure cultures and comparing their residue profiles with library data are carried out with the help of both private identification program by image recognition method as well as a manuals of determinative bacteriology.
On mass-phragmentograms the marker peak areas were integrated automatically according to task program with using of internal standard. Then these data were input into account program prepared in electronic EXCEL tables. By 12-hydroxystearic acid a calibration results were used for numerical computation. The calculation of structure and number of effective microbial cells.
Calculation of community structure and effective microbial cells number. For composition of analysis program, identification algorithm as well as numerical calculation of effective number of cells as natural microbial community components are used an information about chemical composition of individual and collective markers of more than 500 species of microorganisms, assumed members of community.
Different microorganisms are known to have in total in cell envelope lipid structure about 200 of fatty acids differed it from plant and animal cells. Some compounds was appeared to associate with one taxon only. Cell number or weight of such microorganisms were accounted by marker compound concentration using known information about fatty acid content into microbial cell taking in consideration preparing and device calibration conditions.
Marker peak area is proportionate to its concentration and consequently to following microorganism content and so it is define as a cell number N₁ per unit of a volume or weight by a formula:
N₁ = A_i [M_st/(q₂×M_sam×A_st)]/Ri₁,
where expression within square brackets is permanent coefficient:
k = M_st/q₂/M_sam/A_st = M_st (mg)/5.1×10^(-15)g/M_sam(mg)/A_st
In this formulas   A_i — marker peak area; M_st — standard amount in mg, injected into the probe; M_sam — probe amount accordingly; A_st — standard peak area; R_i1 — marker part in percents with index i in fatty acid profile of defining microbe with number 1 (N₁); q₂ — coefficient which is equal 5.1×10^(-15) g and involve the basic magnitude for evaluation in cell number (this one is 5.9×10¹² microorganism cells included in 1 g of microbial biomass) as well as fatty acid amount per cell taken for 3 % in average.
Accordingly cell number af any following microorganism may be accounted by the same way N₂ = A_i×k/R_i2 and so on multiplying peak area of A_i marker on the coefficient k and dividing on marker part in percents (%) within fatty acid structure of this microorganism. Here we shall not concern the case when the same fatty acid originates from different microorganisms and then a more complex calculations are required for division of components. If a compound does not have marker property, i.e. may be  belong to pair or more taxons, then in this case a contributing part of every microorganism may be account if to use a resolution of equation system for two or more compounds.

3. RESULTS

3.1. Magnet-ordered compounds producing bacteria
Five strains of iron reducing bacteria g.Bacillus were isolated from VDW rocks: four cultures — from the depth 2575 m and two from 2805 m. However only two cultures produced strongly magnetic minerals in above mentioned media under anaerobic conditions (table 1). Interestingly that magnetic parameters of dry biomass exceeded those for rocks and positively correlated with increasing of rocks magnetic saturation degree along with its burial depth. It is necessary to note that strain of Bacillus sp.4 was single, which one produced magnet-ordered compounds in small amount under aerobic conditions (c_par.= 16,5×10^-9 I.U., s_sat.= 84,3×10^-3 A×m^-2×kg^-1).
Table 1
Magnetic parameters of rocks from VDW as well as isolated iron reducing bacteria

Thus, microbiological analysis of VDW rocks supports the opportunity of involvement of iron-reducing bacteria in genesis of rock magnetization that is agreed with results of recent microbiological studying the cores from Ural superdeep well⁷.
3.2. Fatty acid composition and approach for microbial community structure decoding
The structure of detected lipid components is shown on the table 2. There are an organisms  which may have identified compounds.
Table 2
The fatty acid structure inside of VDW  rock samples in reference to microorganisms having it more frequently 

