Virgibacillus olivae sp. nov., isolated from waste wash-water from processing of Spanish-style green olives

Abstract

Four bacterial strains (E 30 8 T , E 55 49, I 30 77 and N 30 129) were isolated from the residual wash-water produced during the processing of Spanish-style green table olives. The isolates were subjected to a polyphasic taxonomic study using phenotypic, phylogenetic and genotypic methods. The bacteria were Gram-positive, spore-forming rods. Moreover, they were heterotrophs that were able to utilize cellobiose, glucose, mannose and rhamnose as carbon sources. The G+C content of their genomic DNA ranged from 30.7 to 33.4 mol%. The major cellular fatty acids found in strain E 30 8 T were iso-C 15 : 0 , anteiso-C 15 : 0 , iso-C 17 : 0 and anteiso-C 17 : 0 . DNA-DNA hybridization shows 76.2-88.3 % relatedness among the four strains. The 16S rRNA gene sequence of isolate E 30 8 T shows that it belongs to the genus Virgibacillus, with the highest sequence similarity (99 %) to Virgibacillus marismortui 123 T . However, phenotypic differences and DNA-DNA relatedness between strain E 30 8 T and V. marismortui ATCC 700626 T of less than 47 % suggest the placement of these strains within a novel species of the genus Virgibacillus. The name Virgibacillus olivae sp. nov. is proposed, with strain E 30 8 T (=LMG 23503 T =DSM 18098 T ) as the type strain.During the preparation of edible olives according to the procedure used to give so-called 'Spanish green olives', residual wash-water (EWW, edible-olive wash-water) is generated as a waste product. In this process, the olives are treated with brines which contain 10-13 % (w/v) NaCl, in which they undergo lactic fermentation (Brenes & de Castro, 1997). The brines represent a large proportion of the total contaminating discharges from the olive industry. However, specific treatments for these wash-waters are either nonexistent or ineffective (Romero Barranco et al., 2001). To obtain a better insight into the microbial community present in EWW, we analysed the microbial content. In the present study, we report the description of a novel aerobic, mesophilic, endospore-forming organism isolated from EWW that was able to grow in 0-20 % NaCl.Initially, 180 strains were isolated from samples of EWW (provided by AgroSevilla S.A., La Roda, Sevilla, Spain) obtained at different stages of the edible olive processing. Isolation was performed by the standard dilutionplating technique on HM medium (Ventosa et al., 1982) supplemented with a balanced mixture of sea salts with 10 % (w/v) NaCl (Rodríguez-Valera et al., 1981) at 30 u C. Strains were maintained on HM medium (Ventosa et al., 1982) supplemented with 5 % (w/v) sea-salt solution (referred to here as HM 5 % sea salts medium) (Rodríguez-Valera et al., 1981). The predominant strains present in this wash-water were Gram-positive, endospore-forming bacilli. For this study, of the 180 isolated strains, four were selected on the basis of their similar physiological characteristics.Cell morphology was examined by light microscopy and transmission electron microscopy using cells from exponentially growing cultures. Endospores were stained accor...

Virgibacillus xinjiangensis sp. nov., isolated from a Salt Lake of Xin-jiang Province in China

“…The major lipoquinone, major fatty acids, and G+C content were in line with those of members of the genus Virgibacillus Heyrman et al, 2003;Quesada et al, 2007;Chen et al, 2008;Hua et al, 2008;Wang et al, 2008). Typical phenotypes of strain SL6-1 T are summarized and compared with those of the type strains of closely related taxa (Table 1).…”

“…Virgibacillus olivae E308 T(Quesada et al, 2007); 3, Virgibacillus kekensis YIM kkny16 T; 4, Virgibacillus marismortui DSM 12325 T(Arahal et al, 1999); 5, Virgibacillus pantothenticus IAM 11061 T(Heyndrickx et al, 1999). +, positive; -, negative; w, weakly positive; v, variable; E, ellipsoidal; S, spherical; ST, subterminal;…”

Virgibacillus kapii sp. nov., isolated from Thai shrimp paste (Ka-pi)

“…These chemotaxonomic properties are commonly found in other members of genus Virgibacillus (Arahal et al, 1999(Arahal et al, , 2000Heyrman et al, 2003;Quesada et al, 2007;Hua et al, 2008). The strain contained anteiso-C 15 : 0 (44.01 %), anteiso-C 17 : 0 (41.30 %), iso-C 15 : 0 (6.12 %), iso-C 16 : 0 (4.89 %), iso-C 17 : 0 (1.56 %) and iso-C 14 : 0 (1.04 %) in the cellular fatty acid profile (Table S1).…”

“…Strain KN3-8-4 T grew in the presence of 0-15 % (w/v) NaCl, at 11-42 8C and at pH 4.5-10 (optimally in 1-5 % NaCl, at 37 8C, and at pH 7.5). The strain was sensitive to (mg unless otherwise stated): amikacin (30) T was 43.5 mol%, which was in the range of the genus Virgibacillus (Lee et al, 2006;An et al, 2007;Quesada et al, 2007), although it was higher than the closely related type strains (Table 1). Strain T contained meso-diaminopimelic acid in Virgibacillus kapii sp.…”

Virgibacillus litoralis sp. nov., a moderately halophilic bacterium isolated from saline soil

“…At the time of writing, the genus comprised 14 recognized species, i.e. Virgibacillus pantothenticus (Heyndrickx et al , 1999, Virgibacillus proomii (Heyndrickx et al 1999), Virgibacillus carmonensis, Virgibacillus necropolis, Virgibacillus marismortui, and Virgibacillus salexigens (Heyrman et al 2003), Virgibacillus halodenitrificans (Yoon et al 2004), Virgibacillus dokdonensis (Yoon et al 2005), Virgibacillus koreensis (Lee et al 2006), Virgibacillus olivae (Quesada et al 2007), Virgibacillus halophilus , Virgibacillus chiguensis , Virgibacillus kekensis and Virgibacillus salarius (Hua et al 2008). During an investigation of the microbial diversity of Naozhou Island, Leizhou Bay, South China Sea Xiao et al 2009), a moderately halophilic strain, designated JSM 089168 T , was isolated from a saline soil sample.…”