Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa

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Abstract
Earthworms emit nitrous oxide (N2O) via the activity of bacteria in their gut. Four N2O-producing facultative aerobes, ED1T, ED5T, MH21T and MH72, were isolated from the gut of the earthworm Aporrectodea caliginosa. The isolates produced N2O under conditions that simulated the microenvironment of the earthworm gut. ED1T and ED5T were Gram-negative, motile rods that carried out complete denitrification (i.e. the reduction of nitrate to N2) and contained membranous c-type cytochromes. ED1T grew optimally at 30 °C and pH 7. ED1T oxidized organic acids and reduced (per)chlorate, sulfate, nitrate and nitrite. The closest phylogenetic relative of ED1T was Dechloromonas agitata. ED5T grew optimally at 25 °C and pH 7. ED5T grew mainly on sugars, and nitrate and nitrite were used as alternative electron acceptors. The closest phylogenetic relatives of ED5T were Flavobacterium johnsoniae and Flavobacterium flevense. MH21T and MH72 were motile, spore-forming, rod-shaped bacteria with a three-layered cell wall. Sugars supported the growth of MH21T and MH72. Cells of MH21T grew in chains, were linked by connecting filaments and contained membranous b-type cytochromes. MH21T grew optimally at 30–35 °C and pH 7·7, grew by fermentation and reduced low amounts of nitrite to N2O. The closest phylogenetic relatives of MH21T were Paenibacillus borealis and Paenibacillus chibensis. Based on morphological, physiological and phylogenetic characteristics, ED1T (=DSM 15892T=ATCC BAA-841T), ED5T (=DSM 15936T=ATCC BAA-842T) and MH21T (=DSM 15890T=ATCC BAA-844T) are proposed as type strains of the novel species Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov. and Paenibacillus anaericanus sp. nov., respectively. MH72 is considered a new strain of Paenibacillus terrae.

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