Genomic Analysis of Carbon Monoxide Utilization and Butanol Production by Clostridium carboxidivorans Strain P7T

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7T possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO2 fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7T chromosome. The functionality of these pathways was also confirmed by growth of P7T on CO and production of CO2 as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7T was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7T genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas

Etude de gènes fimbriaux de souches d'Haemophilus responsables d'infections génitales, materno-foetales et néonatales, et développement d'une méthode de typage moléculaire pour les souches d'Haemophilus

L'opéron ghf a été caractérisé et son expression étudiée chez 6 souches cryptiques d'Haemophilus responsables d'infections génitales, materno-fœtales et néonatales. L'opéron est complet et son organisation génétique est identique à celle de l'opéron hif codant les fimbriae LKP mais il ne s'exprime pas en raison d'altérations dans sa région promotrice. Ceci a été confirmé par microscopie électronique et par l'étude des propriétés d'hémagglutination des souches cryptiques. La recherche d'autres gènes codant les fimbriae de ces souches a montré l'absence de gènes similaires à des gènes fimbriaux déjà décrits. La recherche de gènes nouveaux par mutagenèse aléatoire par transposition a été initiée. La diversité des profils obtenus en PCR avec une amorce issue de la séquence répétée IDS, quasi spécifique du genre Haemophilus et susceptible d'encadrer des gènes de virulence, nous a amenés à utiliser cette technique, appelée IDS-PCR, comme méthode de typage pour les souches d'Haemophilus.The fimbrial ghf gene cluster was characterized in 6 strains of a cryptic genospecies of Haemophilus responsible for genital, mother-infant and neonatal infections. The ghf cluster is complete and homologous to the hif cluster which encodes LKP-type fimbriae but is not expressed in the cryptic genital strains due to the major alterations of the region which includes the ghf genes promoters. These results were confirmed by hemagglutination properties and electron microscopy. We showed in our cryptic genital strains, the lack of genes related to fimbrial genes which were previously described in other bacteria and we initiated the identification of new fimbrial genes by in vivo transposition. Finally, the results obtained by PCR using a primer designed from the Haemophilus specific repeated sequence IDS, which has been found flanking virulence genes, encourage us to develop this technique, called IDS-PCR, as a typing method for Haemophilus strains.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Fimbrial ghf Gene Cluster of Genital Strains of Haemophilus spp.

We analyzed the LKP fimbrial gene clusters of six piliated strains of a cryptic genospecies of Haemophilus isolated from the genital tracts of adult patients (five strains) and from an infected neonate. In a group of 19 genital strains, LKP-like genes have been found in only these 6 strains. In addition to the ghfA, ghfD, and ghfE genes previously described, we characterized two genes, designated ghfB and ghfC, encoding the putative chaperone and assembly platform proteins. All six strains had a complete and unique LKP-like gene cluster consisting of the five genes ghfA to ghfE, homologous to genes hifA to hifE of Haemophilus influenzae. The sequences of the coding and intergenic regions of the ghf clusters of the six strains were remarkably homologous. Unlike hif clusters, which are inserted between purE and pepN, the ghf cluster was inserted between purK and pepN on the chromosome. Analysis of the flanking regions of the ghf cluster identified a large deletion, identical in the 5′ end regions of all strains, including the whole purE gene and much of the purK gene. Ultrastructural observations, an attempt at enriching LKP fimbriae, and hemagglutination experiments demonstrated that none of the strains had LKP-type fimbriae. Nevertheless, reverse transcription (RT)-PCR showed that ghf genes were transcribed in four of the six strains. Sequencing of the intergenic ghfA-ghfB regions, including the ghf gene promoters, showed that the absence of transcripts in the remaining two strains was due to a decrease in the number of TA repeats (4 or 9 repeats rather than 10) between the −10 and −35 boxes of the two overlapping and divergent promoters. The other four strains, which had ghf transcripts, had the optimal 10 TA repeats (one strain) or 5 repeats associated with putative alternative −35 boxes (three strains). The absence of 10 repeated palindromic sequences of 44 or 45 nucleotides upstream of ghfB induces an increased instability of mRNA, as quantified by real-time RT-PCR, and may explain why the LKP fimbrial gene cluster is not expressed in these strains

Typing of Nonencapsulated Haemophilus Strains by Repetitive-Element Sequence-Based PCR Using Intergenic Dyad Sequences

