Katsutoshi Hori

Coaggregation Facilitates Interspecies Hydrogen Transfer between Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus

ABSTRACT A thermophilic syntrophic bacterium, Pelotomaculum thermopropionicum strain SI, was grown in a monoculture or coculture with a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH. Microscopic observation revealed that cells of each organism were dispersed in a monoculture independent of the growth substrate. In a coculture, however, these organisms coaggregated to different degrees depending on the substrate; namely, a large fraction of the cells coaggregated when they were grown on propionate, but relatively few cells coaggregated when they were grown on ethanol or 1-propanol. Field emission-scanning electron microscopy revealed that flagellum-like filaments of SI cells played a role in making contact with ΔH cells. Microscopic observation of aggregates also showed that extracellular polymeric substance-like structures were present in intercellular spaces. In order to evaluate the importance of coaggregation for syntrophic propionate oxidation, allowable average distances between SI and ΔH cells for accomplishing efficient interspecies hydrogen transfer were calculated by using Ficks diffusion law. The allowable distance for syntrophic propionate oxidation was estimated to be approximately 2 μm, while the allowable distances for ethanol and propanol oxidation were 16 μm and 32 μm, respectively. Considering that the mean cell-to-cell distance in the randomly dispersed culture was approximately 30 μm (at a concentration in the mid-exponential growth phase of the coculture of 5 × 107 cells ml−1), it is obvious that close physical contact of these organisms by coaggregation is indispensable for efficient syntrophic propionate oxidation.

Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa.

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.

Construction of self-disruptive Bacillus megaterium in response to substrate exhaustion for polyhydroxybutyrate production.

Abstract. In order to establish a novel recovery system for polyhydroxyalkanoates, a self-disruptive strain of Bacillus megaterium that responds to substrate exhaustion was constructed. A gene cassette carrying the lysis system of Bacillus amyloliquefaciens phage – holin and endolysin – was inserted into the Escherichia coli-Bacillus subtilis shuttle vector pX under the control of a xylose-inducible expression system, xylR-xylA′. In this system, the expression of a target gene is induced by xylose but inhibited by glucose, which acts as an anti-inducer. B. megaterium was transformed with pX conveying the phage lysis system, which was integrated into the amyE locus of chromosomal DNA of B. megaterium by homologous recombination. The lysis system caused self-disruption of the transformant cells effectively even when expression of the lysis genes was induced during stationary phase. For the production of polyhydroxybutyrate (PHB), the transformant was grown in a medium containing glucose as a substrate in the presence of xylose. When the glucose concentration approached zero, self-disruption was spontaneously induced, releasing intracellularly accumulated PHB into the culture broth. This system realizes timely cell disruption immediately after the PHB content in the cell reaches a maximum level.

Two Morphological Types of Cell Appendages on a Strongly Adhesive Bacterium, Acinetobacter sp. Strain Tol 5

ABSTRACT Two morphological types of appendages, an anchor-like appendage and a peritrichate fibril-type appendage, have been observed on cells of an adhesive bacterium, Acinetobacter sp. strain Tol 5, by use of recently developed electron microscopic techniques. The anchor extends straight to the substratum without branching and tethers the cell body at its end at distances of several hundred nanometers, whereas the peritrichate fibril attaches to the substratum in multiple places, fixing the cell at much shorter distances.

Fungal contribution to in situ biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film in soil.

Abstract. The contribution of fungi to the microbial degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films in soil was studied. Various traces, cavities, and grooves observed on the dented surface of PHBV films demonstrated that the degradation was a concerted effect of a microbial consortium colonizing the film surface, including fungi, bacteria, and actinomycetes. The succession of microbial consortia in the soil around the PHBV films during the degradation showed a distinctive increase in the fungal population, resulting in its dominance. Comparison of the degradation ability of microbial strains isolated from soil where PHBV films were degraded, revealed that fungi showed the highest contribution to PHBV degradation, growing very rapidly along the film surface with their high degradation ability and then expanding their hyphae in a three-dimensional manner.

Carbon Fiber as an Excellent Support Material for Wastewater Treatment Biofilms

Fibrous materials made of carbon fiber (CF), aromatic polyamide (AP), preoxidized polyacrylonitrile (PAN), and polyethylene (PE), which are widely used in the textile industry, were evaluated as biofilm supports for wastewater treatment. We found that CF has a high capacity for adsorbing nitrifying bacterial sludge. The adhesion rate of four pure strains-Cytophaga hutchinsonii, Alcaligenes faecalis, Bacillus subtilis, and Escherichia coli-was highest to CF. The ζ-potentials of the fibrous supports, and the cell surface potentials of these bacteria on the basis of the soft particle theory, were experimentally determined. Bacterial cell adhesion to the fibrous supports could be explained by Derjaguin-Landau-Verwey-Overbeek theory. Interaction energy profiles based on this theory indicated the disappearance of the energy barrier in bacterial cell adhesion to the CF support, whereas an insurmountable energy barrier was observed in the adhesion to the other fibrous supports. This result was attributed to the less negative ζ-potential of CF and the relatively large Hamaker constant for the CF/bacterium interaction in water; through simulations, the latter factor was found to make a greater contribution to lowering the energy barrier. In practice and theory, CF is an excellent material as a microbial and biofilm support for wastewater treatment.

Effects of culture conditions on molecular weights of poly(3-hydroxyalkanoates) produced byPseudomonas putida from octanoate

SummaryTime-dependent changes in the molecular weights of poly(3-hydroxyalkanoates) (P(3HA)) produced byPseudomonas putida from octanoate were investigated. The mole ratio of carbon to nitrogen sources (C/N), incubation temperature, concentration of octanoate, and pH of culture solution were varied to study the effects of culture conditions on the yield and molecular weights of P(3HA). The molecular weights of P(3HA) decreased with time to reach a constant value during incubation. When the incubation temperature and the concentration of octanoate were low, P(3HA) polymers of high molecular weights were produced.

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

Identification of biofoulant of membrane bioreactors in soluble microbial products.

To reveal primary biofoulant in soluble microbial products (SMP) and/or soluble extracellular polymeric substances (EPS), after removal of sludge particles, activated sludge samples were subjected to microfiltration tests in a submerged MBR. Filtration resistance directly correlates with the saccharide concentration. Saccharides in wastewater from several sources contained uronic acids, which increased the filtration resistance. When the microfiltration test liquids contained saccharides over 80mg l(-1), a gelatinous mass remained on the membrane surface after filtration and contained concentrations of saccharides and uronic acids 50 times higher than the original test liquid while only trace amounts of these substances were contained in the filtrate. The gelatinous mass contained high molecular weight substances of 10(6)-10(8)Da, suggesting the presence of polysaccharides. However, molecules of this size were calculated to be much smaller than the pore size of the membrane. Ethylenediaminetetraacetic acid decreased filtration resistance, suggesting that polysaccharides containing uronic acid units could undergo intermolecular or intramolecular ionic cross-linking by polyvalent cations and form the gel, thus clogging the membrane pores as an actual biofoulant.

Bacillus amyloliquefaciens phage endolysin can enhance permeability of Pseudomonas aeruginosa outer membrane and induce cell lysis

To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P. aeruginosa was analyzed. Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin. Neither E15H nor T32E substitution induced enzymatic and antibacterial activities. These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme. The addition of purified E15H and T32E proteins to P. aeruginosa cells induced the release of periplasmic β-lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane. However, the addition of E15H and T32E proteins to P. aeruginosa cells did not induce the release of cytoplasmic ATP from the cells. These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P. aeruginosa outer membrane and N-terminal enzymatic activity.