Mohr et al. Microb Cell Fact (2018) 17:108 https://doi.org/10.1186/s12934-018-0954-3 Microbial Cell Factories

RESEARCH Open Access CO‑dependent hydrogen production by the facultative anaerobe Parageobacillus thermoglucosidasius Teresa Mohr1,4* , Habibu Aliyu1, Raphael Küchlin1, Shamara Polliack2, Michaela Zwick1, Anke Neumann1, Don Cowan2 and Pieter de Maayer3

Abstract Background: The overreliance on dwindling fossil fuel reserves and the negative climatic efects of using such fuels are driving the development of new clean energy sources. One such alternative source is hydrogen (H­ 2), which can be generated from renewable sources. Parageobacillus thermoglucosidasius is a facultative anaerobic thermophilic bacte- rium which is frequently isolated from high temperature environments including hot springs and compost. Results: Comparative genomics performed in the present study showed that P. thermoglucosidasius encodes two evolutionary distinct ­H2-uptake [Ni-Fe]-hydrogenases and one ­H2-evolving hydrogenases. In addition, genes encod- ing an anaerobic CO dehydrogenase (CODH) are co-localized with genes encoding a putative H­ 2-evolving hydroge- nase. The co-localized of CODH and uptake hydrogenase form an enzyme complex that might potentially be involved in catalyzing the water-gas shift reaction (CO ­H2O ­CO2 ­H2) in P. thermoglucosidasius. Cultivation of P. thermo- glucosidasius DSM 2542­ T with an initial gas atmosphere+ → of 50%+ CO and 50% air showed it to be capable of growth at elevated CO concentrations (50%). Furthermore, GC analyses showed that it was capable of producing hydrogen at an equimolar conversion with a fnal yield of 1.08 ­H2/CO. Conclusions: This study highlights the potential of the facultative anaerobic P. thermoglucosidasius DSM 2542­ T for developing new strategies for the biohydrogen production. Keywords: Biohydrogen production, Parageobacillus thermoglucosidasius, Carbon monoxide dehydrogenase, Hydrogenase, Water-gas shift reaction

Background Hydrogen ­(H2) has recently become prominent as In the next 30 years, the global energy demand will a very attractive clean and sustainable energy source, expand by ca. 30% and the vast majority (ca. 85%) of the especially when generated via ‘eco-friendly’ strategies. energy resources required to ofset the rising demand will In comparison to other fuels, ­H2 has the highest energy come from non-renewable sources such as natural gas content (141.9 MJ/kg higher heating value) [2]. Addi- and crude oil [1]. Tis will result in increased pressure on tionally, its complete combustion with pure oxygen pro- the dwindling fossil fuel reserves and in greater emission duces only water (2 ­H2 + O2 → 2 ­H2O) as a by-product. of greenhouse gases into the Earth’s atmosphere. Tere is Te majority of current industrial ­H2 production strat- thus an urgent need for further development and imple- egies, such as coal gasifcation, steam reformation and mentation of clean and renewable alternative energy partial oxidation of oil, are unsustainable, harmful to sources [2, 3]. the environment, energy intensive and expensive [4, 5]. As such, over the past few years, the production of *Correspondence: [email protected] ­H via microbial catalysis has drawn increasing inter- 4 Section II: Technical Biology, Institute of Process Engineering in Life 2 Science, Karlsruhe Institut für Technologie (KIT), Kaiserstrasse 12, est. Several diferent strategies to produce biohydro- 76131 Karlsruhe, Germany gen, such as photofermentation of organic substances Full list of author information is available at the end of the article

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mohr et al. Microb Cell Fact (2018) 17:108 Page 2 of 12

by photosynthetic bacteria, bio-photolysis of water by Methods algae and dark fermentation of organic substances by Microorganisms anaerobic microorganism, have been explored [6]. Tese Te production of ­H2 by P. thermoglucosidasius when strategies have the advantage of lower energy expendi- grown in the presence of CO was tested using P. ther- ture, lower cost and higher yields than the industrial moglucosidasius DSM ­2542T. Two related strains, methods [7]. Another advantage is the potential to use Geobacillus thermodenitrifcans DSM ­465T and P. toe- cheap feedstocks, such as lignocellulosic waste bio- bii DSM 14590­ T, which lack orthologues of the three mass, which can be converted into a gas mixture termed hydrogenase loci as well as the CODH locus, were ‘synthesis gas’. Tis gas consists primarily of carbon included as controls. All strains were obtained from monoxide (CO), carbon dioxide ­(CO2) and ­H2 [8]. In a the DSMZ (Deutsche Sammlung von Mikroorganismen further step, the CO can react with water to generate und Zellkulturen GmbH, Braunschweig, Germany). ­H2 via a biologically- or chemically-mediated water-gas shift (WGS) reaction: CO + H2O → CO2 + H2. During the biologically mediated reaction, a carbon monoxide Culture conditions and media dehydrogenase (CODH) oxidizes CO and electrons are Pre-cultures and cultures were grown aerobically in released. Subsequently, a coupled hydrogenase reduces mLB (modifed Luria–Bertani) medium containing the released electrons to molecular hydrogen [9]. Sev- tryptone (1% w/v), yeast extract (0.5% w/v), NaCl (0.5% eral mesophilic, anaerobic prokaryotic taxa, including w/v), 1.25 ml/l NaOH (10% w/v), and 1 ml/l of each of Rhodospirillum rubrum and Rhodopseudomonas palus- the flter-sterilized stock solutions: 1.05 M nitrilotri- tris, are known for the ability to perform the WGS reac- acetic acid, 0.59 M ­MgSO4·7H2O, 0.91 M ­CaCl2·2H2O tion [10]. and 0.04 M FeSO­ 4·7H2O. Te frst pre-culture was It has been observed that higher yields of ­H2 can be inoculated from glycerol stock (20 µl in 20 ml mLB) and obtained in higher temperature fermentations [11]. cultivated for 24 h at 60 °C and rotation at 120 rpm in Thus, there has been increasing interest in the use of an Infors Termotron (Infors AG, Bottmingen, Swit- thermophilic anaerobic bacteria, such as Carboxydo- zerland). A second pre-culture was inoculated from the thermus hydrogenoformans and Thermosinus carboxy- frst one to an ­OD600 of 0.1 and incubated as above for divorans [12, 13], as well thermophilic archaea (e.g. 12 h. For the experiments, 250 ml serum bottles were Thermococcus onnurineus) [7]. prepared with 50 ml mLB and a gas phase of 50% CO An industrial process utilizing CO-oxidizing bacte- and 50% atmospheric air at 1 bar atmospheric pressure, ria for biohydrogen production has not yet been real- which were inoculated with 1 ml from the second pre- ized, although many CO-using hydrogenogenic species culture. Te experiments were conducted in quadrupli- have been isolated. This may largely be attributed to cate for a total duration of 84 h. the sensitivity of both the hydrogenase and CODH enzymes to oxygen [6, 14]. For example, the hydro- genase of Thermotoga maritima lost 80% of its activ- Analytical methods ity after flushing with air for 10 s [15]. Removal of O­ 2 Samples were taken at diferent time points during the from industrial waste gases or from bioreactors is pro- experimental procedure. Before and after the sampling hibitively expensive, making the use of strictly anaero- the pressure was measured using a manometer (GDH bic CO-oxidizing hydrogenogens unfeasible [16]. Here, 14 AN, Greisinger electronic, Regenstauf, Germany). we have analysed the hydrogenogenic capacity of the To monitor the growth of the cultures, 1 ml of the cul- facultative anaerobe Parageobacillus thermoglucosi- ture was aspirated through the stopper and absorb- dasius. Comparative genomics revealed the presence ance was measured at OD­ 600 using an Ultrospec 1100 of three distinct hydrogenases, two uptake hydroge- pro spectrophotometer (Amersham Biosciences, USA). nases as well as one ­H2-evolving hydrogenase, which Te determination of the gas compositions at diferent is linked to an anaerobic CODH. Evolutionary analy- time points was conducted using a 3000 Micro GC gas sis showed that this combination of hydrogenases is analyzer (Infcon, Bad Ragaz, Switzerland) with the col- unique to P. thermoglucosidasius and suggests that umns Molsieve and PLOT Q. A total of 3 ml was sam- ­H2 plays a pivotal in the bioenergetics of this organ- pled from the head space and injected into the GC. A ism. Furthermore, fermentations and downstream GC constant temperature of 80 °C was maintained during analysis showed that this facultative anaerobe is capa- the total analysis time of 180 s. Te gas compositions at ble of utilizing CO in the WGS reaction to generate an the diferent sampling points were calculated using the equimolar amount of ­H2 once most of the oxygen in formulas in Additional fle 1. the medium has been exhausted. Mohr et al. Microb Cell Fact (2018) 17:108 Page 3 of 12

