Diverse bacteria isolated from microtherm oil-production water

In total, 435 pure bacterial strains were isolated from microtherm oil-production water from the Karamay Oilfield, Xinjiang, China, by using four media: oil-production water medium (Cai medium), oil-production water supplemented with mineral salt medium (CW medium), oil-production water supplemented with yeast extract medium (CY medium), and blood agar medium (X medium). The bacterial isolates were affiliated with 61 phylogenetic groups that belong to 32 genera in the phyla Actinobacteria, Firmicutes, and Proteobacteria. Except for the Rhizobium, Dietzia, and Pseudomonas strains that were isolated using all the four media, using different media led to the isolation of bacteria with different functions. Similarly, nonheme diiron alkane monooxygenase genes (alkB/alkM) also clustered according to the isolation medium. Among the bacterial strains, more than 24 % of the isolates could use n-hexadecane as the sole carbon source for growth. For the first time, the alkane-degrading ability and alkB/alkM were detected in Rhizobium, Rhodobacter, Trichococcus, Micrococcus, Enterococcus, and Bavariicoccus strains, and the alkM gene was detected in Firmicutes strains.     

Mercury-resistant rhizobial bacteria isolated from nodules of leguminous plants growing in high Hg-contaminated soils

A survey of symbiotic bacteria from legumes grown in high mercury-contaminated soils (Almadén, Spain) was performed to produce a collection of rhizobia which could be well adapted to the environmental conditions of this region and be used for restoration practices. Nineteen Hg-tolerant rhizobia were isolated from nodules of 11 legume species (of the genera Medicago, Trifolium, Vicia, Lupinus, Phaseolus, and Retama) and characterized. Based on their growth on Hg-supplemented media, the isolates were classified into three susceptibility groups. The minimum inhibitory concentrations (MICs) and the effective concentrations that produce 50% mortality identified the patterns of mercury tolerance and showed that 15 isolates were tolerant. The dynamics of cell growth during incubation with mercury showed that five isolates were unaffected by exposure to Hg concentrations under the MICs. Genetic analyses of the 16S rRNA gene assigned ten strains to Rhizobium leguminosarum, six to Ensifer medicae, two to Bradyrhizobium canariense, and one to Rhizobium radiobacter. Inoculation of host plants and analysis of the nodC genes revealed that most of them were symbiotically effective. Finally, three isolates were selected for bioremediation processes with restoration purposes on the basis of their levels of Hg tolerance, their response to high concentrations of this heavy metal, and their genetic affiliation and nodulation capacity.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Free-living heterotrophic nitrogen-fixing bacteria isolated from fuel-contaminated antarctic soils

Five bacterial isolates enriched from fuel-contaminated Antarctic soils fixed nitrogen in the dark heterotrophically and nonsymbiotically. Two isolates utilized jet fuel vapors and volatile hydrocarbons for growth but not in N-deficient medium. Bacteria such as these may contribute to in situ biodegradation of hydrocarbons in Antarctic soils.     

Detection of cellulolytic bacteria from the human colon

The main representatives of bacteria in the human colon were investigated by specific PCR and denaturing gradient gel electrophoresis (DGGE). Prevalent in both cases were species of Bifidobacterium, Clostridium, Bacteroides, Faecalibacterium and Eubacterium. Simultaneously, cellulolytic bacteria were isolated from the human feces. The largest proportion was represented by ruminococcus-like isolates. Their presence was confirmed both by PCR and DGGE methods; the latter one was able to give more comprehensive data about the composition of bacterial population in the human colon chyme.     

Stress induced phosphate solubilization in bacteria isolated from alkaline soils

Phosphate solubilizing bacteria NBRI0603, NBRI2601, NBRI3246 and NBRI4003 were isolated from the rhizosphere of chickpea and alkaline soils. All four strains demonstrated diverse levels of phosphate solubilization activity under in vitro conditions in the presence of various carbon and nitrogen sources. Acid production may have contributed to phosphate solubilization, but was not the only reason for phosphate release into the medium. Among the four strains, NBRI2601 was the most efficient strain in terms of its capability to solubilize phosphorus in the presence of 10% salt, pH 12, or 45 degrees C. The strains showed varied levels of phosphate solubilization when the effects of different sources of nitrogen were examined during growth. The presence of low levels of Ca(2+) and EDTA in the medium enhanced phosphate solubilization.     

