Reductive metabolism of the important atmospheric gas isoprene by homoacetogens

Isoprene is the most abundant biogenic volatile organic compound (BVOC) in the Earth’s atmosphere and plays important roles in atmospheric chemistry. Despite this, little is known about microbiological processes serving as a terrestrial sink for isoprene. While aerobic isoprene degrading bacteria have been identified, there are no known anaerobic, isoprene-metabolizing organisms. In this study an H2-consuming homoacetogenic enrichment was shown to utilize 1.6 μmoles isoprene h−1 as an electron acceptor in addition to HCO3−. The isoprene-reducing community was dominated by Acetobacterium spp. and isoprene was shown to be stoichiometrically reduced to three methylbutene isomers (2-methyl-1-butene (>97%), 3-methyl-1-butene (≤2%), 2-methyl-2-butene (≤1%). In the presence of isoprene, 40% less acetate was formed suggesting that isoprene reduction is coupled to energy conservation in Acetobacterium spp. This study improves our understanding of linkages and feedbacks between biogeochemistry and terrestrial microbial activity.

In the atmosphere, isoprene reaction products modulate the oxidizing potential, which leads to a prolonged lifetime of greenhouse gases such as methane [12,[22][23][24]. Additionally, isoprene oxidation products cause secondary organic aerosols (SOA) formation [25], which affects the climate directly by scattering absorbance and indirectly via nuclei cloud formation [26,27].
In the present study, the fate of isoprene in anoxic environments was investigated for the first time. Given the environmental abundance and ubiquity of isoprene, we hypothesized that it is available to anaerobic microorganisms. Samples taken from different environments were used to enrich for anaerobic isoprene utilizing microorganisms with the aim of determining the fate of isoprene in anaerobic microbial systems in order to better understand its global degradation pathways. We show for the first time that obligate anaerobes also transform isoprene. Moreover, whereas isoprene primarily serves as an electron donor in aerated soils, we provide evidence that is used as an electron acceptor to support homoacetogenesis.

Inocula
Sewage sludge samples were obtained from St. Marys Sewage Treatment Plant Sydney, Australia and stored anaerobically in the dark at 4°C. Soil samples from Botany Bay, Sydney, Australia were core drilled from 3.6 meters beneath the surface and stored in the dark at room temperature in anaerobic media. Soil samples from 5 cm beneath the surface of Colo River area in Wollemi National Park, Blue Mountains Australia, were stored in the dark at room temperature.

Initial enrichment cultures for isoprene reduction
Sludge (50 µl, 2 × 10 9 cells ml -1 ) was added to sealed culture flasks (120 ml) containing 80 ml anaerobic minimal media. D/L-Lactate was supplied at~10 mM from a sterile stock solution and isoprene was added as an electron acceptor from a 99.9% stock solution by using a 100 µl glass syringe to a final concentration of 1.3 mM in liquid media. Dilution to extinction series (10 −1 -10 −6 ) were performed using the same culture condition except in 60 ml flask containing 40 ml anaerobic minimal media.

Growth in H 2 and HCO 3 − containing media
Anaerobic culture flask (120 ml) containing 80 ml minimal media were supplied with 0.5 bar sterile filtered H 2 and 30 mM NaHCO 3 . Isoprene was added from a 99.9% stock solution by using a 100 µl glass syringe to a final concentration of 1.3 mM in liquid media. In the case of ethylene it was added from a 99.5% gas stock via a 5 ml and 0.5 ml gas tight syringe to 0.38 or 0.02 mM in liquid media. Cultures were inoculated with isoprene reducing enrichment culture (1 ml). In case of fructose it was added from a 1 M anaerobic stock solution to a final concentration of 20 mM.

