Organic Geochemistry 65 (2013) 118–126

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Organic Geochemistry

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Anaerobic biodegradation of the isoprenoid biomarkers pristaneand phytane

0146-6380/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.orggeochem.2013.10.010

⇑ Corresponding author. Present address: California Institute of Technology,Division of Geological and Planetary Sciences, Pasadena, CA 91125, USA. Tel.: +1 626395 6894; fax: +1 626 568 0935.

E-mail address: kdawson@caltech.edu (K.S. Dawson).

Katherine S. Dawson ⇑, Irene Schaperdoth, Katherine H. Freeman, Jennifer L. MacaladyPennsylvania State University, Department of Geosciences, University Park, PA 16802, USA

a r t i c l e i n f o

Article history:Received 26 June 2013Received in revised form 27 September 2013Accepted 16 October 2013Available online 22 October 2013

a b s t r a c t

Isoprenoids, a diverse class of compounds synthesized by all three domains of life, comprise many of thebiomarker compounds used in paleoenvironmental and paleoecological reconstruction of Earth history.These biomarkers include hopanoids, sterols and archaeal membrane lipids. While changes in hydrocar-bon profiles in anoxic sediments and oilfields indicate that anaerobic microbial metabolism is involved inthe disappearance or alteration of isoprenoids, direct links between specific compounds and their micro-bial degraders are lacking. Here we describe pristane (Pr) and phytane (Ph) degradation associated withNO�3 reduction. We confirmed isoprenoid conversion to CO2 using 13C-labeled Ph. After 120 days, dis-solved inorganic carbon (DIC) produced in incubations grown with 13C-labeled Ph had a d13C value of+76.7 ± 11.9‰, significantly higher than values for incubations with unlabeled Ph (�35.7 ± 2.0‰) andthose without an added carbon substrate (�30.0 ± 2.1‰). Additional incubations, displayed NO�3 reduc-tion after amendment with archaeal diphytanyl glycerol diether (DGD) core lipids, but not in thoseamended with glycerol diphytanyl glycerol tetraether (GDGT) core lipids. Both 16S rRNA clone librariesand whole cell rRNA-targeted fluorescent in situ hybridization (FISH) indicated that the likely Pr and Phdegrading Bacteria were Gamma proteobacteria, with > 99% similarity to Pseudomonas stutzeri.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Several reviews have highlighted progress in understanding themicrobiology and biochemistry associated with anaerobic hydro-carbon degradation (Spormann and Widdel, 2000; Lovley, 2001;Head et al., 2003; Widdel et al., 2006; Heider, 2007; Foght, 2008;Grossi et al., 2008; Widdel et al., 2010; Mbadinga et al., 2011).Additional studies have investigated biodegradation of hydrocar-bon mixtures in subsurface petroleum reservoirs and contami-nated aquifers (Head et al., 2003; Larter et al., 2003; Townsendet al., 2003; Jones et al., 2008; Gieg et al., 2010). Most studies havefocused on the anaerobic biodegradation of pollutants such as ben-zene and toluene and gaseous hydrocarbons such as methane andpropane. In contrast, anaerobic biodegradation of linear and cyclicisoprenoid hydrocarbons has received less attention (Hylemon andHarder, 1998) despite the relevance of these compounds as essen-tial biomarkers for taxonomic groups (Brocks and Pearson, 2005;Peters et al., 2005; Dutkiewicz et al., 2006) and paleoenvironmen-tal conditions such as temperature and salinity (Wuchter et al.,2006; Turich and Freeman, 2011), as well as petroleum maturity(Petrov and Abryutina, 1989). Isoprenoid hydrocarbons can repre-

sent up to 1% of a crude oil, with a major contribution from pris-tane (Pr) and phytane (Ph; Tissot and Welte, 1978).

Hydrocarbons in general are recalcitrant to anaerobic biodegra-dation and require activation by conversion to more oxidized inter-mediates prior to catabolism via b-oxidation (Heider et al., 1998;Heider, 2007). Variation in susceptibility to biodegradation de-pends on features such as chain length, branching and cyclization(Head et al., 2003; Peters et al., 2005). Isoprenoids derive from lip-ids made by organisms from all three domains of life and are syn-thesized from branched, C5 isoprene units. Branching can impartadditional recalcitrance by hindering b-oxidation (Cantwell et al.,1978; Schaeffer et al., 1979). In studies of aerobic branched alkanedegradation, this hindrance is overcome through additional deca-rboxymethylation steps (Seubert and Fass, 1964; Pirnik et al.,1974; Pirnik and McKenna, 1977; Cantwell et al., 1978). The needfor repeated decarboxymethylation or alternative demethylationsteps before b-oxidation can proceed effectively reduces the meta-bolic energy yield from isoprenoid substrates and may explain whydegradation in natural environments appears slower than forequivalent non-isoprenoid hydrocarbons (Head et al., 2003). Inaddition, iso-methyl branching at the chain ends (e.g. Pr, archaeol)likely prevents some activation mechanisms, such as fumarateaddition (Heider, 2007), due to steric hindrance, while low solubil-ity likely reduces bioavailability (Fichan et al., 1998; Hylemon andHarder, 1998).

