High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates.

This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species.

Parapedobacter pyrenivorans sp. nov., isolated from a pyrene-degrading microbial enrichment, and emended description of the genus Parapedobacter.

A novel pyrene-degrading, Gram-negative bacterium, designated strain P-4(T), was isolated from a polycyclic aromatic hydrocarbon-degrading enrichment of polluted soils from a coking chemical plant. Cells of strain P-4(T) were non-motile rods. Strain P-4(T) grew at 15-45 °C (optimum, 37 °C), pH 6.0-10.0 (optimum, pH 8.5) and 0-4 % (w/v) NaCl. Analysis of the 16S rRNA gene sequence showed that strain P-4(T) was related phylogenetically to members of the genus Parapedobacter, with sequence similarity of 93.7-95.1 %. The cellular fatty acids of strain P-4(T) were iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C17 : 0 3-OH, summed feature 9 (iso-C17 : 1ω9c and/or 10-methyl C16 : 0), anteiso-C15 : 0, iso-C15 : 0 3-OH, C16 : 0, iso-C15 : 1 G, C16 : 0 3-OH and C17 : 0 2-OH. Cells contained menaquinone 7 as the major quinone. The polyamine of strain P-4(T) was homospermidine, and the main polar lipids were phosphatidylethanolamine and a sphingolipid. The G+C content of the DNA was 45.4 mol%. Strain P-4(T) showed a range of phenotypic characteristics that differentiated it from previously recognized Parapedobacter species, particularly its ability to use pyrene as a sole carbon source for growth and its alkaline optimal pH for growth (pH 8.5). On the basis of these results, it is concluded that strain P-4(T) represents a novel species of the genus Parapedobacter, for which the name Parapedobacter pyrenivorans (type strain P-4(T) = NBRC 109113(T) = CGMCC 1.12195(T)) is proposed. An emended description of the genus Parapedobacter is also provided.

Reconstruction of metabolic networks in a fluoranthene-degrading enrichments from polycyclic aromatic hydrocarbon polluted soil.

Microbial degradation of polycyclic aromatic hydrocarbons (PAHs) is the primary process of removing PAHs from environments. The metabolic pathway of PAHs in pure cultures has been intensively studied, but cooperative metabolisms at community-level remained to be explored. In this study, we determined the dynamic composition of a microbial community and its metabolic intermediates during fluoranthene degradation using high-throughput metagenomics and gas chromatography-mass spectrometry (GC-MS), respectively. Subsequently, a cooperative metabolic network for fluoranthene degradation was constructed. The network shows that Mycobacterium contributed the majority of ring-hydroxylating and -cleavage dioxygenases, while Diaphorobacter contributed most of the dehydrogenases. Hyphomicrobium, Agrobacterium, and Sphingopyxis contributed to genes encoding enzymes involved in downstream reactions of fluoranthene degradation. The contributions of various microbial groups were calculated with the PICRUSt program. The contributions of Hyphomicrobium to alcohol dehydrogenases were 62.4% in stage 1 (i.e., when fluoranthene was rapidly removed) and 76.8% in stage 3 (i.e., when fluoranthene was not detectable), respectively; the contribution of Pseudomonas were 6.6% in stage 1 and decreased to 1.2% in subsequent stages. To the best of the author's knowledge, this report describes the first cooperative metabolic network to predict the contributions of various microbial groups during PAH-degradation at community-level.