Double-Sided Microwells with a Stepped Through-Hole Membrane for High-Throughput Microbial Assays.

To improve the throughput of microwell arrays for identifying immense cellular diversities even at a single-bacteria level, further miniaturization or densification of the microwells has been an obvious breakthrough. However, controlling millions of nanoliter samples or more at the microscale remains technologically difficult and has been spatially restricted to a single open side of the microwells. Here we employed a stepped through-hole membrane to utilize the bottom as well as top side of a high-density nanoliter microwell array, thus improving spatial efficiency. The stepped structure shows additional effectiveness for handling several millions of nanoliter bacterial samples in the overall perspectives of controllability, throughput, simplicity, versatility, and automation by using novel methods for three representative procedures in bacterial assays: partitioning cells, manipulating the chemical environment, and extracting selected cells. As a potential application, we show proof-of-concept isolation of rare cells in a mixed ratio of 1 to around 106 using a single chip. Our device can be further applied to various biological studies pertaining to synthetic biology, drug screening, mutagenesis, and single-cell heterogeneity.

Kluyvera georgiana MCC 3673: A Novel Electrogen Enriched in Microbial Fuel Cell Fed with Oilseed Cake.

A novel electrogenic bacterial species, Kluyvera georgiana MCC 3673, was isolated by enrichment in microbial fuel cells (MFC) using oilseed cake as a growth substrate. CHNS analyses showed that oilseed cakes are rich in carbon and nitrogen content. Utilization of these compounds by bacteria was evident from 50% reduction in chemical oxygen demand. The maximum power density of 379 ± 8 mW/m2 was produced from sesame seed cake media with mixed consortia inoculum from lake sediment. Enrichment was carried out for over 15 cycles by renewing the media periodically on drop of the voltage. A pure culture of enriched electrogen was isolated by dilution plate technique. Physiological and biochemical studies were performed on the isolate as per standard methods. Genetic analysis by 16S rDNA sequencing revealed that this organism is closely related to Kluyvera georgiana. When inoculated in MFC as pure culture, the maximum power density of 158 ± 11 mW/m2 and 172 ± 13 mW/m2 was produced with sesame and groundnut oilseed cake media, respectively. The performance increased in LB media producing maximum power density of 394 ± 6 mW/m2. This bacterium has also scope for application in wide range of MFC as it can produce electricity even in suspended culture. To our knowledge, this is the first report on bio-electricity generation using oilseed cake as substrate in MFC.

Excellent N-fixing and P-solubilizing traits in earthworm gut-isolated bacteria: A vermicompost based assessment with vegetable market waste and rice straw feed mixtures.

Vermicomposting is a dependable waste recycling technology which greatly augments N and P levels mainly through microbial action. This paper aims to identify efficient N-fixing (NFB) and P-solubilizing (PSB) bacteria from earthworm intestines. Various combinations of vegetable market waste, rice straw, and cowdung were fed to two earthworm species (Eisenia fetida and Perionyx excavatus). Total organic C decreased, pH shifted towards neutrality, and NPK availability, and microbial (NFB, PSB, and total bacteria) population increased remarkably during vermicomposting with E. fetida. Therefore, 45 NFB and 34 PSB strains isolated from Eisenia gut were initially screened, their inter-dominance assessed, and 8 prolific strains were identified through 16SrRNA sequencing. Interestingly, two novel N-fixing strains of Kluyvera ascorbata emerged as an efficient biofertilizer candidate. Moreover, both N-fixing and P-solubilizing strains of Serratia and Bacillus were isolated from earthworm gut. All the isolated strains significantly improved soil health and facilitated crop growth as compared to commercial biofertilizers.

Isolation and characterization of Kluyvera georgiana strain with the potential for acrylamide biodegradation.

Worldwide contamination by acrylamide, a neurotoxicant and carcinogen in animals, is becoming a significant problem. We isolated three novel acrylamide-degrading bacteria from domestic wastewater in Chonburi, Thailand. Using biochemical characteristics and 16S rRNA gene sequencing, the strains were identified as Klebsiella pneumoniae, Kluyvera georgiana and Enterococcus faecalis. K. georgiana strain No. 2 was selected for further characterization due to its degradation potential of high concentrations of acrylamide at the mesophilic temperatures. The strain grew well in the presence of acrylamide at concentrations to 0.5 % (w/v), pH 5.0 to 7.0 and 37°C. Degradation of acrylamide to acrylic acid began after 30 min of cultivation as a biomass-dependent manner. Mass balance analysis revealed 92.3 % conversion of acrylamide to acrylic acid and two lower polarity compounds. Strain No. 2 degraded many aliphatic amides but not iodoacetamide and thioacetamide. High degradation level (>80 %) was found with propionamide, cyanoacetamide and acetamide. Moderate degradation was obtained in the order of formamide > butyramide > lactamide > urea while sodium azide provided 34 % degradation. These findings render this novel bacterium attractive for biodegradation of acrylamide and other aliphatic amides in the environment.

