A three-dimensional (3D) printing approach to fabricate an isolation chip for high throughput in situ cultivation of environmental microbes.

The full plethora of environmental bacteria is often poorly represented in vitro as the majority remain difficult, if not impossible, to culture under standard laboratory settings. These bacteria often require native conditions for the formation of cell masses that collectively have higher chances of survival. With that, a 3D-printed version of the isolation chip (iChip) was used to cultivate bacteria from a tropical peat swamp in situ prior to growth and maintenance in vitro. Briefly, plates made from either acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), or epoxy resin were tested in terms of their usability and durability under acidic conditions similar to those of peat matter. The epoxy resin plates were then found to be most optimal for the sampling conditions. Peat soil samples were collected from the base of a Koompassia malaccensis tree and reconstituted in molten 10% (wt/vol) tryptone soy agar (TSA) prior to inoculation. The iChips were subsequently assembled and buried in the site of origin. As a comparison, bacteria from the same soil sample were cultivated directly on TSA and incubated at 28 °C for two weeks. Thereafter, agar plugs from the iChip were transferred to TSA plates to allow microcolonies within each plug to grow. Each pure isolate from both cultivation approaches that grew was then pooled and extracted for total DNA prior to 16S rRNA gene amplification and sequencing via Illumina MiSeq. Taxonomic abundance comparison revealed that the bacterial taxa at the level of order were significantly different between the two approaches, particularly in the orders, Burkholderiales, Xanthomonodales, Enterobacteriales, and Actinomycetales (differences of 12.0, 7.1, 8.0, and 4.2%, respectively). This indicated that the 3D-printed iChips present a possible low-cost tool for the isolation of bacterial genera that may not be able to grow on media directly in vitro.

Chitinophaga extrema sp. nov., isolated from subsurface soil and leaf litter in a tropical peat swamp forest.

A Gram-negative, filamentous aerobic bacterium designated as strain Mgbs1T was isolated on 12 April 2017 from the subsurface soil and leaf litter substrate at the base of a Koompassia malaccensis tree in a tropical peat swamp forest in the northern regions of the state of Selangor, Malaysia (3° 39' 04.7' N 101° 17' 43.7'' E). Phylogenetic analyses based on the full 16S rRNA sequence revealed that strain Mgbs1T belongs to the genus Chitinophaga with the greatest sequence similarity to Chitinophaga terrae KP01T (97.65 %), Chitinophaga jiangningensis DSM27406T (97.58 %), and Chitinophaga dinghuensis DHOC24T (97.17 %). The major fatty acids of strain Mgbs1T (>10 %) are iso-C15 : 0, C16 : 1 ω5c and iso-C17 : 0 3-OH while the predominant respiratory quinone is menaquinone-7. Strain Mgbs1T has a complete genome size of 8.03 Mb, with a G+C content of 48.5 mol%. The DNA-DNA hybridization (DDH) score between strain Mgbs1T and C. jiangningensis DSM27406T was 15.9 %, while in silico DDH values of strain Mgbs1T against C. dinghuensis DHOC24T and C. terrae KP01T were 20.0 and 19.10% respectively. Concurrently, Average Nucleotide Identity (ANI) scores between strain Mgbs1T against all three reference strains are 73.2 %. Based on the phenotypic, chemotaxonomic, and phylogenetic consensus, strain Mgbs1T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga extrema sp. nov. is proposed (=DSM 108835T=JCM 33276T).