Chemical and Sensory Changes Associated with Microbial Flora of Mediterranean Boque (Boops boops) Stored Aerobically at 0, 3, 7, and 10°C

The development of a microbial population and changes in the physicochemical and sensorial characteristics of Mediterranean boque (Boops boops), called gopa in Greece, stored aerobically at 0, 3, 7, and 10°C were studied. Pseudomonads and Shewanella putrefaciens were the dominant bacteria at the end of the storage period, regardless of the temperature tested. Enterobacteria and Brochothrix thermosphacta also grew, but their population density was always 2 to 3 log₁₀ CFU g⁻¹ less than that of pseudomonads. The concentration of potential indicators of spoilage, glucose and lactic acid, decreased while that of the α-amino groups increased during storage. The concentrations of these carbon sources also decreased on sterile fish blocks inoculated with strains isolated from fish microbial flora. The organic acid profile of sterile fish blocks inoculated with the above-mentioned bacteria and that of naturally spoiled fish differed significantly. An excellent correlation (r = −0.96) between log₁₀ counts of S. putrefaciens or Pseudomonas bacteria with freshness was observed in this study.     

Siderophore-Mediated Iron Sequestering by Shewanella putrefaciens

The iron-sequestering abilities of 51 strains of Shewanella putrefaciens isolated from different sources (fish, water, and warm-blooded animals) were assessed. Thirty strains (60%) produced siderophores in heat-sterilized fish juice as determined by the chrome-azurol-S assay. All cultures were negative for the catechol-type siderophore, whereas 24 of the 30 siderophore-producing strains tested positive in the Csáky test, indicating the production of siderophores of the hydroxamate type. Siderophore-producing S. putrefaciens could to some degree cross-feed on the siderophores of other S. putrefaciens strains and on compounds produced by an Aeromonas salmonicida strain under iron-limited conditions. The siderophores of S. putrefaciens were not sufficiently strong to inhibit growth of other bacteria under iron-restricted conditions. However, siderophore-producing Pseudomonas bacteria were always inhibitory to S. putrefaciens under iron-limited conditions. Growth of siderophore-producing strains under iron-limited conditions induced the formation of one major new outer membrane protein of approximately 72 kDa. Two outer membrane proteins of approximately 53 and 23 kDa were not seen when iron was restricted.     

Survey of Plant Drought-Resistance Promoting Bacteria from Populus euphratica Tree Living in Arid Area

Two hundred and thirty-two bacterial strains were isolated from the rhizospheric soil of Populus euphratica which is the dominant tree living in extreme arid regions in northwest China. Some strains with plant growth-promoting bacteria related metabolic characteristics were able to promote drought resistance in plants after inoculation. Ten strains with the greatest effects increased the dry weight of wheat shoots from 0.5 to 34.4 %, and the surface area of the root systems from 12.56 to 212.17 % compared to the control after drought treatment whereas no obvious promoting effect was observed in normal water conditions. These 10 strains were identified to be of the genera Pseudomonas, Bacillus, Stenotrophomonas and Serratia by 16S rRNA (rrs) gene sequence alignment. Among these strains, Serratia sp. 1-9 and Pseudomonas sp. 5-23 were the two most effective strains. Both of them produced auxin and the production increased significantly when cultured under simulated drought conditions which are inferred to be the most plausible mechanism for their plant growth-promoting effect under drought stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0479-3) contains supplementary material, which is available to authorized users.     

Bacterial synergism or antagonism in a gel cassette system

The growth and the metabolic activity of Shewanella putrfaciens, Brochothrix thermosphacta, and Pseudomonas sp., when cultured individually or in all possible combinations in gel cassettes system supplemented with 0.1% glucose at 5 degrees C, were investigated. The overall outcome was that the coexistence of the above-mentioned microorganisms affected not only each growth rate but also their type of metabolic end products compared to the control cultures. These effects were varied and depended on the selection of the combination of the tested bacteria. For example, the growth of Pseudomonas sp. strains cocultured with either B. thermosphacta or S. putrefaciens strains resulted in different effects: a promoting one for the first and an inhibitory one for the second. Moreover, the production of formic acid and two unidentified organic acids (peaks a and b) was characteristic in all cases in which S. putrefaciens was cultured.     