Types and properties of some bacteria isolated from hypersaline soils

Five rhizosphere soil samples from the dominant xerophytic plants, and nearby root-free soil samples were obtained from a series of hypersaline soils (5.0–10.7% NaCl) from sites near Alicante in Spain. Physico-chemical analyses were made, and the bacterial flora estimated using three different plating media. Counts from rhizosphere soil were always significantly higher than those from root-free soils. A total of 211 strains isolated were purified and identified to genus level; 12 could not be classified. The range of salt concentration allowing growth was determined for each isolate, but this did not correlate with the salt content of the soil habitat. Most isolates appeared to be typical moderate halophiles (with optimum growth between 5 and 15% salts), but about half of them grew on normal media with only 0.9% naCl, a notable difference from moderately halophilic aquatic bacteria. Extreme halophiles were rare but this may have been due to an insufficient incubation period.     

Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria isolated from PAH-contaminated soils were analyzed genotypically and phenotypically for their capacity for metabolism of naphthalene and other PAH substrates. The methods used for the analyses were DNA hybridization using NAH7-derived gene probes, PAH spray plate assays, ¹⁴C-PAH mineralization assays, and dioxygenase activity assays. The results of the analyses showed a dominant number of PAH-degrading bacteria with a NAH7-like genotype. The results support the continued use of the nahA probe for contaminated soils to monitor the genetic potential of indigenous microorganisms to degrade PAHs. However, the finding of non-it nahA-hybridizing PAH-degrading bacteria show the limitation of NAH7-derived gene probes. Fifteen percent (13/89) of PAH-degrading bacteria isolated were not detected with the nahA gene probe. Four isolates (designated A5PH1, A8AN3, B1PH2, and B10AN1) did not hybridize with any of the NAH7-derived gene probes ( nahA, nahG, nahH, and nahR) used in this study. Considering the numerous unculturable microorganisms in nature and their potential genotypes, NAH7-derived gene probes may underestimate the microbial potential to catabolize PAHs. This necessitates development of new gene probes for enumeration and isolation of PAH-degrading bacteria to better understand the in situ microbial potential to degrade PAHs.     

Screening of bacteria, isolated from PAH-contaminated soils, for production of biosurfactants and bioemulsifiers

Fifty-seven bacterial strains were isolated from PAH-contaminated soils using PAH-amended minimal medium. The isolates were screened for their production of biosurfactants and bioemulsifiers when grown in liquid media containing selected PAHs. The results suggest that many, but not all, of the isolates are able to produce biosurfactants or bioemulsifiers under the experimental conditions. The majority of the strains isolated on phenanthrene, pyrene, and fluoranthene were better emulsifiers than surface tension reducers and the stability of the formed emulsions was in general high. The strains isolated on anthracene were in general better in lowering the surface tension than in forming emulsions. In all strains, reduction of surface tension and emulsion formation did not correlate. However, in the majority of strains the two factors were associated with the bacterial cell surfaces, rather than the culture supernatants. Nevertheless, supernatants from selected surfactant-producing anthracene isolates increased the aqueous solubility of anthracene. Although a significant potential for surfactant and emulsifier production in the microbiota of the PAH-contaminated soils was found in this study, the ability of individual strains to mineralize PAHs did not coincide with production of surface-active compounds.     

Diverse endophytic bacteria isolated from a leguminous tree Conzattia multiflora grown in Mexico

Conzattia multiflora is a leguminous tree present only in Mexico and Guatemala. There is no record about its symbiotic or pathogenic microbes. In this study, we found that numerous bacteria with 10⁴–10⁶ individuals per gram of fresh epidermis were distributed in the tissue of this plant. All the bacteria isolated from the Conzattia epidermis were Gram-negative, facultative anaerobic rods and formed yellow or colorless colonies. They were identified as endophytes by inoculation tests. Some of the bacteria could significantly promote the growth of Conzattia seedlings. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes. The existence of plant-borne lineages of Salmonella indicates that the unexplored plants may harbor some unknown microbes.     

Genetic and phenotypic diversity of carbofuran-degrading bacteria isolated from agricultural soils

Thirty-seven carbofuran-degrading bacteria were isolated from agricultural soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize carbofuran as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Rhodococcus, Sphingomonas, and Sphingobium, including new types of carbofuran-degrading bacteria, Bosea and Microbacterium. Among the 37 isolates, 15 different chromosomal DNA patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. Five of the 15 representative isolates were able to degrade carbofuran phenol, fenoxycarb, and carbaryl, in addition to carbofuran. Ten of the 15 representative isolates had 1 to 8 plasmids. Among the 10 plasmid-containing isolates, plasmid-cured strains were obtained from 5 strains. The cured strains could not degrade carbofuran and other pesticides anymore, suggesting that the carbofuran degradative genes were on the plasmid DNAs in these strains. When analyzed with PCR amplification and dot-blot hybridization using the primers targeting for the previously reported carbofuran hydrolase gene (mcd), all of the isolates did not show any positive signals, suggesting that their carbofuran hydrolase genes had no significant sequence homology with the mcd gene.     