Standards
Standards were prepared in 120 ml flasks with 80 ml anaerobic minimal medium. Isoprene was added from stock solution with a glass syringe to prepare standards reaching from 0.4 to 5 mM nominal concentrations. 3-methyl-1butene, 2-methyl-2-butene, and 2-methyl-1-butene were added from each stock solution with a glass syringe and combined in one flask as a standard reaching from 0.4 to 5 mM nominal concentrations. H 2 was added from a H 2 generator with standards reaching from 0.2 to 20 mM nominal concentrations. Ethane, ethylene, and methane standards were prepared from a gas mixture (33.33% gas each) to prepare standards reaching from 0.1 to 12 mM nominal concentrations. All gases were added with different sizes of gas tight syringes. Dimensionless Henry constants for isoprene, ethylene, ethane, methylbutenes, H 2, and methane were calculated from Sander [36].
Isoprene, H 2 , HCO 3 − , and hydrocarbon analysis Isoprene, methylbutenes, methane, H 2, CO 2 and hydrocarbon gases were monitored by gas chromatography (GC) using a Shimadzu GC-2010. Isoprene, methylbutenes and methane were analyzed by using a GasPro PLOT column (60 m x 0.32 mm, Agilent Technologies) with Helium as a carrier gas (3 ml min −1 ) and flame ionization detection (FID). The oven temperature was 150°C for 30 s and was increased by 20°C min −1 to a final temperature of 250°C. Gas samples (100 µl) were withdrawn from the flask via a pressure-lockable gas tight syringe and directly injected into the GC. H 2 was analyzed using a HP-PLOT Molesieve column (30 m × 0.32 mm × 0.25 mm, Agilent Technologies, Australia) and pulsed discharged ionization detector (PDD). The carrier gas was Helium (3 ml min −1 ) and the oven temperature was applied at 50°C for 1.2 min. Gas samples (20 µl) were withdrawn from the flask via a gas lock syringe and directly injected into the Shimadzu GC-2010. HCO 3 − concentrations were measured by acidification of media (100 µl) in a sealed flask resulting in the release of HCO 3 − in form of CO 2 . CO 2 was measured by GC-PDD using a HP-PLOT Q column (30 m x 0.32 mm, Agilent Technologies, Australia). The carrier gas was Helium (3 ml min −1 ) and the oven temperature was applied from 50°C for 1 min to 54.5°C with a rate of 3.5°C. Gas samples (40 µl) were withdrawn from the flask via a gas lock syringe and directly injected into a Shimadzu GC-2010. Formate, acetate, butyrate, and propionate were analyzed as their ethyl ester derivative by GC-FID using a DB-FFAP column (30 m x 0.32 mm × 0.25 mm, Agilent Technologies) at 40°C for 6 min with helium as the carrier gas (3 ml min −1 ). Samples (500 µl media) were supplied with 100% ethanol (200 µl) and undiluted sulfuric acid (200 µl) for esterification, sealed immediately and incubated at 60°C for 45 min. Before injection into the GC samples were incubated at 80°C for 5 min at 500 rpm, 250 µl of headspace sample was withdrawn from the flask via an automatic sampler (Shimadzu AOC-5000 plus) and directly injected into a Shimadzu Plus GC-2010 at 500 µl s −1 .
Ethene and ethane amounts were measured by GC-FID using a GS-Q column (30 m x 0.32 mm × 0.25 mm) at 100°C for 2 min with helium as the carrier gas (3 ml min −1 ). Gas samples (100 µl) were withdrawn from the flask via a gas lock syringe and directly injected into a Shimadzu GC-2010.

D/L-Lactate analysis
Lactate concentrations were monitored by using the D/L-lactic acid kit from Megazyme following manufacturer's instructions.

DNA extraction and Illumina sequencing
DNA was extracted from 300 µl culture using standard phenol-chloroform extraction method. Lysis buffer [37] was added to the sample and the tube was mechanically agitated in FastPrep Lysis Matrix A tubes (MP Biomedicals). DNA was extracted with sequential phenol (phenolchloroform-isoamyl alcohol (25:24:1), 7.5 M ammonium acetate, chloroform and isopropanol treatments, precipitated with ethanol using a general protocol, resuspended in 20 µl H 2 O and stored at −20°C until further analysis. Regions of 16S rDNA gene were amplified by PCR from extracted DNA with the Q5 high-fidelity DNA polymerase (New England BioLabs) using the universal primers 926F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACA G -[AAA CTYAAAKGAATTGRCGG]-3′) and 1392R (5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA G -[ACG GGC GGT GTG TRC-3′) targeting bacteria and archaea [38]. The samples were sequenced on an Illumina MiSeq Sequencer (Illumina, USA) using V3 chemistry at the Next Generation Sequencing Facility at Western Sydney University's Hawkesbury Institute for the Environment (Sydney, Australia). In all, 16S rRNA gene amplicon sequences were analyzed with QIIME2 (https://qiime2.org) [39] utilizing the dada2 pipeline [40]. Sequencing quality was first visualized with FastQC (www.bioinformatics.ba braham.ac.uk) resulting in forwards and reverse reads being trimmed at 290 base pairs and 240 base pairs, respectively. Forward and reverse sequences that passed the default quality control were merged and non-overlapping sequences were discarded. Chimeras were analyzed and removed via the consensus method within the dada2 pipeline. Remaining sequences had taxonomy assigned with the RDP classifier [41] using the Greengenes 13_8 database [42]. Taxa present at <2% abundance were removed for clarity.

