Formation of highly twisted ribbons in a carboxymethylcellulase gene-disrupted strain of a cellulose-producing bacterium.

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state (13)C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

Decolorization of oxygen-delignified bleaching effluent and biobleaching of oxygen-delignified kraft pulp by non-white-rot fungus Geotrichum candidum Dec 1.

Decolorization of oxygen-delignified bleaching effluent (abbreviated as OBE) and biobleaching of oxygen-delignified kraft pulp (OKP) were conducted using a non-white rot fungus Geotrichum candidum Dec 1 (abbreviated as Dec 1) which has ability to decolorize various synthetic dyes and molasses. Dec 1 decolorized up to 77% of OBE for 6 days. In addition, Dec 1 increased the brightness of OKP from 47.8% to 51.2% and decreased the kappa value of OKP from 12.4 to 10.4 points during a 6-day incubation period at a 25% of pulp-concentration. At 2% pulp-concentration, the brightness of OKP increased by 13% and the kappa value of OKP decreased by 4 points only for a 3-day incubation period. When the decolorized OBE was used for bleaching of OKP, the brightness of OKP increased to 62.7% under the shaking culture to a 2% pulp-concentration using culture fluid of decolorized OBE. It was revealed that Dec 1 is a potential to apply for decolorization of wastewater and biobleaching of pulp in paper-mills.

Degradation pathway of an anthraquinone dye catalyzed by a unique peroxidase DyP from Thanatephorus cucumeris Dec 1.

The reactants produced by action of a purified unique dye-decolorizing peroxidase, DyP, on a commercial anthraquinone dye, Reactive Blue 5, were investigated using electrospray ionization mass spectrometry (ESI-MS), thin-layer chromatography (TLC), and (1)H- and (13)C- nuclear magnetic resonance (NMR). The results of ESI-MS analysis showed that phthalic acid, a Product 2 (molecular weight 472.5), and a Product 3 (molecular weight 301.5), were produced. Product 2 and Product 3 were generated by usual peroxidase reaction, whereas phthalic acid was generated by hydrolase- or oxygenase-catalyzed reaction. One potential associated product, o-aminobenzene sulfonic acid, was found to be converted to 2,2'-disulfonyl azobenzene by ESI-MS and NMR analyses. From these results, we propose, for the first time, the degradation pathway of an anthraquinone dye by the enzyme DyP.

Solid state fermentation of lipopeptide antibiotic iturin A by using a novel solid state fermentation reactor system.

A new solid state fermentation reactor (SSFR) for solid substrate was used for the production of lipopeptide antibiotic iturin A using Bacillus subtilis RB14-CS. Solid state fermentation (SSF) is the technique of cultivation of microorganisms on solid and moist substrates in the absence of free water. SSF has shown much promise in the development of several bioprocesses and products because of their several advantages like absence of free water that allows simplified downstream processing and low cost. SSFR allows agitation of the SSF culture with improved temperature control and air supply. Interestingly, when okara, the widely available waste product from the tofu industries, was used as the solid substrate for the SSFR, no iturin A production was observed. However, without agitation, production of iturin A was observed in the SSFR but the production level remained low. The low production of iturin A was found to be due to the heat generation and excess temperature rise inside the reactor system during the fermentation process. Maintaining the temperature within a range of 25-30°C, production of iturin A was significantly improved in the SSFR. This was comparable to the laboratory scale production, and signifies the potential application of the SSFR for SSF.

Removal of COD and color from livestock wastewater by the Fenton method.

The Fenton method was applied to the removal of chemical oxygen demand using chromate (CODcr) and color from high-strength livestock wastewater in which the initial CODcr was 5000-5700 mg/L. The optimum ratio of H2O2 (mg/L) to the initial CODcr was 1.05 and the optimum molar ratio of H2O2/Fe2+ was 2. The optimum initial pH and the optimum reaction time were 3.5-4 and 30 min, respectively. Under optimal conditions, the removal ratios of CODcr and color of the supernatant after static precipitation of the produced sludge were 88 and 95.4%, respectively. Addition of Fenton's reagents in several aliquots did not affect the efficiencies of CODcr and color removal.

Removal of o-xylene using biofilter inoculated with Rhodococcus sp. BTO62.