Comparative genomic analyses 1d uptake hydrogenase (E-value = 0.0). Tis unidirec- Te large hydrogenase subunits were identifed from the tional, membrane-bound, O­ 2-tolerant hydrogenase is annotated genome of P. thermoglucosidasius DSM 2542­ T present in a broad range of obligately aerobic and facul- (CP012712.1) by comparison against the Hydrogenase tatively anaerobic soil-borne, aquatic and host-associated DataBase (HyDB) [17]. Te full hydrogenase loci were taxa such as Ralstonia eutropha, Escherichia coli and identifed by searching the genome up- and downstream Wolinella succinogenes [25, 26]. Te H­ 2 molecules con- of the large subunit gene, extracted and mapped against sumed by group 1d hydrogenases are coupled to aerobic the genome using the CGView server [18]. Te proteins respiration ­(O2 as electron acceptor) or to respiratory encoded on the genome were compared by BlastP against reduction of various anaerobic electron acceptors includ- 3− 2− the NCBI non-redundant (nr) protein database to iden- ing ­NO , ­SO4 , fumarate and CO­ 2. Te P. thermoglu- tify orthologous loci. Full loci were extracted from the cosidasius DSM ­2542T hydrogenase locus incorporates comparator genomes and all loci were structurally anno- genes coding for both small (PhaA; ALF10692; 324 aa) tated using Genemark.hmm prokaryotic v.2 [19]. Te and large (PhaB; ALF10693; 573 aa) catalytic hydroge- resultant protein datasets were compared by local BlastP nase subunits. Te strain also encodes seven additional with Bioedit v 7.2.5 [20] to identify orthologues, where proteins involved in hydrogenase formation, maturation orthology was assumed for those proteins sharing > 30% and incorporation of the Ni-Fe metallocenter, includ- amino acid identity over 70% sequence coverage. ing a third hydrogenase subunit (PhaC) which is pre- A maximum likelihood (ML) phylogeny was con- dicted to serve as cytochrome b orthologue and links structed based on the amino acid sequences of three the hydrogenase to the quinone pools of the respiratory commonly used housekeeping markers: translation ini- chains (Fig. 1; Additional fle 2) [26]. Te pha genes are tiation factor IF-2 (InfB), DNA recombination and repair fanked at the 5′ end by two genes coding for orthologues protein RecN RNA polymerase subunit B (RpoB). Te of the Twin-arginine translocation (Tat) pathway proteins proteins were individually aligned using M-Cofee [21], TatA and TatC (Fig. 1; Additional fle 2). Tese have been the alignments concatenated and poorly aligned regions shown to form part of the membrane targeting and trans- were trimmed using Gblocks [22]. Finally, the trimmed location (Mtt) pathway which targets the fully folded alignment was used to generate a ML phylogeny using hydrogenase heterodimer to the membrane [27]. PhyML-SMS, using the optimal amino acid substitution Te Phb locus (chromosomal position 2,488,614– model as predicted by the Smart Model Selection tool 2,503,714; 15.1 kb in size), located ~ 19 kb downstream [23, 24]. Similarly, ML phylogenies were constructed of the Pha locus, comprises 16 protein coding sequences on the basis of the concatenated orthologous proteins (NCBI Accession ALF10723-738; PhbA-PhbP) (Fig. 1; encoded on the Pha, Phb, Phc and CODH loci. Additional fle 2). Te predicted catalytic subunit (ALF10727—PhbE) compared against HydDB classifes the product of this locus as a [Ni-Fe] group 2a uptake Results hydrogenase (E-value = 0.0) [17]. Members of this group The genome of P. thermoglucosidasius encodes three of uptake hydrogenases are widespread among aero- distinct hydrogenases bic soil bacteria and Cyanobacteria and play a role in Analysis of the complete, annotated genome sequence of recycling ­H2 produced by nitrogenase activity and fer- T P. thermoglucosidasius DSM 2542­ showed the presence mentative pathways [28, 29]. Te recycled H­ 2 is used in of three putative [Ni-Fe]-hydrogenase loci on the chro- hydrogenotrophic respiration with O­ 2 serving as termi- mosome. Two of these hydrogenases are encoded on the nal electron acceptor, and thus group 2a hydrogenases forward strand, while the third is located on the reverse are often O­ 2-tolerant [26]. Tis locus encodes both large strand (Fig. 1). Given the convoluted nomenclature of (PhbE; ALF10727; 544 aa) and small (PhbD; ALF10726; hydrogenase genes, we have termed these loci as Para- 317) [Ni-Fe] hydrogenase subunits and eight additional geobacillus hydrogenase a, b and c, in accordance with proteins with predicted roles in hydrogenase formation, their chromosomal locations), to distinguish between maturation and incorporation of the Ni-Fe metal center them. in the large subunit (Fig. 1; Additional fle 2) [26]. Fur- Te Pha locus (chromosomal position 2,456,963– thermore, this locus encodes six proteins whose role 2,469,832; 12.9 kb in size) comprises eleven protein in hydrogenase biosynthesis and functioning remains coding sequences (NCBI Accession ALF10692-10702; unclear. Tese include a tetratricopeptide-repeat (PhbH) PhaA-PhaK) (Fig. 1; Additional fle 2). Comparison of and NHL repeat (PhbK) containing protein, which also the amino acid sequence of the predicted catalytic subu- occur in the [Ni-Fe] group 2a hydrogenase loci of Nos- nit (ALF10727—PhaB) against HydDB [17] classifes the toc punctiforme ATCC 29133 and Nostoc sp. PCC 7120, hydrogenase produced by this locus as a [Ni-Fe] group where they are co-transcribed with the hydrogenase Mohr et al. Microb Cell Fact (2018) 17:108 Page 4 of 12

P. thermoglucosidasius DSM 2542T

Phc

Phb Pha

2 kb

phaE phaG phaI Pha -[Ni-Fe] group 1d atlF tatA tatC phaA phaB phaC phaD phaF phaH phaJ phaK thyA uptake hydrogenase

phbB phbG grpB phbA phbC phbD phbE phbF phbH phbI phbJ phbK phbL phbM phbN phbO phbP hsp70aarF Phb -[Ni-Fe] group 2a uptake hydrogenase

phcI Phc -[Ni-Fe] group 4a mdaB cooC cooS cooF phcA phcB phcC phcD phcE phcF phcG phcH phcJ phcK phcL empB