Intergenic dyad sequences (IDS) are short repeated elements that have been described for several Haemophilus genomes and for only two other bacterial genera. We developed a repetitive-element sequence-based PCR using an IDS-specific primer as a typing method (IDS-PCR) for nonencapsulated Haemophilus strains and compared this technique with pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. IDS-PCR was rapid, easy to perform, and reproducible, with a high discriminatory capacity for nontypeable Haemophilus influenzae (NTHI) strains. The 69 NTHI strains tested generated 65 different banding patterns. Epidemiologically related strains gave similar or identical fingerprints, and all of the unrelated strains except two showed different patterns. These results were in agreement with those obtained by PFGE. For 20 genital strains usually identified as being biotype IV NTHI and belonging to a cryptic genospecies of Haemophilus with remarkable genetic homogeneity, four bands were significantly present and six bands were significantly absent from the fingerprints. The 20 strains were gathered in 11 closely related profiles, whereas PFGE provided no band when DNA was treated with SmaI. IDS-PCR improved the differentiation previously obtained within this species by ribotyping and multilocus enzyme electrophoresis. Our findings suggest that IDS-PCR is a rapid, reliable, and discriminatory method for typing NTHI strains and is currently the most efficient method for distinguishing strains within the cryptic genospecies of Haemophilus

Carbon dioxide conversion to C1 - C2 compounds in a microbial electrosynthesis cell with in situ electrodeposition of nickel and iron

Highlights • Continuous CH4 bioelectrosynthesis from CO2 demonstrated with 80% or higher Coulombic Efficiency • At pH values below 8 CH4 cathodic off-gas contains up to 85% CH4 • At pH above 8.5, production of acetate and then ethanol (up to 8 g L−1) was obtained • Coulombic efficiency remained above 80% • 16S sequencing showed proliferation of Clostridium, Methanosaeta, Methanobrevibacter and Methanobacterium spp at the cathode This study demonstrates the continuous conversion of CO2 to methane, acetate, and ethanol in a Microbial Electrosynthesis Cell (MESC) with a carbon felt biocathode. The MESC was inoculated with a mixed anaerobic microbial consortium and operated at a mesophilic temperature of 30 °C. In situ deposition of Ni and Fe was achieved by introducing 0.2 g L−1 of NiSO4 or FeSO4, respectively, into the cathode compartment influent stream. In response, a considerable improvement in MESC performance was observed with a current density of 6.4 mA cm−2 (per separator area) and a CH4 production of 0.83 L (LR d)−1 (R = cathode volume). Once Ni and Fe were removed from the influent solution, the performance remained unchanged. Electron dispersive spectroscopy confirmed Ni and Fe electrodeposition. A shift from CH4 to acetate and ethanol production with concentrations reaching 5 and 8 g L−1, respectively, was observed upon increasing the cathode compartment pH to 8.5–9.0. 16S rRNA gene sequencing showed significant changes in the bacterial population at the cathode with Clostridia representing almost two-thirds of the population. Methanosaeta, Methanobrevibacter, and Methanobacterium species dominated the archaeal community

Specific inhibitors for identifying pathways for methane production from carbon monoxide by a nonadapted anaerobic mixed culture

Specific inhibitors such as 2-bromoethanesulfonate (BES) and vancomycin were employed in activity batch tests to decipher metabolic pathways that are preferentially used by a mixed anaerobic consortium (sludge from an anaerobic digester) to transform carbon monoxide (CO) into methane (CH\u2084). We first evaluated the inhibitory effect of both BES and vancomycin on the microbial community, as well as the efficiency and stability of vancomycin at 35 \ub0C, over time. The activity tests with CO\u2082\u2013H\u2082, CO, glucose, acetate, formate, propionate, butyrate, methanol, and ethanol showed that vancomycin does not inhibit some Gram-negative bacteria, and 50 mmol/L BES effectively blocks CH\u2084 production in the sludge. However, when sludge was incubated with propionate, butyrate, methanol, or ethanol as the sole energy and carbon source, methanogenesis was only partially inhibited by BES. Separate tests showed that 0.07 mmol/L vancomycin is enough to maintain its inhibitory efficiency and stability in the population for at least 32 days at 35 \ub0C. Using the inhibitors above, it was demonstrated that CO conversion to CH\u2084 is an indirect, 2-step process, in which the CO is converted first to acetate and subsequently to CH\u2084.Peer reviewed: YesNRC publication: Ye

Biomethanation Of Syngas Using Anaerobic Sludge: Shift In The Catabolic Routes With The CO Partial Pressure Increase