Rhodococcus sp. BTO62 was isolated from activated sludge from a wastewater treatment plant as an o-xylene-degrading microorganism. BOT62 degraded not only o-xylene, but also benzene, toluene, ethylbenzene, m- and p-xylenes and styrene (BTEXS). A laboratory scale biofilter packed with Biosol as packing material, which is made from foamed waste glass mixed with corrugated cardboard, was inoculated with strain BTO62 and operated to remove relatively high loading of o-xylene at different space velocities under non-sterile and sterile conditions. The o-xylene elimination capacity to maintain more than 90% removal efficiency was 41g/m3/h under sterile condition, but it enhanced to 160g/m3/h under non-sterile condition. This indicates possibilities of the role of other contaminants for degradation of o-xylene and the degradation of intermediate products of o-xylene by contaminants. Quick recovery of o-xylene degradation was observed after shutdown of o-xylene gas supply and mineral medium circulation for 10-30 days.

Treatment of concentrated leachate with low greenhouse gas emission in two-stage membrane bioreactor bio-augmented with Alcaligenes faecalis no. 4.

Methane (CH4) and nitrous oxide (N2O) emissions from two-stage membrane bioreactor (MBR) bio-augmented by Alcaligenes faecalis no. 4 during municipal solid waste leachate treatment were investigated. The system was operated at hydraulic retention time (HRT) of 2.5 and 1 days in each reactor under the presence and absence of sludge recirculation. Alcaligenes faecalis no. 4 bio-augmentation helped improving organic carbon and nitrogen removals while reducing CH4 and N2O emissions. CH4 and N2O emissions were decreased by 46% and 85% when A. faecalis no. 4 was introduced at HRT of 2.5 days. Under the presence of A. faecalis no. 4, the operation of two-stage MBR with sludge recirculation could reduce CH4 and N2O emissions by 51% and 54% as compared to its operation without sludge recirculation. An operation under short HRT of 1 day also yielded high organic carbon and nitrogen removals of more than 85% while emitting lower CH4 and N2O emission of 6.7% C and 0.04% N when operated with sludge recirculation. Implications: A two-stage membrane bioreactor was effectively applied to the treatment of concentrated leachate (BOD~20,000 mg/L) at a short hydraulic retention time of 2.5 days and 1 day. About 80% of CH4 and N2O was emitted from the anaerobic and aerobic reactors, respectively. Introduction of Alcaligenes faecalis no. 4 reduced CH4 and N2O emissions in both reactors as it became the predominant microorganism under an elevated pH condition. Lower CH4 and N2O emissions were achieved under a sludge recirculation operation, as Alcaligenes faecalis no. 4 could suppress methanogenic activities in the anaerobic reactor and converted a majority of nitrogen into its cell mass, thus reducing N2O production through a biological nitrification-denitrification pathway.

Biofilm fermentation of iturin A by a recombinant strain of Bacillus subtilis 168.

Bacillus subtilis 168 produces thin and fragile biofilm in the static culture, however, it was found out that its transformant B. subtilis RM/iSd16 containing wild sfp, itu operon and degQ, which produced lipopeptide antibiotic iturin A, produced thick and much stable biofilm. Production of iturin A by RM/iSd16 in biofilm was almost two times higher compared to that in the submerged culture at 28 degrees C.

Improvement in ammonium removal efficiency in wastewater treatment by mixed culture of Alcaligenes faecalis no. 4 and L1.

To improve ammonium removal efficiency in wastewater treatment, a mixed culture of Alcaligenes faecalis no. 4 and its mutant L1, both of which have heterotrophic nitrification and aerobic denitrification abilities, was performed. In a batch culture, no. 4 has a higher denitrification ability than L1, but its ammonium removal rate was lower. In a mixed continuous culture in the ammonium loading range of 750 to 3500 mg-N/l/d, the average ammonium removal rate and the average denitrification ratio were 61 mg-N/l/h and 31%, respectively. In the mixed culture, the ammonium removal rate was twofold higher than that in a single culture of no. 4, the rate was similar to that in a single culture of L1, and the denitrification ratio was very high compared with that in the single culture of L1.

Production of dye-decolorizing peroxidase (rDyP) from complex substrates by repeated-batch and fed-batch cultures of recombinant Aspergillus oryzae.

We examined production levels of dye-decolorizing peroxidase (rDyP) by recombinant Aspergillus oryzae using wheat bran and rice bran powders in repeated-batch and fed-batch cultures. Similar average rDyP productivities were observed in repeated-batch cultures using wheat bran powder and rice bran powder. Average rDyP productivities in fed-batch cultures were slightly lower than those in repeated-batch cultures. The rDyP production was affected by the addition of K(2)HPO(4) in the repeated-batch and fed-batch cultures using wheat bran powder. All average rDyP productivities in this study were significantly higher than those for any other peroxidases previously reported.