H2-evolving hydrogenase Fig. 1 Schematic diagram of the [Ni-Fe] hydrogenase loci and their localization on the chromosome of P. thermoglucosidasius DSM 2542­ T

genes and have been suggested to play a role in protein– enterobacteria associated with animal intestinal tracts protein interactions and Fe–S cluster biogenesis (PhbJ) [25, 26]. FHL-1 couples the reduction of protons from which may mediate electron transport to redox partners water to the anaerobic oxidation of formate to form ­CO2 in downstream reactions [30]. and ­H2 [26, 31]. Te Phc locus (chromosomal position 2,729,489– BlastP and tBlastN analyses of the protein sequences 2,741,372), ~ 226 kb downstream of the Phb locus is encoded in the P. thermoglucosidasius DSM ­2542T hydro- 11.9 kb in size and encodes 12 distinct proteins (PhcA- genase loci showed that the Pha, Phb and Phc loci are PhcL) (Fig. 1; Additional fle 2). Tese include a small universally present in eight other P. thermoglucosida- (PhcE; ALF10919; 247 aa) and large (PhcG; ALF19021; sius strains for which genomes are available (Additional 574 aa) [Ni-Fe]-hydrogenase catalytic subunit and ten fle 2: Table S1). Tese loci are highly syntenous and the additional proteins involved in hydrogenase formation encoded proteins share average amino acid identities of and maturation (Fig. 1; Additional fle 2). Te HydDB 99.73% ([Ni-Fe]-group 1d hydrogenase Pha—13 pro- classifes the Phc hydrogenase as a [Ni-Fe] group 4a teins), 99.61% ([Ni-Fe]-group 2a hydrogenase Phb—16 hydrogenase or formate hydrogenlyase complex I (FHL- proteins) and 99.22% ([Ni-Fe]-group 4a hydrogenase 1) [17]. Members of this group are oxygen-sensitive, Phc—12 proteins) with those of DSM 2542­ T, respec- membrane-bound and are largely restricted to the fac- tively. Pairwise BlastP analyses showed limited orthology ultatively fermentative Proteobacteria, particularly between the two uptake hydrogenase loci, with 36.29% Mohr et al. Microb Cell Fact (2018) 17:108 Page 5 of 12

average amino acid identity in nine proteins encoded NUB3621, with an average amino acid identity of on the two loci. Te group 1d (Pha) and group 2a (Phb) 92.37% (13 proteins) with the DSM ­2542T Pha pro- uptake hydrogenase loci share 33.40 and 62.32% average teins. A phylogeny on the basis of nine conserved Pha amino acid identity for three proteins with the H­ 2 evolv- proteins (PhaABCDGHIJK) showed a similar branch- ing hydrogenase (Phc) locus. Te higher level of orthol- ing pattern (Fig. 3a) as observed for the phylogeny ogy for Phb and Phc loci proteins can be correlated with housekeeping protein (InfB-RecN-RpoB) phylogeny, the HypA-like (PhbB and PhcK) and the HypB-like (PhbC suggesting that this is an ancestral locus that has been and PhcL) proteins, which share 75.22 and 86.08% amino vertically maintained. Tis is supported by the low level acid identity, respectively, and are predicted to play a of discrepancy in G+C content for the P. thermoglu- role in the incorporation of nickel into the hydrogenase cosidasius strains, which are on average 0.87% above enzyme [32]. Limited orthology is observed between the genomic G+C content. Larger discrepancies are, the hydrogenase catalytic subunits or other hydroge- however, evident among the Bacteroidetes, where G+C nase formation and maturation proteins, suggesting dis- contents for the locus are on average 4.43% above that tinct evolutionary histories for the two uptake and one of the genome, and the absence of Pha loci in other ­H2-evolving hydrogenases in P. thermoglucosidasius. Parageobacillus spp. including P. toebii (fve genomes available) and P. caldoxylosilyticus (four genomes avail- P. thermoglucosidasius contains a unique profle able) and Geobacillus spp. suggest a more complex evo- of hydrogenases with distinct evolutionary histories lutionary history for the group 1d hydrogenase. Te proteins encoded by the Pha, Phb and Phc loci were Orthologous [Ni-Fe] group 2a uptake hydrogenase used in BlastP comparisons against the NCBI non- (Phb) loci are also common among the Firmicutes, but redundant (nr) protein database and HydDB (catalytic show a more restricted distribution within the fam- subunits) to identify orthologous loci in other bacterial ily Bacillaceae, with only Aeribacillus pallidus 8m3 and taxa. Tis revealed that, aside from the α-proteobacteria Hydrogenibacillus schlegelii DSM ­2000T containing Azospirillum halopraeferens DSM ­3675T and Rhodopseu- orthologues outside the genus Parageobacillus. Highly domonas palustris BAL398, the combination of [Ni-Fe] conserved and syntenous loci are, however, present in group 1- 2- 4 hydrogenases appears to be unique to P. three non-thermoglucosidasius strains: Parageobacillus thermoglucosidasius (Fig. 2). In these two proteobacte- sp. NUB3621, Parageobacillus sp. W-2 and P. toebii DSM rial taxa the group 2a uptake hydrogenase is, however, 18751 (Fig. 3b; Additional fle 3). Orthologous loci are replaced by a group 2b uptake hydrogenase. Group 2b present across a much wider range of phyla than the Pha uptake hydrogenases do not have a direct role in energy locus, including members of the Chlorofexi, Gemmati- transduction but are fanked by a PAS domain protein monadetes, Actinobacteria, Proteobacteria, Nitrospirae which accepts the hydrogenase-liberated electrons, mod- and Deinococcus-Termus (Fig. 2). Te latter is of inter- ulating the activity of a two-component regulator that est as Termus thermophilus SG0.5JP17-16 clusters with upregulates the expression of other uptake hydrogenases, the Firmicutes in a phylogeny of ten conserved proteins thereby serving as ­H2-sensing system [33, 34]. (PhbBCDEFHJLMN—72.76% average amino acid iden- Te Pha uptake hydrogenase locus is relatively well tity with P. thermoglucosidasius DSM 2542­ T) (Fig. 3b), conserved among members of the Firmicutes, includ- but is phylogenetically disparate from the Firmicutes. ing a number of taxa belonging to the Classes Bacilli, Te T. thermophilus locus is present on the plasmid Clostridia and Negativicutes, as well as the phyla pTHTHE1601 (NC_017273) suggesting that this locus Proteobacteria and Bacteroidetes (Fig. 2; Additional forms part of the mobilome. Furthermore, the G+C con- fle 3). However, the more distantly related taxa retain tent of the Phb locus difers by an average of 4.55% from little synteny with the Pha locus in P. thermoglucosi- the average genomic G+C among the eight compared P. dasius (Fig. 3a). Orthologues of the Pha are present thermoglucosidasius strains, suggesting recent horizontal in one other Parageobacillus spp., namely genomosp. acquisition of this locus.

(See fgure on next page.) Fig. 2 Prevalence of [Ni-Fe] hydrogenases orthologous to those in P. thermoglucosidasius among other bacterial taxa. The ML phylogeny was constructed on the basis of the trimmed alignment (1597 amino acids in length) of the concatenated InfB, RecN and RpoB amino acid sequences. Diferent taxa and branch colours indicate the diferent phyla/classes. Values on the branches indicate bootstrap values (n 500 replicates) and = the tree was rooted on the midpoint. The presence of [Ni-Fe] group 1d, group 2a and group 4a hydrogenases is represented by dark blue, red and green blocks, respectively. Where [Ni-Fe] hydrogenases belonging to the same groups but not the same subtype as those in P. thermoglucosidasius are present they are indicated by light blue ([Ni-Fe] group 1 hydrogenases), pink ([Ni-Fe] group 2 hydrogenases) and light green ([Ni-Fe] group 4 hydrogenases) blocks, respectively Mohr et al. Microb Cell Fact (2018) 17:108 Page 6 of 12

UPTAKE EVOLVING

1 24 P. thermoglucosidasius YU P. thermoglucosidasius ZCTH02-B4 P. thermoglucosidasius B4168 P. thermoglucosidasius Y4.1MC1 486 P. thermoglucosidasius TNO09.020 P. thermoglucosidasius M10EXG

425 P. thermoglucosidasius GT23 P. thermoglucosidasius DSM 2542T

500 P. thermoglucosidasius C56-YS93 Parageobacillus sp. W-2

495 P. toebii DSM 18751 342 Parageobacillus sp. NUB3621 Anoxybacillus thermarum DSM 17141T 500 Anoxybacillus flavithermus DSM 2641T Bacilli 373 Bacillus solimangrovi DSM 27083T 316 Bacillus azotoformans DSM 1046T Bacillus drentensis DSM 15600T 500 Aeribacillus pallidus 8m3 1a Ureibacillus thermosphaericus A1 458 Anaerobacillus arseniciselenatis DSM 15340T

T 422 Sporolactobacillus laevolacticus DSM 442 Aneurinibacillus terranovensis DSM 18919T Firmicutes 318 Planifilum fulgidum DSM 44945T

323 Alicyclobacillusmacrosporangiidus CPP55 493 Kyrpidia tusciae DSM 2912T 448 Effusibacillus lacus DSM 27172T 4f Hydrogenibacillus schlegelii DSM 2000T Heliobacterium modesticalcum DSM 9504T

