A simple strategy for investigating the diversity and hydrocarbon degradation abilities of cultivable bacteria from contaminated soil

The use of indigenous bacterial strains is a valuable bioremediation strategy for cleaning the environment from hydrocarbon pollutants. The isolation and selection of hydrocarbon-degrading bacteria is therefore crucial for obtaining the most promising strains for site decontamination. Two different media, a minimal medium supplemented with a mixture of polycyclic aromatic hydrocarbons and a MS medium supplemented with triphenyltetrazolium chloride, were used for the isolation of bacterial strains from two hydrocarbon contaminated soils and from their enrichment phases. The hydrocarbon degradation abilities of these bacterial isolates were easily and rapidly assessed using the 2,6-dichlorophenol indophenol assay. The diversity of the bacterial communities isolated from these two soil samples and from their enrichment phases was evaluated by the combination of a bacterial clustering method, fluorescence ITS-PCR, and bacterial identification by 16S rRNA sequencing. Different PCR-based assays were performed in order to detect the genes responsible for hydrocarbon degradation. The best hydrocarbon-degrading bacteria, including Arthrobacter sp., Enterobacter sp., Sphingomonas sp., Pseudomonas koreensis, Pseudomonas putida and Pseudomonas plecoglossicida, were isolated directly from the soil samples on minimal medium. The nahAc gene was detected only in 13 Gram-negative isolates and the sequences of nahAc-like genes were obtained from Enterobacter, Stenotrophomonas, Pseudomonas brenneri, Pseudomonas entomophila and P. koreensis strains. The combination of isolation on minimal medium with the 2,6-dichlorophenol indophenol assay was effective in selecting different hydrocarbon-degrading strains from 353 isolates.     

Isolation and Partial Characterization of Bacterial Strains on Low Organic Carbon Medium from Soils Fertilized with Different Organic Amendments

A total of 720 bacterial strains were isolated from soils with four different organic amendment regimes on a low organic carbon (low-C) agar medium (10 µg C ml^?1) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in dissolved organic carbon contents in these soils. There were negative correlations between total and dissolved organic carbon content and the number of isolates on low-C agar medium, whereas these correlations were absent for bacterial strains isolated from the same soil on high-C agar medium (1,000 µg C ml^?1). Repeated transfers (up to ten times) of the isolates from low-C agar medium to fresh low- and high-C agar media were done to test for exclusive growth under oligotrophic conditions. The number of isolates exclusively growing under oligotrophic conditions dropped after each subsequent transfer from 241 after the first to 98 after the third transfer step. Identification on the basis of partial 16S rRNA gene sequences revealed that most of the 241 isolates (as well as the subset of 98 isolates) belong to widespread genera such as Streptomyces, Rhizobium, Bradyrhizobium, and Mesorhizobium, and the taxonomic composition of dominant genera changed from the first transfer step to the third. A selected subset of 17 isolates were further identified and characterized for exclusive growth on low-C agar medium. Two isolates continued to grow only on low-C agar medium up to the tenth transfer step and matched most closely with Rhizobium sullae and an uncultured bacterium on the basis of the almost full-length 16S rRNA gene. It was concluded that the vast majority of strains which are isolated on low-C agar media belong to the trophic group of microorganisms adapted to a “broad range” of carbon concentrations, including well-known and widespread bacterial genera. Oligotrophy is a physiological, not a taxonomic property, and can only be identified by cultural means so far. We showed that true oligotrophs that are unable to grow on high carbon media are rare and belong to genera that also contain broad-range and copiotrophic strains.     

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil,shale oil or diesel fuel

The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.     