K.S. Dawson et al. / Organic Geochemistry 65 (2013) 118–126 119

Several studies have implicated microbially catalyzed degrada-tion of isoprenoids, including alkenes (Harder and Probian, 1995;Bonin et al., 2002), acids (Hylemon and Harder, 1998) and alcohols(Grossi, 1998) in anoxic environments and anaerobic cultures.Indirect evidence of a microbial role includes the simultaneous dis-appearance of electron acceptors [SO2�

4 , Fe(III) and NO�3 ], gas evolu-tion and changing hydrocarbon profiles (Grossi et al., 2000; Boninet al., 2002; Head et al., 2003; Massias et al., 2003). Additionally, afew laboratory microcosm studies have indicated anaerobic degra-dation of the isoprenoid alkane, Pr, associated with NO�3 and SO2�

4

reduction. These include enrichments from a diesel fuel-contami-nated aquifer under NO�3 reducing conditions (Bregnard et al.,1997) and from anoxic marine sediment under SO2�

4 reducing con-ditions (Grossi et al., 2000). Direct attribution of degrading organ-isms is, however, lacking because neither study provided aphylogenetic description of the associated microbial community.Knowledge of the phylogeny of degrading organisms should helpadvance studies of both mechanistic pathways and rate of isopren-oid loss under anoxic conditions.

Here we have investigated Pr and Ph degradation in laboratoryenrichments under NO�3 reducing conditions. 16S rRNA gene clonelibraries and whole-cell rRNA-targeted fluorescent in situ hybrid-ization (FISH) revealed the dominant organisms in the enrichmentto be closely related to the NO�3 reducer Pseudomonas stutzeri andto a denitrifying, polyhydroxyalkanoate (PHA) degrading Simplici-spira sp. We confirmed the biodegradation using enrichments sup-plemented with 13C-labeled Ph (2,6,10,14-tetramethylhexadecane)and subsequent measurement of the dissolved inorganic carbon(d13CDIC) produced during microbial growth. The ratio of 13C/12Cin DIC showed a significant difference between incubations grownwith 13C-labeled Ph vs. those unlabeled Ph or with no added carbonsubstrate.

2. Material and methods

2.1. Source of Archaea and Bacteria

Samples were obtained from an activated sludge unit at thePennsylvania State University wastewater treatment plant. Solidmaterial was pelleted by centrifugation for 5 min at 3000g, andthe supernatant was used as an inoculum. For GDGT experiments,Thermoplasma acidophilum (DSMZ 1728) was grown as describedby Langworthy et al. (1972). For DGD and Ph experiments, Halofe-rax sulfurifontis strain SD1 (L. Krumholz, University of Oklahoma)was grown as described by Elshahed et al. (2004).

2.2. Enrichment of isoprenoid degrading bacteria

Anaerobic cultivation was carried out using a modified mediumdescribed by Widdel and Bak (1992). Trace element, vitamin andselenate–tungstate solutions were prepared as described (Widdeland Bak, 1992). The unbuffered basal medium was modified tocontain (g l�1): 1 g NaCl; 0.4 g MgCl2; 0.1 g CaCl2�2H20; 0.5 g Na2

SO4; 0.25 g NH4Cl; 0.2 g KH2PO4; 0.5 g KCl; 0.25 g KNO3. The med-ium was initially at pH 7.0 and increased to pH 8.0 over the courseof incubation. All experiments were incubated in the dark, at roomtemperature without shaking. Initial enrichments were carried outin 200 ml serum bottles with a N2 headspace, and butyl rubberstoppers (Bellco, Vineland, NJ) using 30 ml medium, 10 ml acti-vated sludge supernatant and 120 mg Pr (2,6,10,14-tetramethyl-pentadecane, 98.9%, Chem Service Inc., West Chester, PA). Initialenrichments were carried out in triplicate and included inoculatedserum bottles with Pr and without Pr, as well as an uninoculatedcontrol with Pr. Further enrichments were carried out in triplicate(2% inoculum v/v) in 60 ml serum bottles prepared under anoxic

conditions with a 1 atm N2 headspace. Following sterilization ofthe medium, 10 mg Pr or 10 mg Ph were introduced via a gas-tightsyringe as the sole source of carbon. Controls included inoculatedbottles with no added substrate, which were controls in alltransfers.

Enrichment cultures were transferred to new bottles followingthe consumption of 2–3 mM NO�3 . Aliquots of the third transferfrom the initial activated sludge were preserved at �80 �C in 25%glycerol and future cultures were initiated from these stocks. To re-duce carryover of glycerol to the Pr and Ph degradation experi-ments, the glycerol preserved cells were washed with 1�phosphate buffered saline (PBS). Cultures revived from glycerolstocks were then transferred once before use in Pr or Ph degrada-tion experiments. Comparison of NO�3 consumption between inoc-ulated cultures containing Pr or Ph and controls with no addedsubstrate was used to confirm the absence of residual glycerol.

13C-labeled Ph was prepared as described in Section 2.4. Addi-tional controls for the 13C-labeled Ph incubation included uninoc-ulated bottles, inoculated bottles with no added carbon sourceand inoculated bottles with unlabeled Ph (99%, Ultra, Kingstown,RI). The carbon isotope signature of the substrates was analyzedwith a Costech elemental analyzer (EA) coupled to a Thermo Finn-igan Delta Plus XP irMS mass spectrometer (details in Section 2.4):Pr �27.1‰; unlabeled Ph �29.0‰; 13C-labeled Ph ca. 700‰.Labeled Ph (22.3 mg) was diluted with unlabeled Ph (28.3 mg) toprovide sufficient material for triplicate incubations. The d13Cvalue of the Ph mixture added to cultures was determined fromisotope mass balance to be ca. 292‰. The isotope mass balancefor incubation was calculated as follows: d13CPh = [(d13Cunlabeled Ph

�Munlabeled Ph) + (d13Clabeled Ph �Mlabeled Ph)]/MPh, where mass (M)was the amount of Ph added to the mixture.