314 Moorella thermoacetica DSM 2955T

393 Moorella thermoacetica DSM 12993

500 Moorella thermoacetica Y72 364 Moorella thermoacetica DSM 11768 500 Moorella thermoacetica DSM 21394 500 Moorella glycerini NMP Thermanaeromonas toyohensis DSM 14490T 1a 497 385 Calderihabditans maritimus DSM 26464T 1a 296 Thermoacetogenium phaeum DSM 12270T 1a Clostridia Sulfobacillus acidophilus DSM 10332T 1a Megamonas hypermegale DSM 1672T 500 Acetonema longum DSM 6540T 4f 429 500 Caldanaerobacillus subterraneus subsp. yonseiensis DSM 13777T Caldanaerobacter subterraneus subsp. tengcongensis DSM 15242T 500 Thermanaeromonas toyohensis DSM 14490T 1a 500 Roseiflexus castenholzii DSM 13941T 1f T Chloroflexi 348 Chloroflexus aggregans DSM 9485 500 Mycobacterium smegmatis DSM 43756T 1h

500 T Gordoniarhizosphera DSM 44383 1h Actinobacteria Pseudonocardia thermophila DSM 43832T 1h 500 Streptomyces thermoautotrophicus DSM 41605T 1h Thermus thermophilus SG0.5JP17-16 Deinococcus-Thermus 500 Yersinia enterocolitica 8081 1c 500 Escherichia coli K-12 str. MG1655 363 Methylomonas methanica MC09 2c

T 500 Uliginosibacterium gangwonense DSM 18521 1e 500 Paludibacterium yongneupense DSM 18731T Acidithiobacillus thiooxidans CLST 499 383 Rhodobacter sphaeroides WS8N 2c Novosphingobium pentaromativorans DSM 17173T Proteobacteria 500 Bradyrhizobium japonicum 22 2d 500 Bradyrhizobium canariense UBMA171 500 303 500 Rhodopseudomonas palustris BAL198 2b 500 Azorhizobium caulinodans DSM 5975T 2b Methylocapsa acidiphila DSM 13967T Azospirillum halopraeferens DSM 3675T 2b Nitrospira moscoviensis DSM 10035T Nitrospira Coprobacter fastidiosus DSM 26242T 500 Barnesiella intestinihominis DSM 21032T

500 Parabacteroides goldsteinii CL02T12C30 290 Mucilaginibacter paludis DSM 18603T Bacteroidetes Niastella koreensis DSM 17620T 267 499 Flavobacterium johnsoniae DSM 2604T Gemmatimonas aurantiaca DSM 14586T Gemmatimonadetes

0.2 Mohr et al. Microb Cell Fact (2018) 17:108 Page 7 of 12

a [Ni-Fe] group 1d hydrogenase -Pha

T 500 P. thermoglucosidasius DSM 2542 2 kb 500 Parageobacillus sp. NUB 3621 Anoxybacillus thermarum DSM 17141T P. thermoglucosidasius DSM 2542T 430 phaE phaG phaI 500 Anoxybacillus flavithermus DSM 2641T atlF tatA tatC phaA phaB phaC phaD phaF phaH phaJ phaK thyA

T 447 Bacillus drentensis DSM 15600 T 385 Bacillus azotoformans DSM 1046 328 Anaerobacillus arseniciselenatis DSM 15340T 434 P. fulgidum DSM 44945T Bacillus solimangrovi DSM 27083T phaE phaG phaI 500 nikA phaA phaB phaC phaD phaF phaH phaJ phaK trpB Planifilum fulgidum DSM 44945T 499 Sporolactobacillus laevolacticus DSM 442T 500 Hydrogenibacillus schlegelii DSM 2000T 498 Aneurinibacillus terranovensis DSM 18919T H. modesticaldum DSM 9504T phaD phaG phaI 317 Heliobacterium modesticalcum DSM 9504T hyp phaA phaB phaC phaF phaH phaJ phaK trpB Acetonema longum DSM 6540T 500 Megamonas hypermegale DSM 1672T

481 Bradyrhizobium japonicum 22 B. japonicum 22 phaD hupI phaG phaI Methylomonas methanica MC09 hupV phaA phaB phaC hupF hupG hupH hupJ hupK phaF phaH phaJ phaK hypX 500 Rhodobacter sphaeroides WS8N 500 Azorhizobium caulinodans DSM 5975T Parabacteroides goldsteinii CL02T12C30 500 B. intestinihominis DSM 21032T Coprobacter fastidiosus DSM 26242T met phaG phaF phaH phaI phaJ phaK phaA phaB phaC phaD hyp Barnesiella intestinihominis DSM 21032T

T 500 500 Niastella koreensis DSM 17620 Mucilaginibacter paludis DSM 18603T 500 Flavobacterium johnsoniae DSM 2604T

0.2 b [Ni-Fe] group 2a hydrogenase -Phb 2 kb

T. thermophilus SG0.5JP17-16

hyp hypF hypD phbB phbG tnp phbL phbM phbN gmhA phbO phbP pigL sigK rskA tnp hypE hupN phbA phbC phbD phbE phbF phbH phbI phbJ phbK taq

P. thermoglucosidasius DSM 2542T P. thermoglucosidasius DSM 2542T phbB phbG Aeribacillus pallidus 8m3 grpB phbA phbC phbD phbE phbF phbH phbI phbJ phbK phbL phbM phbN phbO phbP hsp70aarF 500 Parageobacillus sp. NUB3621

275 Parageobacillus sp. W-2 295 P. toebii DSM 18751 480 U. thermosphaericus A1 T Aneurinibacillus terranovensis DSM 18919 phbB phbG 450 tnpphbA phbC phbD phbE phbF phbH phbI phbJ phbL phbM phbN phbP nikA Effusibacillus lacus DSM 27172T 439 Alicyclobacillus macrosporangiidus CPP55 275 Ureibacillus thermosphaericus A1 491 S. acidophilus DSM 10332T Hydrogenibacillus schlegelii DSM 2000T 450 phbB phbG hupF Kyrpidia tusciae DSM 2912T hyp phbC phbA phbD phbE phbF phbH phbJ phbK phbL phbM phbN gmhA phbO phbP tnp 500 Thermus thermophilus SG0.5JP17-16 Sulfobacillus acidophilus DSM 10332T T 395 Roseiflexus castenholzii DSM 13941T M. smegmatis DSM 43756 phbB 500 T hyp Chloroflexus aggregans DSM 9485 araC phbA phbD phbE phbF phbH phbJ phbK hyppphbC phbP hbL phbM phbN ligB 495 Gemmatimonas aurantiaca DSM 14586T Mycobacterium smegmatis DSM 43756T 500 Gordonia rhizosphera DSM 44383T B. canariense UBMA171 499 Streptomyces thermoautotrophicus DSM 41605T hyp phbG hyp phbB atoC phbD phbE hyp phbF phbH hyp phbJ phbK phbP phbL gmhA phbM phbN phbC nicO 500 Pseudonocardia thermophila DSM 43832T Novosphingobium pentaromativorans DSM 17173T

389 Acidithiobacillus thiooxidans CLST 500 Nitrospira moscoviensis DSM 10035T Bradyrhizobium canariense UBMA171 Methylocapsa acidiphila DSM 13967T

0.2 c [Ni-Fe] group 4a hydrogenase -Phc 2 kb

P. thermoglucosidasius DSM 2542T P. thermoglucosidasius DSM 2542T phcI mdaB cooC cooS cooF phcA phcB phcC phcD phcE phcF phcG phcH phcJ phcK phcL empB 500 500 Moorella glycerini NMP Moorella thermoacetica DSM 21394