The fate of lignin and lignin‐derived compounds in anaerobic environments

During ODP Leg 201 microbial communities in Eastern Equatorial Pacific Ocean and Peru Margin sediments were investigated. The sediment layers sampled extended down to 420 m below the sea floor, with estimated ages of up to 40 million years. Contamination-free anoxic slurries were inoculated into media containing different substrate combinations, all at micromolar concentration. These culture media were designed for a broad spectrum of physiological groups. A total of 162 pure cultures were isolated that could be grouped into 19 different phylotypes based on 16S rRNA gene analysis. The isolates belonged to the Alpha-, Gamma- and Deltaproteobacteria, the Firmicutes, Actinobacteria, and Bacteroidetes. The genera most frequently isolated were Bacillus (68 isolates) and Rhizobium (40 isolates). Comparison of strains with the same phylotypes by enterobacterial repetitive intergenic consensus (ERIC-PCR) analysis revealed the presence of several subgroups that did not correlate with medium, sediment depth or sampling site. The majority of the isolates, although obtained from anoxic environments and isolated under strictly anoxic conditions, turned out to be facultativly aerobic. Physiologically, the isolates were characterized as generalists, able to utilize a broad variety of electron donors with either oxygen, nitrate and in some cases manganese oxides as electron acceptors. The diversity inferred from physiological tests was even higher than that on the phylogenetic or genomic level. The outcome of the contamination tests, the isolation of close relatives of already known subsurface bacteria, the repeated finding of the same phylotype from different sites and the level of diversity present in the culture collection strongly suggest that indigenous deep-biosphere bacteria had been isolated.     

Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes

A collection of 18 previously unstudied strains isolated from root nodules of Genista germanica (German greenweed) grown in southeast Poland was evaluated for the level of genetic diversity using the BOX-PCR technique and the phylogenetic relationship based on both core (16S rRNA, dnaK, ftsA, glnII, gyrB, recA, rpoB) and nodulation (nodC and nodZ) gene sequences. Each of the 18 G. germanica root nodule isolates displayed unique BOX-PCR patterns, indicating their high level of genomic heterogeneity. Based on the comparative 16S rDNA sequence analysis, 12 isolates were affiliated to the Bradyrhizobium genus and the other strains were most similar to Rhizobium species. Phylogenetic analysis of the core gene sequences indicated that the studied Bradyrhizobium bacteria were most closely related to Bradyrhizobium japonicum, whereas Rhizobium isolates were most closely related to Rhizobium lusitanum and R. leguminosarum. The phylogenies of nodC and nodZ for the Rhizobium strains were incongruent with each other and with the phylogenies inferred from the core gene sequences. All Rhizobium nodZ gene sequences acquired in this study were grouped with the sequences of Bradyrhizobium strains. Some of the studied Rhizobium isolates were placed in the nodC phylogenetic tree together with reference Rhizobium species, while the others were closely related to Bradyrhizobium bacteria. The results provided evidence for horizontal transfer of nodulation genes between Bradyrhizobium and Rhizobium. However, the horizontal transfer of nod genes was not sufficient for Rhizobium strains to form nodules on G. germanica roots, suggesting that symbiotic genes have to be adapted to the bacterial genome.     

The isolation of heavy-metal resistant culturable bacteria and resistance determinants from a heavy-metal-contaminated site

In this study we performed a phylogenetic analysis of a culturable bacterial community isolated from heavymetal-contaminated soil from southwest Slovakia using 16S rRNA (16S rDNA) and heavy-metal resistance genes. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and a trace amount of cadmium (<0.25 mg/kg). A total of 100 isolates were grown on rich (Nutrient agar No. 2) or minimal (soil-extract agar medium) medium. The isolates were identified by phylogenetic analysis using partial sequences of their 16S rRNA (16S rDNA) genes. Representatives of two broad taxonomic groups, Firmicutes and Proteobacteria, were found on rich medium, whereas four taxonomic groups, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, were represented on minimal medium. Forty-two isolates grown on rich medium were assigned to 20 bacterial species, while 58 bacteria grown on minimal medium belonged to 49 species. Twenty-three isolates carried czcA- and/or nccA-like heavy-metal-resistance determinants. The heavy-metalresistance genes of nine isolates were identified by phylogenetic analysis of their protein sequences.     