Second stage production of iturin A by induced germination of Bacillus subtilis RB14.

Bacillus subtilis RB14, a dual producer of lipopeptide antibiotics iturin A and surfactin undergoes sporulation in the submerged fermentation and the production of these secondary metabolites becomes halted. In this study, production of lipopeptide antibiotics was investigated by induced germination of the spores by heat-activation and nutrient supplementation. The induced spores became metabolically active vegetative state and produced lipopeptide antibiotic iturin A that added up the total production at the end of the fermentation. However, additional production of surfactin was not observed. This second time iturin A production by the germinated cells from the spores was defined as second stage production.

Piggery wastewater treatment using Alcaligenes faecalis strain No. 4 with heterotrophic nitrification and aerobic denitrification.

Alcaligenes faecalis strain No. 4, which has heterotrophic nitrification and aerobic denitrification abilities, was used to treat actual piggery wastewater containing high-strength ammonium under aerobic conditions. In a continuous experiment using a solids-free wastewater (SFW) mixed with feces, almost all of the 2000 NH4+ -N mg/L and 12,000 COD mg/L in the wastewater was removed and the ammonium removal rate was approximately 30 mg-N/L/h, which was 5-10 times higher than the rates achieved by other bacteria with the same abilities. The denitrification ratio was more than 65% of removed NH4+ -N, indicating that strain No. 4 exhibited its heterotrophic nitrification and aerobic denitrification abilities in the piggery wastewater.

Removal of p-xylene with Pseudomonas sp. NBM21 in biofilter.

As a p-xylene (p-Xyl)-degrading microorganism, Pseudomonas sp. NBM21 was isolated from an activated sludge of a wastewater treatment plant. NBM21 degraded p-Xyl, m-xylene, benzene and toluene, but not o-xylene, ethylbenzene (Eb) and styrene. NBM21 was inoculated to a biofilter with Biosol as a packing material and p-Xyl removal was operated for 105 d under sterile and nonsterile conditions. The maximum elimination capacities for p-Xyl at higher than 90% removal efficiency were 160 g/m3/h and 150 g/m3/h under nonsterile and sterile conditions, respectively. A high load of Eb adversely affected to the removal of xylene.

Enhancement of styrene removal efficiency in biofilter by mixed cultures of Pseudomonas sp. SR-5.

The styrene-degrading bacterium Pseudomonas sp. SR-5 exhibited a high styrene removability in a biofilter. However, the styrene removal efficiency (RE) of SR-5 decreased with time. We carried out styrene gas removal in a biofilter inoculated with mixed cultures of SR-5 and other microorganisms to determine the possibility of obtaining an enhanced RE for a long period. The following three inocula were carried out: (i) styrene-degrading bacteria, strains 1 and 3, (ii) a benzoic acid-degrading bacterium Raoultella sp. A, and (iii) wastewater from a chemical company dealing with styrene. These biofilters with mixed SR-5 showed an enhanced RE compared with those with a single culture of SR-5. The complete styrene elimination capacities for ensuring 100% styrene removal in those mixed cultures were 151, 108 and 124 g/m(3)/h, compared with a single culture of SR-5.

Effect of shutdown on styrene removal in a biofilter inoculated with Pseudomonas sp. SR-5.

Styrene gas removal was carried out in a biofilter inoculated with a styrene-degrading Pseudomonas sp. SR-5 using a mixed packing material of peat and ceramic under the non-sterile condition. More than 86% removal efficiency was obtained at styrene load of 5-93 g m(-3) h(-1) for 62 days operation period and 78% carbon of removed styrene was converted to CO2. Thereafter, three kinds of styrene shutdown experiments were conducted: (i) air and mineral medium were supplied for 4 days, (ii) complete shutdown, namely no styrene, air and moisture supply was conducted for 3 days, and (iii) only air was supplied for 11 days. When styrene gas was re-supplied after (i) and (iii) shutdown experiments, styrene removal efficiency rapidly recovered, but after (ii) shutdown, recovery of styrene removal was significantly delayed. Supply of air during shutdown period was found to be enough to resume microbial activity to degrade styrene.

Precipitation diagram and optimization of crystallization conditions at low ionic strength for deglycosylated dye-decolorizing peroxidase from a basidiomycete.