Correlation of fluoroquinolone resistance with expression of qnrA gene in Kluyvera spp.

The objective of the present study was to examine whether the expression of qnrA may contribute to a high level of resistance among parent and induced strains of Kluyvera spp. Two clinical isolates of ciprofloxacin-resistant Kluyvera spp. were obtained from livers of diseased chickens, and upon induction with ciprofloxacin, six strains with increased resistance were produced. Point mutations in qnrA, aac(6')-Ib-cr, gyrA, gyrB, parC, and parE were investigated by polymerase chain reaction (PCR) amplification and DNA sequencing, and expression levels of acrAB and qnrA in all strains were investigated by quantitative real-time PCR (qRT-PCR). The induced strains contained the same mutations in quinolone resistance-determining region as those of the parent strains. qRT-PCR showed that the expression of the acrA gene was not detected in any strain and acrB gene expression was unchanged between induced and parental strains. However, difference in expression of qnrA was observed, which correlated well with the level of quinolone resistance in the parent and induced strains. The induced high resistance was not affected by mutations in qnrA and aac(6')-Ib-cr, by new mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE, or by the expression level of acrAB. These data suggest that the expression of qnrA may be a factor contributing to the high level of resistance among parent and induced strains of Kluyvera spp.

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.

Sugarcane bagasse mild alkaline/oxidative pretreatment for ethanol production by alkaline recycle process.

In order to decrease the alkali and water consumptions in the sugarcane bagasse alkaline/oxidative pretreatment for ethanol production, an alkaline recycle process was carried out. Two recycles of NaOH/H2O2 pretreatment did not decrease the pretreatment and enzymatic hydrolysis efficiencies and the consumptions of NaOH and water would be saved by 26% and 40%, respectively. A simultaneous saccharification and fermentation (SSF) culture with pretreated bagasse as substrate was developed giving 25 g ethanol l(-1) with a yield of 0.2 g g(-1) bagasse and productivity of 0.52 g l(-1) h(-1).

Phylogenetic relationships of the genus Kluyvera: transfer of Enterobacter intermedius Izard et al. 1980 to the genus Kluyvera as Kluyvera intermedia comb. nov. and reclassification of Kluyvera cochleae as a later synonym of K. intermedia.

In order to assess the relationship between the genus Kluyvera and other members of the family Enterobacteriaceae, the 16S rRNA genes of type strains of the recognized Kluyvera species, Kluyvera georgiana, Kluyvera cochleae, Kluyvera ascorbata and Kluyvera cryocrescens, were sequenced. A comparative phylogenetic analysis based on these 16S rRNA gene sequences and those available for strains belonging to several genera of the family Enterobacteriaceae showed that members of the genus Kluyvera form a cluster that contains all the known Kluyvera species. However, the type strain of Enterobacter intermedius (ATCC 33110T) was included within this cluster in a very close relationship with the type strain of K. cochleae (ATCC 51609T). In addition to the phylogenetic evidence, biochemical and DNA-DNA hybridization analyses of species within this cluster indicated that the type strain of E. intermedius is in fact a member of the genus Kluyvera and, within it, of the species Kluyvera cochleae. Therefore, following the current rules for bacterial nomenclature and classification, the transfer of E. intermedius to the genus Kluyvera as Kluyvera intermedia comb. nov. is proposed (type strain, ATCC 33110T=CIP 79.27T=LMG 2785T=CCUG 14183T). Biochemical analysis of four E. intermedius strains and one K. cochleae strain independent of the respective type strains further indicated that E. intermedius and K. cochleae represent the same species and are therefore heterotypic synonyms. Nomenclatural priority goes to the oldest legitimate epithet. Consequently, Kluyvera cochleae Muller et al. 1996 is a later synonym of Kluyvera intermedia (Izard et al. 1980) Pavan et al. 2005.