A method adapting microarray technology for signature-tagged mutagenesis of Desulfovibrio desulfuricans G20 and Shewanella oneidensis MR-1 in anaerobic sediment survival experiments

Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.     

Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter column

AIMS: To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized. METHODS AND RESULTS: Geosmin-degrading bacteria from a biologically active sand filter column were enriched by their growth in a minimal medium supplemented with geosmin as the sole carbon source. By day 51, 21.7 mg l(-1) of geosmin had been degraded as determined by solid-phase microextraction gas chromatography/mass spectrometry, and was accompanied by a 2.12 log(10) increase in active bacterial numbers as measured using the BacLight(TM) bacterial viability kit and flow cytometric enumeration. During the onset of geosmin degradation, the predominance of three bacteria, most similar to previously cultured species of Sphingopyxis alaskensis, Novosphingobium stygiae and Pseudomonas veronii based on 16S rRNA gene sequences was detected by denaturing gradient gel electrophoresis. Subsequent isolation of these organisms revealed that degradation of geosmin, when present as either the sole carbon source (ranging from 40 ng l(-1) to 20 mg l(-1)) or when spiked into sterile reservoir water (37 and 131 ng l(-1)), occurred only when all three isolates were present. None of the isolates was shown to be capable of degrading geosmin either individually or in any combination of two. CONCLUSIONS: This study has reported, for the first time, the cooperative degradation of geosmin by a consortium comprising three gram-negative bacteria isolated from a biologically active sand filter column. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are important for researchers currently employing molecular-based approaches to further understand the biodegradation of geosmin by bacteria, as such studies may be complicated by the discovery of geosmin degradation occurring by a consortium. This study also advances the knowledge surrounding the types of bacteria capable of degrading the taste and odour compound, as investigations to date regarding this are limited.     

Methodology for Estimating Numbers of Free-Living and Attached Bacteria in Estuarine Water

A fundamental problem in estuarine microbiology studies is the accurate determination of the density in the water column of both free-living bacteria and those attached to suspended particulate matter. When a water sample is filtered and the filter is viewed by epifluorescence microscopy, counts can be made of the numbers of bacteria which are seen on the filter background (free-living) and those which appear to lie on sediment particles (both free-living and attached). With only the additional knowledge of the proportion of the filter area covered by particles (a quantity that is straightforwardly determined by stereological point counting), results from geometric probability were used to determine the expected number of bacteria which are hidden by particles and hence to provide an estimation scheme for the true densities of free-living and attached bacteria. Variance equations based on a Taylor series are given, and a partial check of the method is attempted with controlled mixtures of bacteria and sediment. An alternative procedure is also proposed, in which the natural attached/free-living ratio is altered by an intervention experiment, allowing an estimation which is less model dependent but more labor intensive. Both methods are applied to a series of samples from the Tamar estuary, United Kingdom, taken in April 1985. A notable conclusion is that there are always more free-living than attached bacteria in the water column throughout the estuary.     

A Method Adapting Microarray Technology for Signature-Tagged Mutagenesis of Desulfovibrio desulfuricans G20 and Shewanella oneidensis MR-1 in Anaerobic Sediment Survival Experiments

Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.     

The effects of Fe(II) and Fe(III) concentration and initial pH on microbial leaching of low-grade sphalerite ore in a column reactor

In this study the effects of initial concentration of Fe(II) and Fe(III) ions as well as initial pH on the bioleaching of a low-grade sphalerite ore in a leaching column over a period of 120 days with and without bacteria were investigated. Four different modifications of medium were used as column feed solutions to investigate the effects of initial concentration of Fe(II) and Fe(III) ions on zinc extraction. The experiments were carried out using a bench-scale, column leaching reactor, which was inoculated with mesophilic iron oxidizing bacteria, Acidithiobacillus ferrooxidans, initially isolated from the Sarcheshmeh chalcopyrite concentrate (Kerman, Iran). The effluent solutions were periodically analyzed for Zn, total Fe, Fe(II) and Fe(III) concentrations as well as pH values. Bacterial population was measured in the solution (free cells). Maximum zinc recovery in the column was achieved about 76% using medium free of initial ferrous ion and 11.4 g/L of ferric ion (medium 2) at pH 1.5. The extent of leaching of sphalerite ore with bacteria was significantly higher than that without bacteria (control) in the presence of ferrous ions. Fe(III) had a strong influence in zinc extraction, and did not adversely affect the growth of the bacteria population.     