Isolation of rhizospheric and roots endophytic actinomycetes from Leguminosae plant and their activities to inhibit soybean pathogen, Xanthomonas campestris pv. glycine

In this study, actinomycetes from roots and rhizospheric soils of leguminous plants were isolated using starch casein agar supplemented with antifungal and antibacterial antibiotics. Three hundred and seventeen actinomycetes were isolated with 77 isolates obtained from plant roots and 240 isolates from rhizospheric soils. Analysis of whole-organism hydrolysates showed that 289 strains were rich in the LL-isomer of diaminopimelic acid, a result consistent with their assignment to the streptomycetes. The remaining 28 strains were assigned to non-streptomycetes based on the presence of meso-isomer of diaminopimelic acid in cell wall. Sixty-four isolates (20.2 %) showed antagonistic activity against soybean pathogen Xanthomonas campestris pv. glycine by agar overlay method. Isolate RM 365 showed the highest activity with an inhibition ratio of 3.79, with no inhibitory activity on the growth of Rhizobium japonicum TISTR 079, Rhizobium sp. TISTR 061 and Rhizobium sp. TISTR 063. The 16S rRNA gene sequence analysis revealed that isolate RM 365 shared 99.28 % similarity to Streptomyces caeruleatus GIMN4^T (GQ329712). In addition, isolates which contained meso-DAP were also identified by 16S rRNA gene sequence analysis. The results showed that they were members of the genus Amycolatopsis, Isoptericola, Micromonospora, Microbispora, Nocardia, Nonomuraea, Promicromonospora and Pseudonocardia.     

Biological nitrogen fixation and phosphate solubilization by bacteria isolated from tropical soils

Introduction

In addition to fixing atmospheric nitrogen, some bacterial isolates can also solubilize insoluble phosphates, further contributing to plant growth.

Aims

The objectives of this study were the following: isolate, select, and identify nodulating bacteria in the cowpea that are efficient not only in biological nitrogen fixation (BNF) but also in the solubilization of insoluble inorganic phosphates; identify and quantify the organic acids produced; and establish the relationship between those acids and the solubilizing capacity.

Methods

The bacteria were captured from two soils containing high concentrations of insoluble phosphorus from the cities of Lavras and Patos de Minas, using the cowpea [Vigna unguiculata (L.) Walp.] as bait. We obtained 78 strains, which were characterized according to their cultural attributes in culture medium 79 with the strains UFLA 03-84, INPA 03-11B, and BR3267 (approved by the Ministry of Livestock and Supply Agriculture—MAPA, as inoculants for the cowpea) and Burkholderia cepacia (LMG1222^T), which was used as a positive control for phosphate solubilization. Strains that were selected for their efficiency in both processes were identified by 16S rDNA sequence analysis. We evaluated the symbiotic efficiency (BNF) in a greenhouse and the solubilization efficiency of CaHPO₄, Al(H₂PO₄)₃, and FePO₄.2H₂O in solid and liquid GELP media. Strains that excelled at the solubilization of these phosphate sources were also evaluated for the production of the following organic acids: oxalic, citric, gluconic, lactic, succinic, and propionic.

Results

The presence of Acinetobacter, Bacillus, Firmicutes, Microbacterium, Paenibacillus, and Rhizobium was detected by 16S rDNA sequencing and analysis. Bacterial strains obtained from cowpea nodules varied greatly in the efficiency of their BNF and phosphate solubilization processes, especially in the strains UFLA 03-09, UFLA 03-10, UFLA 03-12, and UFLA 03-13, which were more efficient in both processes. More strains were able to solubilize insoluble inorganic calcium and iron phosphates in liquid medium than in solid medium. The production of organic acids was related to the solubilization of CaHPO₄ and FePO₄.2H₂O for some strains, and the type and concentration of the acid influenced this process.

Conclusions

These are the first results obtained with bacterial isolates from tropical soils in which the production of organic acids was detected and quantified to examine the solubilization of insoluble inorganic phosphates.     

