Feasibility of sulfur oxidizing bacteria–assisted electrolysis for low-voltage H2 evolution with autotrophic CO2 fixation

Autotrophic bacteria can assimilate atmospheric carbon dioxide (CO2) and convert CO2 into organic carbon. The CO2 fixation by autotrophic bacteria is important for the improvement of carbon sequestration in agricultural soils. However, the effect of soil texture on autotrophic CO2 fixation bacteria and their CO2 fixation capacity is still unknown. Here, two paddy soils with different textures (loamy clay soil and sand clay loam soil) were incubated with continuous 14C-CO2 in a glass chamber. The two soils were developed from the same parent. At the end of 110 days incubation, the 14C-CO2 incorporated in soil organic carbon (14C-SOC), microbial biomass carbon (14C-MBC) and dissolved organic carbon (14C-DOC) were measured to explore the effects of soil texture on the autotrophic bacterial CO2 fixation rates. The effect of soil texture on the composition and diversity of autotrophic CO2 fixation bacterial community was investigated using cloning and sequencing of the cbbL gene, which encodes ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO) in the Calvin cycle. The results showed that the average contents of 14C-SOC, 14C-MBC and 14C-DOC were 133.81, 40.16 and 8.10 mg·kg-1 in loamy clay soil, respectively, which were significantly higher than their corresponding contents in sand clay loam soil (P<0.05). This suggested that soil texture not only affected the amounts of autotrophic bacteria CO2 fixation but also had an effect on the transformation of microbial assimilated 14C in soil. The cbbL gene libraries of two soils were significantly different as revealed by libshuff analyses (P<0.05). Phylogenetic analysis showed that cbbL sequences from the loamy clay soil were closely affiliated with known cultures such as Rhodoblastus acidophilus, Blastochloris viridis, Thauera humireducens, Mehylibium sp.and Variovorax sp., whereas these sequences belonging to the sand clay loam soil were related to branching lineages originating from Rhizobiales and Actinomycetales.Rarefaction curve, clone library coverage and diversity index analysis based on bacterial cbbL clone libraries indicated that the loamy clay soil had higher cbbL gene diversity compared to the sand clay loam soil. These results suggested that soil texture had a pronounced effect on the composition and diversity of autotrophic CO2 fixation bacterial communities. The higher clay content, nutrient availability and cation exchange capacity may stimulate the growth and activity of autotrophic bacteria, and result in the higher amounts of 14C in loamy clay soil. These data broaden the understanding and knowledge of mechanisms of microbial carbon fixation and their influencing factors in agricultural soils.

The microbial community composition and its functionality was assessed for hydrothermal fluids and volcanic ash sediments from Haungaroa and hydrothermal fluids from the Brothers volcano in the Kermadec island arc (New Zealand). The Haungaroa volcanic ash sediments were dominated by epsilonproteobacterial Sulfurovum sp. Ratios of electron donor consumption to CO2 fixation from respective sediment incubations indicated that sulfide oxidation appeared to fuel autotrophic CO2 fixation, coinciding with thermodynamic estimates predicting sulfide oxidation as the major energy source in the environment. Transcript analyses with the sulfide-supplemented sediment slurries demonstrated that Sulfurovum prevailed in the experiments as well. Hence, our sediment incubations appeared to simulate environmental conditions well suggesting that sulfide oxidation catalyzed by Sulfurovum members drive biomass synthesis in the volcanic ash sediments. For the Haungaroa fluids no inorganic electron donor and responsible microorganisms could be identified that clearly stimulated autotrophic CO2 fixation. In the Brothers hydrothermal fluids Sulfurimonas (49%) and Hydrogenovibrio/Thiomicrospira (15%) species prevailed. Respective fluid incubations exhibited highest autotrophic CO2 fixation if supplemented with iron(II) or hydrogen. Likewise catabolic energy calculations predicted primarily iron(II) but also hydrogen oxidation as major energy sources in the natural fluids. According to transcript analyses with material from the incubation experiments Thiomicrospira/Hydrogenovibrio species dominated, outcompeting Sulfurimonas. Given that experimental conditions likely only simulated environmental conditions that cause Thiomicrospira/Hydrogenovibrio but not Sulfurimonas to thrive, it remains unclear which environmental parameters determine Sulfurimonas’ dominance in the Brothers natural hydrothermal fluids.