Effects of Surface Area and Flow Rate on Marine Bacterial Growth in Activated Carbon Columns

The colonization of granular activated carbon columns by bacteria can have both beneficial and potentially detrimental consequences. Bacterial growth on the carbon surface can remove adsorbed organics and thus partially regenerate the carbon bed. However, growth can also increase the levels of bacteria in the column effluents, which can adversely affect downstream uses of the treated water. This study of a sand column and several activated carbon columns demonstrated that considerable marine bacterial growth occurred in both sand and carbon columns and that this growth increased the number of bacteria in column effluents. Activated carbon supported approximately 50% more bacteria than did sand. Bacterial growth on activated carbon was reduced by increasing the flow rate through a carbon column and increasing the carbon particle size. Scanning electron micrographs showed that bacteria preferred to attach in the protected crevices on both the sand and carbon surface. The results of this study indicated that the colonization of activated carbon by marine bacteria was enhanced because of carbon's high surface area, its rough surface texture, and its ability to absorb organic materials.     

A procedure for anaerobic column chromatography employing an anaerobic freter-type chamber

A technique is described for the anaerobic fractionation of oxygen-sensitive material. A Freter-type chamber in which the oxygen concentration is maintained at 2 to 5 μl/liter is used in conjunction with anaerobic chromatographic columns that are exterior to the chamber. The column inlet and outlet are connected via thick-walled polyethylene tubing to access ports in the chamber wall. Anaerobic buffer inside the chamber is pumped from the chamber to equilibrate the column. The oxygen-labile sample then is pumped onto the anaerobic column followed by the elution gradient buffer. Column eluate is returned to a fraction collector inside the chamber. At no stage is the sample exposed to air. This technique has been used effectively for fractionation of highly oxygen-sensitive enzymes from methanogenic bacteria where use of other methods failed.     

Enhanced Toluene Degradation Under Chlorate-Reducing Conditions by Bioaugmentation of Sand Columns with Chlorate- and Toluene-Degrading Enrichments

Chlorate was examined as a potential electron acceptor for enhancing toluene degradation. Most chlorate respiring bacteria (CRB) use nitrate as an electron acceptor, and toluene is known to be degraded under denitrifying conditions. Therefore, it was hypothesized that there would be bacteria that could degrade toluene using chlorate as an electron acceptor, and that chlorate could be used to stimulate toluene degradation. Repeated tests and different approaches in batch tests failed to produce an enrichment capable of toluene degradation supported by chlorate reduction. However, the addition of chlorate increased the overall rate of toluene degradation in bioaugmented columns that were fed chlorate vs. a control column. Toluene removal at an influent toluene concentration of 11 mg/L was 93±5%, which was larger by a factor of 1.95 than toluene removal in a nonbioaugmented control column. Following the discontinued feed of chlorate, toluene removal decreased to 69±4%, demonstrating that chlorate could be used to produce a 1.36-fold increase in toluene removal.     

Effect of temperature and active biogas process on passive separation of digested manure

The objective of the study was to identify the optimum time interval for effluent removal after temporarily stopping stirring in otherwise continuously stirred tank reactors. Influence of temperature (10 and 55 degrees C) and active biogas process on passive separation of digested manure, where no outside mechanical or chemical action was used, within the reactor was studied in three vertical settling columns (100 cm deep). Variations in solids and microbial distribution at top, middle and bottom layers of column were assessed over a 15 day settling period. Results showed that best solids separation was achieved when digested manure was allowed to settle at 55 degrees C with active biogas process (pre-incubated at 55 degrees C) compared to separation at 55 degrees C without active biogas process (autoclaved at 120 degrees C, for 20 min) or at 10 degrees C with active biogas process. Maximum solids separation was noticed 24h after settling in column incubated at 55 degrees C, with active biogas process. Microbiological analyses revealed that proportion of Archaea and Bacteria, absent in the autoclaved material, varied with incubation temperature, time and sampling depth. Short rod shaped bacteria dominated at 55 degrees C, while long rod shaped bacteria dominated at 10 degrees C. Methanosarcinaceae were seen more abundant in the surface layer at 55 degrees C while it was seen more common in the top and bottom layers at 10 degrees C. Thus, passive separation of digester contents within the reactor can be used effectively as an operating strategy to optimize biogas production by increasing the solids and biomass retention times. A minimum of 1-2h "non-stirring" period appears to be optimal time before effluent removal in plants where extraction is batch-wise 2-4 times a day.     

