C-13 analysis of fatty acid fragments by gas chromatography mass spectrometry for metabolic flux analysis

Wu, WC; Dijkstra, P; Dippold, MA
2020 | Geochim. Cosmochim. Acta | 284 (92-106)
biosynthesis , fatty-acids , fragment c-13 analysis , glucose , isotopomer distributions , lipopolysaccharides , mass isotopomer distribution , metabolic flux analysis , microbial communities , pathways , phospholipids , quantification , soil-microorganisms , turnover
When multiple metabolic pathways lead to the same product, compound-specific isotope analysis may not provide enough information to quantify the activities of the contributing pathways. Instead, identification of where in the molecule the C-13 is incorporated is required. Here we show how knowledge of position-specific (13) C incorporation in fatty acids (FA) and FA fragments can be used to quantitatively estimate the fluxes through the central C metabolic network. We developed a method to measure C-13 enrichment of FA and FA fragments (ethanoate, propionate) using electron impact GC-MS. We tested the accuracy and repeatability of the measurements using natural abundance and positionspecific C-13 labelled standards and FA extracted from Bacillus licheniformis and Pseudomonas fluorescens grown with labelled and unlabelled glucose. The molecular ions of FA generally reflected theoretical predictions of mass isotopomer distributions for natural abundance values, but that of the associated FA fragments deviated from expected values, likely associated with McLafferty rearrangements of hydrogen. After correction for naturally occurring isotopes, C-13 enrichments of FA and FA fragments showed good agreement with expected isotope composition of FA standards (root mean square error < 0.044 at %; delta C-13 of similar to 40%), natural abundance and labelled glucose. The unsaturated FA extracted from P. fluorescens deviated from expected values likely associated with problems of co-elution and ion suppression and were excluded from analysis. The ratio of glucose-1-C-13 to glucose-3-C-13 incorporation into FA fragments was high for B. licheniformis, but low for P. fluorescens. Metabolic flux modelling based on the C-13 enrichment of ethanoate and propionate fragments showed that B. licheniformis used Embden-Meyerhof-Parnas and pentose phosphate pathway (66% and 30%, respectively), whereas P. fluorescens utilized Entner-Doudoroff and pentose phosphate pathway (72% and 27%, respectively). FA fragment analysis is therefore a promising tool to study central C metabolic network activities of co-occurring groups of microbes in intact and complex environmental communities. (C) 2020 Elsevier Ltd. All rights reserved.

Intact Ether Lipids in Trench Sediments Related to Archaeal Community and Environmental Conditions in the Deepest Ocean

Xu, YP; Wu, WC; Xiao, WJ; Ge, HM; Wei, YL; Yin, XR; Yao, HM; Lipp, JS; Pan, BB; Hinrichs, KU
2020 | J. Geophys. Res.-Biogeosci. | 125 (7)
ammonia-oxidizing archaea , archaea , biomarker , carbon , challenger deep , diversity , gdgt , hadal zone , intact polar lipid , mass spectrometry , membrane-lipids , organic-matter , oxygen , polar lipids , tetraether lipids , tex86
Archaea play an important role in marine biogeochemical cycle; however, their phylogenetic distribution and lipid composition in the hadal zone (6-11 km water depth) are poorly known. Here, we analyzed archaeal membrane lipids and 16S rRNA gene sequences in sediments from Mariana Trench (MT), Massau Trench (MS), and New Britain Trench (NBT), varying from 1,560 to 10,840 m depth. Forty-two intact polar lipids (IPLs) were identified, including glycerol dialkyl glycerol tetraethers (GDGTs), OH-GDGTs, glycerol dialkyl diethers (GDDs), and archaeol (AR) with polar headgroups of monohexose (1G), dihexose (2G), trihexose (3G), and hexose-phosphohexose (HPH). Compositional and spatial distribution patterns of archaeal lipids suggest benthic Thaumarchaeota as a major source for IPLs, consistent with the predominance of Thaumarchaeota genes (>80%). The redundancy analysis (RDA) based on lipid and 16S rRNA data separates samples into three groups: extremely deep water (MT), significant terrestrial influence (NBT 1, 4, and 6), and predominant marine influence (MS, NBT 2, 3, 7, and 10). 1G-GDDs and 1G-AR positively correlate with water depth, likely reflecting the adaptation of benthic archaea to elevated hydrostatic pressure or variation of archaeal community in trench sediments. Bathyarchaeota are more abundant in sediments receiving terrestrial input; this pattern was attributed to their capability of utilizing terrestrial organic matter as an energy source. Our study highlights important environmental influences (e.g., pressure and organic matter quality and quantity) on benthic archaeal community and archaeal IPL compositions, which should be considered when IPLs and core lipids are applied as chemotaxonomic markers and paleo-proxies.