The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H2 and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO2 fixation. The following enzymes were found [values in parentheses = μmol min-1 (mg protein)-1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F420-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F420-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F420 (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO2 fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N2O with NO as intermediate (0.1 μmol N2O formed per min and mg protein), showing that - at least in principle - F. placidus has a denitrifying capacity.

In Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO2 fixation via the Calvin cycle is regulated by repression/derepression. During growth of the organism on fructose alone, the synthesis of ribulosebisphosphate carboxylase (RuBPCase) remained fully repressed, both in batch culture and in fructose-limited continuous cultures at various dilution rates. Growth in batch culture on a mixture of fructose and formate resulted in the simultaneous utilization of both substrates. Under these conditions we observed synthesis of RuBPCase up to high levels, indicating that formate did not merely function as an ancillary energy source in the metabolism of fructose, but stimulated autotrophic CO2 fixation via the Calvin cycle. In subsequent experiments growth of P. oxalaticus on mixtures of fructose and formate was studied in carbon source-limited continuous cultures. In these experiments further evidence was obtained that fructose is a poor source of (co-)repressor molecules for the synthesis of RuBPCase in the presence of formate. Thus, addition of formate to the medium reservoir of a fructose-limited continuous culture resulted in derepression of RuBPCase synthesis at (relatively) high ratios of fructose over formate. In the reverse experiment the specific activity of RuBPCase decreased with increasing concentrations of fructose in the medium reservoir. However, it can be calculated that the total capacity of RuBPCase in the culture to fix CO2 remained constant. In these experiments the dry weight produced on the various mixtures equalled the sum of the dry weight values obtained during growth on the same amounts of the two substrates separately. This indicated that, once RuBPCase was present, autotrophic and heterotrophic carbon assimilation pathways functioned simultaneously and independently of each other. Possible explanations for the low repressing effect of fructose on autotrophic CO2 fixation in P. oxalaticus are discussed.

The involvement of reactions of the tricarboxylic acid cycle in autotrophic CO2 fixation in Methanobacterium thermoautotrophicum was investigated. The incorporation of succinate into glutamate (= alpha-ketoglutarate), aspartate (= oxaloacetate) and alanine (= pyruvate) was studied. The organism was grown on H2 plus CO2 at pH 6.5 in the presence of 1 mM [U-14C-]succinate. Significant amounts of the dicarboxylic acid were incorporated into cellular material under these conditions. Alanine, aspartate, and glutamate were isolated and their specific radioactivities were determined. Only glutamate was found to be labelled. Degradation of glutamate revealed that C-1 of glutamate was derived from CO2 and C-2--C-5 from succinate indicating that in M. thermoautotrophicum alpha-ketoglutarate is synthesized via reductive carboxylation of succinyl CoA. The finding that succinate was not incorporated into alanine and aspartate excludes that oxaloacetate and pyruvate are synthesized from alpha-ketoglutarate via isocitrate or citrate. This is taken as evidence that a complete reductive carboxylic acid cycle is not involved here in autotrophic CO2 fixation.

Autotrophic CO2 fixation is the most important biotransformation process in the biosphere. Research focusing on the diversity and distribution of relevant autotrophs is significant to our comprehension of the biosphere. In this study, a draft genome of a bacterium from candidate phylum SBR1093 was reconstructed with the metagenome of an industrial activated sludge. Based on comparative genomics, this autotrophy may occur via a newly discovered carbon fixation path, the hydroxypropionate-hydroxybutyrate (HPHB) cycle, which was demonstrated in a previous work to be uniquely possessed by some genera from Archaea. This bacterium possesses all of the thirteen enzymes required for the HPHB cycle; these enzymes share 30∼50% identity with those in the autotrophic species of Archaea that undergo the HPHB cycle and 30∼80% identity with the corresponding enzymes of the mixotrophic species within Bradyrhizobiaceae. Thus, this bacterium might have an autotrophic growth mode in certain conditions. A phylogenetic analysis based on the 16S rRNA gene reveals that the phylotypes within candidate phylum SBR1093 are primarily clustered into 5 clades with a shallow branching pattern. This bacterium is clustered with phylotypes from organically contaminated environments, implying a demand for organics in heterotrophic metabolism. Considering the types of regulators, such as FnR, Fur, and ArsR, this bacterium might be a facultative aerobic mixotroph with potential multi-antibiotic and heavy metal resistances. This is the first report on Bacteria that may perform potential carbon fixation via the HPHB cycle, thus may expand our knowledge of the distribution and importance of the HPHB cycle in the biosphere.

