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.

Biotreatment of restaurant wastewater with an oily high concentration by newly isolated bacteria from oily sludge.

High concentration restaurant oily wastewater from restaurants and food processing industries discharged into water environment usually results in environment pollution and inhibits the activity of microorganisms in biological wastewater treatment systems. In this study, 75 strains from oily sludge were isolated with oil degradation activity for edible oil-contained wastewater. Eight isolates were able to grow well in liquid cultures with edible oil as the sole carbon source and discovered with high efficient oil-degrading ability. Seven out of eight isolates were identified as Acinetobacter and one isolate as Kluyvera cryocrescens, based on their 16S rRNA gene sequences. Three highly efficient oil degrading bacteria (Acinetobacter dijkshoorniae LYC46-2, Kluyvera cryocrescens LYC50-1a and Acinetobacter pittii LYC73-4b) were selected and their degradation characteristic were examined, the results showed that the three isolates were effective under pH range from 7.0 to 10.0, and temperature from 25 to 35 °C. For degradation of 2-4% (v/v) of vegetable oil, > 85% degradation percentage were obtained within 30 h. Degradation of the higher concentration oil (6-8%, v/v) result in 50-70% degradation percentage within 72 h, and the degradation percentage for the isolated strains were decreased about 50% for the degradation of 10% oil (< 45%) compared to 2% oil. Different type of oils were also tested, > 90% of degradation percentage were obtained by the three isolates, implied that these strains are capable of removing various oils efficiently. These results suggested that Acinetobacter dijkshoorniae LYC46-2, Kluyvera cryocrescens LYC50-1a and Acinetobacter pittii LYC73-4b are potential species could be efficiently used for high concentration restaurant oily wastewater treatment and might be applicable to a wastewater treatment system for the removal of oil.

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.