CO2 conversion to methane and biomass in obligate methylotrophic methanogens in marine sediments

Yin, XR; Wu, WC; Maeke, M; Richter-Heitmann, T; Kulkarni, AC; Oni, OE; Wendt, J; Elvert, M; Friedrich, MW
2019 | ISME J. | 13 (8) (2107-2119)
anaerobic oxidation , carbon-dioxide , genome sequence , intact polar lipids , interspecies electron-transfer , mass spectrometry , methanococcoides-burtonii , microbial-populations , ribosomal-rna , sulfate-reducing bacteria
Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires C-13-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60-86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways (similar to 40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Although methane formation from methanol is the primary pathway of methanogenesis, the observed high DIC incorporation into lipids is likely linked to CO2-dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.

Origin of hydroxyl GDGTs and regular isoprenoid GDGTs in suspended particulate matter of Yangtze River Estuary

Lu, XX; Chen, JL; Han, TW; Yang, H; Wu, WC; Ding, WH; Hinrichs, KU
2019 | Org. Geochem. | 128 (78-85)
dialkyl glycerol tetraethers , east china seas , igdgts , intact polar , membrane-lipid composition , oh-gdgts , organic-matter , oxygen minimum zone , shelf circulation , suspended particle matter , variability , water column , yangtze river estuary , yellow-river
Hydroxylated isoprenoid glycerol dialkyl glycerol tetraethers (OH-GDGTs) were found having the potential to track past sea surface temperatures (SST), in analogy to the TEX86- a paleothermometer based on isoprenoid GDGTs (iGDGTs). Especially in estuarine regions with high terrestrial input, the ring index of OH-GDGTs (RI-OH) was more robust to trace the summer SST than TEX86. In order to clarify the potential sources of sedimentary GDGTs and to further elucidate the potential of OH-GDGTs as SST proxy, we investigated the seasonal distribution of core OH-GDGTs and iGDGTs in the suspended particulate matter in a transition section in the Yangtze River Estuary. The concentrations of OH-GDGTs and iGDGTs were higher in the estuary than those in the lower Yangtze River, which suggested the OH-GDGTs and iGDGTs in estuarine sediment mainly came from marine autochthonous organisms, whereas the terrestrial contribution was relatively low. The higher content of OH-GDGTs and iGDGTs in summer than in winter indicated that sedimentary OH-GDGTs and iGDGTs mainly originated from the summer SPM deposition. In addition, the ratio of OH-GDGTs versus iGDGTs suggested that OH-GDGTs were relatively abundant in the upper water layer compared to iGDGTs, regardless of season. Furthermore, the distributions of iGDGTs and OH-GDGTs were found to be influenced by hydrodynamics. The increasing warm bias of iGDGT and OH-GDGT reconstructed temperatures with water depth suggested that either GDGTs in deeper waters derive mainly from surface water or GDGTs cyclization in deeper waters was influenced by reduced ammonium oxidation rates in response to the limited supply of ammonium and oxygen, especially in summer. (C) 2018 Elsevier Ltd. All rights reserved.

Production and turnover of microbial organic matter in surface intertidal sediments