Cloning
In all, 16S rDNA gene fragment cloning was performed on DNA samples from the isoprene reducing culture after 29 days [ Fig. 7]. Part of the Acetobacterium 16S rDNA fragment was PCR-amplified with the Aceto572F and Aceto784R [43] and a constant annealing temperature of 59°C for 34 cycles. All products were cloned into the pCR™2.1-TOPO® vector with TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Plasmid DNA was extracted from overnight Escherichia coli (DH5α™-T1®) cultures using the PureYield™ Plasmid Miniprep System (Promega, Fitchburg, WI).

Quantitative real-time PCR
Quantitative real-time PCR targeting Acetobacterium was performed on a Biorad real-time PCR system by using QuantiTect SYBR green PCR mastermix (Qiagen, Germany) and Acetobacterium primers Aceto572F and Aceto784R. The thermocycling program was as follows: initial denaturation for 3 min at 98°C; 39 cycles of [95°C for 20 s, 59°C annealing for 50 s] and a final melting curve analysis from 60 to 99°C. The standard curve was generated with serial dilutions of a known quantity of 16S rDNA Acetobacterium gene-contained in plasmids generated as described above. Five copies of 16S rRNA genes per Acetobacterium genome were calculated based on the sequenced genome of A. woodii DSM 1030 to convert gene copies to cell numbers [44].
In the first round of enrichment cultures, isoprene depleted at a rate of~0.3 μmoles per day after a 50 day lag period, with concomitant production of 15 μmoles methylbutenes after 200 days [ Fig. 1a, b]. The predominant isoprene reduction product was 2-methyl-1butene (98%, 12.3 μmoles), with lesser amounts of 3-methyl-1-butene (2%). Lactate (900 μmoles) was completely consumed after 50 days in the presence or absence of isoprene [ Fig. 2a] and was fermented to acetate, propionate (400 μmoles each), H 2 with associated biomass generation [ Fig. 2b-e]. Isoprene reduction occurred Fig. 1 Depletion of isoprene (a) and subsequent production of methylbutenes (b) was only observed in microcosms containing sludge, lactate, and isoprene. In control samples supplemented with only lactate or isoprene, no isoprene depletion or methylbutene formation was detected. Methane (c) production only occurred in cultures supplied with lactate. Error bars represent one standard deviation (n = 4) subsequent to lactate consumption (after 50 days). Cultures without lactate amendment generated low quantities of fatty acids (<6 μmoles) and H 2 (~3−10 μmoles) presumably derived from biomass supplied in the inoculum, but did not convert isoprene to methylbutenes. Methane production was not detected in the presence of isoprene and lactate, however in the presence of lactate alone methane production commenced after 48 days and increased to 700 μmoles within 100 days with associated depletion of acetate and propionate [ Figs. 1c and 2c, d]. Autoclaved and uninoculated controls did not show any depletion of isoprene or formation of methylbutenes.

Community analysis of lactate driven anaerobic isoprene transformations
Bacterial and archaeal community analysis was performed using 16S rRNA gene amplicons derived from DNA extracted from the lactate and isoprene fed cultures sampled before and after isoprene consumption [ Fig. 3]. In the isoprene fed cultures, Acetobacterium, Geobacter, and in one replicate Anaeromusa where found to be the dominant bacterial genera [Fig. 3a]. Cultures supplied with lactate but without isoprene were dominated by a consortium of methanogenic archaea and bacteria Geobacter, Clostridium, Acetobacterium, and Anaeromusa [ Fig. 3b].