Syngas generated by thermal gasification of biomass or coal can be steam reformed and purified into methane, which could be used locally for energy needs, or re-injected in the natural gas grid. As an alternative to chemical catalysis, the main components of the syngas (CO, CO2, and H2) can be used as substrates by a wide range of microorganisms, to be converted into gas biofuels, including methane. This study evaluates the carboxydotrophic (CO-consuming) methanogenic potential present in an anaerobic sludge from an upflow anaerobic sludge bed (UASB) reactor treating waste water, and elucidates the CO conversion routes to methane at 35±3˚C. Kinetic activity tests under CO at partial pressures (pCO) varying from 0.1 to 1.5 atm (0.09-1.31 mmol/L in the liquid phase) showed a significant carboxydotrophic activity potential for growing conditions on CO alone. A maximum methanogenic activity of 1 mmol CH4 per g of volatile suspended solid and per day was achieved at 0.2 atm of CO (0.17 mmol/L), and then the rate decreased with the amount of CO supplied. The intermediary metabolites such as acetate, H2 and propionate started to accumulate at higher CO concentrations. Inhibition experiments with 2-bromoethanesulfonic acid (BES), fluoroacetate, and vancomycin showed that in a mixed culture CO was converted mainly to acetate by acetogenic bacteria, which was further transformed to methane by acetoclastic methanogens, while direct methanogenic CO conversion was negligible. Methanogenesis was totally blocked at high pCO in the bottles (≥ 1 atm). However it was possible to achieve higher methanogenic potential under a 100% CO atmosphere after acclimation of the sludge to CO. This adaptation to high CO concentrations led to a shift in the archaeal population, then dominated by hydrogen-utilizing methanogens, which were able to take over acetoclastic methanogens, while syntrophic acetate oxidizing (SAO) bacteria oxidized acetate into CO2 and H2. The disaggregation of the granular sludge showed a negative impact on their methanogenic activity, confirming that the acetoclastic methanogens were the most sensitive to CO, and a contrario, the advantage of using granular sludge for further development towards large-scale methane production from CO-rich syngas

Dataset of anaerobic acidogenic digestion for hydrogen production using xylose as substrate : biogas production and metagenomic data

This paper presents the raw data of biogas production and composition (relative pressures and concentrations of each of the biogas constituents) for batch experiments to evaluate the anaerobic digestion of xylose. Also, metagenomic sequencing data and analysis were reported. All data is available at Mendeley Data. 16S DNA sequencing data and metadata is available at MG-RAST (metagenomics.anl.gov/linkin.cgi?project = 9961). For further discussion, please refer to the scientific article entitled "Effect of acidic and thermal pretreatments on a microbial inoculum for hydrogen and volatile fatty acids production through xylose anaerobic acidogenic metabolism" (Mockaitis et al., 2020)26FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/18172-2; 2010/18463-9; 2009/15984-

Natural rubber based films integrating Zosteric acid analogues as bioactive monomers

International audienc

Acidic and thermal pre-treatments for anaerobic digestion inoculum to improve hydrogen and volatile fatty acid production using xylose as the substrate

Xylose is a by-product of lignocellulosic biomass processing for production of second-generation biofuels and could be suitable for bioproduct manufacturing. This paper describes an innovative approach that enables the system to achieve high yielding for hydrogen production. The study compared 4 physico-chemical pre-treatments performed in an anaerobic mixed culture (acidic, thermal, acidic-thermal and thermal acidic) to achieve an inoculum with a high-efficiency xylose to hydrogen conversion under mesophilic conditions (30 degrees C). The acidic pre-treatment was the most efficient to select microorganisms able to produce hydrogen and volatile acid from xylose. Kinetics has shown that acidic pre-treatment had a hydrogen/xylose molar yielding factor of 1.57 (molar base) and a hydrogen maximum production rate of 253 mL H-2 h(-1). Mass balance considered all possible metabolic pathways using xylose as a substrate. Anaerobic degradation of ethanol was the most active pathway for hydrogen production in all experiments, except for the control. Each pre-treatment performed for the original inoculum resulted in different microbiological profiles, but the genus Clostridium was the most abundant in all assays. Acidic pre-treatment stimulated the growth of organisms from the genera Peptostreptococcaceae, Truepera and Kurthia, which could be related to the better results in hydrogen production found in this condition14513881398FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/18172-2; 2010/18463-9; 2009/15984-