An additional experiment was carried out in triplicate to com-pare the ability of the Pr-degrading enrichment to grow on Pr,archaeal DGD core lipids and archaeal GDGT core lipids. DGDs(2.25 mg) or GDGTs (0.5 mg) were added in a small volume of hex-ane to triplicate serum bottles. Hexane was evaporated under astream of N2 prior to the addition of sterilized medium and a N2

headspace. Pr (10 mg) was added to an additional triplicate set ofserum bottles via a gas-tight syringe. Cultures were inoculated asdescribed above, including triplicate controls with no added sub-strate. The ability of the Pr-degrading enrichment to use thesealternate substrates was determined by monitoring the loss ofNO�3 .

2.3. Archaeal lipid extraction and analysis

GDGTs, including caldarchaeol and analogues containing one tofive cyclopentane rings, were prepared from laboratory cultures ofT. acidophilum (Swain et al., 1997). DGDs were obtained from cul-tures of H. sulfurifontis, which produces C20–20 DGDs (Elshahedet al., 2004). Lipids were obtained from lyophilized cell pelletsusing a modified Bligh-Dyer extraction described by Dawsonet al. (2012). Briefly, the lipids were extracted with CH3OH/CHCl3/H2O (2:1:0.8). Neutral lipids including pigments were removedfrom H. sulfurifontis total lipid extracts by overnight precipitationof polar lipids with several volumes of cold acetone (Choquetet al., 1992). Core lipids were prepared from the polar lipids bystrong acid methanolysis (10:1:1 CH3OH/CHCl3/HCl) at 100 �C for1 h. Methanolysed lipids were extracted with hexane/CHCl3 (4:1),dried over Na2SO4 and evaporated to dryness with N2. GDGT andDGD core lipids were purified using silica gel chromatography withSupelclean LC-Si SPE tubes (6 ml, Supelco, Bellefonte, PA).

GDGTs and DGDs were analyzed using high performance liquidchromatography-atmospheric pressure chemical ionization-massspectrometry (HPLC-APCI-MS). Methods followed those developedby Hopmans et al. (2000), using an Agilent 6310 ion trap.

120 K.S. Dawson et al. / Organic Geochemistry 65 (2013) 118–126

Separation was achieved with a Prevail Cyano column(2.1 � 150 mm 3 lm; Alltech, Deerfield, IL) at 30 �C. GDGTs andDGDs were eluted isocratically with 99% hexane:1% isopropanolfor 5 min, followed by a linear gradient to 98.4% hexane:1.6% iso-propanol over 40 min. After each analysis the column was cleanedwith 90% hexane:10% isopropanol for 10 min followed by a 10 minreequilibration to 99% hexane:1% isopropanol. The flow rate was0.2 ml/min. Instrument conditions were: corona, 5000 nA, capillary3500 V, nebulizer 60 psi, dry gas �6 l/min, dry temperature 200 �C,vaporizer 400 �C.

2.4. Preparation of 13C-labeled Ph from DGD lipids

13C-labeled archaeal lipids were prepared from H. sulfurifontisSD1 grown in the modified liquid medium described by Elshahedet al. (2004) for growth on sugars. The medium contained(g l�1): 150.0 g NaCl; 20.0 g MgCl2; 12.0 g Hepes; 5.0 g K2SO4;0.1 g CaCl2; 0.1 g yeast extract; 0.5 g D-glucose. Glucose addedto cultures was a mixture of 1% U–13C6 (99%, Cambridge IsotopeLaboratories, Andover, MA) and 99% unlabeled glucose for d13Cca. 885‰.

The expected 13R and 13F for glucose could be calculated fromthe d13C of the unlabeled glucose and the mole fraction of labeledglucose. Fractional abundance is defined as 13F = 13C/(13C + 12C) andthe carbon isotope ratio is defined as 13R = 13C/12C. Using therelationship, 13Ra = 13RVPDB(da/1000 + 1), we converted the d13Cvalue of unlabeled glucose to an isotopic ratio where13RVPDB = 0.0112373. The isotopic ratio was converted to 13F usingthe relationship, 13F = 13R/(13R + 1). The expected 13F of the addedglucose was determined using a mass balance relationship forthe labeled and unlabeled glucose, 13FT = 13Flabeled v + 13Funla-

beled(1 � v) where v is the mole fraction of labeled glucose and13Flabeled � 0.99.

Ph was prepared from extracted core diphytanyl glyceroldiether (DGD) lipids of H. sulfurifontis SD1 (Fig. S-1). Preparationof alkanes from archaeal lipids followed a combination of the pro-tocols described by Nishihara et al. (1989) and Koga and Morii(2006). Briefly, core lipids were refluxed for 2 h at 100 �C with3 ml 55% HI (EM Scientific, Carson City, NV) to generate phytyl io-dide. The latter was refluxed for 2 h at 120 �C with 30.0 mg of Zndust (98%, Alfa Aesar, Ward Hill, MA) and 1 ml CH3CO2H. Thisreductive dehalogenation generated Ph that was purified by elu-tion through silica using Supelclean LC-Si SPE tubes (6 ml, Supelco,Bellefonte, PA). Additional details on Ph purity and yield areprovided in the Supplementary information.