Community analysis of H 2 driven anaerobic isoprene transformations
Illumina sequencing of 16S rRNA gene amplicons from H 2 supplied, isoprene reducing enrichment cultures revealed enrichment of Acetobacterium to 92-100% relative abundance [ Figs. 4a and 6]. Comamonadaceae were also present (2-7%). There was no notable difference between bacterial community compositions in the presence vs. absence of isoprene [ Fig. 6].
To further probe an isoprene dependent difference in community composition a new set of cultures was monitored over a longer period by resupply of isoprene, H 2 , and HCO 3 − when depleted [ Fig. 7]. Isoprene was depleted within 3-5 days after each resupply at a similar rate as observed before (1.6 μmoles h −1 ) [ Fig. 7a]. Methylbutene accumulated up to 800 μmoles, respectively [ Fig. 7b]. Illumina sequencing of 16S rRNA amplicons again showed no appreciable difference in microbial community composition when comparing cultures with or without isoprene. Both communities were once again dominated by Acetobacterium spp. [ Supplementary Fig. S3]. Additionally, Acetobacterium cell numbers were similar after 29 days (i.e., 4.8 × 10 7 ± 1.3 × 10 7 cells ml −1 for cultures with isoprene and 5.6 × 10 7 ± 2.8 × 10 7 cells ml −1 for those without) [ Fig. 7e]. Importantly, however, there was a significant difference in the amount of acetate produced in cultures with and without isoprene. After day 16, cultures with isoprene produced~250 μmoles of acetate while those without produced~400 μmoles [ Fig. 7c].