Comparative genome analysis of the Sphaerotilus-Leptothrix group supports the unification of the genera Sphaerotilus and Leptothrix into a single emended genus Sphaerotilus.

Chlorobium limicola has been proposed to assimilate CO2 autotrophically via a reductive tricarboxylic acid cycle rather than via the Calvin cycle. This proposal has been a matter of considerable controversy. In order to determine which pathway is operative, the bacterium was grown on a mineral salts medium with CO2 as the main carbon source supplemented with specifically labeled 14C-pyruvate, and the incorporation of 14C into alanine (≙intracellular pyruvate), aspartate (≙oxaloacetate), glutamate (≙α-ketoglutarate), and glucose (≙hexosephosphate) was measured in exponentially growing cells in long term labeling experiments. During growth in presence of pyruvate, 20% of the cell carbon were derived from pyruvate in the medium, 80% from CO2. Since pyruvate was not oxidized to CO2, only those compounds should become labeled which were synthesized from CO2 via pyruvate.

Thiobacillus A2 was grown in chemostat culture under four distinct types of substrate limitation: chemolithoautotrophically with limitation by thiosulphate or CO2; heterotrophically with limitation by glucose; and mixotrophically with dual limitation by both thiosulphate and glucose. Under mixotrophic conditions energy was obtained from the oxidation of both thiosulphate and glucose, and carbon was derived both from CO2 fixation by the Calvin cycle and from glucose. Ribulosebisphosphate carboxylase (RuBP carboxylase) activity was negligible and chemolithotrophic thiosulphate oxidation and autotrophic CO2 fixation were apparently repressed in bacteria which had been grown heterotrophically. Conversely, under autotrophic conditions the ability to oxidize glucose was repressed. Growth yields from mixotrophic cultures were the sum of those obtained under single substrate limitation. Intermediate activities of RuBP carboxylase were detected in mixotrophic cultures, but more glucose was assimilated mixotrophically than heterotrophically. Glucose was metabolized by the Entner-Doudoroff (85 to 90%) and pentose phosphate (10 to 15%) pathways under both heterotrophic and mixotrophic conditions, with slight involvement also of the Embden-Meyerhof pathway (&lt; 9%) heterotrophically. RuBP carboxylase activity in autotrophic cultures was enhanced four- or fivefold by CO2 limitation. Repression of RuBP carboxylase activity and thiosulphate-oxidizing ability during the transition from autotrophy to heterotrophy and the activities of carbohydrate-metabolizing enzymes in autotrophic, heterotrophic and mixotrophic cultures are described.

The regulation of C1-metabolism in Xanthobacter strain 25a was studied during growth of the organism on acetate, formate and methanol in chemostat cultures. No activity of methanol dehydrogenase (MDH), formate dehydrogenase (FDS) or ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisC/O) could be detected in cells grown on acetate alone over a range of dilution rates tested. Addition of methanol or formate to the feed resulted in the immediate induction of MDH and FDH and complete utilization (D=0.10 h-1) of acetate and the C 1-substrates. The activities of these enzymes rapidly dropped at the higher growth rates, which suggests that their synthesis is further controlled via repression by “heterotrophic” substrates such as acetate. Synthesis of RuBisC/O already occurred at low methanol concentrations in the feed, resulting in additive growth yields on acetate/methanol mixtures. The energy generated in the oxidation of formate initially allowed an increased assimilation of acetate (and a decreased dissimilation), resulting in enhanced growth yields on the mixture. RuBisC/O activity could only be detected at the higher formate/acetate ratios in the feed. The data suggest that synthesis of RuBisC/O and CO2 fixation via the Calvin cycle in Xanthobacter strain 25 a is controlled via a (de)repression mechanism, as is the case in other facultatively autotrophic bacteria. Autotrophic CO2 fixation only occurs under conditions with a diminished supply of “heterotrophic” carbon sources and a sufficiently high availability of suitable energy sources. The latter point is further supported by the clearly more pronounced derepressing effect exerted by methanol compared to formate.