Dehydrogenase activity of pseudomonas species

Single-strain cultures of Pseudomonas fragi, P. fluorescens, P. putrefaciens, and strains of two marine species, Pseudomonas type I and Pseudomonas type II, were found to be capable of reducing added acetaldehyde, proprionaldehyde, and butyraldehyde to the corresponding alcohols at 21 C. All species studied reduced propionaldehyde at 6 C. P. fragi, Pseudomonas type I, and Pseudomonas type II reduced butanone at 6 and 21 C. P. fragi and Pseudomonas type II reduced acetone at 21 C. Dehydrogenase activity was found in some cultures in which growth was not evident. Under aerobic conditions, a strain of P. fragi reduced added propionaldehyde to n-propanol quantitatively within 36 hr at 21 C.     

Applicability of an Arrhenius Model for the Combined Effect of Temperature and CO2 Packaging on the Spoilage Microflora of Fish

The temperature behavior of the natural microflora on the Mediterranean fish red mullet (Mullus barbatus) was examined as a case study. The growth of the spoilage bacteria Pseudomonas spp., Shewanella putrefaciens, Brochothrix thermosphacta, and lactic acid bacteria was modeled as a function of temperature and the concentration of carbon dioxide in modified atmosphere packaging. Combined models were developed and comparatively assessed based on polynomial, Belehradek, and Arrhenius equations. The activation energy parameter of the Arrhenius model, E_A, was independent of the packaging atmosphere and ranged from 75 to 85 kJ/mol for the different bacteria, whereas the preexponential constant decreased exponentially with the packaging CO₂ concentration. We evaluated the applicability of the models developed by using experimental bacterial growth rates obtained from 42 independent experiments performed with three Mediterranean fish species and growth rates predicted from the models under the same temperature and packaging conditions. The accuracy factor and bias factor were used as statistical tools for evaluation, and the developed Arrhenius model and the Belehradek model were judged satisfactory overall.     

Quantification of Frankia Strains and Other Root-Associated Bacteria in Pure Cultures and in the Rhizosphere of Axenic Seedlings by High-Performance Liquid Chromatography-Based Muramic Acid Assay

Application of a high-performance liquid chromatography-based muramic acid assay with precolumn fluorescence derivatization to quantification of root-associated bacteria was studied both in pure cultures and in the rhizosphere of axenic Festuca rubra seedlings. Quantities of muramic acid from acid-hydrolyzed cells of Frankia strains, Streptomyces griseoviridis, Enterobacter agglomerans, Klebsiella pneumoniae, Pseudomonas sp., and Bacillus polymyxa were mostly proportional to the respective cell protein and carbon quantities, but in some strains, culture age and particularly sporulation affected these ratios considerably. The muramic acid/cell protein ratio was generally 2 to 4 times higher in strains of the two actinomycete genera, Frankia and Streptomyces, than in the rest of the strains. Quantification of Frankia strains, S. griseoviridis, E. agglomerans, and Pseudomonas sp. was also attempted from the rhizosphere of F. rubra seedlings which had been inoculated with pure cultured bacteria and incubated briefly. It was possible to quantify Frankia cells by use of the muramic acid assay from both the root and the growth medium, whereas cells of the rest of the bacterial genera could only be detected in the medium. The detection limit for muramic acid was about 10 ng/ml hydrolysis volume, and from the Festuca rhizosphere, 28 to 63% of the muramic acid in the Frankia inoculum was recovered.     