Compound names: 17:1 — 17 is a number of carbon atoms; figure after colon is a quantity of double links; h — oxy-acid; a, i at the beginning means ramification of carbon chain; alc at the symbol end means alcohol. For example: ha17 is the 3-oxy-anteisoheptadecanoic acid, 2h24alc means the 2-oxy-tetracozanol alcohol.
VDW rock fatty acid structure research revealed some components which characterized procaryotic microbial community. In studied samples the main products of methanolysis lipid fraction were a fatty acids with saturated and unsaturated right chains, branched fatty acids and hydroxy acids (table 2). Finding in probes of fatty acids with 12 up 19 of carbon atoms are thought to be a bacterial biomass sign^8,9,10. Moreover analysis shown the presence those and only those fatty acids which belong to microorganisms what exclude a possibility its alternative origin as well an absence of  a following possible (after microorganisms death) chemical processes.
Separately remark the finding in rocks of 3-hydroxy acids, being exclusively bacteria sign. They testify about presence in ancient community of gramnegative microorganisms  which cell walls are included in. Cyanobacteria are also represented in gramnegative bacteria group and may be a main suppliers of these biomarkers. A fatty acid structure of studied rocks is typical for microbial community but no single microorganism. Some of fatty acids may be  refer to quite definite genera or species of modern microorganisms. For example, hydroxyacid type shows on the presence of pseudomonads related to Pseudomonas putida or representatives of g. Acinetobacter because there is 2-hydroxylauric acid as well as hydroxymerisric and hydropalmitic acids are typical for P. cepacea and cyanobacteria¹¹. Other possible candidates from marker table (Bacteroides, Cytophaga, etc.) should not regard because the branched oxyacids with 15 and 17 carbon atoms are absent. Hydroxystearic acid is known for two organisms: Acinetobacter diazotrophicus and cyanobacterium Gloebacter violaceous. 10-hydroxystearic acid  is characteristic for Clostridium perfringens and some others bacteria (including nocardia) which are capable to oxidize the oleic acid in a rich organic matter substrates. From the other characteristic fatty acids the 10-methyldecanoic (10Me16) should be mentioned because it is usually considered as sulfate reducing bacteria of a g. Desulfobacter. Here the 10Me16 in union with 10Me14 are obviously belong to actynomicetes¹². Remarkable amount of branched acids including evens (iso-meristic, iso-palmetic) and having in a view their ratio should be refer to bacteria gg. Bacillus, Kurthia, Arthrobacter, Cellulomonas. Thus, received fatty acid profiles of rocks testify about presence of fatty acids — residues of microbial community that didn’t undergo any changes through rapid conservation during mummification.
Profiles of two samples and its quality structures are very close that suggest a relationship between two rock microbial communities. Exclusion is linoleic acid (18:2) which is more in sample from the depth of 2575 m. This acid is most probably belong to microscopic fungi. In the sample from the depth 2805 m there are more gramnegative microorganisms evolving in its structure oxyacids 2h12, 2h14, h16, h18 (names are in table 2) as well as 11,12-hexadecenoic, heptadecanoic and cys-vaccenoic.
Information about lipid component structure being used for microorganism chemo-differentiation allows to refer a natural sample fatty acids to definite taxonomic groups: genera and sometimes species. A list of more probable microorganisms having this sign is in the last column of table 2. Since in most cases  a relation is not simple a following step of a preferable taxon choice has been done accordingly to information about physiological and trophic abilities of inhabitants in studied ecological niche as well as a habitat ecological conditions: temperature, pH, salinity, mineralization, oxygen concentration and etc. An objects of clinical microbiology which are for our opinion not appropriate to studied conditions are not included in table.
As it was mentioned above the oxyacids are the most specific markers of gramnegative bacteria. Here hydroxypalmitic (h16) and hydroxystearic (h18) acids are distinguished by concentrations. Some part of h16 acid may be relative to Pseudomonas cepacea (h14 is a marker) but it main amount is belong to other organisms: bateroids or cyanobacteria, for example, g. Gloebacter¹³. The presence of  a- and b-oxyacids (oxy-decanoic and oxy-lauric) definitely direct to availability of pseudomonads, Acinetobacter sps., probably Beggiatoa. The 10h18 acid attributes to Clostridium perfringens or to 2 — 3 other bacteria which are capable to produce this product as the result of oleic acid fermentative oxidation.
The simple acid explanation has less definiteness than in case with its hydroxic derivatives. However,  it may be supposed that moreover a branched acids with 13 carbon atoms refer to bacilli, i14 — to thermophilic Bacillus caldolyticus, 14:1D9 — to Clostridium, i17:1 — to Desulfovibrio or Desulfohalobium. Bacilli, actynomycetes and corynebacteria may be supposed to contribute the essential part of branched acids: i,a,15; i,a,17 and i16. 10Me-branched fatty acid refer to Rhodococcus (10Me16) or Thermoactinomyces (10Me14). More exact ascribing may be done after community structure forming and creating data base of fatty acid profiles for it members.
For example, the fatty acid balance optimization allowed to reveal that decanoic (10:0) and heptadecenoic acids in present communities most likely belong to additional two rhodococci groups than to streptococci or mycobacteria accordingly.
Data for specific fatty acids were taken from papers, cited above, or other numerous publications of C.W.Moss laboratory, E.Yantzen papers, other scientists who measured cellular fatty acids by gas chromatography and our own investigation. Some substances are characteristic for the whole majority of microorganisms in community. They are palmitic, stearic, mirystic, oleic, lauric acids and other substances. These acids are not specific and are used only for equation fitting. If community members were chosen correctly, the sum of partial include from each member to given substance should be equal to measured concentration of this substance. Negative difference, when occurs, means that some microbe is not present in the community. Positive differens means that some microorganisms are not included in the community list, and the set of such positives formulates the fatty acid profile of forgotten microbe. Sometimes this procedure leads to discovery of previously unknown species^1,6. 
A tentative reference of lipid components to concrete taxons (table 2) shows that there may be introduced bacteria, actynomecetes, microscopic fungi and other microorganisms, gramnegative and grampositive, aerobic and anaerobic. The latter may come into a staff of microbial communities which is considered as aerobic accordingly their properties. Alternatively, aerobes are discovered in strictly anaerobic conditions. For example, bacteroids and methanogenes are found in aerating active sludge as well as pseudomonads and bacilli — in methane-tank content. Exactly like that, it is possible a coexistance of sulfure oxidizing with sulfate reducing and methanogenesis with methylotrophy both in micro-zones of unit community.
3.3. What could detected community do in subsurface?
As the result of fatty acid analysis we have any reconstruction of microbial communities inside the VDW rocks (table 3). Compositions of both communities are very close that correlates with the similar conditions at two depths  There are some typical oligotrophic species such as representatives of gg. Caulobacter, Corynebacterium and Rhodococcus, that are known to utilize dispersed organic matter. The presence of obligatory (Desulfovibrio Clostridium) and facultative anaerobes (Capnocytophaga, Bacillus, Corynebacterium) as well as microaerophilic Bacteroides does not contradict a reductive
Table 3
Reconstruction of microbial community structures from VDW rocks
(species content in %)