T 427 Thermanaeromonas toyohensis DSM 14490 M. glycerini NMP phcI 276 Caldanaerobacter subterraneus subsp. tengcongensis DSM 15242T rocR cooC cooS cooF phcA phcB phcC phcD phcE phcF phcG phcH phcJ hnoB hypA 457 C.s subterraneus subsp. yonseiensis DSM 13777T 500 500 Thermoanaerobacter sp. YS13 M. thermoacetica DSM 21394 Moorella thermoacetica DSM 11768 phcI phcK rocR hypcooScooFpphcA phcB hcCphcDphcEphcF phcG phcH phcJ phcL hypF Moorella thermoacetica DSM 12993 Moorella thermoacetica Y72 448 Moorella thermoacetica DSM 12797 C. subterraneus subsp. yonseiensis DSM 13777T phcI 500 500 Moorella thermoacetica DSM 6867 pspF cooC cooS cooF phcA phcB phcC phcD phcE phcF phcG phcH phcJ hypinfC 493 Moorella thermoacetica ATCC 39073 Moorella thermoacetica DSM 2955T T 306 Moorella thermoacetica DSM 7417 M. thermoacetica DSM 2955 500 phcI arsR fdhC fdhH hydN phcA phcB phcC phcD phcE phcF phcG phcH phcJ hypB hypF hypC hypD hypE fdhD mobA 271 Moorella thermoacetica DSM 103132 429 Moorella thermoacetica DSM 103284 Calderihabditans maritimus DSM 26464T T. phaeum DSM 12270T Thermacetogenium phaeum DSM 12270T phcI xre hyp hyp fdhH hydN phcA phcB phcC phcD tnp phcF phcG phcH phcJ phoE 487 Uliginosibacterium gangwonense DSM 18521T

500 Paludibacterium yongneupense DSM 18731T Yersinia enterocolitica 8081 Y. enterocolitica 8081 hypC phcI 500 Escherichia coli K-12 str. MG1655 sanA hypA hypB phcA phcB phcE phcC phcD phcF phcG phcH phcJ hydN fdhH fhlA focA mglC Azospirillum halopraeferens DSM 3675T 490 Rhodopseudomonas palustris BAL198 A. halopraeferens DSM 3675T hypC phcI sanA hypD hypB hypA moaA fdhH hydN phcA phcB phcE phcC phcD phcF phcG phcH phcJ focA eutQ 0.1 Mohr et al. Microb Cell Fact (2018) 17:108 Page 8 of 12

(See fgure on previous page.) Fig. 3 Prevalence and synteny of the P. thermoglucosidasius-like [Ni-Fe] hydrogenases. a [Ni-Fe] group 1d orthologues. The ML phylogeny was determined on the basis of the trimmed alignment of nine Pha locus proteins (PhaABCDGHIJK—2206 amino acids in length). Hydrogenase genes are coloured in light blue (dark blue for large and small catalytic subunits), tatAE genes in purple and fanking genes in yellow in the synteny diagrams. b [Ni-Fe] group 2a orthologues. The ML phylogeny was determined on the basis of the trimmed alignment of 10 Phb locus proteins (PhbBCDEFHJLMN—2348 amino acids in length). Hydrogenase genes are coloured in red (dark red for large and small catalytic subunits), genes of no known function in biosynthesis and functioning of the hydrogenase in white and fanking genes in yellow in the synteny diagrams. c [Ni-Fe] group 4a orthologues. The ML phylogeny was determined on the basis of the trimmed alignment of nine Phc locus proteins (PhcABCDFGHIJ—2744 amino acids in length). Hydrogenase genes are coloured in light green (dark green for large and small catalytic subunits), anaerobic CODH genes in purple, formate dehydrogenase-related genes in blue and fanking genes in yellow in the synteny diagrams. Values on all trees refect bootstrap analyses (n 500 replicates) and all trees were rooted on the midpoint =

Te [Ni-Fe] group 4a H­ 2-evolving hydrogenase (Phc) Phc locus, suggesting the P. thermoglucosidasius FdhH locus shows the most restricted distribution of the three protein does not function together with the [Ni-Fe] loci among the Firmicutes, with orthologous loci only group 4a hydrogenase. Instead, the P. thermoglucosida- present in the eight compared P. thermoglucosidasius sius Phc hydrogenase may form a novel complex with the strains and members of the clostridial family Termoa- adjacent anaerobic CODH locus. naerobacteraceae (Fig. 2). Further, Phc-like loci appear to be restricted to members of the Proteobacteria. High The P. thermoglucosidasius [Ni‑Fe] group 4a hydrogenase levels of synteny and sequence conservation can be forms a novel complex with the anaerobic (Coo) CO observed among the Phc loci in both phyla, with the dehydrogenase, with a distinct evolutionary history exception of the PhcK and PhcL proteins, which are only Te three genes located just upstream of the Phc present in the P. thermoglucosidasius and Moorella ther- hydrogenase locus, cooC, cooS and cooF code for a CO moacetica DSM 21394 Phc loci (Fig. 3c). BlastP analy- dehydrogenase maturation factor (Figs. 3c, 4), a CO dehy- ses indicate that PhcK and PhcL show highest orthology drogenase catalytic subunit and CO dehydrogenase Fe–S with PhbB and PhbC in the Phb locus and may have been protein, respectively. Together these proteins catalyse the derived through gene duplication events. oxidation of CO to generate ­CO2 (CO + H2O → CO2 + 2 + It is notable that the P. thermoglucosidasius Phc locus ­H + 2ē). Te electrons are then used in reduction clusters with a subset of the Termoanaerobacteraceae reactions, including sulphate reduction, heavy metal in the concatenated Phc protein phylogeny, including reduction, acetogenesis, methanogenesis and hydrogeno- Moorella glycerini NMP, M. thermoacetica DSM 21394, genesis [38, 39]. Termoanaeromonas toyohensis DSM 14490­ T, Caldan- Te CODH locus is also co-localised with the Phc aerobacter subterraneus subsp. tencogensis DSM 15242­ T hydrogenase locus and highly conserved among the eight and subsp. yonseiensis DSM ­13777T and Termoanaero- other P. thermoglucosidasius genomes (99.36% average bacter sp. YS13 (Fig. 3c). Tese difer from the remaining amino acid identity with CooCSF in P. thermoglucosi- Termoanaerobacteraceae taxa and the proteobacterial dasius DSM ­2542T), while no CODH orthologues are orthologous loci in that they are fanked by three genes, encoded on the genomes of any other Parageobacillus cooCSF, coding for an anaerobic carbon monoxide (CO) or Geobacillus spp. A phylogeny on the basis of the con- dehydrogenase, rather than genes coding for a formate served CooS and CooF proteins (Fig. 4) showed that, as dehydrogenase (FdhH) as is typical for the [Ni-Fe] group with the Phc locus phylogeny (Fig. 3c), those taxa where 4a hydrogenases [25]. Tese are generally accompanied cooCFS fanks the Phc hydrogenase locus cluster together by fanking genes coding for the formate dehydrogenase and show extensive synteny in both the coo and phc accessory sulfurtransferase protein FdhD, electron trans- gene clusters. Tis would suggest the co-evolution of the porter HydN, transcriptional activator FhlA and formate anaerobic CODH and Phc [Ni-Fe] group 4a hydrogenase transporters FdhC and FocA, which together with FdhH loci. However, diferences in the G+C contents could be drive the anaerobic oxidation of formate (Fig. 3c) [26, observed among the P. thermoglucosidasius coo (average 35–37]. G+C content 46.97%) and phc (average G+C content BlastP analysis with the FdhH protein of M. ther- 49.02%) loci. Tis is even more pronounced among the moacetica DSM 2955­ T (AKX95035) shows that an ortho- Termoanaerobacteraceae with this CODH-Phc arrange- T logue is present in P. thermoglucosidasius DSM ­2542 ment, where the G+C contents of the two loci difers (ALF09582). Te latter protein, however, shares lim- by an average of 6.62% and is particularly evident in C. ited orthology (39% amino acid identity; Bitscore: 497; subterraneus subsp. tencongensis where the G+C con- E-value: 6e−614) with its M. thermoacetica counterpart tents of the coo and phc loci difer by 11.77%, suggesting and is furthermore localised ~ 1.5 Mb upstream of the independent evolution of these two loci. Tis is further Mohr et al. Microb Cell Fact (2018) 17:108 Page 9 of 12

2 kb P. thermoglucosidasius DSM 2542T phcI mdaB cooC cooS cooF phcA phcB phcC phcD phcE phcF phcG phcH phcJ phcK phcL empB

