Article pubs.acs.org/est
Methanospirillum Respiratory mRNA Biomarkers Correlate with Hydrogenotrophic Methanogenesis Rate during Growth and Competition for Hydrogen in an Organochlorine-Respiring Mixed Culture Annette R. Rowe,†,§,* Cresten B. Mansfeldt,‡ Gretchen L. Heavner,‡ and Ruth E. Richardson‡ †
Field of Microbiology, Cornell University, Ithaca, New York, United States Department of Civil and Environmental Engineering, Cornell University, Ithaca, New York, United States
‡
S Supporting Information *
ABSTRACT: Molecular biomarkers hold promise for inferring rates of key metabolic activities in complex microbial systems. However, few studies have assessed biomarker levels for simultaneously occurring (and potentially competing) respirations. In this study, methanogenesis biomarkers for Methanospirillum hungatei were developed, tested, and compared to Dehalococcoides mccartyi biomarkers in a wellcharacterized mixed culture. Proteomic analyses of mixed culture samples (n = 4) confirmed expression of many M. hungatei methanogenesis enzymes. The mRNAs for two oxidoreductases detected were explored as quantitative biomarkers of hydrogenotrophic methanogenesis: a coenzyme F420-reducing hydrogenase (FrcA) and an iron sulfur protein (MvrD). As shown previously in D. mccartyi, M. hungatei transcript levels correlated linearly with measured (R = 0.97 for FrcA, R = 0.91 for MvrD; n = 7) or calculated respiration rate (R = 0.81 for FrcA, R = 0.62 for MvrD; n = 35) across two orders of magnitude on a log−log scale. The average abundance of MvrD transcripts was consistently two orders of magnitude lower than FrcA, regardless of experimental condition. In experiments where M. hungatei was competing for hydrogen with D. mccartyi, transcripts for the key respiratory hydrogenase HupL were generally less abundant per mL than FrcA and more abundant than MvrD. With no chlorinated electron acceptor added, HupL transcripts fell below both targets. These biomarkers hold promise for the prediction of in situ rates of respiration for these microbes, even when growing in mixed culture and utilizing a shared substrate which has important implications for both engineered and environmental systems. However, the differences in overall biomarker abundances suggest that the strength of any particular mRNA biomarker relies upon empirically established quantitative trends under a range of pertinent conditions.
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INTRODUCTION In anaerobic environments, methanogenesis serves as a major terminal electron accepting process driving the degradation of organic biomass.1 Although the majority of environmentally produced methane is consumed by methane oxidizers, the portion of methane that escapes to the atmosphere (∼0.4 Gigatons a year )1 acts as a potent greenhouse gas.2 As methane also serves as an important energy source, synthesis and collection of methane has many important industrial applications, including anaerobic digestion. The productivity of anaerobic digesters communities has been linked to phylogenetic composition,3 although currently we have limited resolution with respect to specific methanogenic activities (i.e., acetoclastic vs hydrogenotrophic methanogenesis) in mixed communities. Inferring instantaneous fluxes, and in particular fluxes associated with specific groups or types of methanogenesis, could have important applications for facilitating industrial processes and/or better understanding methanogenesis in environmental systems. © 2012 American Chemical Society
A pertinent example is illustrated by competition between organochlorine-respiring Dehalococcoides species and hydrogenotrophic methanogens.4−8 Competition with methanogens has been implicated in incomplete or stalled remediation.9 Additionally, many remediation approaches apply biostimulation (often in conjunction with bioaugmentation) to supply fermentable electron donors to stimulate interspecies hydrogen transfer to Dehalococcoides in the contaminant plume, which when provided in excess can have undesired effects. Namely, stimulating biomass growth in groundwater systems, which, in turn can lead to biofouling and the accumulation of metabolic end-products like methane (a flammable greenhouse gas), in some cases to potentially explosive levels. 10 Molecular Received: Revised: Accepted: Published: 372
July 27, 2012 November 6, 2012 November 15, 2012 November 15, 2012 dx.doi.org/10.1021/es303061y | Environ. Sci. Technol. 2013, 47, 372−381
Environmental Science & Technology
Article