Three transposon Tn5-induced mutants deficient in autotrophic CO2 fixation were isolated from a megaplasmid pHG1-cured strain of Alcaligenes eutrophus H16. Their phenotypes were initially characterized by their ability to form both key enzymes of the Calvin cycle, ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK). Since the transposon insertions were at different sites within the chromosomal cluster of cfx genes encoding Calvin cycle enzymes, the individual mutants showed different inactivation patterns for Rubisco and PRK synthesis. These data together with already known sequence data and the arrangement of cfx genes suggested that the Rubisco, fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase and PRK genes are constituents of the same operon. This was further confirmed by trans complementation analyses which indicated that the very similarly organized pHG1-encoded cfx genes additionally present in wild-type strain H16 are functional and also form a common operon. Each operon may also include a glyceraldehyde-3-phosphate dehydrogenase gene. Thus, the duplicated cfx operons of A. eutrophus H16 are large transcriptional units comprising at least about 8 kilobase pairs (kb) and possibly as much as 11 kb.

Bio-conversion of CO2 into biofuels and other value-added chemicals via metabolic engineering

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO2, H2, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H2S. Enzymes of the autotrophic CO2 assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 μmol · min-1 · mg cell protein-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO2 fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO2 fixation, two different autotrophic pathways occur in the same physiological group.

An increasing number of strict anaerobic bacteria are being found which use an alternative pathway to the ubiquitous Calvin cycle for CO2 fixation into cell compounds and the ubiquitous Krebs cycle for acetyl CoA oxidation to CO2. The principles of this non-cyclic pathway, the acetyl CoA pathway, have long been studied in acetogenic bacteria. These bacteria can catalyze the exergonic reduction of 2 CO2 with 8 reducing equivalents to acetate. In this pathway, CO2 reduction is part of a catabolic redox process which functions to accept reducing equivalents from a variety of dehydrogenated substrates. This process yields net ATP generated by electron transport phosphorylation. Acetyl CoA is an intermediate, formed from one CO2 via a tetrahydropteridine-bound 1-carbon unit (methyl group of acetate), and from another CO2 via a bound carbon monoxide (carboxyl group of acetate). The most characteristic and complex enzyme involved in acetyl CoA synthesis is carbon monoxide dehydrogenase (‘acetyl CoA synthase’). The enzymes of this acetyl CoA pathway not only participate in (1) acetate synthesis in energy metabolism of acetogenic bacteria, but also mediate (2) acetyl CoA oxidation in sulfate-reducing bacteria and possibly other anaerobes; (3) acetate disproportionation to CO2 and CH4 in the energy metabolism of many methanogenic bacteria; (4) autotrophic CO2 fixation in autotrophic acetogenic, methanogenic, and most autotrophic sulfate-reducing bacteria; (5) assimilation and/or dissimilation of 1-carbon compounds in many anaerobes; (6) CO oxidation to CO2 in anaerobes. A specialized group of anaerobes performs acetate synthesis from CO2 or from C1 units via a different pathway, the glycine synthase/glycine reductase pathway. Glycine is an intermediate which is formed from 2 C1 compounds, and is then reduced to acetate. The principal features of the two pathways and some open questions are discussed in this review. Emphasis is placed upon the acetyl CoA pathway in acetogenic bacteria, but important advances in the study of other strict anaerobes are also considered.

The Calvin-Benson-Bassham (CBB) cycle assimilates CO2 for the primary production of organic matter in all plants and algae, as well as in some autotrophic bacteria. The key enzyme of the CBB cycle, ribulose-bisphosphate carboxylase/oxygenase (RubisCO), is a main determinant of de novo organic matter production on Earth. Of the three carboxylating forms of RubisCO, forms I and II participate in autotrophy, and form III so far has been associated only with nucleotide and nucleoside metabolism. Here, we report that form III RubisCO functions in the CBB cycle in the thermophilic chemolithoautotrophic bacterium Thermodesulfobium acidiphilum, a phylum-level lineage representative. We further show that autotrophic CO2 fixation in T. acidiphilum is accomplished via the transaldolase variant of the CBB cycle, which has not been previously demonstrated experimentally and has been considered unlikely to occur. Thus, this work reveals a distinct form of the key pathway of CO2 fixation.