Joris Mergaert

Phylogenetic position of phytopathogens within the Enterobacteriaceae

The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Erwinia may be divided into three phylogenetic groups. Cluster I represents the true erwinias and comprises E. amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila. We propose to unite the species of cluster II, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. carotovora subsp. odorifera, E. carotovora subsp. wasabiae, E. cacticida, E. chrysanthemi and E. cypripedii in the genus Pectobacterium respectively as P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii. The species E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis, comprising cluster III, are being classified into a new genus Brenneria gen. nov. respectively as B. alni comb. nov., B. nigrifluens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

Microbiological aspects of biowaste during composting in a monitored compost bin

Aims: To determine the microbial succession of the dominating taxa and functional groups of microorganisms and the total microbial activity during the composting of biowaste in a monitored process.

TRANSFER OF ERWINIA-ANANAS (SYNONYM, ERWINIA-UREDOVORA) AND ERWINIA-STEWARTII TO THE GENUS PANTOEA EMEND AS PANTOEA-ANANAS (SERRANO 1928) COMB-NOV AND PANTOEA-STEWARTII (SMITH 1898) COMB-NOV, RESPECTIVELY, AND DESCRIPTION OF PANTOEA-STEWARTII SUBSP INDOLOGENES SUBSP NOV.

Eight Erwinia stewartii strains, 11 Erwinia ananas strains, and 7 Erwinia uredovora strains, as well as 9 phenotypically similar Erwinia herbicola strains and Enterobacter agglomerans LMG 5342, were compared by examining electropherograms prepared from their soluble proteins and were grouped into nine protein electrophoretic groups. The levels of DNA relatedness among these electrophoretic groups were determined spectrophotometrically from the renaturation rates at 74°C of the DNAs of 13 selected strains. The representatives from five protein electrophoretic groups, including E. ananas LMG 2665T (T = type strain) and E. uredovora LMG 2667T, exhibited 76 to 100% DNA binding to each other and constituted DNA hybridization group 2665. All E. stewartii strains (including LMG 2715T) were electrophoretically very similar; representatives of this species exhibited 93 to 99% DNA binding to each other and constituted DNA hybridization subgroup 2715. The strains belonging to the remaining three protein electrophoretic groups exhibited 94 to 96% DNA binding to each other and formed DNA hybridization subgroup 2632. The latter two subgroups were 60 to 83% (average, 73%) interrelated and exhibited 30 to 39% DNA binding to group 2665. It is proposed that E. ananas and E. uredovora should be united in a single species, which should be classified in the genus Pantoea as Pantoea ananas (Serrano 1928) comb. nov.; its type strain is strain LMG 2665 (= NCPPB 1846). This species also includes DNA hybridization group VI of Brenner et al. (D. J. Brenner, G. R. Fanning, J. K. Leete Knutson, A. G. Steigerwalt, and M. I. Krichevsky, Int. J. Syst. Bacteriol. 34:45-55, 1984). The transfer of E. stewartii to the genus Pantoea and the creation of two separate subspecies within Pantoea stewartii (Smith 1898) comb. nov. are also proposed. Pantoea stewartii subsp. stewartii (Smith 1898) comb. nov. (synonym, Erwinia stewartii) contains the strains belonging to subgroup 2715, and its type strain is strain LMG 2715 (= NCPPB 2295); and Pantoea stewartii subsp. indologenes subsp. nov. contains the strains belonging to subgroup 2632, and its type strain is strain LMG 2632 (= NCPPB 2280). As determined by principal-component analysis of the cellular fatty acid compositions, P. ananas, P. stewartii subsp. stewartii, and P. stewartii subsp. indologenes are separated from each other mainly by differences in the relative contents of cis-9-hexadecenoic acid (C16:1 cis 9), cyclo-heptadecanoic acid (C17:0 cyclo), and straight-chain octodecenoic acids (C18:1). P. stewartii subsp. stewartii can also be differentiated from P. ananas and P. stewartii subsp. indologenes by its inability to produce indole, to utilize citrate, to grow on cis-aconitate, and to form acid from seven carbohydrates. P. stewartii subsp. indologenes can also be separated from P. ananas by its inability to form acid from sorbitol and α-methyl-D-mannoside. Descriptions of P. ananas and P. stewartii and its two subspecies are given, and the description of the genus Pantoea is emended.

Bioremediation of diesel oil-contaminated soil by composting with biowaste

Soil spiked with diesel oil was mixed with biowaste (vegetable, fruit and garden waste) at a 1:10 ratio (fresh weight) and composted in a monitored composting bin system for 12 weeks. Pure biowaste was composted in parallel. In order to discern the temperature effect from the additional biowaste effect on diesel degradation, one recipient with contaminated soil was hold at room temperature, while another was kept at the actual composting temperature. Measurements of composting parameters together with enumerations and identifications of microorganisms demonstrate that the addition of the contaminated soil had a minor impact on the composting process. The first-order rate constant of diesel degradation in the biowaste mixture was four times higher than in the soil at room temperature, and 1.2 times higher than in the soil at composting temperature.