Two naphthalene degrading bacteria belonging to the genera Paenibacillus and Pseudomonas isolated from a highly polluted lagoon perform different sensitivities to the organic and heavy metal contaminants

Two bacterial strains were isolated in the presence of naphthalene as the sole carbon and energy source from sediments of the Orbetello Lagoon, Italy, which is highly contaminated with both organic compounds and metals. 16S rRNA gene sequence analysis of the two isolates assigned the strains to the genera Paenibacillus and Pseudomonas. The effect of different contaminants on the growth behaviors of the two strains was investigated. Pseudomonas sp. ORNaP2 showed a higher tolerance to benzene, toluene, and ethylbenzene than Paenibacillus sp. ORNaP1. In addition, the toxicity of heavy metals potentially present as co-pollutants in the investigated site was tested. Here, strain Paenibacillus sp. ORNaP1 showed a higher tolerance towards arsenic, cadmium, and lead, whereas it was far more sensitive towards mercury than strain Pseudomonas sp. ORNaP2. These differences between the Gram-negative Pseudomonas and the Gram-positive Paenibacillus strain can be explained by different general adaptive response systems present in the two bacteria.     

Characterization and identification of compost bacteria based on 16S rRNA gene sequencing

The aim of this study was to isolate and characterize bacteria from the compost of fruit and vegetable waste (FVW) for plant growth-promoting (PGP) activities and investigate the pro-active influence of bacterial isolates on wheat growth. Fourteen bacterial strains (RHC-1 to RHC-14) were isolated and purified in tryptic soya agar (TSA). In addition to being biochemically characterized, these bacterial strains were also tested for their PGP traits, such as phosphate (P)-solubilization, nifH gene amplification, indole-3-acetic acid (IAA) quantification and the production of ammonia, oxidase and catalase. Based on 16S rRNA gene sequencing, these bacterial strains were identified as belonging to species of Bacillus, Lysinibacillus, Lysobacter, Staphylococcus, Enterobacter, Pseudomonas and Serratia. All bacterial strains solubilized tri-calcium phosphate and produced IAA. Two bacterial strains RHC-8 (Enterobacter sp.) and RHC-13 (Pseudomonas sp.) solubilized the maximum amount of tri-calcium phosphate, i.e. 486 and 464 μg/ml, respectively. P-solubilization was associated with a significant drop in the pH of the broth culture from an initial pH of 7 to pH 4.43. In addition to P-solubilization and IAA production, six bacterial strains also carried the nifH gene and were further evaluated for their effect on wheat (Triticum aestivum) growth under controlled conditions. All six bacterial strains enhanced wheat growth as compared to uninoculated control plants. Two of the bacterial strains, RHC-8 and RHC-13, identified as Enterobacter aerogenes and Pseudomonas brenneri, respectively, were assessed as potential PGP rhizobacteria due to exhibiting characteristics of four or more PGP traits and enhancing wheat growth though their specific mechanism of action.     

Enhanced Growth of Wheat and Soybean Plants Inoculated with Azospirillum brasilense Is Not Necessarily Due to General Enhancement of Mineral Uptake

The capacity of Azospirillum brasilense to enhance the accumulation of K⁺, P, Ca²⁺, Mg²⁺, S, Na⁺, Mn²⁺, Fe²⁺, B, Cu²⁺, and Zn²⁺ in inoculated wheat and soybean plants was evaluated by using two different analytical methods with five A. brasilense strains originating from four distinct geographical regions. A Pseudomonas isolate from the rhizosphere of Zea mays seedlings was included as a control. All A. brasilense strains significantly improved wheat and soybean growth by increasing root and shoot dry weight and root surface area. The degree of plant response to inoculation varied among the different strains of A. brasilense. All strains were capable of colonizing roots, but the best root colonizer, Pseudomonas sp., had no effect on plant growth. The numbers of organisms of Brazilian strains Sp-245 and Sp-246 colonizing roots were similar regardless of the host plant. Numbers of organisms for the other strains were directly dependent on the host plant. The main feature characterizing mineral accumulation in inoculated plants was that all inoculation treatments changed the mineral balance of the plants, but in an inconsistent manner. Enhancement of mineral uptake by plants also varied among strains to a great extent and was directly dependent on the strain-plant combination; i.e., a strain capable of increasing accumulation of a particular ion in one plant species or cultivar often lacked the ability to do so in another. Minerals in inoculated plants were not evenly distributed in different plant tissues, and the changes varied among groups of plants within each bacterial strain inoculation treatment. We suggest that, although A. brasilense strains are capable of changing the mineral balance and content of plants, it is unlikely that this ability is a general mechanism responsible for plant improvement by A. brasilense.     