biomarkers for in situ microbial activities could be employed at such remediation sites and other environmental systems for monitoring effects of competing or problematic methanogen populations enabling more efficient biostimulation while mitigating unintended environmental consequences. Several studies have focused on development of Dehalococcoides biomarkers, particularly DNA markers, and their application to field sites undergoing in situ bioremediation.11 Relationships between the abundance of mRNA biomarkers and organochlorine-respiration have highlighted potential quantitative biomarkers.12−19 Characterization of similar relationships in other hydrogen-consuming organisms will allow assessment of differential activities among organisms, and will potentially prove useful for understanding microbial interactions in myriad environmental systems, as well as for facilitating cost-effective and efficient bioremediation. In a methanogenic and organochlorine-respiring enrichment community (Donna II) maintained for over 15 years on tetrachloroethene (PCE) and butyrate, syntrophically produced hydrogen is consumed by both Dehalococcoides mccartyi (formerly Dehalococcoides ethenogenes) str. 195 and a Methanospirillum hungatei strain.7,8,20 In this culture, several candidate Dehalococcoides biomarkers have been tested at the mRNA transcript level. These include several reductive dehalogenases, enzymes that catalyze reduction of organochlorines, and the well conserved and highly expressed NiFe-hydrogenase Hup, thought to be important for oxidation of hydrogen (Dehalococcoides only known electron donor).21−23 As hydrogen is the electron donor for which D. mccartyi and M. hungatei compete, enzymes involved in hydrogen utilization were the focus of the current study. In hydrogenotrophic methanogenesis, the deazaflavin F420 serves as an important electron carrier. In M. hungatei, an F420reducing NiFe-hydrogenase (Frc) has been shown to catalyze hydrogen-oxidation with F420-reduction (Figure 1, Reaction 11).24 The last step in methanogenesis has previously been shown to utilize a methyl-viologen reducing hydrogenase (Mvr).25,26 M. hungatei contains one of the important subunits (MvrD) that has been linked to shuttling electrons to heterodisulfide reductase (Hdr) for the regeneration of CoMSH and CoB-SH from heterodisulfide (Figure 1, Reaction 9).26 In this work, mixed-culture proteomics was used to identify important respiratory enzymes present in M. hungatei. qRTPCR data for key oxidoreductase transcripts (MvrD and FrcA) were used to examine correlations between mRNA levels and methanogenesis rate across a range of culture conditions. Previous mRNA expression work in mixed culture,20 or coculture with Syntrophobacter f umaroxidans 27 has suggested that these transcripts may serve as viable biomarkers. However, previous culturing conditions did not allow for direct quantitative comparison of M. hungatei mRNA abundance and rates of methanogenesis. As such, the first goal of this work was to develop biomarker abundance versus methanogenesis rate relationships for M. hungatei targets in the Donna II mixed culture. Quantification of mRNA expression via qRTPCR was applied in cultures spanning a wide range of hydrogenotrophic methanogenesis rates. Relationships between observed M. hungatei mRNA levels and methanogenesis were then compared with previously described D. mccartyi mRNA-respiration relationships from the same biological samples. In addition to characterizing biomarkers suitable for tracking methanogenesis, comparison of the behavior of these molecules in phylogenetically distinct organism provides insight into the variability in across organism trends and the viability of using respective D.
Figure 1. Schematic of M. hungatei methanogenesis pathway. Specific cofactors utilized are abbreviated: methanofuran (MFR), Ferredoxin (Fd), tetrahydromethanopterin (H2MPT), Coenzyme F420 (F420), Coenzyme M (CoM), and Coenzyme B (CoB). Reduced redox state is indicated by (red) or H2 and oxidized by (ox). Enzymes involved in catalysis of these reactions are indicated with numbers in grayed-in circles indicating detection in shotgun proteomic experiments. White circles with gray outlines and numbers (2−3) represent enzymes which were not detected in the mixed culture proteome: formylmethanofurantetrohydromethanopterin formyltransferase and F420-independent methylenetetrahydromethanopterin dehydrogenase. Numbers also listed with corresponding enzymes in Table 2. Reactions 1 and 8 are potentially coupled, and form a complex with currently unknown oxidative subunit.27 This and other putative reactions (formate oxidation to F420 and reverse electron transport reduction of Fd) are highlighted with gray dashed arrows.
mccartyi and M. hungatei mRNA biomarker abundances to infer metabolic rates of these populations.