Diversity of 746 heterotrophic bacteria isolated from microbial mats from Ten Antarctic lakes

Microbial mats, growing in Antarctic lakes constitute unique and very diverse habitats. In these mats microorganisms are confronted with extreme life conditions. We isolated 746 bacterial strains from mats collected from ten lakes in the Dry Valleys (lakes Hoare and Fryxell), the Vestfold Hills (lakes Ace, Druzhby, Grace, Highway, Pendant, Organic and Watts) and the Larsemann Hills (lake Reid), using heterotrophic growth conditions. These strains were investigated by fatty acid analysis, and by numerical analysis, 41 clusters, containing 2 to 77 strains, could be delineated, whereas 31 strains formed single branches. Several fatty acid groups consisted of strains from different lakes from the same region, or from different regions. The 16S rRNA genes from 40 strains, representing 35 different fatty acid groups were sequenced. The strains belonged to the alpha, beta and gamma subclasses of the Proteobacteria, the high and low percent G+C Gram-positives, and to the Cytophaga-Flavobacterium-Bacteroides branch. For strains representing 16 fatty acid clusters, validly named nearest phylogenetic neighbours showed pairwise sequence similarities of less than 97%. This indicates that the clusters they represent, belong to taxa that have not been sequenced yet or as yet unnamed new taxa, related to Alteromonas, Bacillus, Clavibacter, Cyclobacterium, Flavobacterium, Marinobacter, Mesorhizobium, Microbacterium, Pseudomonas, Saligentibacter, Sphingomonas and Sulfitobacter.

Biodiversity of microorganisms that degrade bacterial and synthetic polyesters.

The biodiversity and occurrence in nature of bioplastic-degrading microorganisms are exemplified by the identification of 695 strains, isolated from different environments, such as soils, composts, natural waters, and sludge, that are able to degrade the bacterial polyester poly(3-hydroxybutyrate)in vitro. These microorganisms belong to at least 57 different taxa, including Gram-negative and Gram-positive bacteria, streptomycetes, and moulds. The literature on the biodiversity of poly(3-hydroxybutyrate)-degrading microorganisms is reviewed. The degrading abilities of 171 streptomycete strains were investigated on four different bacterial poly(3-hydroxyalkanoates), and the synthetic polyesters poly(ε-caprolactone) and BIONOLLE, and most of these strains degraded at least three different polymers.

Characterization of facultative oligotrophic bacteria from polar seas by analysis of their fatty acids and 16S rDNA sequences

One hundred and seventy three bacterial strains, isolated previously after enrichment under oligotrophic, psychrophylic conditions from Arctic (98 strains) and Antarctic seawater (75 strains), were characterized by gas-liquid chromatographic analysis of their fatty acid compositions. By numerical analysis, 8 clusters, containing 2 to 59 strains, could be delineated, and 8 strains formed separate branches. Five clusters contained strains from both poles, two minor clusters were confined to Arctic isolates, and one cluster consisted of Antarctic isolates only. The 16S rRNA genes from 23 strains, representing the different fatty acid profile clusters and including the unclustered strains, were sequenced. The sequences grouped with the alpha and gamma Proteobacteria, the high percent G+C gram positives, and the Cytophaga-Flavobacterium-Bacteroides branch. The sequences of strains from 4 clusters and of 7 unclustered strains were closely related (sequence similarities above 97%) to reference sequences of Sulfitobacter mediterraneus, Halomonas variabilis, Alteromonas macleodii, Pseudoalteromonas species, Shewanella frigidimarina, and Rhodococcus fascians. Strains from the other four clusters and an unclustered strain showed sequence similarities below 97% with nearest named neighbours, including Rhizobium, Glaciecola, Pseudomonas, Alteromonas macleodii and Cytophaga marinoflava, indicating that the clusters which they represent form as yet unnamed taxa.

Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor

A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics. Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria. One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed. Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing. Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed. The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed. The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon. Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample. The other groups occurred either in the lower or upper part, or in both samples. They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas.

Reclassification of non-pigmented Erwinia herbicola strains from trees as Erwinia billingiae sp. nov.

Twenty-two Erwinia-like strains, isolated from trees since the late fifties and belonging to a distinct phenotypic group with resemblance to Pantoea agglomerans, were further characterized by conventional biochemical tests, the BIOLOG metabolic fingerprinting system and fatty acid analysis. Their phylogenetic positions were determined by comparing the 16S rRNA gene sequence of a representative strain to available sequences of Erwinia, Pantoea, Pectobacterium and Brenneria species. The strains were shown to belong to the genus Erwinia, with Erwinia rhapontici and Erwinia persicina as the closest phylogenetic relatives. The name Erwinia billingiae sp. nov. is proposed (type strain LMG 2613T) and a description of the species is given.

Isolation and identification of cellulolytic bacteria involved in the degradation of natural cellulosic fibres.

In search for bacterial cultures that are able to rapidly degrade cellulosic plant fibres in vitro, 77 cellulolytic strains were isolated from Belgian and Czech soils after enrichment on flax or sisal fibres as sole sources of carbon. The strains were characterized using fatty acid analysis, and 74 strains were grouped into three major clusters by numerical analysis. The first major cluster contained Cellulomonas strains. Within this cluster three subclusters could be delineated by principal component analysis, that were recognized by their fatty acid compositions as Cellulomonas gelida, Cellulomonas biazotea and Cellulomonas cellulans, containing 9, 8 and 13 strains respectively. The second major cluster, with 9 strains, was assigned to Flavobacterium johnsoniae. The 34 strains of the third cluster could not be identified by commercial identification systems on the basis of their fatty acid profiles and API 20NE profiles. On the basis of their phenotypic characteristics they met the description of the genus Cellvibrio, their fatty acid profiles were similar to those of four authentic Cellvibrio mixtus strains, and the 16S rRNA genes from four representatives showed up to 97.8% sequence similarity to 16S rDNA from Cellvibrio mixtus ACM 2603. Three non-clustered strains were assigned to Curtobacterium flaccumfaciens, Achromobacter piechaudii and Pseudomonas mendocina. Two strains assigned to Cellvibrio were able to degrade several flax, broom and cotton fibres very rapidly in a standardized in vitro test, causing mass losses of 40 to 86% within 13 days of incubation, but not jute.