Isoprene serves as an electron acceptor
Even though isoprene is a highly abundant, energy rich metabolite, little is known about its anaerobic metabolism. In this study we tested the utility of isoprene as both an electron donor for inorganic oxide reduction and as an electron acceptor where lactate and molecular H 2 were electron donors.
Anaerobic isoprene oxidation coupled to inorganic oxide reduction (SO 4 2− , NO 3 2− , or Fe 3+ ) could not be demonstrated in any of the tested inocula after 1 year of incubation. Under standard conditions, anaerobic isoprene oxidation is exergonic, considering the theoretical stoichiometries of isoprene mineralization and SO 4 2− , Fe 3+ , or NO 3 − reduction (isoprene energy of formation calculated as 197 kJ mol −1 [45,46].
Evidently the enzyme systems required for the above transformations are either non-existent, extremely rare or inhibited or not induced under the conditions applied.
Acetobacterium spp. reduce isoprene Illumina sequencing targeting archaeal and bacterial 16S rRNA gene amplicons revealed that acetogenic Acetobacterium spp. dominated the isoprene reducing enrichment culture. Acetogens are anaerobic bacteria that use CO 2 as a terminal electron acceptor for energy conservation and carbon fixation utilizing the reductive acetyl coenzyme A (acetyl-CoA) pathway or Wood-Ljungdahl pathway [48,49]: They are a phylogenetically and metabolically diverse group using a variety of different electron donors and acceptors [50]. Reducing equivalents can be generated from oxidation of H 2 , C 1 -compounds like methanol and formate, sugars, organic acids, and alcohols [51]. Besides CO 2 , acetogens can also use alternative electron acceptors such as Fig. 6 Composition of the bacterial populations at Family level and if possible on Genus level of the isoprene reducing culture during growth on H 2 + HCO 3 − + isoprene and on H 2 + HCO 3 − controls at different time points 0, 46, and 96 h. Other category is the sum of all classifications with <2% abundance. Error bars represent one standard deviation (n = 3). Classifications in the legend are clustered according to their phylum (from top to bottom); Firmicutes, Proteobacteria, Actinobacteria, Euryarchaeota acrylate derivatives [52], fumarate [53], nitrate [54], chlorethenes, chlorethanes [55], and brominated/aromatic compounds [44]. The best studied example for CO 2 -alternative electron acceptors in acetogens is the reduction of the carbon-carbon double bond in phenylacrylates (e.g., caffeate) by the model organism Acetobacterium woodii [52,56,57].
As shown in this study, Acetobacterium spp. appear also to utilize isoprene as an alternative electron acceptor to CO 2 . Isoprene reduction to methylbutenes depended on the presence of H 2 and CO 2 [ Fig. 4], common substrates for acetogens, which can otherwise only be metabolized by methanogens under anaerobic conditions. Methanogens Fig. 7 Depletion of isoprene (a) and reciprocal production of total methylbutenes (b) in anaerobic cultures containing an enriched isoprene reducing culture amended with H 2 and HCO 3 − with and without isoprene. Cultures amended with isoprene produced 40% less acetate (c) while still consuming the same amount of H 2 (d). Acetobacterium cells ml −1 (e) calculated from 16S rDNA gene copies ml −1 demonstrate equivalent growth in both conditions. ***p-value < 0.0001, **pvalue = 0.005 analyzed by a two-way ANOVA. Error bars represent one standard deviation (n = 4) were not present in isoprene amended enrichment cultures and no methane formation was observed. In cultures resupplied with H 2 , HCO 3 − , and isoprene, 40% less acetate was formed compared to H 2 and HCO 3 − supplied cultures [ Fig. 7c] suggesting that the Acetobacterium spp. transferred electrons from H 2 to isoprene at the expense of CO 2 reduction. In cultures without isoprene, 400 μmoles acetate were produced, which required oxidation of 1600 μmoles H 2 (Eq. 5) or 3200 μmoles of electrons. However, cultures with isoprene produced only 240 μmoles of acetate requiring 960 μmoles H 2 . In addition 800 μmoles H 2 were required to reduce 800 μmoles of isoprene. Altogether that totals 1760 μmoles H 2 or 3520 μmoles of electrons transferred in cultures supplied with isoprene. Since similar amounts of electrons were transferred in cultures with (3520 μmoles) and without isoprene (3200 μmoles) and similar cell densities in each condition were observed, it can be concluded that the shortage of acetate in isoprene amended microcosms results from the reduction of isoprene instead of CO 2 . Isoprene reduction might, therefore, also be coupled to energy conservation. Similar results were found by Hansen et al. [58]  Considering the ΔG°values of the isoprene/methylbutene couple (Eq. 4), isoprene reduction is energetically favored over CO 2 reduction (Eq. 5). This could lead to preferential use of isoprene over CO 2 . However, in the case of caffeate (caffeate/hydrocaffeate), its reduction and acetogenesis from H 2 and CO 2 were either catalyzed simultaneously or CO 2 was preferred over caffeate [56,58]. Tschech and Pfennig [59] observed that caffeate was preferentially used over CO 2 as an electron acceptor in the presence of methanol (methanol/caffeate 1:3). In the enrichment culture generated in this study, it remains to be tested whether isoprene reduction also occurs simultaneously catalyzed by a dominating Acetobacterium spp. by multiple or if one species only reduces isoprene and not CO 2 .
It was unexpected that pure Acetobacterium isolates (A. woodii DSM 1030, A. malicum DSM 4132, and A. wieringae DSM 1911) showed no isoprene reduction activity. However, Acetobacterium are a particularly tight phylogenetic group with 96-99% 16S gene sequence similarity between species [60]. It is possible that an uncultured species of Acetobacterium is responsible for isoprene reduction, or the gene enabling the reduction was acquired via horizontal gene transfer. These possibilities will be investigated in future studies. Also, Comamonadaceae cannot be conclusively excluded from involvement in isoprene reduction as Illumina sequencing data did not reveal the genus of organisms present. There is a possibility that Hydrogenophaga lineages, some of which can oxidize H 2 and fix CO 2 [61], contribute to isoprene reduction.
Bioenergetics and growth efficiencies of acetogens can be evaluated by using the acetate-to-biomass ratios [62]. Even though less acetate was formed in isoprene amended cultures, similar cell densities were achieved. This suggests that isoprene reduction is coupled to ATP synthesis as shown for caffeate reduction by A. woodii [58]. The current  [44] to be 197 kJ mol -1 model in A. woodii suggests that electrons flow from H 2 to NAD + and ferredoxin, which are reduced by a electronbifurcating hydrogenase [58]. A Na + -translocating ferredoxin: NAD + -oxidoreductase then oxidizes ferredoxin (Rnf complex) and generates a Na + gradient over the cytoplasmic membrane for ATP synthesis [57,63,64]. It is possible that some of these enzymes might be involved in isoprene reduction. But in contrast to phenylacrylates, fumarate and chlorethenes, isoprene is an unsubstituted alkene making nucleophilic attack more difficult.