Interactions among cellulolytic bacteria from an anaerobic digester

High cellulolytic activity of particular strains did not cause dominance of one, or a few, species of fiber-digesting bacteria in a cattlewaste anaerobic digester. The population contained a large number of species and varieties with different cellulolytic and fiber-digesting activities. Although mixed cultures of some of these bacteria showed no intereffects, with others, cellulolysis was less or in some cases greater than that shown by individual components of the cultures. The interactions were probably related to effects on growth of the bacteria rather than on activities of components of the cellulase enzyme complex, and culture filtrates of two of the more numerous cellulolytic species ofClostridium affected growth of other cellulolytic bacteria. The inhibitory factor(s) appeared to be of bacteriocin type, but the stimulatory factor(s) was unknown. It was suggested that these interactions are localized or short-lived in the digester, and so the population remains in a dynamic steady state.Some inhibitions of growth of rumen cellulolytic bacteria were caused by the digester bacteria, but it was suggested that factors other than these inhibitions are responsible for the absence of rumen bacteria from anaerobic digesters.     

Degradation of anthracene by bacteria isolated from oil polluted tropical soils

Four bacteria, identified as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus subtilis and Micrococcus luteus were isolated from crude oil polluted soils using anthracene as the sole carbon and energy source. All the organisms utilized n-hexadecane, n-tetradecane, diesel oil, engine oil and naphthalene as sole carbon sources. None could utilize hexane, cycloheptane, xylene, benzene, toluene, phenol, fluoranthene,and kerosene as carbon sources. Highest cell density obtained with 0.1% (w/v) anthracene were 4.5 x 10(7) (cfu/ml), 8.6 x 10(6) (cfu/ml), 5.4 x 10(6) and 2.4 x 10(6) (cfu/ml) respectively, for P. aeruginosa, A. eutrophus, B. subtilis and M. luteus after 30 days incubation. Growth of the organisms on a Nigerian crude oil resulted in a residual oil concentration of 22.2%, 33.3%, 39.3%, 44% and 91.7% respectively, for P. aeruginosa, A. eutrophus, B. subtilis, M. luteus and the noninoculated control on the 14 th day. Ring fission enzymes of the meta pathway were detected in induced cells of P. aeruginosa and A. eutrophus while ortho pathway enzymes were detected in B. subtilis and M. luteus. P. aeruginosa and A. eutrophus had specific catechol-2,3-dioxygenase activities of 3.8 +/- 0.183 and 0.64 +/- 0.032 micromol/min x mg protein respectively while catechol-1,2-dioxygenase activities of 1.95 +/- 0.029 and 1.89 +/- 0.026 micromol/min x mg protein were detected in B. subtilis and M. luteus respectively. This work, highlights the capability of these unreported tropical strains of A. eutrophus, B. subtilis and M. luteus as anthracene degraders.     

Anaerobic cellulolytic bacteria from wetwood of living trees

Obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from the wetwood of elm and maple trees. The isolation of these bacteria involved inoculation of selective enrichment cultures with increment cores taken from trees showing evidence of wetwood. Cellulolytic bacteria were present in the cores from seven of nine trees sampled, as indicated by the disappearance of cellulose from enrichment cultures. With two exceptions, cellulolytic activity was confined to the darker, wetter, inner section of the cores. Cellulolytic bacteria were also present in the fluid from core holes. The cellulolytic isolates were motile rods that stained gram negative. Endospores were formed by some strains. The physiology of one of the cellulolytic isolates (strain JW2) was studied in detail. Strain JW2 fermented cellobiose, d-glucose, glycerol, l-arabinose, d-xylose, and xylan in addition to cellulose. In a defined medium, p-aminobenzoic acid and biotin were the only exogenous growth factors required by strain JW2 for the fermentation of cellobiose or cellulose. Acetate and ethanol were the major nongaseous end products of cellulose fermentation. The guanine-plus-cytosine content of the DNA of strain JW2 was 33.7 mol%. Cellulolytic bacteria have not previously been reported to occur in wetwood. The isolation of such bacteria indicates that cellulolytic bacteria are inhabitants of wetwood environments and suggests that they may be involved in wetwood development.     

Isolation and characterization of cellulolytic bacteria from the Stain house Lake,Antarctica

The main aim was to evaluate the occurrence of cellulolytic bacteria from the Stain house Lake, located at Admiralty Bay, Antarctica. Thick cotton string served as a cellulose bait for the isolation of bacteria. A total of 52 bacterial isolates were recovered and tested for their cellulase activity, and two of them, isolates CMAA 1184 and CMAA 1185, showed significant cellulolytic activity on carboxymethylcellulose agar plates. Phylogenetic analysis placed the isolates into the Bacillus 16S ribosomal RNA gene subclade. Both isolates produced a cold-active cellulase which may play a crucial role in this extreme environment.     