Ph was analyzed for purity with a Hewlett-Packard 5972GCMS instrument equipped with a fused silica column (J&WDB-5; 30 m � 0.25 mm i.d.; film thickness 0.25 lm) (Fig. S-2).The injector temperature was 320 �C. The oven was held at65 �C for 1 min before the temperature was increased by 6 �C/min to 320 �C (held 20 min). The d13C value of enriched Ph wasdetermined via continuous flow (He; 120 ml/min) with a Costechelemental analyzer (EA) by oxidation at 1020 �C over chromium(III) oxide and silvered cobalt (II, III) oxide, followed by reductionover elemental Cu at 650 �C. The resulting CO2 was passedthrough a water trap and then a 5 Å molecular sieve GC instru-ment at 50 �C to separate N2 from CO2. The CO2 was diluted withHe in a Conflo III interface/open split prior to analysis. d13C valueswere measured with a Thermo Finnigan Delta Plus XP irMSinstrument. Measured d13C values were corrected for sample sizedependency and then normalized to the Vienna Peedee Belemnite(VPDB) scale with a two-point calibration (Coplen et al., 2006).Error was determined by analyzing independent standards acrossall EA runs. Accuracy was ± 0.02‰ (n = 54) and precisions ± 0.02‰

(n = 88; 1r).

2.5. Monitoring NO�3 reduction and DIC production

Growth in enrichment cultures was monitored using changesin the concentrations of terminal electron acceptors. NO�3 ;NO�2 ;Cl�and SO2�

4 concentrations were measured using an ICS2500 Dionex ion chromatography (IC) system and an IonPacAS18 column (Dionex, Sunnyvale, CA) with an isocratic elution pro-gram using 30 mM KOH at 1 ml/min and oven temperature 31 �C.Anion concentrations were normalized to Cl� concentration, whichwas considered an inert reference ion between sampling points.For DIC analysis, 1 ml aliquots of culture were taken at variouspoints during growth and transferred to pre-combusted 8 ml ser-um bottles flushed with He and sealed with butyl rubber stoppers.Subsequently, these aliquots were acidified with 2 drops H3PO4

(12 N). Samples were analyzed using an SRI 310 gas chromato-graph (SRI Instruments, Menlo Park, CA) equipped with a thermalconductivity (TCD) detector and a PoraPak Q packed column(0.91 m � 3.18 mm) at 50 �C with a He carrier phase.

2.6. Stable isotope analysis of CO2

Aliquots of CO2 for DIC analysis were cryogenically distilled toremove water vapor (propan-2-ol and dry ice) and to isolate CO2

from non-condensable gases (liquid N2) prior to isotope analysis.The 13C/12C ratio of the CO2 was measured with a Finnigan MAT252 dual inlet mass spectrometer using a micro-volume inlet. Eachsample was analyzed twice and the statistical standard deviationof measurements was always better than 0.8‰ standard deviation.

2.7. Phylogenetic analysis of enrichment cultures

DNA extraction, PCR amplification and cloning were carried outon the 3rd (PiaDegr, Figs. 3 and 4) and two parallel 6th transfers ofthe Pr degrading enrichment (NP13 and NP6D, Figs. 3 and 4) andthe Ph degrading incubation (13Cphyt, Figs. 3 and 4) as describedby Macalady et al. (2008). Clones were sequenced at the Penn StateUniversity Biotechnology Center using T3 and T7 plasmid-specificprimers. CodonCode Aligner v.1.2.4 (CodonCode Corp., Dedham,MA, USA) was used to assemble sequences and manually checkfor ambiguity. Assembled gene sequences were compared withthe public databases using BLAST (Altschul et al., 1990) andchimera checked with Bellerophon 3 (Huber et al., 2004) andCHIMERA_CHECK v.2.7 (Cole et al., 2003). Putative chimeras wereexcluded from subsequent sequence analysis. Non-chimeric se-quences were aligned using the NAST aligner (DeSantis et al.,2006), added to an existing alignment in ARB (Ludwig et al.,2004), and manually refined.

2.8. Nucleotide sequence accession numbers

The 16S rRNA gene sequences determined were submitted tothe GenBank database under accession numbers GU222220-GU222257, GU250847-GU250871 and JF834284-JF834313.

2.9. FISH analysis of community composition

Aliquots of enrichment cultures for FISH were fixed in 3 vol-umes of 4% (w/v) paraformaldehyde in 1� PBS for 2–3 h at 4 �Cand stored in 1:1 PBS/EtOH at �20 �C. FISH experiments were car-ried out as described by Hugenholtz et al. (2001) using probeslisted in Table S1. Populations of Simplicispira sp. (CTE659) (Schle-ifer et al., 1992), P. stutzeri (Pseu15) (Demaneche et al., 2008) andBacteroidales (CFB719b) were determined from counts of between1000–2000 DAPI-stained cells for each sample/probe combination.The probe CFB719 (Weller et al., 2000) was modified to CFB719busing the probe design tools in the ARB software package to

K.S. Dawson et al. / Organic Geochemistry 65 (2013) 118–126 121

increase specificity for the Bacteroidales clones identified. Oligonu-cleotide probes were synthesized and labeled at the 50 end with theindocarbocyanine dye CY3 (Sigma-Aldrich, St. Louis, MO).