P. thermoglucosidasius DSM 2542T CODH Ni-Fe Hyd 4a

CODH 500 Moorella glycerini NMP Ni-Fe Hyd 4a M. glycerini NMP phcI Moorella thermoacetica DSM 21394 CODH Ni-Fe Hyd 4a 489 rocR cooC cooS cooF phcA phcB phcC phcD phcE phcF phcG phcH phcJ hnoB hypA 498 Caldanaerobacillus subterraneus subsp. yonseiensis DSM 13777T CODH Ni-Fe Hyd 4a

500 Caldanaerobacter subterraneus subsp. tengcongensis DSM 15242T CODH Ni-Fe Hyd 4a C. subterraneus subsp. tengongensis DSM 15242T 500 Thermanaeromonas toyohensis DSM 14490T CODH Ni-Fe Hyd 4a phcI T pspF cooC cooS cooF phcA phcB phcC phcD phcE phcF phcG phcH phcJ cbiM 494 Carboxydothermus hydrogenoformans DSM 6800 (CODH-2) CODH Orphan

T CODH Orphan 498 Desulfurispora thermophila DSM 16022 Desulfovirgula thermocuniculi DSM 16036T NAD/FAD Dehyd CODH 500 C. hydrogenoformans DSM 6800T CODH-2 339 Desulfotomaculum alcoholivorax DSM 16058T NAD/FAD Dehyd CODH 499 hlyD cooF cooS crp Sporomusa silvacetica DSM 10669T CODH Fe-Fe Hyd A

C. hydrogenoformans DSM 6800T (CODH-4) CODH NAD/FAD Dehyd C. hydrogenoformans DSM 6800T CODH-4 281 Carboxydocella sporoproducens DSM 16521T Ni-Fe Hyd 4c CODH NAD/FAD 496 ydcE cooF cooS dehyd yotD Thermosinus carboxydivorans DSM 14886T Ni-Fe Hyd 4c CODH 495 Carboxydothermus hydrogenoformans DSM 6800T (CODH-1) Ni-Fe Hyd 4c CODH

Calderihabitans maritimus DSM 26464T Ni-Fe Hyd 4c CODH 278 C. hydrogenoformans DSM 6800T CODH-1 Geobacter pickeringi DSM 17153T Ni-Fe Hyd 4c CODH cooX crp cooC cooM cooK cooL cooU cooH hypA cooF cooS hyp dedA 500 Rhodopseudomonas palustris BisB18 Ni-Fe Hyd 4c CODH 382 475 Rhodospirillum rubrum DSM 467T Ni-Fe Hyd 4c CODH

492 Citrobacter amalonaticus Y19 CODH Ni-Fe Hyd 4c S. enterica subsp. enterica Senftenberg SS209 hyp hypA cooX Salmonella enterica subsp. enterica Senftenberg SS209 CODH Ni-Fe Hyd 4c tnpchypE hypD hypC hypB hyp cooC ooScooFchypC cooM cooK ooLcooUcooH crp

Desulfitobacterium hafniense DSM 10664T CODH Orphan

499 Clostridium pasteurianum BC1 CODH Fe-Fe Hyd B C. pasteurianium BC1 Alkaliphilus metalliredigens QYMF CODH Orphan 500 atoC cooF cooS cooC hydS hoxN tnp Selenihalanaerobacter shriftii ATCC BAA-73T CODH Orphan

0.1 Fig. 4 Prevalence and synteny of the P. thermoglucosidasius-like CODH loci. A phylogeny was constructed on the basis of the concatenated alignments of two proteins (CooFS—692 amino acids in length). Boostrap analysis (n 500 replicates) was performed and the tree was rooted on = the mid-point. In the synteny diagrams the CODH genes are coloured in purple (dark purple for the catalytic subunit gene cooS), the [Ni-Fe] group 4c hydrogenase genes in blue (dark blue for catalytic subunits), the [Ni-Fe] group 4a hydrogenase genes in green (dark green for catalytic subunits), NAD/FAD oxidoreductase gene in orange, [Fe-Fe] hydrogenase group A genes in red, [Fe-Fe] hydrogenase group B genes in purple and fanking genes in yellow

support by the phylogeny (Fig. 4), where the CODH- anaerobic CODH, were included as controls. Te cultiva- Phc loci cluster with CODHs which appear on their own tion of P. thermoglucosidasius DSM 2542­ T in serum bot- and those fanked by an NAD/FAD oxidoreductase are tles with a gas atmosphere consisting of 50% CO and 50% thought to play a role in oxidative stress response [40]. air showed that this strain was able to efectively grow in Te Energy Conserving Hydrogenase (ECH–[Ni-Fe] the presence of 50% CO, reaching a maximum absorb- group 4c hydrogenase)-CODH complex, which has been ance of 0.82 after 6 h of cultivation (Fig. 5). A fractional shown to couple CO oxidation to proton reduction to amount of CO was consumed at the beginning of the ­H2 in C. hydrogenoformans and Rhodospirillum rubrum, experiment, when oxygen was still available, by P. toebii clusters more distantly from the CODH-[Ni-Fe] group DSM ­14590T (0.37 mmol) and G. thermodenitrifcans 4a complex [41, 42]. Overall, the results suggest that the DSM ­465T (0.216 mmol), respectively. Tis suggests that CODH and [Ni-Fe] group 4a hydrogenase have evolved these strains may possess an alternative mechanism, such independently, but may form a complex linking CO oxi- as an aerobic CO dehydrogenase, where CO oxidation dation to reduction of protons to produce ­CO2 and ­H2. is coupled to an electron transport chain which fnally reduces oxygen [38]. For instance, a predicted aerobic The CODH‑[Ni‑Fe] group 4a hydrogenase complex CODH is present (CoxMSL—OXB91742-744) in P. toebii efectively couples CO oxidation to hydrogenogenesis DSM ­14590T but is absent from G. thermodenitrifcans Te predicted function of the co-localized genes encod- DSM ­465T. ing the anaerobic CODH and ­H2-evolving hydrogenase While the two control strains tolerated the presence (Fig. 3c) was tested using P. thermoglucosidasius DSM of CO, no H­ 2 production was observed for either strain ­2542T. Two related strains, Geobacillus thermodenitri- (Figs. 6 and 7). By contrast GC analyses revealed the T T T fcans DSM ­465 and P. toebii DSM ­14590 , which lack production of H­ 2 by P. thermoglucosidasius DSM 2542­ both orthologues of the three hydrogenases and the after ~ 36 h (Fig. 8). Tis corresponds with O­ 2 reaching a Mohr et al. Microb Cell Fact (2018) 17:108 Page 10 of 12

1.0 DSM 465 1.0 6

5 0.8 0.8 [mmol]

4 2

0.6 H 0.6 ; 2 3 CO OD [-]

OD [-] 0.4

0.4 2 CO ; ; 2 N

0.2 ; 1 2 0.2 O

0.0 0 020406080 0.0 020406080 Time [h] Time [h] O2 [mmol] DSM 465 N2 [mmol] DSM 2542 DSM 14590 CO [mmol] CO2 [mmol] T Fig. 5 Growth curves of P. thermoglucosidasius DSM ­2542 , P. H2 [mmol] toebii DSM 14590­ T and G. thermodenitrifcans DSM ­465T. All strains OD [-] were grown in quadruplicate in stoppered serum bottles with Fig. 7 Gas phase composition during the cultivation of G. an initial gas atmosphere composition of 50% CO and 50% air. P. thermodenitrifcans DSM 465. Initial gas composition was 50% CO thermoglucosidasius DSM ­2542T reached a maximum absorbance and 50% air. ­O (dark blue) decreased from 0.83 to ~ 0.03 mmol after (OD­ 0.82) after 6.01 h, by the end of the cultivation the ­OD 2 600 = 600 24.01 h. CO (dark red) decreased fractionally about 0.22 mmol. No increased to a value of 0.71. A maximum absorbance for P. toebii DSM hydrogen (dark grey) was detected. ­CO (dark yellow) increased ­14590T was reached after 9.12 h ­(OD 0.73). The ­OD decreased 2 600 = 600 during the cultivation to 0.49 mmol. After 6.04 h a maximum during the cultivation to a fnal value of 0.24. For G. thermodenitrifcans absorbance ­(OD in black) with a value of 0.64 could be detected DSM 465 the highest OD­ 0.64 was observed after 6.04 h. The 600 600 = ­OD600 decreased to a fnal value of 0.40