Indole-3-Acetic Acid (IAA) Production in Symbiotic and Non-Symbiotic Nitrogen-Fixing Bacteria and its Optimization by Taguchi Design

Production of Indole-3-acetic acid (IAA) in 35 different symbiotic and non-symbiotic nitrogen-fixing bacteria strains isolated from soil and plant roots was studied and assayed by chromatography and colorimetric methods. These bacteria included Agrobacterium, Paenibacillus, Rhizobium, Klebsiella oxytoca, and Azotobacter. The best general medium and synergism effects of isolates for IAA production were investigated. Effects of different variables containing physical parameters and key media components and optimization of condition for IAA production were performed using the Design of Experiments. Qualitek-4 (W32b) software for automatic design and analysis of the experiments, both based on Taguchi method was used. The results showed that Rhizobium strains, symbiotic, and Paenibacillus non-symbiotic bacteria yielded the highest concentrations of IAA (in the range of 5.23–0.27 and 4.90–0.19 ppm IAA/mg biomass, respectively) and IAA production was increased by synergism effect of them. Yeast Extract Mannitol medium supplemented with l-tryptophan was the best general medium for IAA production. The analysis of experimental data using Taguchi method indicated that nitrogen source is very prominent variable in affecting the yield and mannitol as carbon source, potassium nitrate (1%), and l-tryptophan (3 g/l) as nitrogen sources after 72-h incubation at 30°C were the optimum conditions for production of IAA. 5.89 ppm IAA/mg biomass was produced under these optimal conditions.     

Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia

Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei, and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter, Agrobacterium, Burkholderia, Cronobacter, Enterobacter, Mesorhizobium, Novosphingobium, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, and Variovorax. Seven isolates were Gram-positive bacteria belonging to the genera Bacillus, Paenibacillus, Planomicrobium, and Rhodococcus. Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA, glnII, rpoB, and 16S rRNA gene sequences, Rhizobium, Mesorhizobium, and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter, Rhizobium sp., Rhizobium phaseoli, Mesorhizobium sp., and putative new Rhizobium species. The strains from R. phaseoli, Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, and Serratia, were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.     

Cultivation of Denitrifying Bacteria: Optimization of Isolation Conditions and Diversity Study

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.     

Indigenous PAH-degrading bacteria from oil-polluted sediments in Caleta Cordova,Patagonia Argentina

Indigenous bacteria with the capability to degrade polycyclic aromatic hydrocarbons (PAH) were isolated from polluted sediment samples recovered from Caleta Cordova by using selective enrichment cultures supplemented with phenanthrene. Bacterial communities were evaluated by denaturing gradient gel electrophoresis (DGGE) in order to detect changes along enrichment culture and relationships with the representative strains subsequently isolated. Members of these communities included marine bacteria such as Lutibacter, Polaribacter, Arcobacter and Olleya, whose degradation pathway of PAH has not been studied yet. However, isolated bacteria obtained from this enrichment comprised the genus Pseudomonas, Marinobacter, Salinibacterium and Brevibacterium. The ability of isolates to grow and degrade naphthalene, phenanthrene and pyrene was demonstrated by detection of the residual substrate by HPLC. Archetypical naphthalene and catechol dioxygenase genes were found in two isolates belonging to genus Pseudomonas (Pseudomonas monteilii P26 and Pseudomonas xanthomarina N12), suggesting biodegradation potential in these sediments. The successful bacterial isolation with the ability to degrade PAH in pure culture suggest the possibility to study and further consider strategies like growth stimulation in situ, in order to increase the intrinsic bioremediation opportunities in the polluted Caleta Cordova harbor.     

Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity

Rhizobium sp. strain WR1001, isolated from the Sonoran Desert by Eskew and Ting, was found to be able to grow in defined medium containing NaCl up to 500 mM, a concentration approaching that of sea water. Therefore, it is a valuable strain for studying the biochemical basis of salt tolerance. Intracellular free glutamate was found to increase rapidly in response to osmotic stress by NaCl. It accounted for 88% of the amino acid pool when the bacterium was grown in 500 mM NaCl. The role of glutamate dehydrogenase in glutamate biosynthesis was examined in several Rhizobium strains. Both NADH- and NADPH-dependent glutamate dehydrogenase activities in various Rhizobium strains were observed. The range of activity differed considerably depending on the particular strain. KCl (500 mM) did not stimulate glutamate dehydrogenase activity, as reported in a number of bacterial strains by Measures. The low activity of glutamate dehydrogenase in Rhizobium sp. strain WR1001 apparently cannot fulfill a biosynthetic function of glutamate formation in response to medium NaCl concentrations.     

