Boosting hydrogen production in Rhodospirillum rubrum by syngas-driven photoheterotrophic adaptive evolution.

Rhodospirillum rubrum is a photosynthetic purple non-sulphur bacterium with great potential to be used for complex waste valorisation in biotechnological applications due to its metabolic versatility. This study investigates the production of hydrogen (H2) and polyhydroxyalkanoates (PHA) by R. rubrum from syngas under photoheterotrophic conditions. An adaptive laboratory evolution strategy (ALE) has been carried out to improve the yield of the process. After 200 generations, two evolved strains were selected that showed reduced lag phase and enhanced poly-3-hydroxybutyrate (PHB) and H2 synthesis compared to the parental strain. Genomic analysis of the photo-adapted (PA) variants showed four genes with single point mutations, including the photosynthesis gene expression regulator PpsR. The proteome of the variants suggested that the adapted variants overproduced H2 due to a more efficient CO oxidation through the CO-dehydrogenase enzyme complex and confirmed that energy acquisition was enhanced through overexpression of the photosynthetic system and metal cofactors essential for pigment biosynthesis.

Enhancement of biohydrogen production rate in Rhodospirillum rubrum by a dynamic CO-feeding strategy using dark fermentation.

BACKGROUND: Rhodospirillum rubrum is a purple non-sulphur bacterium that produces H2 by photofermentation of several organic compounds or by water gas-shift reaction during CO fermentation. Successful strategies for both processes have been developed in light-dependent systems. This work explores a dark fermentation bioprocess for H2 production from water using CO as the electron donor. RESULTS: The study of the influence of the stirring and the initial CO partial pressure (pCO) demonstrated that the process was inhibited at pCO of 1.00 atm. Optimal pCO value was established in 0.60 atm. CO dose adaptation to bacterial growth in fed-batch fermentations increased the global rate of H2 production, yielding 27.2 mmol H2 l-1 h-1 and reduced by 50% the operation time. A kinetic model was proposed to describe the evolution of the molecular species involved in gas and liquid phases in a wide range of pCO conditions from 0.10 to 1.00 atm. CONCLUSIONS: Dark fermentation in R. rubrum expands the ways to produce biohydrogen from CO. This work optimizes this bioprocess at lab-bioreactor scale studying the influence of the stirring speed, the initial CO partial pressure and the operation in batch and fed-batch regimes. Dynamic CO supply adapted to the biomass growth enhances the productivity reached in darkness by other strategies described in the literature, being similar to that obtained under light continuous syngas fermentations. The kinetic model proposed describes all the conditions tested.

Genome sequence and characterization of the bcs clusters for the production of nanocellulose from the low pH resistant strain Komagataeibacter medellinensis ID13488.

Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose-producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT-PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT-PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed.

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology.

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis. Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol(OA)/Cmol(lac)) and a higher P(3HB) production (18.88 g(P(3HB))/L) were achieved, comparing to the original recombinant strain (0.11 Cmol(OA)/Cmol(lac) and 7.8 g(P(3HB))/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol (P(3HB))/Cmol(lac) vs 0.10 Cmol(P(3HB))/Cmol(lac) of the original strain).