DSM 2542 1.0 6 DSM 14590 1.0 6 5 0.8

5 [mmol] 0.8 4 2 H

[mmol] 0.6 ; 2 4 2

H 3 ;

0.6 CO 2 OD [-] 3 0.4 CO 2 CO ; OD [-] ;

0.4 2 N CO ;

2 0.2 ; ; 2

2 1 O N

0.2 ; 1 2 O 0.0 0 020406080 0.0 0 Time [h] 020406080

Time [h] O2 [mmol] N [mmol] O [mmol] 2 2 CO [mmol] N [mmol] 2 CO [mmol] CO [mmol] 2 H2 [mmol] CO2 [mmol] OD [-] H2 [mmol] OD [-] Fig. 8 Gas phase composition during the cultivation of P. Fig. 6 Gas phase composition during the cultivation of P. toebii DSM thermoglucosidasius DSM ­2542T. Initial gas composition was 50% CO T ­14590 . Initial gas composition was 50% CO and 50% air. ­O2 (dark and 50% air. ­O2 (dark blue) decreased from 0.85 to ~ 0.03 mmol after blue) decreased from 0.66 to ~ 0.01 mmol after 23.25 h. CO (dark red) 22 h. CO (dark red) decreased until the start of hydrogen production decreased fractionally about 0.34 mmol. No hydrogen (dark grey) (dark grey) from 3.20 to 2.79 mmol (35.89 h). After 84 h the CO was

was detected. ­CO2 (dark yellow) increased during the cultivation to consumed completely and 2.47 mmol hydrogen was produced. ­CO2 0.56 mmol. After 9.12 h a maximum absorbance ­(OD600 in black) with (dark yellow) increased during the cultivation to 2.84 mmol. After a value of 0.73 was reached 6.01 h a maximum absorbance ­(OD600 in black) with a value of 0.82 was reached Mohr et al. Microb Cell Fact (2018) 17:108 Page 11 of 12

plateau value of ~ 0.03 mmol. After 84 h 2.28 mmol CO Additional fles was consumed and 2.47 mmol H­ 2 produced. P. thermo- T glucosidasius DSM 2542­ is thus capable of producing H­ 2 Additional fle 1. Calculation of the gas composition. Description of the at a near equimolar conversion to CO consumption once calculation of the gas composition by using the ideal gas law. most residual oxygen has been exhausted with a fnal Additional fle 2. Annotations of the CODH and [Ni-Fe] hydrogenase loci of P. thermoglucosidasius DSM ­2542T. The locus tags, sizes, protein names yield of 1.08 ­H2/CO. as well as the functions of the proteins in the three [Ni-Fe] hydrogenase loci and the anaerobic CODH locus of P. thermoglucosidasius DSM ­2542T. Discussion BlastP data (locus tag, average amino acid identity, bitscore and e-value) for the closest non-Parageobacillus orthologue and the top conserved Te redox potential and difusion coefcient of molecular domain for each P. thermoglucosidasius DSM ­2542T protein are shown. ­H make it a key component of metabolism and a potent 2 Additional fle 3. Orthologous [Ni-Fe] hydrogenase and anaerobic CODH energy source for many microbial taxa [25]. Te abil- loci in Parageobacillus and other taxa. The locus size, G C content, G C ity to utilize this energy source relies on the production deviation of the orthologous [Ni-Fe] hydrogenase and+ anaerobic CODH+ of various hydrogenase enzymes, which power both the loci of other P. thermoglucosidasius strains and distinct taxa as used in Figs. 2, 3 and 4. The number of protein orthologous and average amino consumption and production of H­ 2 and inextricably cou- acid identity of these proteins to those encoded on the P. thermoglucosi- T ple H­ 2 to energy-yielding pathways such as acetogenesis, dasius DSM ­2542 loci are indicated. methanogenesis and respiration [26, 43]. Our compara- tive genomic analysis revealed that P. thermoglucosidasius Authors’ contributions contains a unique hydrogenase compliment comprised TM planned the experiments, collected and analyzed the experimental of two uptake hydrogenases (group 1d and 2a) and one data. HA, PM, SP and TM conducted the genomic analysis. RK conducted his Bachelor thesis under the supervision of MZ and AN. AN and DC substantially ­H2-evolving hydrogenase (group 4a). Evolutionary analysis contributed to conception and design of the experiments. AN, HA, PM and TM showed that these hydrogenases are derived through three drafted the manuscript. All authors read and approved the fnal manuscript. independent evolutionary events. Tis indicates that H­ 2 is Author details likely to play a pivotal role in P. thermoglucosidasius metab- 1 Section II: Technical Biology, Institute of Process Engineering in Life Sci- olism and bioenergetics in the ecological niches it occupies. ence, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany. 2 Centre By contrast, members of the sister genus Geobacillus lack for Microbial Ecology and Genomics, University of Pretoria, Hatfeld 0028 Pre- toria, South Africa. 3 School of Molecular & Cell Biology, Faculty of Science, Uni- orthologous hydrogenase loci and, aside from P. thermoglu- versity of the Witwatersrand, WITS 2050 Johannesburg, South Africa. 4 Section cosidasius, only the group 1d and 2a uptake hydrogenases II: Technical Biology, Institute of Process Engineering in Life Science, Karlsruhe share orthology in one and three Parageobacillus spp., Institut für Technologie (KIT), Kaiserstrasse 12, 76131 Karlsruhe, Germany. respectively, even though they are frequently isolated from Acknowledgements the same environments. TM was supported by the Federal Ministry of Education and Research (Grant #031B0180). PDM was funded by the National Research Foundation of South Te group 4a ­H2-evolving hydrogenase of P. thermoglu- Africa (Grant #109137). HA acknowledges funding from Alexander von Hum- cosidasius is not found in any other members of the class boldt Foundation. Bacilli and is most closely related to those found in mem- bers belonging to the class Clostridia, particularly the Competing interests The authors declare that they have no competing interests. family Termoanaerobacteraceae. Furthermore, it forms an association with a CODH, which is found in common Availability of data and supporting materials with a more restricted subclade of strict anaerobes within All the data can be found within the article and its additional fles. the family Termoanaerobacteraceae. Our fermentation Consent for publication studies with P. thermoglucosidasius in the presence of CO All the authors agree to submission. showed that P. thermoglucosidasius grows efciently when Ethics approval and consent to participate exposed to high concentrations of CO and that the CODH- Not applicable. group 4a hydrogenase complex can efectively couple CO Funding oxidation to ­H2 evolution, P. thermoglucosidasius can do We acknowledge support by Deutsche Forschungsgemeinschaft and Open so at a near-equimolar conversion. Furthermore, unlike Access Publishing Fund of Karlsruhe Institute of Technology. other CO oxidizing hydrogenogenic bacteria, which are strict anaerobes, P. thermoglucosidasius is a facultative Publisher’s Note anaerobe capable of frst removing residual oxygen from Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional afliations. CO gas sources prior to producing ­H2 via the water-gas shift reaction. Te combination of these features makes Received: 8 May 2018 Accepted: 2 July 2018 P. thermoglucosidasius an attractive target for potential incorporation in industrial-scale production strategies of biohydrogen. Mohr et al. Microb Cell Fact (2018) 17:108 Page 12 of 12