Phylogenetic and phenotypic diversity of 4-chlorobenzoate-degrading bacteria isolated from soils

Twenty numerically dominant 4-chlorobenzoate (4-CBA)-degrading bacteria were isolated from agricultural soils. The isolates were able to utilize 4-CBA as a sole source of carbon and energy. A total of 65% of the isolates was identified to the species level by fatty acid methyl ester (FAME) analysis, and the isolates were strains of Micrococcus, Pseudomonas, Oerskovia, Cellulomonas, and Arthrobacter species. The chromosomal DNA patterns of the isolates obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences were distinct from each other. Most of the isolates grew rapidly in 4-CBA medium, but their substrate utilization capabilities were generally restricted. Plasmid DNAs were detected from 55% of the isolates, and one strain, HR7, was shown to have self-transmissible, 4-CBA degradative plasmids. 4-CBA degradative enzymes were inducible by the presence of 4-CBA and most of the isolates appeared to mineralize it through 4-hydroxybenzoate rather than 4-chlorocatechol.     

Adhesive properties of five mesophilic, cellulolytic Clostridia isolated from the same biotope

Abstract Adhesion to cellulose of five strains of mesophilic, cellulolytic clostridia , isolated from a municipal waste digestor, was found to be a reversible phenomenon. The type of attachment for the five strains conformed to a multilayer adhesion. In a first step, attachment to the adhesion site occurred by cell-cellulose interaction. In a second step, cell-cell interactions were identified. The five strains adhered slightly better to magazine paper and Whatman No. 1 filter paper than to newspaper and cardboard. Two strains, C401 and A22, were studied in more detail. The two strains, harvested in stationary phase, presented a heterogeneous population which could be separated: (i) as 'unbound' cells, corresponding to cells remaining in suspension from cellulose-grown cultures; and (ii) as 'bound' cells, coming from two successive washes with 50 mM Tris HCl, pH 7.0, which released 'bound' cells. In adhesion measurements, eluted cells ('bound' cells) adhered better to the cellulose than the 'unbound' cells. Strain C401 adhered better than strain A22 to the cellulose: 1.9-fold for the 'bound' cells and 3.6-fold for the 'unbound' cells. Adhesion of the two isolates was enhanced by the presence of calcium (10 mM). Cellobiose and glucose had no effect on strain A22 adhesion. Conversely, adhesion of strain C401 to cellulose was enhanced by cellobiose at a concentration of 1.5 g I⁻¹, but 85% inhibited by a concentration of 5.0 g I⁻¹. The two strains adhered to the same site on Whatman filter paper and unspecific interactions between the two strains occur.     

Characterization of fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated soils and sediments

Sixteen environmental samples, from the United States, Germany and Norway, with histories of previous exposure to either creosote, diesel fuel or coal tar materials, were screened for bacteria which could degrade high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A modified version of the spray plate technique was used for the isolations. Using fluoranthene (FLA) and pyrene (PYR) as model HMW PAHs, we isolated 28 strains on FLA and 21 strains on PYR. FLA degraders were defined as able to grow on FLA but not PYR. PYR degraders grew on both PAHs. All PYR degraders were found to be Gram-positive and all FLA degraders were Gram-negative. GC-FAME analysis showed that many of the PYR degraders were Mycobacterium spp and many of the FLA degraders were Sphingomonas spp. Comparison of the metabolic characteristics of the strains using the spray plate technique and direct growth studies revealed that more than half of the FLA degraders (59%) were able to cometabolize PYR (ie, they produced clearing zones or colored metabolites on spray plates but did not grow on the PAH) and the ability of many of these strains to cometabolize fluorene, anthracene, benzo[b]fluorene, benzo[a]anthracene and benzo[a]pyrene was significantly affected by pre-exposure to phenanthrene. Studies on the metabolic products produced from PYR cometabolism by strain EPA 505 suggested the possibility of attack at two different sites on the PYR molecule. However, the inability to derive degradable carbon from initial opening of one of the PYR rings probably accounted for the lack of growth on this PAH by the FLA-degrading strains. The PYR degraders on the other hand, were less able to cometabolize HMW PAHs, even following pre-exposure to PHE. Characterization of the FLA degradation pathway for several of the Sphingomonas isolates indicated oxidation and ring opening through to acenaphthenone as the principle metabolite. Strain CO6, however, also oxidized FLA through fluorenone, suggesting a dual attack on the FLA molecule, similar to that observed by others in Mycobacterium spp. Journal of Industrial Microbiology & Biotechnology (2000) 24, 100–112. Received 01 May 1999/ Accepted in revised form 01 November 1999     

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, β-d-glucosidase, cellobiohydrolase, β-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated β-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal–Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.