properties of subsurface atmosphere. But a reach diversity of aerobes from gg. Acetobacter, Acinetobacter, Sphingomonas, Pseudomonas, Caulobacter, Rhodococcus, Streptomyces with abundance of copiotrophs like clostridia looks at least strange.
To solve our doubts let’s look at dominants in detected microbial community. Two main genera prevail in it. These are Clostridium and Rhodococcus. What can they do in deep rocks and how they could coexist with each other?
The role of rhodococci in deep subsurface has been proved by microbiological researches in field of oil and gas search¹⁴. It has been shown that rhodococci are oligocarbophilic bacteria, which are capable to utilize volatile hydrocarbons as a single source of carbon and energy. It had been found that Rhodococcus rhodochrous prevails over co-existing species of gas consuming rhodococci in soils of oil-bearing regions and it is a single in underground waters above oil deposit¹⁴. This microorganism is observed to dominate in our reconstructed community too which is located in rarefied zone where hard methane homologies predominate among hydrocarbon gases³. Moreover, it is known that oxidizing of propane or butane under anaerobic conditions the rhodococci release a lot of free aminoacids  into environment¹⁴. Having in a view this fact it should pay attention that the second dominant microorganism  Clostridium putrificum is known to be capable to grow utilizing no less than two pure aminoacids¹⁵. Therefore there is a syntrophic association which should supply other members by nutrition probably the following way: : subsurface  fluid hydrocarbons ® it oxidizing rhodococci ® free aminoacids and biomass proteins (products of rhodococci metabolism) ® it fermenting clostridia.
Really the lactate and acetate are common products of any organic matter fermentation under anaerobic conditions. So, isolated iron reducing bacteria could receive the necessary carbon and energy sources in situ. At table 3 is shown that bacteria of  g. Bacillus represent the essential enough part of microbial communities in VDW rocks: 6,8 % — in the samples from depth 2575 m and 4,6 % — 2805 m. Moreover, there is a certain conformity between abundance of bacilli in reconstructed community and diversity of isolated strains. Although the bacilli spores are known to be preserved during billion years in subsurface it should be agree that our reconstructed community has all possibilities to live in modern geochemical situation.
Thus, using the fatty acid biomarkers analysis of Archean basement crystalline
rocks we reconstructed a deep subsurface microbial communities which can successfully exist in modern circumstances and support of rock magnetization. However these are very differ from the one described by Pedersen in deep granitic rock¹⁶.
SUMMARY

• Evidence of microbes at 2575 — 2805 m in rocks of Archean crystalline basement (2.65 bln years) from area of  impact structure formed about  180 mln years ago
• Micro-dispersed structure of magnetite was discovered in ancient rock material and some pure cultures producing magnet-ordered compound were isolated and identified as Bacillus sp.
• Microbial cell markers (34 specific fatty acids) were detected in samples by using gas chromatograthy — mass-spectrometry
• Reconstruction of ancient microbial community was made and revealed the presence of 22 taxons
• Rhodococcus and Bacilluswere recovered as predominate groups

ACKNOWLEDGMENTS
Present research was supported by State Department of Natural Resources of Russian Federation.
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