Wu, WC; Meador, T; Hinrichs, KU
2018 | Org. Geochem. | 121 (104-113)
carbon fixation , deep pore waters , endospore abundance , fatty-acids , heavy-water , heterotrophic inorganic carbon assimilation , hydrogen isotopes , intertidal flat , microbial fatty acids , microbial production , stable isotope probing , sulfate-reducing bacteria , tidal-flat sediments , vertical-distribution , wadden sea
Benthic microorganisms play a significant role in the remineralization of sedimentary organic matter (OM). In order to constrain the growth rate, turnover and carbon metabolism of microbial communities in anoxic sandy sediments, we quantified the rate of inorganic carbon (IC) assimilation and of lipid production via a recently developed dual isotope-labelling assay that can differentiate autotrophic vs. heterotrophic production in a 22 cm sediment core from the Janssand tidal flat (Wadden Sea, Germany). Despite an extremely low concentration of total organic carbon (TOC) in the majority of samples (<0.3%), the concentration of total fatty acids (TFAs) was in the range 4.5 to 28.4 mu g/mg TOC, suggestive of a high contribution of fresh microbial and algal biomass to the TOC pool. This was corroborated by a production rate of microbial FAs, which ranged from 0.3 to 4.7 mu g TFAs/g(dw)/yr (where dw = dry wt) and the mean turnover time of microbial FAs was 6 +/- 5 yr. The enhanced production rate of iso- and anteiso-branched FAs was consistent with the presence of an active population of sulfate reducing Deltaproteobacteria. The assimilation rate of IC into bacterial lipids was relatively low (0.16 +/- 0.07 mu g C/g(dw)/yr) in OM-lean sandy sediments (i.e. upper 17 cm), such that the IC assimilation to lipid production ratio values was typically <0.3, indicating that heterotrophic bacteria were dominant and dark IC fixation played a minor role at the study site. The measured rates of FA production converted to an anaerobic heterotrophic C demand of 0.4-1.8 mu g C/g(dw)/d, assuming a bacterial growth efficiency of 10%. Such high biomass proportion, production rate and C demand suggested that dissolved OM must play a vital role in sustaining the active heterotrophic microbial populations in these sandy sediments. (C) 2018 Elsevier Ltd. All rights reserved.

Growth of sedimentary Bathyarchaeota on lignin as an energy source

Yu, TT; Wu, WC; Liang, WY; Lever, MA; Hinrichs, KU; Wang, FP
2018 | Proc. Natl. Acad. Sci. U.S.A. | 115 (23) (6022-6027)
bathyarchaeota , carbon fixation , group lineage , lignin degradation , lipid , marine-sediments , mass spectrometry , mcg archaea , methane , microbial communities , miscellaneous crenarchaeotal group , organic-matter , sedimentary carbon cycling , subseafloor sediments
Members of the archaeal phylum Bathyarchaeota are among the most abundant microorganisms on Earth. Although versatile metabolic capabilities such as acetogenesis, methanogenesis, and fermentation have been suggested for bathyarchaeotal members, no direct confirmation of these metabolic functions has been achieved through growth of Bathyarchaeota in the laboratory. Here we demonstrate, on the basis of gene-copy numbers and probing of archaeal lipids, the growth of Bathyarchaeota subgroup Bathy-8 in enrichments of estuarine sediments with the biopolymer lignin. Other organic substrates (casein, oleic acid, cellulose, and phenol) did not significantly stimulate growth of Bathyarchaeota. Meanwhile, putative bathyarchaeotal tetraether lipids incorporated C-13 from C-13-bicarbonate only when added in concert with lignin. Our results are consistent with organoautotrophic growth of a bathyarchaeotal group with lignin as an energy source and bicarbonate as a carbon source and shed light into the cycling of one of Earth's most abundant biopolymers in anoxic marine sediment.

Production rates of bacterial tetraether lipids and fatty acids in peatland under varying oxygen concentrations