The growth of suitably sized protein crystals is essential for protein structure determination by X-ray crystallography. In general, crystals are grown using a trial-and-error method. However, these methods have been modified with the advent of microlitre dispensing-robot technology and of protocols that rapidly screen for crystal nucleation conditions. The use of one such automatic dispenser for mixing protein drops (1.3-2.0 microl in volume) of known concentration and pH with precipitating solutions (ejecting 2.0 microl droplets) containing salt is described here. The results of the experiments are relevant to a crystallization approach based on a two-step procedure: screening for the crystal nucleation step employing robotics followed by optimization of the crystallization conditions using incomplete factorial experimental design. Large crystals have successfully been obtained using quantities as small as 3.52 mg protein.

Performance of a styrene-degrading biofilter inoculated with Pseudomonas sp. SR-5.

Styrene removal was studied for 3 months in a laboratory-scale biofilter packed with a mixed packing material of peat and ceramic at a ratio of 1 to 1 on a dry-weight basis and inoculated with Pseudomonas sp. SR-5. More than 90% removal efficiency (RE) was attained at 1-140 g/m3/h styrene loads under nitrogen-source limitation. When RE decreased to 70% after 30 d with an increase in styrene load, readdition of SR-5 and washing of the filter packing material restored the RE to more than 90% by maintaining the population of SR-5 at 1-10% of the total cell number. The maximum elimination capacity (EC) by kinetic analysis was estimated to be 290 g/m3/h. High conversion of the removed styrene carbon to CO2, and significantly small production of cell mass from the removed carbon were confirmed.

Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4.

Alcaligenes faecalis no. 4 has heterotrophic nitrification and aerobic denitrification abilities. By taking the nitrogen balance under different culture conditions, 40-50% of removed NH4+-N was denitrified and about one-half of removed NH4+-N was converted to intracellular nitrogen. The maximum ammonium removal rate of no. 4 (28.9 mg-N/l/h) and its denitrification rate at high-strength NH4+-N of about 1200 ppm in aerated batch experiments at a C/N ratio of 10 were 5-40 times higher than those of other bacteria with the same ability. Only a few percent of the removed ammonium was converted to nitrite, and the main denitrification process was speculated to be via hydroxylamine which was produced by ammonium oxidation.

Bacterial cellulose production by fed-batch fermentation in molasses medium.

Batch and fed-batch fermentations for bacterial cellulose (BC) production using molasses as a carbon source by Acetobacter xylinum BPR2001 were carried out in a jar fermentor. For improvement of BC production, molasses was subjected to H2SO4-heat treatment. The maximum BC concentration by this treated molasses increased 76%, and the specific growth rate increased 2-fold compared with that by untreated molasses. In batch fermentation, when the initial sugar concentrations of H2SO4-heat-treated molasses were varied from 20 to 70 g/L, the highest value of maximum BC concentration of 5.3 g/L was observed at 20 g/L. BC production in intermittent fed-batch (IFB) fermentation was conducted referring to the data in batch fermentation, and the highest BC production of 7.82 g/L was obtained when 0.2 L of molasses medium was added five times. When continuous fed-batch (CFB) fermentations were conducted, maximum BC concentration was obtained with a feeding rate of 6.3 g-sugar/h, which was derived from the optimal IFB experiment.

Change in broth culture is associated with significant suppression of Escherichia coli death under high magnetic field.

When Escherichia coli B was cultivated under an inhomogeneous magnetic field of 5.2-6.1 T, a significant 100,000-fold suppression of cell death was observed [Bioelectrochemistry 53 (2001) 149]. The limited magnetic field exposure for 12 h after logarithmic growth phase was sufficient to observe similar suppressive effects on cell death [Bioelectrochemistry 54 (2001) 101]. These results suggest some possible changes in either the medium or the cells during the magnetic field exposure. When the cell-free filtrate of the broth cultured under the magnetic field for 10 h and the cells of E. coli cultivated under the geomagnetic field for 30 h were mixed, and the mixture was subsequently cultivated under the geomagnetic field, the number of cells observed in the filtrate exposed to the high magnetic field was 20,000 times higher than that in the filtrate exposed to the geomagnetic field. When the cells cultivated under the magnetic field for 10 h and the cell-free filtrate of the broth culture exposed to the geomagnetic field were mixed, only a 50-fold difference in the number of cell between under the magnetic field and under the geomagnetic field was observed. This suggests that the filtrate of the broth culture exposed to the magnetic field is primarily responsible for the cell death suppression. It was also revealed that the small difference in pH of the filtrates of the broth culture between under the magnetic field and under the geomagnetic field was critical for the cell death suppression.