3. Results

3.1. Enrichment of NO�3 reducing, isoprenoid-degrading bacteria

Experiments showed consumption of NO�3 concomitant withproduction of NO�2 and DIC in enrichments containing Pr(Fig. 1A–D). Over 6 months, the initial enrichment cultures con-sumed ca. 14.5 mM NO�3 (80 lM/day) vs. ca. 0.38 mM in a Pr-freeinoculated control and 0.48 mM in an uninoculated control con-taining Pr. Subsequent transfers (2% inoculum, v/v) showed some-what reduced rates of NO�3 reduction, 50 lM/day, in enrichmentcultures containing Pr. After 40 days growth, the consumption of2.0 mM NO�3 resulted in the production of 1.9 mM NO�2 and1.1 mM DIC (Fig. 1A–D). Inoculated controls without added carbonsubstrate (Fig. 2 D) did not show a significant loss of NO�3 over thecourse of the initial 6 month experiment or in subsequent trans-fers. Additionally, SO2�

4 concentration remained constant over thecourse of all experiments.

3.2. Confirmation of isoprenoid degradation with 13C-labeled Ph

Incubations with 13C-labeled Ph showed significantly a reducedrate of NO�3 reduction (15 lM/day) vs. rate in the Pr degradingenrichments (p = 0.0013). NO�3 loss and DIC production were de-tected in the controls with no substrate added. After the first50 days, the rate of NO�3 loss in the controls decreased to 2.5 lM/day. IC and DIC analysis showed production of NO�2 and DIC asso-ciated with the NO�3 loss in all incubations including the controls.

Fig. 1. NO�3 reducing enrichments with Pr (A–D) and Ph (E–H), showing loss of (A and E) Nsubstrate degradation (D and H). d13C values of DIC (I) respired from 13C-labeled Ph (d13C±1 standard deviation.

SO2�4 concentration remained constant and no CH4 production

was detected. From 20–120 days, consumption of 1.2 mM NO�3 re-sulted in production of 0.99 mM NO�2 and 0.34 mM DIC (Fig. 1E–G).Over the same period, the control experiment with no added sub-strate consumed 0.46 mM NO�3 and produced 0.27 mM DIC.

The carbon sources in the 13C-labeled Ph and control incuba-tions were elucidated by measuring the 13C/12C ratio of the DICproduced by microbial respiration. After 120 days, DIC was en-riched (Fig. 1 I) in 13C in the labeled Ph incubations (d13C+76.7 ± 11.9‰) compared with initial values (d13C �31.0 ± 2.9‰).In contrast, no isotopic enrichment was observed in DIC producedduring incubations with unlabeled Ph or with no added carbonsubstrate. The DIC produced in the control experiments variedfrom d13C �36.0 ± 2.1‰ at 0 days to �35.7 ± 2.1‰ at 120 days forunlabeled Ph and d13C �38.7 ± 0.6 at 0 days to �30.0 ± 2.0‰ at120 days for no added carbon substrate. Enrichment of DIC pro-duced in the labeled Ph experiments was significant relative to ini-tial DIC values (p = 5.4 � 10�5) and compared with the no addedcarbon substrate and unlabeled Ph control experiments(p = 5.3 � 10�5 and 4.4 � 10�5 respectively).

In the 13C-labeled Ph incubations, respired DIC was enriched in13C vs. incubations with unlabeled Ph or no added carbon sub-strate. At the start of the incubation, labeled Ph (22.3 mg) was di-luted with unlabeled Ph (28.3 mg). Based on purity (98.5% fromGC–MS; Fig. S-2), of 13C-labeled Ph, ca. 0.33 mg represented resid-ual compounds from the preparation and purification process, andadditional 0.28 mg of impurity was present in the unlabeled Ph(99% purity). Based on the addition of 6.3 mg of the 13C-labeledPh mixture to each bottle, we estimated the presence of ca.0.041 mg of potentially labeled impurity and 0.035 mg unlabeledimpurity in the added substrate. In the 13C-labeled Ph incubations,the production of 0.85 mg DIC (0.17 mg C) accounted for 2.2� the

O�3 , production of (B and F) NO�2 and (C and G) DIC, and stoichiometrically predicted+292‰). Data points represent average of 3 replicate incubations and error bars are

Fig. 2. NO�3 loss in anaerobic enrichments with (A) Pr and (B) DGD core lipids.Inoculated cultures containing (C) GDGT core lipids or (D) no added carbonsubstrate show only minimal loss of NO�3 over the same period. Data pointsrepresent average of 3 replicate incubations and error bars are ± 1 standarddeviation.

122 K.S. Dawson et al. / Organic Geochemistry 65 (2013) 118–126

amount of total impurity and 4� the amount of labeled impuritypotentially in any individual bottle. Thus, 13C enrichment observedin the DIC is primarily due to utilization of 13C-labeled Ph. Residual13C-labeled compounds, if completely mineralized, accounted forno more than 20% of DIC.

DIC production without added substrate suggested a back-ground source of carbon. This additional source could be derivedfrom dead biomass or PHA inclusions within cells. A Keeling plot(Pataki et al., 2003) was used to confirm the contribution of DICfrom 13C-labeled Ph as part of a two-component mixture of sub-strate sources of DIC in these enrichments (Fig. S-4). We calculatedthe amount of DIC released from oxidation of background carbonusing an isotope and mass balance mixing model. We appliedend member value for background carbon (d13C �40‰) based onthe incubation experiments with no added carbon substrate andfor 13C-labeled Ph (d13C +292‰) estimated from our preparationmethod. In Fig. 1G and H, we plotted the 13C-labeled Ph-derivedDIC and the Ph lost over 120 days of incubation. Ph concentrationwas calculated from the ratio of Ph:DIC inferred from a thermody-namic prediction of stoichiometry (see Supplementary informationfor calculations). Based on the calculations, during the 120 dayincubations, 42.7 ± 5.0 lM 13C-labeled Ph was degraded, resultingin the production of 341.4 ± 40.0 lM DIC.