Huguet, A; Meador, TB; Laggoun-Defarge, F; Konneke, M; Wu, WC; Derenne, S; Hinrichs, KU
2017 | Geochim. Cosmochim. Acta | 203 (103-116)
branched gdgts , dialkyl glycerol tetraethers , environmental controls , fatty-acids , lake-sediments , lipid biosynthesis , mass spectrometry , membrane-lipids , microbial communities , peat , soil , sphagnum peatlands , stable isotope probing , temperature
Interpretations of the abundance and distribution of branched glycerol dialkyl glycerol tetraether (brGDGT) lipids have been increasingly applied to infer changes in paleoenvironment and to estimate terrigenous organic matter inputs into estuarine and marine sediments. However, only preliminary information is known regarding the ecology and physiology of the source organisms of these biomarkers. We assessed the production rates of brGDGTs under different redox conditions in peat, where these lipids are found in high concentrations, particularly at greater depths below the fluctuating water table. The incorporation of hydrogen relative to carbon into lipids observed in our dual stable isotope probing assay indicates that brGDGTs were produced by heterotrophic bacteria. Unexpectedly, incubations with stable isotope tracers of the surface horizon (5-20 cm) initiated under oxic conditions before turning suboxic and eventually anoxic exhibited up to one order of magnitude higher rates of brGDGT production (16-87 ng cm(-3) y(-1)) relative to the deeper, anoxic zone (20-35 cm; ca. 7 ng cm(- 3) y(-1)), and anoxic incubations of the surface horizon (< 3 ng cm(-3) y(-1)). Turnover times of brGDGTs in the surface horizon ranged between 8 and 41 years in the incubations initiated under oxic conditions, in contrast to 123 742 years in anoxic incubations. As brGDGTs were actively produced during both anoxic incubations and those exposed to oxygen, we conclude that their source organisms are likely facultative aerobic heterotrophs that are particularly active in the peat acrotelm. Production rates of bacterial fatty acids (ca. 2 mu g cm(-3) y(-1)) were roughly two orders of magnitude higher than those of brGDGTs, suggesting that brGDGT producers are a minor constituent of the microbial community or that brGDGTs are a small component of the microbial cell membrane in comparison to fatty acids, despite the typically high brGDGT concentrations observed in peat. Multivariate analysis identified two branched fatty acids that shared a similar production pattern as brGDGTs among the experimental treatments and may be associated with brGDGT biosynthesis. (C) 2017 Elsevier Ltd. All rights reserved.

Source and distribution of glycerol dialkyl glycerol tetraethers along lower Yellow River-estuary-coast transect

Wu, WC; Ruan, JP; Ding, S; Zhao, L; Xu, YP; Yang, H; Ding, WH; Pei, YD
2014 | Mar Chem | 158 (17-26)
bit , branched gdgts , gdgts , intact polar , lower amazon river , membrane-lipids , organic carbon , sediments , soil , terrestrial organic-matter , tex86 , tex86 paleothermometer , transport , yellow-river
To assess the source of glycerol dialkyl glycerol tetraethers (GDGTs) and their usefulness as proxies for terrestrial organic matter inputs and temperature in the Yellow River-dominated margin, we measured isoprenoid and branched GDGT concentrations in surface sediments along a lower Yellow River-estuary-coast transect. Branched GDGTs dominated over isoprenoid GDGTs in the riverbed sediments and had similar compositions from river to coast. In contrast, isoprenoid GDGTs displayed an increasing abundance and a decreasing GDGT-0 to crenarchaeol ratio (1.6 to 0.6) toward the sea. Such distribution patterns of GDGTs, combined with the result from a principal component analysis (PCA), confirmed the different origin of branched and isoprenoid GDGTs with branched GDGTs being primarily from soil erosion of the Chinese loess plateau (CLP) whereas, in addition to allochthonous terrestrial inputs, aquatic Thaumarchaeota partially contributes to the isoprenoid GDGT pool in estuarine and coastal areas. The branched GDGT-derived temperature (avg. 11 degrees C) is consistent with the annual mean air temperature (MAT) of the CLP in the middle river basin, a major source region for the Yellow River sediments, whereas the isoprenoid-derived temperature (12.7 to 28.4 degrees C) deviated widely from the annual mean temperature in the study region. Application of a binary mixing model based on delta C-13, the branched and isoprenoid tetraether (BIT) index and branched GDGT concentrations showed consistent decreases in the relative amount of terrestrial organic carbon toward the sea, but estimates from the latter two proxies were lower than those from the delta C-13. (C) 2013 Elsevier B.V. All rights reserved.

