Characterization of a thermostable xylanase from a newly isolated Kluyvera species and its application for biobutanol production.

Kluyvera species strain OM3 isolated from spent mushroom substrate could produce a high level of cellulase-free xylanase (5.12 U/mL). This xylanase showed maximum activities at 70 °C and pH 8.0, which could retain 100% and 71% activity after 1h incubation at 60 °C and 70 °C, and maintain stability over a wide range of pHs (5.0-9.0), indicating its thermal and pH stability. Moreover, the xylanase could hydrolyze untreated lignocellulosics (e.g., palm oil fiber) to reducing sugars with a yield of 27.1-46.9 mg/g. A co-culture consisting of Kluyvera sp. strain OM3 and Clostridium sp. strain BOH3 could directly convert birchwood xylan to 1.2g/L butanol, which was comparable to the amount of butanol (1.7 g/L) generated via separate hydrolysis by the xylanase and fermentation by Clostridium sp. strain BOH3. This is the first report on the production, characterization of a xylanase from genus Kluyvera and its application for butanol production directly from hemicelluloses.

Ethanol production from biodiesel-derived crude glycerol by newly isolated Kluyvera cryocrescens.

The rapidly expanding market for biodiesel has increased the supply and reduced the cost of glycerol, making it an attractive sustainable feed stock for the fuel and chemical industry. Glycerol-based biorefinery is the microbial fermentation of crude glycerol to produce fuels and chemicals. A major challenge is to obtain microbes tolerant to inhibitors such as salts and organic solvents present in crude glycerol. Microbial screening was attempted to isolate novel strain capable of growing on crude glycerol as a sole carbon source. The newly isolated bacteria, identified as nonpathogenic Kluyvera cryocrescens S26 could convert biodiesel-derived crude glycerol to ethanol with high yield and productivity. The supplementation of nutrients such as yeast extract resulted in distinguished enhancement in cell growth as well as ethanol productivity under anaerobic condition. When glycerol fermentation is performed under microaerobic condition, there is also a remarkable improvement in cell growth, ethanol productivity and yield, compared with those under strict anaerobic condition. In batch fermentation under microaerobic condition, K. cryocrescens S26 produced 27 g/l of ethanol from crude glycerol with high molar yield of 80% and productivity of 0.61 g/l/h.

Beta-lactam induction of ISEcp1B-mediated mobilization of the naturally occurring bla(CTX-M) beta-lactamase gene of Kluyvera ascorbata.

ISEcp1B is an insertion element associated with the emerging expanded-spectrum beta-lactamase bla(CTX-M) genes in Enterobacteriaceae. Because ISEcp1B-bla(CTX-M)positive strains may be identified from humans and animals, the ability of this insertion sequence to mobilize the bla(CTX-M-2) gene was tested from its progenitor Kluyvera ascorbata to study the effects of amoxicillin/clavulanic and cefquinome as enhancers of transposition. These beta-lactam molecules are administered parenterally to treat infected animals. ISEcp1B-mediated mobilization of the bla(CTX-M-2) gene from K. ascorbata to a plasmid location in Escherichia coli J53 was studied. Transposition assays were performed with overnight cultures with amoxicillin/clavulanic acid and cefquinome at concentrations expected to mimic those found in feces after parenteral administration (0.4-0.008 mg L(-1) and 0.32-0.064 mg L(-1), respectively). Amoxicillin/clavulanic acid and cefquinome did not modify the transposition frequency (1.85+/-1.7 x 10(-7)) whereas ceftazidime (0.5 mg L(-1)), used as a control, did (5.2+/-2.7 x 10(-5)). Therefore, it is likely that neither amoxicillin/clavulanic acid nor cefquinome concentrations as found in the gut flora may enhance mobilization of the bla(CTX-M) genes in Enterobacteriaceae.

Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5 in bacterial populations.

The intrinsic stability of IncP-1 plasmid pKJK5 was assessed in both an Escherichia coli and a Kluyvera sp. population maintained in bacterial mats and in liquid nutrient broth without selective pressure. A fluorescence tagging/flow cytometry approach was used to detect and quantify plasmid loss from populations harboring either conjugation-proficient or -deficient pKJK5 derivatives. The results show that the plasmid's ability to conjugate plays an important role in its stable maintenance in populations of both species. This effect was most pronounced in dense bacterial populations and to a far lesser extent during growth in liquid broth. Furthermore, conjugation-proficient plasmids were able to spread infectiously in the bacterial mats initiated with various ratios of plasmid-harboring cells, resulting in a nearly exclusively plasmid-harboring population.

In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring beta-lactamase gene blaCTX-M of Kluyvera ascorbata.

ISEcp1B has been reported to be associated with and to mobilize the emerging expanded-spectrum beta-lactamase blaCTX-M genes in Enterobacteriaceae. Thus, the ability of this insertion sequence to mobilize the blaCTX-M-2 gene was tested from its progenitor, Kluyvera ascorbata. Insertions of ISEcp1B upstream of the blaCTX-M-2 gene in K. ascorbata reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 microg/ml). In those cases, ISEcp1B brought promoter sequences enhancing blaCTX-M-2 expression in K. ascorbata. Then, ISEcp1B-mediated mobilization of the blaCTX-M-2 gene from K. ascorbata to Escherichia coli J53 was attempted. The transposition frequency of ISEcp1B-blaCTX-M-2 occurred at (6.4+/-0.5)x10(-7) in E. coli. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40 degrees C.