Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia

The culturability of bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at 1/100 of its normal concentration (dilute nutrient broth [DNB]) as the growth medium. Three-tube most-probable-number serial dilution culture resulted in a mean viable count that was only 1.4% of the mean microscopically determined total cell count. Plate counts with DNB solidified with agar and with gellan gum resulted in viable counts that were 5.2 and 7.5% of the mean microscopically determined total cell count, respectively. Prior homogenization of the soil sample with an ultrasonic probe increased the viable count obtained by using DNB solidified with gellan gum to 14.1% of the mean microscopically determined cell count. A microscopic examination of the cell aggregates that remained after sonication revealed that the potential CFU count was only 70.4% of the total cell count, due to cells occurring as pairs or in clumps of three or more cells. Staining with SYTO 9 plus propidium iodide indicated that 91.3% of the cells in sonicated soil samples were potentially viable. Together, these findings suggest that the maximum achievable CFU count may be as low as 64.3% of the total cell count. Thirty isolates obtained from plate counting experiments performed with DNB as the growth medium were identified by comparative analysis of partial 16S rRNA gene sequences. A large proportion of these isolates represent the first known isolates of globally distributed groups of soil bacteria belonging to novel lineages within the divisions Actinobacteria, Acidobacteria, Proteobacteria, and Verrucomicrobia.     

Isolation and Characterization of Soil Bacteria That Define Terriglobus gen. nov., in the Phylum Acidobacteria

Bacteria in the phylum Acidobacteria are widely distributed and abundant in soils, but their ecological roles are poorly understood, owing in part to a paucity of cultured representatives. In a molecular survey of acidobacterial diversity at the Michigan State University Kellogg Biological Station Long-Term Ecological Research site, 27% of acidobacterial 16S rRNA gene clones in a never-tilled, successional plant community belonged to subdivision 1, whose relative abundance varied inversely with soil pH. Strains of subdivision 1 were isolated from these never-tilled soils using low-nutrient medium incubated for 3 to 4 weeks under elevated levels of carbon dioxide, which resulted in a slightly acidified medium that matched the pH optima of the strains (between 5 and 6). Colonies were approximately 1 mm in diameter and either white or pink, the latter due to a carotenoid(s) that was synthesized preferentially under 20% instead of 2% oxygen. Strains were gram-negative, aerobic, chemo-organotrophic, nonmotile rods that produced an extracellular matrix. All strains contained either one or two copies of the 16S rRNA encoding gene, which along with a relatively slow doubling time (10 to 15 h at ca. 23°C) is suggestive of an oligotrophic lifestyle. Six of the strains are sufficiently similar to one another, but distinct from previously named Acidobacteria, to warrant creation of a new genus, Terriglobus, with Terriglobus roseus defined as the type species. The physiological and nutritional characteristics of Terriglobus are consistent with its potential widespread distribution in soil.     

Effect of pH on Isolation and Distribution of Members of Subdivision 1 of the Phylum Acidobacteria Occurring in Soil

The pH strongly influenced the development of colonies by members of subdivision 1 of the phylum Acidobacteria on solid laboratory media. Significantly more colonies of this group formed at pH 5.5 than at pH 7.0. At pH 5.5, 7 to 8% of colonies that formed on plates that were incubated for 4 months were formed by subdivision 1 acidobacteria. These colonies were formed by bacteria that spanned almost the entire phylogenetic breadth of the subdivision, and there was considerable congruence between the diversity of this group as determined by the cultivation-based method and by surveying 16S rRNA genes in the same soil. Members of subdivision 1 acidobacteria therefore appear to be readily culturable. An analysis of published libraries of 16S rRNAs or 16S rRNA genes showed a very strong correlation between the abundance of subdivision 1 acidobacteria in soil bacterial communities and the soil pH. Subdivision 1 acidobacteria were most abundant in libraries from soils with pHs of <6, but rare or absent in libraries from soils with pHs of >6.5. This, together with the selective cultivation of members of the group on lower-pH media, indicates that growth of many members of subdivision 1 acidobacteria is favored by slightly to moderately acidic growth conditions.     