References 23. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate 1. International Energy Agency. World Energy Outlook 2017. 2017. https​:// large phylogenies by maximum likelihood. Syst Biol. 2003;52:696–704. www.iea.org/weo20​17. Accessed 26 Mar 2018. 24. Lefort V, Longueville J, Gascuel O. SMS: smart model selection in PhyML. 2. Nikolaidis P, Poullikkas A. A comparative overview of hydrogen produc- Mol Biol Evol. 2017;34:2422–4. tion processes. Renew Sustain Energy Rev. 2017;67:597–611. 25. Greening C, Biswas A, Carere CR, Jackson CJ, Taylor MC, Stott MB, et al. 3. Hosseini S, Wahid M. Hydrogen production from renewable and sustain- Genomic and metagenomic surveys of hydrogenase distribution indicate able energy resources: promising green energy carrier for clean develop- ­H2 is a widely utilised energy source for microbial growth and survival. ment. Renew Sustain Energy Rev. 2016;57:850–66. ISME J. 2016;10:761–77. 4. Claassen PAM, van Lier JB, Lopez Contreras AM, van Niel EWJ, Sijtsma L, 26. Vignais PM, Billoud B. Occurrence, classifcation, and biological function Stams AJM, et al. Utilisation of biomass for the supply of energy carriers. of hydrogenases: an overview. Chem Rev. 2007;107:4206–72. Appl Microbiol Biotechnol. 1999;52:741–55. 27. Sargent F, Bogsch EG, Stanley NR, Wexler M, Robinson C, Berks BC, et al. 5. Kothari R, Buddhi D, Sawhney RL. Comparison of environmental and eco- Overlapping functions of components of a bacterial Sec-independent nomic aspects of various hydrogen production methods. Renew Sustain protein export pathway. EMBO J. 1998;17:3640–50. Energy Rev. 2008;12:553–63. 28. Dutta D, De D, Chaudhuri S, Bhattacharya SK. Hydrogen production by 6. Sokolova TG, Henstra AM, Sipma J, Parshina SN, Stams AJM, Lebedinsky cyanobacteria. Microb Cell Fact. 2005;4:36–46. AV. Diversity and ecophysiological features of thermophilic carboxydo- 29. Esteves-Ferreira AA, Cavalcanti JHF, Vaz MGMV, Alvarenga LV, Nunes-Nesi trophic anaerobes. FEMS Microbiol Ecol. 2009;68:131–41. A, Araújo WL. Cyanobacterial nitrogenases: phylogenetic diversity, regula- 7. Kim MS, Bae SS, Kim YJ, Kim TW, Lim JK, Lee SH, et al. CO-dependent H­ 2 tion and functional predictions. Genet Mol Biol. 2017;40:261–75. production by genetically engineered Thermococcus onnurineus NA1. 30. Holmqvist M, Lindberg P, Agervald Å, Stensjö K, Lindblad P. Transcript Appl Environ Microbiol. 2013;79:2048–53. analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. 8. Barnard D, Casanueva A, Tufn M, Cowan D. Extremophiles in biofuel strain PCC 7120 and Nostoc punctiforme ATCC 29133. BMC Res Notes. synthesis. Environ Technol. 2010;31:871–88. 2011;4:186. 9. Maness PC, Weaver PF. Hydrogen production from a carbon-monoxide 31. Andrews SC, Berks BC, McClay J, Ambler A, Quail MA, Golby P, et al. A oxidation pathway in Rubrivivax gelatinosus. Int J Hydrogen Energy. 12-cistron Escherichia coli operon (hyf) encoding a putative proton-trans- 2002;27:1407–11. locating formate hydrogenlyase system. Microbiology. 1997;143:3633–47. 10. Diender M, Stams AJM, Sousa DZ, Robb FT, Guiot SR. Pathways and bio- 32. Chan K-H, Lee K-M, Wong K-B. Interaction between hydrogenase matura- energetics of anaerobic carbon monoxide fermentation. Front Microbiol. tion factors HypA and HypB is required for [NiFe]-hydrogenase matura- 2015;6:1–18. tion. PLoS ONE. 2012;7:e32592. 11. Wang J, Wan W. Efect of temperature on fermentative hydrogen produc- 33. Lenz O, Friedrich B. A novel multicomponent regulatory system mediates tion by mixed cultures. Int J Hydrogen Energy. 2008;33:5392–7. ­H2 sensing in Alcaligenes eutrophus. Proc Natl Acad Sci. 1998;95:12474–9. 12. Oelgeschlaeger E, Rother M. Carbon monoxide-dependent energy 34. Vignais PM, Elsen S, Colbeau A. Transcriptional regulation of the uptake metabolism in anaerobic bacteria and archaea. Arch Microbiol. [NiFe] hydrogenase genes in Rhodobacter capsulatus. Biochem Soc Trans. 2008;190:257–69. 2005;33:28–32. 13. Techtmann SM, Colman AS, Robb FT. ‘That which does not kill us only 35. Maier T, Binder U, Böck A. Analysis of the hydA locus of Escherichia coli: makes us stronger’: the role of carbon monoxide in thermophilic micro- two genes (hydN and hypF) involved in formate and hydrogen metabo- bial consortia. Environ Microbiol. 2009;11:1027–37. lism. Arch Microbiol. 1996;165:333–41. 14. Merrouch M, Hadj-Saïd J, Domnik L, Dobbek H, Léger C, Dementin S, et al. 36. Mukherjee M, Vajpai M, Sankararamakrishnan R. Anion-selective formate/ ­O2 inhibition of Ni-containing CO dehydrogenase is partly reversible. nitrite transporters: taxonomic distribution, phylogenetic analysis and Chem Eur J. 2015;21:18934–8. subfamily-specifc conservation pattern in prokaryotes. BMC Genomics. 15. Juszczak A, Aono S, Adams MW. The extremely thermophilic eubac- 2017;18:1–19. terium, Thermotoga maritima, contains a novel iron-hydrogenase 37. Thomé R, Gust A, Toci R, Mendel R, Bittner F, Magalon A, et al. A sulfur- whose cellular activity is dependent upon tungsten. J Biol Chem. transferase is essential for activity of formate dehydrogenases in Escheri- 1991;266:13834–41. chia coli. J Biol Chem. 2012;287:4671–8. 16. Tirado-Acevedo O, Chinn MS, Grunden AM. Production of biofuels 38. Ragsdale SW. Life with carbon monoxide. Crit Rev Biochem Mol Biol. from synthesis gas using microbial catalysts. Adv Appl Microbiol. 2004;39:165–95. 2010;70:57–92. 39. Techtmann SM, Lebedinski AV, Colman AS, Sokolova TG, Woyke T, Good- 17. Søndergaard D, Pedersen CNS, Greening C. HydDB: a web tool for hydro- win L, et al. Evidence for horizontal gene transfer of anaerobic carbon genase classifcation and analysis. Sci Rep. 2016;6:1–8. monoxide dehydrogenases. Front Microbiol. 2012;3:1–16. 18. Grant JR, Stothard P. The CGView Server: a comparative genomics tool for 40. Wu M, Ren Q, Durkin AS, Daugherty SC, Brinkac LM, Dodson RJ, et al. Life circular genomes. Nucleic Acids Res. 2008;36:181–4. in hot carbon monoxide: the complete genome sequence of Carboxydo- 19. Besemer J, Lomsadze A, Borodovsky M. GeneMarkS: a self-training thermus hydrogenoformans Z-2901. PloS Genet. 2005;1:e60. method for prediction of gene starts in microbial genomes. Implications 41. Fox JD, Kerby RL, Roberts GP, Ludden PW. Characterization of the for fnding sequence motifs in regulatory regions. Nucleic Acids Res. CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum 2001;29:2607–18. and the gene encoding the large subunit of the enzyme. J Bacteriol. 20. Hall TA. Bioedit: a user-friendly biological sequence alignment editor 1996;178:1515–24. and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 42. Soboh B, Linder D, Hedderich R. Purifcation and catalytic properties of 1999;41:95–8. a CO-oxidizing: ­H2-evolving enzyme complex from Carboxydothermus 21. Wallace IM, Sullivan OO, Higgins DG, Notredame C. M-Cofee: combining hydrogenoformans. Eur J Biochem. 2002;269:5712–21. multiple sequence alignment methods with T-Cofee. Nucleic Acids Res. 43. Schwartz E, Fritsch J, Friedrich B. ­H2-metabolizing prokaryotes. In: 2006;34:1692–9. Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thomson F, editors. The 22. Talavera G, Castresana J. Improvement of phylogenies after removing prokaryotes: prokaryotic physiology and biochemistry. Berlin: Springer; divergent and ambiguously aligned blocks from protein sequence align- 2013. p. 119–99. ments. Syst Biol. 2007;56:564–77.