Isolation and Characterization of Moderately Halophilic Bacteria from Tunisian Solar Saltern

Bacterial screenings from solar saltern in Sfax (Tunisia) lead to the isolation of 40 moderately halophilic bacteria which were able to grow optimally in media with 5–15% of salt. These isolates were phylogenetically characterized using 16S rRNA gene sequencing. Two groups were identified including 36 strains of Gamma-Proteobacteria (90%) and 4 strains of Firmicutes (10%). The Gamma-Proteobacteria group consisted of several subgroups of the Halomonadaceae (52.5%), the Vibrionaceae (15%), the Alteromonadaceae (10%), the Idiomarinaceae (7.5%), and the Alcanivoracaceae (5%). Moreover, three novel species: 183ZD08, 191ZA02, and 191ZA09 were found, show <97% sequence similarity of the 16S rRNA sequences while compared to previously published cultivated species. Most of these strains (70%) were able to produce hydrolases: amylases, proteases, phosphatases, and DNAases. Over the isolates, 60% produced phosphatases, 15.0% proteases, 12.5% amylases and DNAases equally. This study showed that the solar saltern of Sfax is an optimal environment for halophilic bacterial growth, where diverse viable bacterial communities are available and may have many industrial applications.     

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica)

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR?DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.     

Comparative analysis of bacterial diversity in the rhizosphere of tomato by culture-dependent and -independent approaches

The microbiome in the rhizosphere–the region surrounding plant roots–plays a key role in plant growth and health, enhancing nutrient availability and protecting plants from biotic and abiotic stresses. To assess bacterial diversity in the tomato rhizosphere, we performed two contrasting approaches: culture-dependent and -independent. In the culture-dependent approach, two culture media (Reasoner’s 2A agar and soil extract agar) were supplemented with 12 antibiotics for isolating diverse bacteria from the tomato rhizosphere by inhibiting predominant bacteria. A total of 689 bacterial isolates were clustered into 164 operational taxonomic units (OTUs) at 97% sequence similarity, and these were found to belong to five bacterial phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes). Of these, 122 OTUs were retrieved from the antibiotic-containing media, and 80 OTUs were recovered by one specific antibiotic-containing medium. In the culture-independent approach, we conducted Illumina MiSeq amplicon sequencing of the 16S rRNA gene and obtained 19,215 high-quality sequences, which clustered into 478 OTUs belonging to 16 phyla. Among the total OTUs from the MiSeq dataset, 22% were recovered in the culture collection, whereas 41% of OTUs in the culture collection were not captured by MiSeq sequencing. These results showed that antibiotics were effective in isolating various taxa that were not readily isolated on antibiotic-free media, and that both contrasting approaches provided complementary information to characterize bacterial diversity in the tomato rhizosphere.     

Newly Cultured Bacteria with Broad Diversity Isolated from Eight-Week Continuous Culture Enrichments of Cow Feces on Complex Polysaccharides

One of the functions of the mammalian large intestinal microbiota is the fermentation of plant cell wall components. In ruminant animals, the majority of their nutrients are obtained via pregastric fermentation; however, up to 20% can be recovered from microbial fermentation in the large intestine. Eight-week continuous culture enrichments of cattle feces with cellulose and xylan-pectin were used to isolate bacteria from this community. A total of 459 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented: Firmicutes (51.9%), Bacteroidetes (30.9%), Proteobacteria (11.1%), Actinobacteria (3.5%), Synergistetes (1.5%), and Fusobacteria (1.1%). The majority of bacterial isolates had <98.5% identity to cultured bacteria with sequences in the Ribosomal Database Project and thus represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, B. ovatus, and B. xylanisolvens and members of the Porphyromonadaceae family. Many of the Firmicutes and Bacteroidetes isolates were related to species demonstrated to possess enzymes which ferment plant cell wall components; the others were hypothesized to cross-feed these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 98% of the isolates not represented as previously cultured, there are new opportunities to study the genomic and metabolic capacities of these members of the complex intestinal microbiota.     