Hydrogenation of isoprene
In the present study, we have shown that isoprene is hydrogenated to three methylbutene isomers with proportions remaining constant throughout the experiments (2methyl-1-butene 98%, 3-methyl-1-butene 1-2%, and 2methyl-2-butene < 1%) i.e., fully reduced alkanes were not produced [ Supplementary Fig. S2]. Hydrogenation of a double bond is a thermodynamically favorable reaction because it forms a more stable (lower energy) product. The released heat is referred to as the heat of hydrogenation (ΔHh 298°) , which reflects the stability of a molecule. Isoprene, due to its conjugated doubled bonds, has a ΔHh 298°v alue of −55 kcal mol −1 compared to its reduced forms (ΔHh 298°3 -methyl-1-butene; −29.9 kcal mol −1 , 2-methyl-1-butene; −28.24 kcal mol −1 and 2-methyl-2-butene; −26.74 kcal mol −1 [65]. Therefore, reduction of a single double bond requires more activation energy and a different set of enzymes. This could explain why ethylene (H 2 C = CH 2 ) was not reduced to ethane by the active isoprene reducing culture. A few observations of microbial hydrogenation of ethylene to ethane have been made [66][67][68][69] but a pure bacterial culture or responsible enzymes have not been identified. It remains to be tested whether the enzymatic hydrogenation of isoprene to methylbutene occurs directly or via an intermediate hydration product (e.g., 3-Methyl-3-buten-1-ol).
The physiological role of isoprene reductionmetabolic strategies of acetogens Depletion of electron acceptors creates a niche for acetogens and methanogens due to their ability to obtain energy from CO 2 reduction via H 2 oxidation [70,71]. In these environments acetogens compete with methanogens for H 2 .
Thermodynamically, hydrogenotrophic methanogenesis (Eq. 6) is favored over acetogenesis (Eq. 5), therefore acetogens are physiologically less competitive for H 2 when it is a limiting resource. Yet, their ability to use different electron acceptors enhances the in situ competitiveness of acetogens [72,73]. Given that ΔG°values for hydrogenotrophic isoprene reduction (Eq. 4) and bicarbonate reduction to methane (Eq. 6) are equivalent, the use of isoprene as an alternative electron acceptor would enable acetogens to compete with methanogens at similar H 2 threshold concentrations.
According to Lever et al. [70] there are two hypotheses for how the wide metabolic spectrum of acetogens enables them to coexist with sulfate reducers and methanogens. Firstly, metabolic versatility leads to niche differentiation with respect to substrate and secondly acetogens can pool energy from a broad range of metabolic reactions (e.g., simultaneous CO 2 /caffeate and now CO 2 /isoprene reduction). Being able to utilize a substrate that inhibits a potential competitor could add a third strategy, whereby environments with isoprene present logically favouring acetogenic organisms.
In this study, isoprene was observed to inhibit methanogenesis. The inhibitory effect of isoprene on methanogenesis in sediment slurries has been observed previously [74] where isoprene (3.6 mM in liquid phase) partially inhibited methanogenesis. Little is known about isoprene concentrations in anaerobic environments and hence it cannot be concluded yet if isoprene mediated inhibition of methanogenesis is ecologically or biogeochemically relevant. Therefore, it is also not yet clear whether isoprene reducers can consume atmospheric isoprene or if they consume isoprene generated in soil sources such as bacteria [20]. Isoprene concentrations used in this and other studies on isoprene biotransformation are in the order of 10 6 times higher than observed under natural conditions [9,30,34,[75][76][77]. Regardless, even at high concentrations the observed capacity for microbes to consume isoprene is far from saturated, suggesting the isoprene consuming microbial community is large relative to the isoprene supply and/ or enzymes involved are highly efficient [75].

Conclusion
This study explored the anaerobic metabolism of isoprene, which is quantitatively the most abundant volatile hydrocarbon emitted by plants. Isoprene was shown to act as an electron acceptor for homoacetogenic bacteria belonging to the Acetobacterium genera and was shown to be reduced to three different methylbutene isomers in an H 2 dependent manner. Isoprene had an inhibitory effect on methanogenesis so there may be a relationship between isoprene emission and methane biosynthesis. The discovery of biohydrogenation of this unsubstituted, unsaturated alkene whose functional group makes up 60% of all Natural Products (isoprenoids or terpenoids) on Earth [78] warrants further investigation. Future experiments should explore the isoprene reduction mechanism, the enzymes involved and its ecological role in biogenic methane sources. Overall, this research demonstrates that isoprene is capable of being reduced in anaerobic environments, implicating a potential previously undiscovered isoprene sink.

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