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.

Heterotrophic nitrification and aerobic denitrification of high-strength ammonium in anaerobically digested sludge by Alcaligenes faecalis strain No. 4.

Alcaligenes faecalis strain No. 4 which is capable of heterogeneous nitrification and aerobic denitrification, was used to remove high-strength ammonium (approximately 1 g NH4(+)-N/l) from digested sludge, the product of an anaerobic digestion reactor, in which methane was produced from excess municipal sewage sludge. Repeated batch operations were conducted at 20°C and 30°C for 550 h, using a jar fermentor. The removal ratios of high-strength ammonium reached 90-100% within 24 h, and the average ammonium removal rate was 2.9 kg-N/m(3)/day, more than 200 times higher than that in conventional nitrification-denitrification processes. During these operations, the cell density was maintained at 10(8)-10(9) cells of A. faecalis strain No. 4/ml. At 3% NaCl in the digested sludge, strain No. 4 exhibited an ammonium removal rate of 3 kg-N/m(3)/day.

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.

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.

Removal of xylene by a mixed culture of Pseudomonas sp. NBM21 and Rhodococcus sp. BTO62 in biofilter.

Xylene (a mixture of o-, m-, p-xylenes and ethylbenzene) gas removal was conducted in the a biofilter inoculated with a mixture of the m- and p-xylene-degraders, Pseudomonas sp. NBM21 and an o-xylene degrader, Rhodococcus sp. BTO62 under non-sterile conditions at 20 degrees C. Elimination capacities of o-, m-, and p-xylenes obtained were 180 g/m(3)/h at 20 degrees C and 100 g/m(3)/h at 10 degrees C, which were significantly higher than the 60-78 g/m(3)/h of previously reported biofilters, indicating that the two bacteria inoculated exhibited an almost total ability to remove xylene although only present in low numbers in the biofilter. In the sterile biofilter, carbon mass balance showed that 11.6% of the removed xylene was converted to cell mass. Among the xylene components, o-xylene was the most resistant to microbial degradation in spite of the low component ratio.

Purification and characterization of two DyP isozymes from Thanatephorus cucumeris Dec 1 specifically expressed in an air-membrane surface bioreactor.

DyP isozymes (DyP2 and DyP3) from the culture fluid of the fungus Thanatephorus cucumeris Dec 1 by air-membrane surface bioreactor were purified and characterized. The characteristics of DyP2 were almost the same as those of a recombinant DyP reported previously, but different from DyP3.

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.

Efficient dye decolorization and production of dye decolorizing enzymes by the basidiomycete Thanatephorus cucumeris Dec 1 in a liquid and solid hybrid culture.

Thanatephorus cucumeris Dec 1, a basidiomycete, is a promising decomposer of several xenobiotics. Air-membrane surface bioreactor culture (AMS culture), a hybrid between solid-state and submerged culture, was used for Dec 1 growth. The optimum temperature for AMS culture (25 degrees C) was lower than that for submerged culture (30 degrees C). Fungal growth was almost the same in AMS culture as in submerged culture at optimum temperatures. However, protein secretion, dye decolorizing peroxidase (DyP) activity, and aryl alcohol oxidase activity, were 18-, 233-, and 36.5-fold higher in AMS culture than in submerged culture, respectively, indicating that AMS culture of Dec 1 was superior to submerged culture. In in vivo dye decolorization tests, 13 of 16 dyes were decolorized by more than 90% within 10 days. Interestingly, under AMS culture, a biofilm was formed; biofilm formation was not, however, essential for DyP and manganese dependent peroxidase (MnP) activities. Although the correlation between DyP activity and water activity was unclear, MnP activity seemed to increase in activity with decreasing water activity, even when no biofilm formation was observed.

Stimulation of anaerobic digestion of thickened sewage sludge by iron-rich sludge produced by the fenton method.

In batch mesophilic anaerobic digestion of a mixture of iron-rich sludge collected from Fenton treatment of molasses wastewater (MWW) and thickened sewage sludge from a municipal wastewater treatment plant, the methane content in the gas from the mixture was significantly higher than that from thickened sewage sludge only. When minerals in iron-rich sludge obtained by incineration were fed to thickened sewage sludge in semi-continuous experiments, the methane content obtained by digestion of thickened sewage sludge only increased approximately from 53% to 64%. These results suggest that iron-rich sludge can enhance methane production for thickened sewage sludge.

Stable repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) from Aspergillus oryzae.