一株海洋放线菌的分离鉴定及活性研究

从大连海域20~30 m深处生长的海绵中分离到一株有很高抗菌活性的链霉菌D164。根据培养和显微形态、生理生化数据、16S rRNA基因序列数据分析,菌株D164鉴定为娄彻氏链霉菌(Streptomyces rochei)。对菌株D164发酵产物进行抗农业病原菌、杀虫和除草活性检测,结果表明,菌株D164发酵产物具有很高的抗农业病原菌活性,同时又具有很高的杀虫和除草活性,其活性化合物值得进一步研究。     

Isolation and Identification of Novel Microcystin-Degrading Bacteria

Of 31 freshwater bacterial isolates screened using the Biolog MT2 assay to determine their metabolism of the microcystin LR, 10 were positive. Phylogenetic analysis (16S rRNA) identified them as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. This is the first report of microcystin degraders that do not belong to the Proteobacteria.A number of studies have reported biological degradation of microcystin in samples from lakes and sediments (3, 4, 12, 15), but only a few bacterial strains with the ability to degrade microcystins have been isolated and characterized (6). Previously identified bacteria belonged to the Proteobacteria, and with the exception of one isolate (Sphingomonas sp. strain CB4), they were all shown to degrade microcystin LR (MC-LR) via the same degradation pathway: formation of linear MC-LR following cleavage at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg bond and hydrolysis at Ala-Leu to yield a tetrapeptide with Adda as the final product detected (1, 10, 11). Genes encoding enzymes involved in this pathway have been characterized and shown to have similarity in all bacterial isolates currently reported to degrade microcystins (2).Recent work indicated a greater diversity of microbes capable of degrading microcystins and nodularin, with the tentative identification of several novel degradation intermediates (5). In the present study, enrichment was used to isolate bacteria from three Scottish water bodies previously shown to contain microflora capable of microcystin degradation (5). The Biolog MT2 assay was used to screen the ability of the isolated bacteria to metabolize MC-LR, since this had previously been shown to be an effective means of demonstrating metabolism of microcystin by Paucibacter toxinivorans (6).The ability to metabolize MC-LR was determined in the Biolog MT screen, with 10 of the bacterial isolates giving a positive result. We subsequently confirmed that they could all degrade MC-LR in batch degradation studies, as evidenced by liquid chromatography-mass spectrometry (LC-MS) analysis. The microcystin-degrading bacteria were identified by using 16S rRNA gene analysis and investigated to determine the presence of mlrA, mlrB, mlrC, and mlrD, the genes previously reported to be involved in the degradation of MC-LR by Sphingomonas sp. strain ACM-3962 (2). We report here isolates identified as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. which have the ability to degrade MC-LR, although none of the previously characterized mlr genes were detected.Surface water samples were collected in sterile Pyrex glass bottles on 26 September 2007 from Loch Rescobie (Ordinance Survey grid reference number NO 52505159), Forfar Loch (NO 293458), and the River Carron (NO 877857), Scotland, United Kingdom. Samples were stored at 4°C overnight and filtered as previously described (5). Aliquots from each water sample (2 × 500 ml) were processed and analyzed by high-performance LC to determine the presence of naturally occurring microcystins (13). Enrichment and shake flask die-away kinetics were monitored in triplicate for each water type (50 ml in sterile 100-ml Erlenmeyer flasks). To enrich bacteria with the ability to degrade a range of different microcystins, three microcystins, selected for their differing polarities, and the pentapeptide nodularin were added to each water sample. MC-LR, MC-RR, MC-LF, and nodularin (Enzo Life Sciences, Lausen, Switzerland) were resuspended in a small volume (100 μl) of methanol and diluted with Milli-Q to a total concentration of 0.4 mg ml⁻¹. The toxin cocktail was sterilized (0.2-μm Dynaguard filter; Fisher, United Kingdom) and added to each flask under aseptic conditions to give a final concentration of 1 μg ml⁻¹of each toxin (i.e., 4 μg ml⁻¹ total concentration). All flasks were incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (2 ml) were removed from each flask under sterile conditions every 2 days, transferred into 4-ml glass vials, and frozen (−20°C) immediately. Die-away kinetics were monitored for 14 days. The frozen samples were freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 minutes. The supernatant (100 μl) was removed for LC-MS analysis (5). Sterile controls (3 × 50 ml) were prepared, incubated, and sampled as described above to confirm whether loss of toxin was a result of microbial activity.After 14 days of enrichment, 1 ml of sample was removed aseptically from each flask, namely, the Loch Rescobie (R), Forfar Loch (F), and River Carron (C) samples. Serial dilutions (to 10⁻⁵) were made using Ringer''s solution (Oxoid Ltd., United Kingdom), and 1 ml of each dilution was removed and mixed with 20 to 25 ml of molten LB agar, poured onto sterile petri dishes, and incubated in the dark at 25°C for 5 days. Colonies with differing morphologies were resuspended in liquid LB medium, and pure cultures were obtained by repeated streaking onto LB agar plates. For the Biolog MT2 assay, a loop of each isolated bacterial strain was transferred to 5 ml of liquid LB medium and incubated overnight in the dark at 25°C. The exponentially growing cultures were then washed twice by