Characterization of Pseudomonas mendocina LR capable of removing nitrogen from various nitrogen-contaminated water samples when cultivated with Cyperus alternifolius L

A new strain of denitrifying bacterium, Pseudomonas mendocina LR, was isolated from rhizosphere soil of aquatic plants living in a river contaminated with industrial wastewater and domestic sewage. The isolate was found to fully remove as much as 613.2 mg nitrate in 60 h under stationary culture conditions. The effects of carbon sources and nitrogen sources on nitrogen removal were investigated using a modified denitrification medium (DM). Sodium citrate was identified as the most effective source of carbon. The ability of LR to adapt to different nitrogen sources, including nitrite, indicated that LR could be used in the purification of wastewater containing different forms of nitrogen. The optimal C/N ratio was 7 for LR, and it was resistant to antibiotics Amp, Chl, Ery, and Str. Plant-microbe bioaugmentation was performed to remove nitrogen dissolved in Hoagland medium and natural wastewater. An increased rate of nitrogen removal was observed when root exudates of Cyperus alternifolius L. were added simultaneously with LR. LR was not able to survive in the natural wastewater unless root exudates from umbrella grass were added. LR cultured with umbrella grass exhibited a maximal nitrogen reduction rate of 95.9% and 97.3% in Hoagland medium and wastewater, respectively. This shows that bioaugmentation utilizing plant-microbe interactions can be an effective and exhaustive means of removing nitrogen and may be an attractive approach to nitrogen reduction in natural environments and wastewater treatment factories.     

A novel enzymatic approach to the massproduction of L-galactose from L-sorbose

Wild-type strain of Pseudomonas cichorii ST-24 was unable to grow on D -psicose and inductively produced D -tagatose 3-epimerase (D -TE) with D -tagatose as an inducer. We have isolated a constitutive mutant, designated strain Ka75, which had acquired a new ability to grow on a mineral salts medium containing D -psicose as a sole carbon source. The D -psicose-metabolizing mutant synthesized a high level of D -TE. When grown on the culture medium supplemented with Mn(2+), the mutant strain produced around 250-fold higher activity than did the parent strain. Enzymatic properties of the constitutive enzyme were similar to those of the wild-type. Using the immobilized D -TE and recombinant L-rhamnose isomerase (L-RhI) from Escherichia coli strain JM109, a two-step enzymatic reaction was performed for massproduction of a rare aldo-hexose monosaccharide, L-galactose, from a common one, L-sorbose. In the first step, L-sorbose was epimerized to L-tagatose in a yield of 28%. The L-tagatose obtained was utilized as a starting material for L-galactose preparation by the immobilized L-RhI. At equilibrium, approximately 30% L-tagatose was isomerized to L-galactose. Finally, 7.5 g of L-galactose was obtained from 100 g of L-sorbose, viz an overall yield of 7.5%. The product obtained was purified and identified to be L-galactose by specific optical rotation and high performance liquid chromatography (HPLC) analysis, and was ultimately confirmed by (13)C nuclear magnetic resonance ((13)C NMR) and IR spectra.     

Characterization of the Enterobacteriaceae community that developed during storage of minced beef under aerobic or modified atmosphere packaging conditions

The whole cell protein and macrorestriction analysis of DNA of Enterobacteriaceae isolates recovered from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO₂-30% O₂-30% N₂ in the presence (MAP+) and absence (MAP−) of oregano essential oil were studied. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles obtained from whole cell protein analysis of the Enterobacteriaceae isolates revealed seven groups. Moreover, application of a modified PFGE protocol with XbaI restriction, resulted into 19 different fingerprints. The Enterobacteriaceae community of fresh meat consisted of Serratia liquefaciens and Serratia proteamaculans. S. liquefaciens strain VK23 was the dominant isolate of Enterobacteriaceae for the most conditions adopted, except 10 °C and 15 °C under MAP + and 10 °C under MAP−. In the latter cases, Hafnia alvei represented the dominant fingerprint. Citrobacter freundii was recovered from minced beef stored aerobically, while H. alvei and Proteus vulgaris were recovered under MAP. Storage conditions affected the Enterobacteriaceae community; modified atmosphere packaging increased both species and strain diversity.     

Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus B-6 grown on Avicel under aerobic conditions

A multienzyme complex, cellulosome, of the facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6 was produced on microcrystalline cellulose (Avicel) under aerobic conditions. During growth on Avicel, the bacterial cells were found to be capable of adhesion to Avicel by scanning electron microscopic (SEM) analysis. The multienzyme complex of P. curdlanolyticus B-6 was isolated from the crude enzyme preparation by gel filtration chromatography on Sephacryl S-300 and affinity purification on cellulose. The isolated multienzyme complex was able to bind to both Avicel and insoluble xylan and consists of cellulolytic and xylanolytic enzymes such as avicelase, carboxymethyl cellulase (CMCase), cellobiohydrolase, β-glucosidase, xylanase, β-xylosidase and α-l-arabinofuranosidase. The molecular mass of the complex was estimated to be 1600 kDa. It composed of at least 12 proteins on SDS-PAGE and 10 CMCases and 11 xylanases on zymograms. The isolated multienzyme complex could degrade the raw lignocellulosic substances effectively.