Hydrogen and Formate Oxidation Coupled to Dissimilatory Reduction of Iron or Manganese by Alteromonas putrefaciens

The ability of Alteromonas putrefaciens to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory Fe(III) or Mn(IV) reduction was investigated. A. putrefaciens grew with hydrogen, formate, lactate, or pyruvate as the sole electron donor and Fe(III) as the sole electron acceptor. Lactate and pyruvate were oxidized to acetate, which was not metabolized further. With Fe(III) as the electron acceptor, A. putrefaciens had a high affinity for hydrogen and formate and metabolized hydrogen at partial pressures that were 25-fold lower than those of hydrogen that can be metabolized by pure cultures of sulfate reducers or methanogens. The electron donors for Fe(III) reduction also supported Mn(IV) reduction. The electron donors for Fe(III) and Mn(IV) reduction and the inability of A. putrefaciens to completely oxidize multicarbon substrates to carbon dioxide distinguish A. putrefaciens from GS-15, the only other organism that is known to obtain energy for growth by coupling the oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). The ability of A. putrefaciens to reduce large quantities of Fe(III) and to grow in a defined medium distinguishes it from a Pseudomonas sp., which is the only other known hydrogen-oxidizing, Fe(III)-reducing microorganism. Furthermore, A. putrefaciens is the first organism that is known to grow with hydrogen as the electron donor and Mn(IV) as the electron acceptor and is the first organism that is known to couple the oxidation of formate to the reduction of Fe(III) or Mn(IV). Thus, A. putrefaciens provides a much needed microbial model for key reactions in the oxidation of sediment organic matter coupled to Fe(III) and Mn(IV) reduction.     

Biochemical and Genetic Analyses of Ferulic Acid Catabolism in Pseudomonas sp. Strain HR199

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding β-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsΩGm and Pseudomonas sp. strain HRechΩKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatΩKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a β-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.     

Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.     

Isolation and characterization of atrazine-degrading Arthrobacter sp. strains from Argentine agricultural soils

Three bacterial strains capable of degrading atrazine were isolated from Manfredi soils (Argentine) using enrichment culture techniques. These soils were used to grow corn and were treated with atrazine for weed control during 3 years. The strains were nonmotile Gram-positive bacilli which formed cleared zones on atrazine solid medium, and the 16S rDNA sequences indicated that they were Arthrobacter sp. strains. The atrazine-degrading activity of the isolates was characterized by the ability to grow with atrazine as the sole nitrogen source, the concomitant herbicide disappearance, and the chloride release. The atrazine-degrader strain Pseudomonas sp. ADP was used for comparative purposes. According to the results, all of the isolates used atrazine as sole source of nitrogen, and sucrose and sodium citrate as the carbon sources for growth. HPLC analyses confirmed herbicide clearance. PCR analysis revealed the presence of the atrazine catabolic genes trzN, atzB, and atzC. The results of this work lead to a better understanding of microbial degradation activity in order to consider the potential application of the isolated strains in bioremediation of atrazine-polluted agricultural soils in Argentina.     

Characterization of the Lipopolysaccharides and Capsules of Shewanella spp.

Electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining and ¹H, ¹³C, and ³¹P-nuclear magnetic resonance (NMR) were used to detect and characterize the lipopolysaccharides (LPSs) of several Shewanella species. Many expressed only rough LPS; however, approximately one-half produced smooth LPS (and/or capsular polysaccharides). Some LPSs were affected by growth temperature with increased chain length observed below 25°C. Maximum LPS heterogeneity was found at 15 to 20°C. Thin sections of freeze-substituted cells revealed that Shewanella oneidensis, S. algae, S. frigidimarina, and Shewanella sp. strain MR-4 possessed either O-side chains or capsular fringes ranging from 20 to 130 nm in thickness depending on the species. NMR detected unusual sugars in S. putrefaciens CN32 and S. algae BrY^DL. It is possible that the ability of Shewanella to adhere to solid mineral phases (such as iron oxides) could be affected by the composition and length of surface polysaccharide polymers. These same polymers in S. algae may also contribute to this opportunistic pathogen's ability to promote infection.     