Functional Profiling and Distribution of the Forest Soil Bacterial Communities Along the Soil Mycorrhizosphere Continuum

An ectomycorrhiza is a multitrophic association between a tree root, an ectomycorrhizal fungus, free-living fungi and the associated bacterial communities. Enzymatic activities of ectomycorrhizal root tips are therefore result of the contribution from different partners of the symbiotic organ. However, the functional potential of the fungus-associated bacterial communities remains unknown. In this study, a collection of 80 bacterial strains randomly selected and isolated from a soil–ectomycorrhiza continuum (oak–Scleroderma citrinum ectomycorrhizas, the ectomycorrhizosphere and the surrounding bulk soil) were characterized. All the bacterial isolates were identified by partial 16S rRNA gene sequences as members of the genera Burkholderia, Collimonas, Dyella, Mesorhizobium, Pseudomonas, Rhizobium and Sphingomonas. The bacterial strains were then assayed for β-xylosidase, β-glucosidase, N-acetyl-hexosaminidase, β-glucuronidase, cellobiohydrolase, phosphomonoesterase, leucine-aminopeptidase and laccase activities, chitin solubilization and auxin production. Using these bioassays, we demonstrated significant differences in the functional distribution of the bacterial communities living in the different compartments of the soil–ectomycorrhiza continuum. The surrounding bulk soil was significantly enriched in bacterial isolates capable of hydrolysing cellobiose and N-acetylglucosamine. In contrast, the ectomycorrhizosphere appeared significantly enriched in bacterial isolates capable of hydrolysing glucopyranoside and chitin. Notably, chitinase and laccase activities were found only in bacterial isolates belonging to the Collimonas and Pseudomonas genera. Overall, the results suggest that the ectomycorrhizal fungi favour specific bacterial communities with contrasting functional characteristics from the surrounding soil.     

Isolation and characterization of root-associated bacteria from agricultural crops in the Kavango region of Namibia

Root-associated bacteria can have beneficial effects on their host plants. Microbial products can promote and stimulate plant growth or lead to bioprotection against pathogens. This study aimed to isolate putatively beneficial bacteria from traditional cereals grown by subsistence farmers in the Kavango of Namibia. Bacteria were isolated from surface-sterilized roots of Pennisetum glaucum, Sorghum bicolor, and Zea mays, and subjected to phenotypic and phylogenetic analyses. A total of 44 root-associated bacterial strains were isolated. From 33 distinct isolates, 22 belonged to Firmicutes and Actinobacteria, while 11 were Proteobacteria. Eleven novel phylotypes were among the isolates. Features known to contribute to plant growth-promotion and biocontrol were tested in vitro and revealed promising candidates with multiple beneficial characteristics. This is the first report on the characterization of native isolates associated with important agriculture crops in the Kavango region of Namibia. Such isolates have the potential for application as inoculants adapted to poor soils and local crops. Desiccation-tolerant or sporulating Gram-positive bacteria are of particular interest for this region, which is characterized by a long dry season.     

Isolation and identification of endophytic bacteria from root tissues of Salvia miltiorrhiza Bge. and determination of their bioactivities

Endophytic bacteria are microorganisms that live in host plants, but do not cause diseases to the hosts. This study examined the occurrence, distribution, growth-promoting and antifungal activities of endophytes in the root of Salvia miltiorrhiza Bge. Six endophytic bacterial strains, which belong to genera of Pseudomonas, Rhizobium, Bacillus and Novosphingobium, were isolated from the root of healthy S. miltiorrhiza. Cell suspension (approx. 10⁹ cell?·?ml^?1) of two isolates and cell-free fermentation filtrate of four isolates substantially promoted the growth of hypocotyl and radicle of muskmelon seeds. The cell-free fermentation filtrate of six isolates had no inhibiting effect on tested pathogenic fungi, namely Fusarium solani, F. oxysporum f. sp. vasinfectum and F. oxysporum. Six compounds were isolated from one of the six endophytic bacteria, namely, Bacillus aryabhattai, and two of these compounds displayed certain antifungal activity against three tested S. miltiorrhiza pathogens. Our work indicates that endophytic bacteria occur in the root of S. miltiorrhiza, and that associated bacterial isolates have growth-promoting effect on muskmelon seeds and are expected to be a potential source for bioactive metabolites.