centrifugation at 1,000 × g for 15 min, the bacterial pellets were resuspended in sterile 0.01 M phosphate-buffered saline, and the cultures were incubated at 25°C for 24 h to deplete residual carbon. The turbidity of all bacterial suspensions was an A₅₉₀ of 0.35. MC-LR was added to Biolog MT2 plates (Technopath, Limerick, Ireland) in triplicate to give final concentrations of 10, 1, 0.1, and 0 μg ml⁻¹. Wells were inoculated with bacterial suspension (150 μl), and plates were incubated at 25°C. Absorbance at 595 nm was recorded by using a Dynex microplate reader (Jencons, Leighton Buzzard, United Kingdom) immediately after inoculation (0 h) and at 3, 6, 15, 18, 24, and 48 h. Metabolism of MC-LR results in the reduction of tetrazolium violet, giving a color reaction that can be quantified spectroscopically (8). Bacterial isolates found by using the Biolog MT screen to metabolize MC-LR were evaluated for their ability to degrade MC-LR. Isolates C1, C3, and C6 (from the River Carron), F3, F7, and F10 (from Forfar Loch), and R1, R4, R6, and R9 (from Loch Rescobie) were grown overnight in LB liquid medium at 25°C. Bacterial isolates were washed and carbon depleted as described above (0.5 ml) and then added to glass universal bottles containing 9 ml of 0.2-μm-filter-sterilized water from their original locations. Aqueous MC-LR (0.5 ml) was added to each bottle under aseptic conditions at a final concentration of 5 μg ml⁻¹. Triplicate samples were prepared for each isolate and incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (0.5 ml) were removed at 24-h intervals under sterile conditions, freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 min. The supernatant (100 μl) was removed for LC-MS analysis performed as previously described (6). Experiments with sterile controls were performed for each water sample. Paucibacter toxinivorans DSMZ-16998 (Braunschweig, Germany) was used as a positive control as it has been reported to degrade MC-LR, MC-YR, and nodularin (16).To identify selected isolates, total DNA was extracted from the pellet by using an UltraClean DNA isolation kit (Mo Bio Laboratories, CA). Sequencing was performed with a BigDye Terminator cycle sequencing reaction kit (202 instrument; Applied Biosystems, United Kingdom) using 8F, 1492, and various other internal primers (518R and 1087R) on an automated DNA sequencer (ABI, United Kingdom) (7, 17). The quality of the sequence was checked by using the sequence analysis software (ABI), and the products of the forward and reverse primers were aligned using Kodon (Applied Maths, Saint-Martens-Latem, Belgium). The analyzed sequences were compared to DNA sequences in public databases using the BLAST function of NCBI (http://www.ncbi.nlm.nih.gov). Individual isolates were classified according to their similarity to sequences in the database. DNA sequences of all isolates, along with those of related bacteria and some known microcystin-degrading bacteria, were used to construct a phylogenetic tree using MEGA4 (18). The sequences were first aligned using Clustal W, and then a phylogenetic tree was constructed by performing neighbor-joining tree analysis with 1,000 bootstrap replicates. Each microcystin-degrading isolate was assayed to determine if mlr genes for the degradation of microcystin could be detected. The PCR method used primers specific for mlrA, mlrB, mlrC, and mlrD with conditions as described before (9). A positive control for these genes was used (Sphingopyxis sp. strain LH21).The Biolog MT2 plates, used to screen 31 isolates, were shown to be an effective means of rapidly identifying bacteria with the ability to metabolize MC-LR (Fig. ​(Fig.1).1). Ten isolates which demonstrated respiration in the presence of MC-LR using the Biolog format were subsequently proven to be microcystin-degrading bacteria in batch studies where MC-LR almost or totally disappeared after 3 days of incubation (Table ​(Table1).1). The results of LC-MS analysis indicated that MC-LR disappeared with no obvious biotransformation or intermediate degradation products. This may be because degradation resulted in only very low concentrations of these compounds and MC-LR is readily utilized by the isolates, as evidenced by respiration in the Biolog assay. Employing the Biolog MT plate enabled rapid (approximately 24 h) selection of bacteria in a high-throughput format (96-well plates) using considerably less microcystin or nodularin, i.e., 5 μg per isolate in the Biolog MT plate compared to 300 μg to follow degradation in die-away kinetics as described herein). Furthermore, following degradation by the latter method requires sample processing and high-performance LC analysis, increasing the time and cost. While the Biolog plates have been widely used for community profiling and bacterial identification, they have yet to be fully exploited in biodegradation studies, where they may facilitate rapid, cost-effective screening of many more bacterial isolates for the ability to utilize a wide range of environmental pollutants.Open in a separate windowFIG. 1.Results of Biolog screen for MC-LR metabolism by bacteria isolated from Loch Rescobie (A), Forfar Loch (B), and the River Carron (C) after 24 h of incubation. Control samples (black bars) contained no additional carbon source. MC-LR was added as the carbon source at 0.1 (open bars), 1 (hatched bars), and 10 (shaded bars) μg ml⁻¹. Error bars represent 1 standard deviation (n = 3).