Variability of tetraether lipids in Yellow River-dominated continental margin during the past eight decades: Implications for organic matter sources and river channel shifts

Wu, WC; Zhao, L; Pei, YD; Ding, WH; Yang, H; Xu, YP
2013 | Org. Geochem. | 60 (33-39)
atchafalaya river , bohai sea , branched gdgts , carbon , lower amazon river , marine , sediment discharge , soil , terrestrial biomarkers , transport
Glycerol dialkyl glycerol tetraethers (GDGTs) and bulk organic geochemical parameters were examined for a short core from the Bohai Sea, a Yellow River-dominated continental margin. A three end member mixing model using branched/isoprenoid tetraethers (BIT) index, delta C-13 and C/N shows that the average fractions of soil, marine and plant organic matter (OM) during the period of 1933-2011 are 67.7% (38-92%), 26.1% (0-58%) and 6.2% (0-23%), respectively. Abrupt changes of sedimentary OM compositions around 1953, 1976 and 1996 are synchronous with the Yellow River mouth relocations. The BIT index values (0.33-0.80) present a stronger correlation with crenarchaeol abundance (R-2 = 0.88) than branched GDGTs abundance (R-2 = 0.27), suggesting that variations of marine Thaumarchaeota abundance rather than soil OM inputs is the first order factor controlling the BIT index values, although this proxy has been widely used for soil OM. The comparison between the BIT index, nutrient status and historical Yellow River sediment load indicates that the high sensitivity of the BIT index to the Yellow River channel shifts cannot be explained by a nutrient stimulation mechanism, but instead is likely caused by the restriction of Thaumarchaeota growth in highly turbid water due to the enormous sediment inputs from Yellow River. Our study demonstrates that local conditions should be considered when applying the BIT index as an environmental proxy. (C) 2013 Elsevier Ltd. All rights reserved.

Sea surface temperature variability in southern Okinawa Trough during last 2700 years

Wu, WC; Tan, WB; Zhou, LP; Yang, H; Xu, YP
2012 | Geophys Res Lett | 39
china sea , climate , east-asian monsoon , holocene , isoprenoid tetraether lipids , membrane-lipids , northwestern pacific , past 2 millennia , proxy , record
Most of the temperature reconstructions for the past two millennia are based on proxy data from various sites on land. Here we present a bidecadal resolution record of sea surface temperature (SST) in Southern Okinawa Trough for the past ca. 2700 years by analyzing tetraether lipids of planktonic archaea in the ODP Hole 1202B, a site under the strong influence of Kuroshio Current and East Asian monsoon. The reconstructed SST anomalies generally coincided with previously reported late Holocene climate events, including the Roman Warm Period, Sui-Tang dynasty Warm Period, Medieval Warm Period, Current Warm Period, Dark Age Cold Period and Little Ice Age. However, the Medieval Warm Period usually thought to be a historical analogue for the Current Warm Period has a mean SST of 0.6-0.8 degrees C lower than that of the Roman Warm Period and Sui-Tang dynasty Warm Period. Despite an increase since 1850 AD, the mean SST in the 20th century is still within the range of natural variability during the past 2700 years. A close correlation of SST in Southern Okinawa Trough with air temperature in East China, intensity of East Asian monsoon and the El-Nino Southern Oscillation index has been attributed to the fluctuations in solar output and oceanicatmospheric circulation.

Significance of long chain iso and anteiso monomethyl alkanes in the Lamiaceae (mint family)

Huang, XY; Meyers, PA; Wu, WC; Jia, CL; Xie, SC
2011 | Org. Geochem. | 42 (2) (156-165)
branched alkanes , carbon-isotope discrimination , cuticular waxes , hydrocarbon constituents , leaves , lower atmosphere , n-alkanes , plants , sediments , series

The potential significance of the contributions of long chain iso and anteiso monomethyl alkanes (LC MMAs) from plants of the Lamiaceae, a family that includes many culinary and aromatic herbs of cultural value, to sediments and soil has been evaluated by analyzing 21 specimens from 16 species of Lamiaceae from different environmental settings in Hubei Province, central China, and comparing the results with those from tobacco plants, which are established to be rich in these compounds. Odd numbered iso-alkanes (i-C-25 to i-C-36) and even numbered anteiso-alkanes (a-C-25 to a-C-36) are abundant in the Lamiaceae (1.9-23.2% and 0.9-23.8% of total alkanes, respectively). The proportions of LC MMAs are relatively high and comparable to those in the tobacco plant. However, chain lengths in the Lamiaceae are longer than those in tobacco plants and compound-specific delta C-13 values are more negative than in the tobacco plants, potentially allowing distinction of their different origins. The results imply that Lamiaceae, in addition to some other land plants, can be important sources of LC iso- and anteiso-alkanes in sediments and soils. (C) 2010 Elsevier Ltd. All rights reserved.

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