3.3. Archaeal core lipid utilization by Pr-degrading enrichment

Degradation of archaeal DGD and GDGT core lipids by the Pr-degrading enrichment was monitored via NO�3 loss. The initialNO�3 concentration in all incubations was 2 mM, with the excep-tion of the GDGT incubations, which had an initial concentrationof 1.6 mM. Incubations with DGD core lipids resulted in the lossof 1764.1 ± 402.1 lM NO�3 in 61 days (44.1 lM/day), which was

comparable with NO�3 loss in Pr incubations 2000 lM(35.1 lM day�1; Fig. 2A and B). Incubations with GDGT core lipidsshowed NO�3 loss similar to inoculated, no substrate added controls(Fig. 2C and D), 116.1 ± 55.4 lM (2.1 lM/day) and 188.2 ± 12.3 lM(3.3 lM/day) respectively.

3.4. Phylogenetic analysis of isoprenoid degrading enrichments

A 16S rRNA gene clone library constructed from the Pr-degrad-ing enrichment after the first transfer had relatively low diversity.No archaeal sequences were retrieved. Most clones (75%) weremembers of a single bacterial phylotype of the Bacteroidales order.Based on BLAST searches and neighbor joining phylogenies con-structed using the phylogenetic software ARB, these clones are re-lated to anaerobic fatty acid degraders and environmental clonesfrom organic-rich and/or anoxic environments. Other clones inthe library are closely related to Gamma- and Betaproteobacteriaisolates and environmental clones associated with hydrocarbonsin organic-rich environments.

Additional 16S rRNA gene clone libraries were constructed fromthe fourth transfer of parallel enrichments on Pr and for a fifthtransfer to incubations with 13C-labeled Ph. The libraries documenta decrease in diversity as well as a strong shift in the most abun-dant phylotypes. The Bacteriodales clones abundant in the firstclone library became a minor component of the subsequent li-braries, representing < 10% of the later clones. In contrast, Gamma-and Betaproteobacteria clones rose in abundance. In particular, thelater clone libraries were dominated by two phylotypes in thePr-degrading enrichment: ca. 50% are related to P. stutzeri (> 99%identity; Fig. 3) and ca. 30% to a denitrifying, PHA degradingSimplicispira isolate (> 99% identity) (Fig. 4). In the 13C-labeledPh-degrading enrichment, ca. 70% of clones were related toP. stutzeri (> 99% identity; Fig. 3) and an additional 25% of clonesshared 97–99.8% identity with Simplicispria isolates previouslyidentified in the Pr-degrading enrichment (Fig. 4).

3.5. Quantitative FISH analysis of enrichment cultures

Oligonucleotide probes specific to the Bacteriodales order(CFB719), the Comamonadaceae family, containing Simplicispira(CTE659), and P. stutzeri (Pseu15) were used in FISH experimentsto obtain quantitative information about the population structureof the isoprenoid degrading enrichments (Fig. S-3). Consistent withthe decline in the number of clones in the Bacteriodales order, thenumber of cells hybridizing to CFB719 decreased from 52.7 ± 11.4%to 0%. Meanwhile, cells hybridizing to the oligonucleotide probePseu15 increased in abundance with successive transfers from of6.1 ± 3.8% to 69.6 ± 10.4% of total bacterial cells and represent themost abundant phylotype in the later enrichments. Cells hybridiz-ing to CTE659 fluctuated in abundance between 75.7 ± 3.4% inearly transfers and 8.8 ± 6.7% in later transfers. The CTE659 hybrid-izing cells were curved rods (1.9 ± 0.3 lm by 0.7 ± 0.1 lm) andwere primarily planktonic. The more abundant, Pseu15 hybridizingcells in the enrichment cultures had rod-shaped morphology(0.9 lm ± 0.2 lm by 0.4 lm ± 0.1 lm) and occurred either asplanktonic cells, or in large flocs (10–20 lm diameter).

4. Discussion

To our knowledge, this study provides the first description ofthe phylogeny of Bacteria associated with anaerobic, aliphatic iso-prenoid degradation. The dominant phylotype in the low diversitycommunity, representing > 70% of the cells identified by FISH, wasof the Gammaproteobacteria, which are closely related to P. stutzeri(> 99%). Strains of P. stutzeri are known NO�3 reducing bacteria, with

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75/-61/-

NJ/MP

0.04 substitutions/site

NP6D_27 (17)

NP13_b18

PiaDegr2 (2)

PiaDegr31

NP13_b24

subsurface water clone (DQ337067)

Isolate strain Y-134 (AB096215)

Isolate strain HdN1 (AF331974)

Petroluem reservoir clone (DQ675028)

Anoxic Lake Tanganyika clone (DQ463702)

Pseudomonas stutzeri SR30 (DQ288950)

Pseudomonas stutzeri strain ATCC17594 (AY905607)

Daqing oil field clone (EV050687)

Nitrite oxidising suspension clone (EV305575)

Pseudomonas stutzeri strain 24a13 (AJ270451)

Biodegraded oil reservoir clone (AY570580)

Hydrocarbon contaminated soil clone (AY571838)

Rancho La Brea heavy oil clone (EF157157)

Polyaromatic contaminated soil clone (AY699600)

13Cphyt_41 (17)

PiaDegr20

13Cphyt_39 (2)

Pseudomonas stutzeri PseudoAeroA1 (EU327524)

Pseudomonas marginalis JH8 (DQ232737)

Pseudomonas stutzeri GKA-13 (EU520400)

NP13_b16 (6)