Recombinant Aspergillus oryzae expressing a dye-decolorizing peroxidase gene (dyp) was cultivated for repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) using maltose as a carbon source. High-level rDyP activity in limitation of carbon and nitrogen sources was maintained stably for 26 cycles of repeated 1-d batches of A. oryzae pellets without any additional pH control. Cultures maintained at 4 degrees C for 20 d resumed rDyP production following a single day of incubation. One liter filtrated crude rDyP containing 4600 U rDyP decolorized 5.07 g RBBR at the apparent decolorization rate of 17.7 mg l(-1) min(-1).

Bioremediation of oil-contaminated soil using Candida catenulata and food waste.

Even though petroleum-degrading microorganisms are widely distributed in soil and water, they may not be present in sufficient numbers to achieve contaminant remediation. In such cases, it may be useful to inoculate the polluted area with highly effective petroleum-degrading microbial strains to augment the exiting ones. In order to identify a microbial strain for bioaugmentation of oil-contaminated soil, we isolated a microbial strain with high emulsification and petroleum hydrocarbon degradation efficiency of diesel fuel in culture. The efficacy of the isolated microbial strain, identified as Candida catenulata CM1, was further evaluated during composting of a mixture containing 23% food waste and 77% diesel-contaminated soil including 2% (w/w) diesel. After 13 days of composting, 84% of the initial petroleum hydrocarbon was degraded in composting mixes containing a powdered form of CM1 (CM1-solid), compared with 48% of removal ratio in control reactor without inoculum. This finding suggests that CM1 is a viable microbial strain for bioremediation of oil-contaminated soil with food waste through composting processes.

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.

DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase family: ASP171 replaces the distal histidine of classical peroxidases.

DyP, a unique dye-decolorizing enzyme from the fungus Thanatephorus cucumeris Dec 1, has been classified as a peroxidase but lacks homology to almost all other known plant peroxidases. The primary structure of DyP shows moderate sequence homology to only two known proteins: the peroxide-dependent phenol oxidase, TAP, and the hypothetical peroxidase, cpop21. Here, we show the first crystal structure of DyP and reveal that this protein has a unique tertiary structure with a distal heme region that differs from that of most other peroxidases. DyP lacks an important histidine residue known to assist in the formation of a Fe4+ oxoferryl center and a porphyrin-based cation radical intermediate (compound I) during the action of ubiquitous peroxidases. Instead, our tertiary structural and spectrophotometric analyses of DyP suggest that an aspartic acid and an arginine are involved in the formation of compound I. Sequence analysis reveals that the important aspartic acid and arginine mentioned above and histidine of the heme ligand are conserved among DyP, TAP, and cpop21, and structural and phylogenetic analyses confirmed that these three enzymes do not belong to any other families of peroxidase. These findings, which strongly suggest that DyP is a representative heme peroxidase from a novel family, should facilitate the identification of additional new family members and accelerate the classification of this novel peroxidase family.

Medium optimization of antifungal lipopeptide, iturin A, production by Bacillus subtilis in solid-state fermentation by response surface methodology.

Iturin A, a lipopeptide antibiotic produced by Bacillus subtilis RB14-CS, suppresses the growth of various plant pathogens. Here, enhancement of iturin A production in solid-state fermentation (SSF) on okara, a soybean curd residue produced during tofu manufacturing, was accomplished using statistical experimental design. Primary experiments showed that the concentrations of carbon and nitrogen sources were the main factors capable of enhancing iturin A production, whereas initial pH, initial water content, temperature, relative humidity, and volume of inoculum were only minor factors. Glucose and soybean meal were the most effective among tested carbon and nitrogen sources, respectively. Based on these preliminary findings, response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen sources in the medium. The maximum iturin A concentration was 5,591 mug/g initial wet okara under optimized condition. Subsequent experiments confirmed that iturin A production was significantly improved under the predicted optimal medium conditions. The SSF product generated under the optimized conditions exhibited significantly higher suppressive effect on the damping-off of tomato caused by Rhizoctonia solani K-1 compared with the product generated under the non-optimized conditions.

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.

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.

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.

Degradation of diesel oil in soil using a food waste composting process.

We investigated the simultaneous degradation of diesel oil in soil and the organic matter in food waste by composting in 8 l reactors. Using a 0.5 l/min air flow rate, and 0.5-1% diesel oil concentrations together with 20% food waste, high composting temperatures (above 60 degrees C) were attained due to the efficient degradation of the food waste. Petroleum hydrocarbons were degraded by 80% after only 15 days composting in the presence of food waste. In a 28 l reactor scale-up experiment using 1% oil, 20% food waste and 79% soil, removal efficiencies of petroleum hydrocarbons and food waste after 15 days were 79% and 77%, respectively.