Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C₁₀H₂₂) to that of tetracontane (C₄₀H₈₂) as a sole carbon source. Two genes ...encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30°C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C₃₂H₆₆) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.
Degradation of alkanes is a widespread phenomenon in nature, and numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing these substrates as a carbon and energy source have ...been isolated and characterized. In this review, we summarize recent advances in the understanding of bacterial metabolism of long-chain n-alkanes. Bacterial strategies for accessing these highly hydrophobic substrates are presented, along with systems for their enzymatic degradation and conversion into products of potential industrial value. We further summarize the current knowledge on the regulation of bacterial long-chain n-alkane metabolism and survey progress in understanding bacterial pathways for utilization of n-alkanes under anaerobic conditions.
A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C₁₀H₂₂) to tetracontane (C₄₀H₈₂) as a sole carbon source was isolated using a system for screening ...microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.
This paper concerns the
in situ compatibilization of immiscible polypropylene/poly(butylene terephthalate) (PP/PBT) blends by a one-step reactive extrusion process. By
one-step reactive extrusion, we ...are referring to functionalization of the PP with a monomer bearing a desired functional group and the subsequent interfacial reaction of this functionalized PP with the PBT, where the whole operation is carried out in
a single extrusion process. Three monomers, acrylic acid (AA), maleic anhydride (MA) and glycidyl methacrylate (GMA), which are potentially reactive towards the car☐ylic and/or hydroxyl groups at the chain ends of the PBT, were melt grafted on to the PP by free-radical reactions. A comparative study showed that GMA is more effective in compatibilizing the PP/PBT blends. Optimization of chemical parameters (initial concentrations of GMA and peroxide) and processing conditions (the sequence of addition of the materials, devolatilization of residual GMA, etc.) allowed us to obtain compatibilized PP/PBT blends with a fifteen- to twentyfold improvement in elongation at break and impact strength over an uncompatibilized PP/PBT blend. The
in situ compatibilization performance of this one-step reactive extrusion process was comparable with that of a classical two-step process. In this latter case, the first extrusion step was to functionalize the PP with GMA, and the second one being the interfacial reaction between the functionalized PP and the PBT.
Summary
It is well established that micro‐organisms colonize a variety of extreme environments, including habitats like oil reservoirs deep inside the earth crust. Here, we present the results of a ...comparative high‐coverage DNA sequencing study of metagenomes derived from two different oil reservoirs, both located about 2.5 km subseafloor below the Norwegian Sea. A previously reported bioinformatic analysis of DNA sequence data derived from one of the reservoirs (Well I) indicated that the community is dominated by bacterial species with a smaller fraction of Archaea. Here, we report results of a similar analysis from another reservoir (Well II) located in the same geographical area, however, according to available geological knowledge lacking direct physical contact with Well I. Interestingly, the Well II community is largely dominated by Archaea with a subordinate fraction of Bacteria. Comparison of the two datasets showed that large fractions of the sequences are extremely similar, both with respect to identity (typically above 98%) and gene organization. We therefore conclude that both wells contain essentially the same organisms, but in different relative abundances. Assuming that the communities have been distinct for long timescales because of physical separation, the results also indicate that microbial growth in the reservoirs is extremely slow.
Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificansα‐carbonic anhydrase (TaCA) has been expressed in Escherichia ...coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon‐dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α‐carbonic anhydrases, TaCA does not display esterase activity towards p‐nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.
Butanol-producing microorganisms are all obligate anaerobes. In this study, a unique symbiotic system TSH06 was isolated to be capable of producing butanol under non-anaerobic condition. Denaturing ...gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) revealed that two strains coexist in TSH06. The two strains were identical to
Clostridium acetobutylicum
and
Bacillus cereus
, respectively. They were isolated individually and named as
C
.
acetobutylicum
TSH1 and
B
.
cereus
TSH2.
C
.
acetobutylicum
TSH1 is a butanol-producing, obligate anaerobic strain. Facultative anaerobic
B
.
cereus
TSH2 did not possess the ability of butanol production; however, it offered
C. acetobutylicum
TSH1 the viability under non-anaerobic condition. Moreover,
B
.
cereus
TSH2 enhanced butanol yield and speed of fermentation. TSH06 produced 12.97 g/L butanol and 15.39 g/L total solvent under non-anaerobic condition, which is 25 and 24 %, respectively, higher than those of
C. acetobutylicum
TSH1. In addition, TSH06 produced butanol faster under non-anaerobic condition than under anaerobic condition. Butanol accounted for more than 80 % of total solvent, which is higher than the known report. TSH06 was stable during passage. In all, TSH06 is a promising candidate for industrialisation of biobutanol with high yield, high butanol proportion, easy-handling and time-saving system. These results demonstrated the potential advantage of symbiosis. This study also provides a promising strategy for butanol fermentation.
Aqueous solutions of a charged hydrophobically modified hydroxyethylcellulose (HM-HEC(−)) exhibit high viscosities even at low polymer concentrations (0.2
wt%), which is an interesting feature in ...connection with enhanced oil recovery. This polymer was synthesized for this work. Effects of temperature and addition of sodium dodecyl sulfate (SDS) or hydroxypropyl-β-cyclodextrin (HP-β-CD) on the viscosity properties of a semidilute solution of HM-HEC(−) are examined. The results for the HM-HEC(−)/SDS system disclose strong interactions between HM-HEC(−) and SDS at low level of SDS addition, and this leads to a significant viscosification of the polymer–surfactant mixture. At higher surfactant concentrations the association complexes are disrupted. A strong temperature effect of the viscosity is observed at moderate levels of SDS addition, with lower values of the viscosity at elevated temperatures because of enhanced polymer chain mobility that breaks up the associations. Addition of HP-β-CD monomers to the HM-HEC(−) solution generates decoupling of associations via inclusion complex formation with the polymer hydrophobic tails and the viscosity decreases. By using temperature and addition of these co-solutes, it is demonstrated that the viscosity of the polymer solution can be tuned over a large range of viscosity values.
Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificansalpha-carbonic anhydrase (TaCA) has been expressed in ...Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized alpha-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold. PUBLICATION ABSTRACT
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