Aquamonas sp. (AM403231)

Subsurface water clone (DQ337007)

Aquamonas vora strain GPTSA 20 (AY544768)

Anammox reactor clone (AB430336)

Oil-shale semi-coke solid waste clone (EF540427)

Chlorobium phaeobacteriodes strain DagowIII (AM050129)

Pseu

dom

onas

Cel

lvib

rio

Pseu

dom

onad

ales

Xant

hom

onad

ales

Fig. 3. Neighbor-joining phylogenetic tree showing Gammaproteobacteria. 13C-labeled Ph and Pr degrading enrichment clones are shown in bold followed by the number ofclones of each phylotype. Maximum parsimony (MP) and neighbor-joining (NJ) bootstrap values > 50% are shown (MP/NJ). Putative Pr (Piadegr, NP13, NP6D) and Ph(13Cphyt) metabolizing clones are annotated by H.

K.S. Dawson et al. / Organic Geochemistry 65 (2013) 118–126 123

the capability of degrading a broad array of compounds, includingaromatic, polycyclic aromatic hydrocarbons and aliphatic hydro-carbons (Lalucat et al., 2006). Several described strains of Pseudo-monas spp. are associated with both aerobic and anaerobichydrocarbon degradation. Of particular note is the association ofone cluster of the Pseudomonas genus, containing P. alcaligenes, P.citronellolis, P. mendocina and P. aeruginosa as well as P. stutzeri,(Yamamoto et al., 2000) with both aerobic and anaerobic degrada-tion of unsaturated isoprenoids and isoprenoid primary alcohols(Cantwell et al., 1978; Harder and Probian, 1995; Bonin et al.,2002). The other major phylotype was a Betaproteobacteria clonewith 97–99.8% similarity to a PHA degrading Simplicispira sp. iso-late (Khan et al., 2002). Members of the Simplicispira genus havebeen isolated from wastewater (Grabovich et al., 2006; Lu et al.,2007) and identified in NO�3 reducing, chlorobenzene degradingenrichments (Nestler et al., 2007) and in a NO�3 treated oil field(Cornish Shartau et al., 2010).

The accumulation of NO�2 in both the Pr and the Ph-degradingenrichments points to inhibition of complete denitrification. Accu-mulation of NO�2 may be due to a shift in pH (Thomsen et al., 1994),the inhibitory effect of NO�3 onNO�2 reduction (Almeida et al., 1995;Kornaros et al., 1996; van Rijn et al., 1996) or to carbon-limitedgrowth (Kornaros et al., 1996). At 25 �C, Pr solubility is estimatedas 1.1 mg l�1 and Ph solubility as 0.3 mg l�1 (Eastcott et al.,1988). Therefore, the low solubility of Pr and Ph in aqueous solu-tion results in low substrate availability.

In order to predict the stoichiometry of Ph degradation, we usedthe thermodynamic calculation approach described by Rittmannand McCarty (2001). The approach utilizes the balance of redoxequations for electron donor and acceptor reactions, as well asfor biomass synthesis. We used equations for partial denitrificationand for the complete oxidation of Ph are coupled to determine thereaction free energy per electron equivalent (DGR). The energy ofcellular synthesis (DGP) is determined from the energy requiredfor Ph conversion to pyruvate. Using a range of energy transfer effi-ciencies typical of anaerobic heterotrophs (e = 0.4–0.7; Rittmannand McCarty, 2001; Xiao and VanBriesen, 2006), the dissimilatoryand assimilatory reactions yield several possible stoichiometriesfor which the ratio of NO�3 to DIC can be compared with the exper-imentally determined value (NO�3 : DIC 3.4; see Supplemental infor-mation for calculations). An energy transfer efficiency of 0.7produces a stoichiometry with the most similar ratio ofNO�3 toDICðNO�3 : DIC3:25Þ. The stoichiometry of Ph degradationpredicted for e = 0.7 is:

C20H42 þ 26NO�3 þ 2:4NHþ4 þ 2:8H2O

! 8HCO�3 þ 2:4C5H7O2Nþ 26NO�2 þ 32Hþ

Under the conditions of our NO�3 reducing enrichments, Pr and Phbiodegradation followed first order kinetics, with Pr having a rateconstant of k = 1.1 yr�1 (r2 0.76, n = 8) to 2.5 yr�1 (r2 0.77, n = 8)and Ph having a rate constant of k = 0.10 yr�1 (r2 0.83, n = 15) to

99/52100/100

89/89

70/71

54/-73/61

99/97

96/71

81/83

97/98 100/99

100/99

89/-52/-

100/100

90/-

84/-

99/87

100/100

75/-88/-

99/-

100/-

92/-

NJ/MP100/100

0.04 substitutions per site

Acidovorax sp. strain R-25212 (AM084022)

Simplicispira sp. strain R-23033 (AM236310)

Aquaspirillum metamorphum strain LMG 4339(AF078757)

Chlorobium phaeobacteriodes strain DagowIII (AM050129)

Denitrifying sewage treatment clone (AY823977)

Rhodocyclus sp. strain HOD 5 (AY691423)Benzene contaminated groundwater clone (AY214205)

Rhodocyclus tenuis strain DSM1C9 (D16208)

Wetlands soil clone vf5 (DQ975218)

Subsurface water clone (DQ337044)

Denitrifying bioreactor clone (DQ836752)Leptothrix sp. strain B2 (AJ867848)

Anoxobacterium dechloraticum (X72724)

Canadian coal bed clone 805 (EU073805)