TABLE 1.

Batch degradation of MC-LR by bacterial isolates in source

Culturability of Stream Bacteria Assessed at the Assemblage and Population Levels

Lotic bacterial communities can be examined at multiple levels: from the assemblage level to populations of individual species. In stream environments, as in many other systems, the percentage of bacteria that are culturable is quite low. In this study, the culturability of the overall bacterial assemblage, as well as the culturability of three common species (Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida), was determined in samples collected from four streams on three dates. Colony hybridization (colonies were grown on modified nutrient agar) and fluorescent in situ hybridization were used to calculate the percentage of cells of a given species that were culturable. Approximately half of the overall assemblage was estimated to be viable but nonculturable cells (VBNC). The culturability of two of the species was low (0.29% for A. calcoaceticus and 0.46% for P. putida), whereas the value for B. cepacia (2.48%) exceeded the overall assemblage level culturability (0.90%). Overall, both bacterial assemblages and populations were dominated by VBNC. These results show quantitatively that not all members of a species that has culturable representatives are culturable when retrieved from natural populations, likely because of interspecific phenotypic and genotypic variability. Thus, the large pool of nonculturable cells includes representatives of species that are, under some circumstances, culturable.     

Linking Sequence to Function in Soil Bacteria: Sequence-Directed Isolation of Novel Bacteria Contributing to Soilborne Plant Disease Suppression

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.     

Method for Radiorespirometric Detection of Bacteria in Pure Culture and in Blood

Methods are described for the detection of low numbers of bacteria by monitoring (14)CO(2) evolved from (14)C-labeled substrates. Cell suspensions are filtered with membrane filters, and the filter is then moistened with 0.1 ml of labeled medium in a small, closed apparatus. Evolved (14)CO(2) is collected with Ba(OH)(2)-moistened filter pads and assayed with conventional radioactivity counting equipment. The kinetics of (14)CO(2) evolution are shown for several species of bacteria. Fewer than 100 colony-forming units of most species tested were detected in 2 h or less. Bacteria were inoculated into blood and the mixture was treated to lyse the blood cells. The suspension ws filtered and the filter was placed in a small volume of labeled medium. The evolved (14)CO(2) was trapped and counted. A key development in the methodology was finding that an aqueous solution of Rhyozyme and Triton X-100 produced lysis of blood but was not detrimental to bacteria.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Isolation and Culture Conditions of Thermophilic Hydrogen Bacteria

Isolation of thermophilic hydrogen bacteria was performed at 50°C using enrichment culture method. One of the four strains isolated, strain TH-1 grew most rapidly. Culture conditions of strain TH-1 were investigated. Optimum temperature and pH for growth proved to be 52°C and 7.0, respectively. There existed a positive correlation between the specific growth rate and the partial pressure of carbon dioxide in the gas phase. Ammonium and nitrate are the good nitrogen sources in that order. Effect of concentrations of nitrogen source, magnesium, ferrous and phosphate ions on the cell growth was also investigated. The maximum specific growth rate (μ_max) of strain TH-1 was determined as 0.68 hr^?1 by the cultivation at 52°C in a jar fermentor containing the optimal medium at pH 7.0.     