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EXPERIMENTAL SECTION Experimental Conditions and Analysis of Metabolites. The Donna II mixed culture has been maintained on PCE and butyrate in a 6-L batch reactor for over 15 years (as described7). Experiments were performed on subcultures constructed in 160 mL serum vials with 100 mL of culture volume. Experimental conditions involved batch and continuous feeding regimes (as described15−17) of electron donors (hydrogen, butyrate, lactate) with and without PCE. Bicarbonate was provided in the basal salts media (BSM7) and through maintaining CO2 in head space gas mix (70%N2/30%CO2). Conditions were varied to alter respiration rate (outlined in Table 1, see Table S1 in Supporting Information (SI) for more detail). Briefly, under continuous feeding conditions soluble substrates were dissolved in BSM at the desired electron equivalence (eeq) ratios and added to cultures via syringe pump.16,17 For continuous feeding conditions, culture volume was periodically removed, maintaining a reactor volume within 100 mL ± 10 mL. The 100 mL reactor volume did not change during batch additions where the total substrates fed were applied at time zero and were not dissolved in media. Gaseous substrates (hydrogen and fluoromethane) were provided in batch, via headspace addition, or 373
dx.doi.org/10.1021/es303061y | Environ. Sci. Technol. 2013, 47, 372−381
Environmental Science & Technology
Article
Table 1. Experimental Parameters for Continuous and Batch Fed Donna II Sub-Cultures Used to Study Protein and mRNA Biomarkers. Parameters grouped by experiment including information on replicates, electron donor, chlorinated electron acceptor types (i.e., PCE, tetrachlorethene [TCE], cis-dichloroethene [cDCE]) and concentrations, as well as average respiration rates and range of aqueous hydrogen concentrations experiment title (continuous feed)
replicates (n)
no substrates (decay) butyrate butyrate + MF
4 2 2 2 3 2 2 3 3
hydrogen PCE hydrogen PCE butyrate
PCE half butyrate PCE lactate PCE no donor
TCE butyrate
cDCE butyrate
chloroethene electron acceptor (EA)
electron donor (ED) butyrate butyrate
1.7 ± 0.4 279 ± 2.1 64 ± 9.8
PCE PCE PCE
H2 H2 H2 butyrate butyrate
167 ± 4.8 1.5 ± 1 1.2 ± 0.15 141 ± 16 102 ± 8.7
2
PCE
butyrate
2 2 3 2 2 2 2 2 2 2 2 2 2
PCE PCE PCE PCE PCE PCE PCE TCE TCE TCE cDCE cDCE cDCE
344 ± 13 323 ± 6 414 ± 26 437 ± 79 0.65 ± 0.05 fermented yeast extract 45.3 ± 6.6 yeast extract 41 ± 1.4 butyrate 420.5 ± 53 butyrate 187 ± 18 butyrate 82.5 ± 9.5 butyrate 143 ± 42 butyrate 161.5 ± 3.5 butyrate 414 ± 26 total ± st.er. electron donor methanogenesis rate (ED) (μeeq/L-hr)
experiment title (batch feed)
replicates (n)
electron acceptor (EA)
batch
4
PCE
431.5 ± 38
butyrate butyrate butyrate lactate
7278 ± 1185
butyrate
continuously through diffusion across low-density polyethylene tubing, as described by 28 (see SI Materials and Methods for details). Constant mixing was maintained throughout all experiments. Methane was quantified from headspace samples using a gas chromatograph (GC) (Perkin-Elmer) equipped with a flame ionizing detector (FID) for levels below 33 μmoles per 160 mL serum vial and a thermal conductivity detector (TCD) for higher methane levels (run conditions described previously). 5 Hydrogen was quantified from headspace samples via GCTCD if above 0.5 μM aqueous concentration (Cw), and below this level using a reduced gas detector (RGD) (Trace Analytical) as previously described.7 The hydrogen detection limit using the RGD is 0.06 μM nominal per reactor (