Changes in Populations of Rhizosphere Bacteria Associated with Take-All Disease of Wheat

Take-all, caused by Gaeumannomyces graminis var. tritici, is one of the most important fungal diseases of wheat worldwide. Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen. Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a Pseudomonas-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA). Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from Pseudomonas to Chryseobacterium species in the rhizosphere of diseased plants. Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria. The functional significance of some of these populations was investigated in vitro. Of those isolated, only a small subset of the most abundant Pseudomonas spp. and a phlD⁺ Pseudomonas sp. showed any significant ability to inhibit G.graminis var. tritici directly. When cultured strains were mixed with the inhibitory phlD⁺ Pseudomonas strain, the Chryseobacterium isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in Chryseobacterium populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producing phlD⁺ pseudomonads.     

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup.     

Metabolic Characterization of cold active Pseudomonas, Arthrobacter, Bacillus, and Flavobacterium spp. from Western Himalayas

Himalayan soils undergo dramatic temporal changes in their microclimatic properties. The soil habitats in the high altitude cold habitats of Himalayas are little explored with respect to bacterial diversity and metabolic potentials of the bacterial species. Soil habitat in Western Himalayas is dominated by the genera of Pseudomonas, Arthrobacter, Bacillus, and Flavobacterium. Strains were found to be diverse in their metabolic potentials to utilize different carbon sources by growing them on media containing 114 different sole carbon sources. Bacillus sp. STL9 was supported by the lowest number (12.3%) of the carbon sources while growth was observed in 73.7% of the carbon sources tested for the Pseudomonas sp. SPS2. Carbohydrates appeared to be preferred carbon sources for these Himalayan isolates followed by amino acids and proteins. These microbes also produced various extra-cellular hydrolytic enzymes having biotechnological potentials, lipase being the one secreted by most strains (85.7%) followed by β-galactosidase (42.8%). Antibiotic resistance profiling for 85 different antibiotics has also been described.     

Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology

Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.     

Bacteria in Crude Oil Survived Autoclaving and Stimulated Differentially by Exogenous Bacteria

Autoclaving of crude oil is often used to evaluate the hydrocarbon-degrading abilities of bacteria. This may be potentially useful for bioaugmentation and microbial enhanced oil recovery (MEOR). However, it is not entirely clear if “endogenous” bacteria (e.g., spores) in/on crude oil survive the autoclaving process, or influence subsequent evaluation of the hydrocarbon-degradation abilities of the “exogenous” bacterial strains. To test this, we inoculated autoclaved crude oil medium with six exogenous bacterial strains (three Dietzia strains, two Acinetobacter strains, and one Pseudomonas strain). The survival of the spore-forming Bacillus and Paenibacillus and the non-spore-forming mesophilic Pseudomonas, Dietzia, Alcaligenes, and Microbacterium was detected using a 16S rRNA gene clone library and terminal restriction fragment length polymorphism (T-RFLP) analysis. However, neither bacteria nor bacterial activity was detected in three controls consisting of non-inoculated autoclaved crude oil medium. These results suggest that detection of endogenous bacteria was stimulated by the six inoculated strains. In addition, inoculation with Acinetobacter spp. stimulated detection of Bacillus, while inoculation with Dietzia spp. and Pseudomonas sp. stimulated the detection of more Pseudomonas. In contrast, similar exogenous bacteria stimulated similar endogenous bacteria at the genus level. Based on these results, special emphasis should be applied to evaluate the influence of bacteria capable of surviving autoclaving on the hydrocarbon-degrading abilities of exogenous bacteria, in particular, with regard to bioaugmentation and MEOR. Bioaugmentation and MEOR technologies could then be developed to more accurately direct the growth of specific endogenous bacteria that may then improve the efficiency of treatment or recovery of crude oil.