Culturability and In Situ Abundance of Pelagic Bacteria from the North Sea

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.     

Detection and Isolation of Novel Rhizopine-Catabolizing Bacteria from the Environment

Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 10⁶ and 10⁷ catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known mocA, mocB, and mocC genes of Sinorhizobium meliloti L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (Arthrobacter, Sinorhizobium, Pseudomonas, Aeromonas, and Alcaligenes) were represented in this set. Only 6 of these 21 isolates could catabolize 3-O-methyl-scyllo-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of Sinorhizobium meliloti. However, these strains are not symbiotically effective on Medicago sativa, and DNA sequences homologous to the nodB and nodC genes were not detected in strains D1 and R3 by Southern hybridization analysis.     

Bacteria of the gamma-subclass Proteobacteria associated with zooplankton in Chesapeake Bay

The seasonal abundance of gamma-subclass Proteobacteria, Vibrio-Photobacterium, Vibrio cholerae-Vibrio mimicus, Vibrio cincinnatiensis, and Vibrio vulnificus in the Choptank River of Chesapeake Bay associated with zooplankton was monitored from April to December 1996. Large (>202- microm) and small (64- to 202- microm) size classes of zooplankton were collected, and the bacteria associated with each of the zooplankton size classes were enumerated by fluorescent oligonucleotide direct count. Large populations of bacteria were found to be associated with both the large and small size classes of zooplankton. Also, the species of bacteria associated with the zooplankton showed seasonal abundance, with the largest numbers occurring in the early spring and again in the summer, when zooplankton total numbers were correspondingly large. Approximately 0.01 to 40.0% of the total water column bacteria were associated with zooplankton, with the percentage of the total water column bacteria population associated with zooplankton varying by season. A taxonomically diverse group of bacteria was associated with zooplankton, and a larger proportion was found in and on zooplankton during the cooler months of the year, with selected taxa comprising a larger percent of the Bacteria in the summer. V. cholerae-V. mimicus and V. vulnificus comprised the bulk of the large and small zooplankton-associated Vibrio-Photobacterium species. In contrast, V. cincinnatiensis accounted for less than 0.1 to 3%. It is concluded that water column and zooplankton bacterial populations vary independently with respect to species composition since no correlation was observed between taxa occurring with highest frequency in the water column and those in association with zooplankton.     

广西沿海地区红树林根系土壤中放线菌的分离与鉴定

本研究通过分离纯培养,从广西北海及防城港红树林根系土壤中分离出放线菌并提取其总DNA,用放线菌通用引物对获得菌株的16S rDNA进行PCR扩增,对获得的扩增产物进行DNA序列测定及菌株鉴定.研究结果表明,从红树林根系土壤样品中分离出15株典型放线菌菌株.16S rDNA测序比对鉴定结果显示,15株典型放线菌菌株中有12株属于链霉菌属(Streptomyces),是常见菌属;3株属于拟诺卡氏菌属(Nocardiopsis),为稀有放线菌.本研究分离纯化获得15株典型放线菌,初步揭示了广西沿海地区红树林土壤中放线菌的多样性.     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Effect of Selected Monoterpenes on Methane Oxidation, Denitrification, and Aerobic Metabolism by Bacteria in Pure Culture

Selected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporium OB3b, Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 mM (−)-α-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was; Escherichia coli (optical density, 0.01), for example, was not affected by up to 7.3 mM (−)-α-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (−)-α-pinene, γ-terpinene, or β-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (−)-α-pinene, (S)-(−)-limonene, (R)-(+)-limonene, and γ-terpinene]. Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, α-pinene oxide, linalool, α-terpineol] and a noncyclic hydrocarbon (β-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature.