Engineering Biocatalytic Solar Fuel Production: The PHOTOFUEL Consortium.

The EU Horizon2020 consortium PHOTOFUEL joined academic and industrial partners from biology, chemistry, engineering, engine design, and lifecycle assessment, making tremendous progress towards engine-ready fuels from CO2 via engineered photosynthetic microbes. Technical, environmental, economic, and societal opportunities and challenges were explored to frame future technology realization at scale.

Versatile transformations of hydrocarbons in anaerobic bacteria: substrate ranges and regio- and stereo-chemistry of activation reactions.

Anaerobic metabolism of hydrocarbons proceeds either via addition to fumarate or by hydroxylation in various microorganisms, e.g., sulfate-reducing or denitrifying bacteria, which are specialized in utilizing n-alkanes or alkylbenzenes as growth substrates. General pathways for carbon assimilation and energy gain have been elucidated for a limited number of possible substrates. In this work the metabolic activity of 11 bacterial strains during anaerobic growth with crude oil was investigated and compared with the metabolite patterns appearing during anaerobic growth with more than 40 different hydrocarbons supplied as binary mixtures. We show that the range of co-metabolically formed alkyl- and arylalkyl-succinates is much broader in n-alkane than in alkylbenzene utilizers. The structures and stereochemistry of these products are resolved. Furthermore, we demonstrate that anaerobic hydroxylation of alkylbenzenes does not only occur in denitrifiers but also in sulfate reducers. We propose that these processes play a role in detoxification under conditions of solvent stress. The thermophilic sulfate-reducing strain TD3 is shown to produce n-alkylsuccinates, which are suggested not to derive from terminal activation of n-alkanes, but rather to represent intermediates of a metabolic pathway short-cutting fumarate regeneration by reverse action of succinate synthase. The outcomes of this study provide a basis for geochemically tracing such processes in natural habitats and contribute to an improved understanding of microbial activity in hydrocarbon-rich anoxic environments.

Co-metabolic conversion of toluene in anaerobic n-alkane-degrading bacteria.

Diverse microorganisms have been described to degrade petroleum hydrocarbons anaerobically. Strains able to utilize n-alkanes do not grow with aromatic hydrocarbons, whereas strains able to utilize aromatic hydrocarbons do not grow with n-alkanes. To investigate this specificity in more detail, three anaerobic n-alkane degraders (two denitrifying, one sulfate-reducing) and eight anaerobic alkylbenzene degraders (five denitrifying, three sulfate-reducing) were incubated with mixtures of n-alkanes and toluene. Whereas the toluene degradationers formed only the characteristic toluene-derived benzylsuccinate and benzoate, but no n-alkane-derived metabolites, the n-alkane degraders formed toluene-derived benzylsuccinate, 4-phenylbutanoate, phenylacetate and benzoate besides the regular n-alkane-derived (1-methylalkyl)succinates and methyl-branched alkanoates. The co-metabolic conversion of toluene by anaerobic n-alkane degraders to the level of benzoate obviously follows the anaerobic n-alkane degradation pathway with C-skeleton rearrangement and decarboxylation rather than the ß-oxidation pathway of anaerobic toluene metabolism. Hence, petroleum-derived aromatic metabolites detectable in anoxic environments may not be exclusively formed by genuine alkylbenzene degraders. In addition, the hitherto largely unexplored fate of fumarate hydrogen during the activation reactions was examined with (2,3-(2) H(2) )fumarate as co-substrate. Deuterium was completely exchanged with hydrogen at the substituted carbon atom (C-2) of the succinate adducts of n-alkanes, whereas it is retained in toluene-derived benzylsuccinate, regardless of the type of enzyme catalysing the fumarate addition reaction.