Isolate strain NOS3 (AB076845)

Denitrifying reactor clone (AJ412674)

Landfill leachate clone (AJ853520)

Pesticide contaminated soil clone (EU037924)

13Cphyt_30 (4)

NP6D_70 (2)

NP13_b36 (11) NP6D_67 (13)

NP6D_61 (2)

NP13_b20

PiaDegr26

NP13_b06

13Cphyt_21

13Cphyt_15 (2)

13Cphyt_22

Lautropia mirablis (X73223)Gas hydrate clone (AY053478)

Activated sludge clone (AB286420)

Com

amon

adac

eae

Bur

khol

deria

les

Bur

khol

deria

ceae

Rho

docy

clac

eae

Rho

docy

clal

esB

urkh

olde

riale

s

Fig. 4. Neighbor-joining phylogenetic tree showing Betaproteobacteria. 13C-labeled Ph and Pr degrading enrichment clones are shown in bold followed by the number ofclones of each phylotype. Maximum parsimony (MP) and neighbor-joining (NJ) bootstrap values > 50% are shown (MP/NJ). Putative Pr (Piadegr, NP13, NP6D) and Ph(13Cphyt) metabolizing clones are annotated by H.

124 K.S. Dawson et al. / Organic Geochemistry 65 (2013) 118–126

0.20 yr�1 (r2 0.83, n = 15) (see Supplementary information for calcu-lations). Both Pr and Ph degradation rates for these enrichments areslower than isoprenoid degradation rates reported by Grossi et al.(2000) for Pr (k = 3.5/yr), squalene (18.5/yr) and phytadienes(6.6 yr�1) in anoxic marine sediment enrichments. Rate constantsfor isoprenoid degradation by both NO�3 reducing (this study) andanoxic marine sediment enrichments (Grossi et al., 2000) are sev-eral orders of magnitude greater than the in situ anaerobic biodeg-radation rate constant for mixed hydrocarbons in oilfields (10�8/yr;Larter et al., 2003), where oxidant and nutrient supplies are limitedby diffusion, and an order of magnitude greater than anaerobic bio-degradation rates in anoxic surface sediment (10�1–10�2/yr; Zen-gler et al., 1999; Larter et al., 2003), where nutrients and oxidantsare not supplemented as in the incubations.

Following the biodegradation simulation detailed by Larteret al. (2003), we estimated the timescale for degradation of the iso-prenoid fraction of a crude oil (ca. 1%; Tissot and Welte, 1978).Based on the degradation rates described above for oilfields, bacte-ria targeting solely isoprenoids would degrade that fraction in1 � 106 yr (Larter et al., 2003). In contrast, in anoxic surface sedi-ment or in our NO�3 reducing incubations, a similar fraction wouldbe degraded in 0.05–1 yr. The increased availability of nutrientsand electron acceptors in laboratory experiments and surface sed-iment vs. oilfields is consistent with the observed difference inrates.

Comparison of results from incubations in the presence of Pr,DGD core lipids and GDGT core lipids provided some insight intothe bioavailability of these compounds. Due to the structural sim-

ilarity in the hydrocarbon chains of DGD and GDGT core lipids to Prand Ph, we hypothesized that the enrichment should be able to uti-lize them at a similar rate. The lack of NO�3 reduction in the pres-ence of GDGT core lipids, and the near equivalent rate of NO�3reduction in the presence of Pr and DGD core lipids suggests thatthe ether linkages between then isoprenoid chain and glycerolmoieties in GDGTs inhibit degradation under the conditions ofour enrichments. Further experiments will be required to identifythe activation mechanism and anaerobic biodegradation pathwayfor Pr, Ph and similar compounds such as archaeal membrane lip-ids. Additionally, alternative environmental inocula or electronacceptors other than NO�3 andSO2�

4 may be able to overcome theenergetic barrier apparently presented by tetraether linkages, toallow GDGT degradation or recycling in anoxic sediments (Takanoet al., 2010; Lin et al., 2012).

5. Conclusions

NO�3 reduction was observed in enrichment cultures supple-mented with the isoprenoid biomarkers Pr and Ph as sole carbonsubstrate. 16S rRNA clone libraries and FISH identified P. stutzerito be the likely isoprenoid degrading bacterium. We confirmed iso-prenoid utilization in incubations with 13C-labeled Ph substrate bymeasuring enrichment in DIC over the course of the incubation.After 120 days, d13CDIC was > 100‰ enriched in incubations with13C-labeled Ph vs. incubations with unlabeled Ph or no added sub-strate. Additional incubations supplemented with structurally sim-ilar DGDs displayed similar rates of NO�3 reduction to the Pr

K.S. Dawson et al. / Organic Geochemistry 65 (2013) 118–126 125

degrading cultures, while NO�3 reduction rate in GDGT amendedincubations was similar to that with no substrate added controls.

Acknowledgments

The research was supported by a grant from American ChemicalSociety Petroleum Research Fund to J.L.M. (48445-AC2) the PennState Biogeochemical Research Initiative for Education (BRIE)(NSF DGE-9972759), the Penn State Astrobiology Research Center(PSARC), NASA NAI (NNA04CC06A) and a Penn State Biogeochem-istry Program fellowship to K.S.D. We thank L. Krumholz for pro-viding a culture of Haloferax sulfurifontis and Z. Zhang and D.Walizer for technical assistance. We also thank C.H. House, J.M. Re-gan and two anonymous reviewers for valuable discussions andcomments towards improving the paper.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.orggeochem.2013.10.010.

Associate Editor – S. Schouten

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