Solvent stress response of the denitrifying bacterium "Aromatoleum aromaticum" strain EbN1.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" strain EbN1 degrades several aromatic compounds, including ethylbenzene, toluene, p-cresol, and phenol, under anoxic conditions. The hydrophobicity of these aromatic solvents determines their toxic properties. Here, we investigated the response of strain EbN1 to aromatic substrates at semi-inhibitory (about 50% growth inhibition) concentrations under two different conditions: first, during anaerobic growth with ethylbenzene (0.32 mM) or toluene (0.74 mM); and second, when anaerobic succinate-utilizing cultures were shocked with ethylbenzene (0.5 mM), toluene (1.2 mM), p-cresol (3.0 mM), and phenol (6.5 mM) as single stressors or as a mixture (total solvent concentration, 2.7 mM). Under all tested conditions impaired growth was paralleled by decelerated nitrate-nitrite consumption. Additionally, alkylbenzene-utilizing cultures accumulated poly(3-hydroxybutyrate) (PHB) up to 10% of the cell dry weight. These physiological responses were also reflected on the proteomic level (as determined by two-dimensional difference gel electrophoresis), e.g., up-regulation of PHB granule-associated phasins, cytochrome cd(1) nitrite reductase of denitrification, and several proteins involved in oxidative (e.g., SodB) and general (e.g., ClpB) stress responses.

Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1.

Anaerobic biodegradation of toluene and ethylbenzene is of environmental concern and biochemical interest due to toxicity and novel reactions, respectively. The denitrifying strain EbN1 is unique in anaerobically degrading both alkylbenzenes via different pathways which converge at benzoyl coenzyme A. The organization of genes involved in both pathways was only recently determined for strain EbN1. In the present study, global expression analysis (DNA microarray and proteomics) indicated involvement of several thus-far-unknown proteins in the degradation of both alkylbenzenes. For example, orf68 and orf57, framing the ebd operon, are implicated in ethylbenzene degradation, and the ebA1932 and ebA1936 genes, located 7.2 kb upstream of the bbs operon, are implicated in toluene degradation. In addition, expression studies were now possible on the level of the complete pathways. Growth experiments demonstrated that degradative capacities for toluene and ethylbenzene could be simultaneously induced, regardless of the substrate used for adaptation. Regulation was studied at the RNA (real-time reverse transcription-PCR and DNA microarray) and protein (two-dimensional-difference gel electrophoresis) level by using cells adapted to anaerobic growth with benzoate, toluene, ethylbenzene, or a mixture of toluene and ethylbenzene. Expression of the two toluene-related operons (bss and bbs) was specifically induced in toluene-adapted cells. In contrast, genes involved in anaerobic ethylbenzene degradation were induced in ethylbenzene- and toluene-adapted cells, suggesting that toluene may act as a gratuitous inducer. In agreement with the predicted sequential regulation of the ethylbenzene pathway, Ebd proteins (encoding subunits of ethylbenzene dehydrogenase) were formed in ethylbenzene- but not in acetophenone-adapted cells, while Apc proteins (subunits of predicted acetophenone carboxylase) were formed under both conditions.

Genes involved in the anaerobic degradation of toluene in a denitrifying bacterium, strain EbN1.

The organization of all genes required for the anaerobic conversion of toluene to benzoyl-CoA was investigated in denitrifying Azoarcus-like strain EbN1. All of these genes are clustered within 25.3 kb of contiguous DNA sequence, which includes only a few intervening sequences. The toluene-catabolic genes are organized in two apparent operons. One contains the genes ( bssCAB) for the three subunits of benzylsuccinate synthase, which initiates anaerobic toluene degradation by converting toluene to ( R)-benzylsuccinate. The BssCAB proteins of strain EbN1 are most similar to those of Thauera aromatica strain K172. The bssCAB genes are part of a larger putative operon ( bssDCABEFGH), which contains the gene bssD, encoding the activase for benzylsuccinate synthase, and four genes ( bssEFGH) encoding proteins of unknown function. RT-PCR experiments showing continuation of transcription over the three largest intergenic regions of the bss operon support the assumed structure. Moreover, BssG was identified as toluene-induced protein. Downstream of the bss genes, another large putative operon ( bbsA- H) was identified that contains all genes required for beta-oxidation of benzylsuccinate to benzoyl-CoA, e.g. bbsEF, encoding succinyl-CoA:( R)-benzylsuccinate CoA-transferase. Immediately upstream of the bss operon, genes for a two-component regulatory system were identified; their products may sense toluene and induce the expression of both catabolic operons. The order and sequences of the bss and bbs genes are highly similar among toluene-degrading denitrifiers. The bss and bbs genes of the Fe(III)-reducing Geobacter metallireducens display less sequence similarity and are organized differently. The genes between the bss and bbs operons and in the flanking regions differ between strain EbN1 and the other strains.