Antimicrobial effects of sanitizers against planktonic and sessile Listeria monocytogenes cells according to the growth phase

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to have potent antibiotic activities to maintain monoxenic condition in its insect host for effective pathogenesis and ultimately for optimal development of its nematode symbiont, Steinernema carpocapsae. In this study we assess its antibacterial activity against plant-pathogenic bacteria and identify its unknown antibiotics. The bacterial culture broth had significant antibacterial activity that increased with development of the bacteria and reached its maximum at the stationary growth phase. The antibiotic activities were significant against five plant-pathogenic bacterial strains: Agrobacterium vitis, Pectobacterium carotovorum subsp. atrosepticum, P. carotovorum subsp. carotovorum, Pseudomonas syringae pv. tabaci, and Ralstonia solanacearum. The antibacterial factors were extracted with butanol and fractionated using column chromatography with the eluents of different hydrophobic intensities. Two active antibacterial subfractions were purified, and the higher active fraction was further fractionated and identified as a single compound of benzylideneacetone (trans-4-phenyl-3-buten-2-one). With heat stability, the synthetic compound showed equivalent antibiotic activity and spectrum to the purified compound. This study reports a new antibiotic compound synthesized by X. nematophila, which is a monoterpenoid compound and active against some Gram-negative bacteria.     

Considerations on the possibility of microbial clogging of re-injection wells of the wastewater generated in a water-dissolved natural gas field

Brine produced from water-dissolved natural gas reservoirs should be returned to the reservoirs after the resources are recovered to prevent land subsidence. However, the ability to re-inject the brine gradually decreases and is only rectified by carrying out backwashing treatment of re-injection wells. Because the brine contains high levels of iodine also, it is also recovered from the brine using sulfuric acid and oxidizing agents. These chemicals may stimulate the growth of microorganisms that may cause the clogging. In this study, we used column experiments to investigate the possibility of the microbial clogging.Significant clogging was observed on the columns that were treated by the brine containing both indigenous microorganisms and dissolved oxygen. In particular, iodide-oxidizing bacteria were detected from the columns and original brine dominantly; therefore, it was assumed to have an important influence on the clogging. Iodine that was produced by iodide-oxidizing bacteria corroded iron in the sand under the presence of dissolved oxygen. Eluted Iron formed ferric hydroxide colloid in the brine and it caused the clogging of the pore spaces.We also demonstrated that deoxidized brine inhibited the iodide-oxidizing bacteria from becoming dominant and the column from the clogging through the column experiments. From these results, we can suggest removing dissolved oxygen as the most feasible countermeasures for the clogging.     

Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing

Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.     

Phylogenetic assessment of heterotrophic bacteria from a water distribution system using 16S rDNA sequencing

Determination of a heterotrophic plate count (HPC) for drinking-water samples alone is not enough to assess possible health hazards associated with sudden changes in the bacterial count. Speciation is very crucial to determine whether the population includes pathogens and (or) opportunistic pathogens. Most of the isolates recovered from drinking water samples could not be allocated to a specific phylogenetic branch based on the use of conventional diagnostic methods. The present study had to use phylogenetic analysis, which was simplified by determining and using the first 500-bp sequence of the 16S rDNA, to successfully identify the type and species of bacteria found in the samples. Gram-positive bacteria alpha-, beta-, and gamma-Proteobacteria were found to be the major groups representing the heterotrophic bacteria in drinking water. The study also revealed that the presence of sphingomonads in drinking water supplies may be much more common than has been reported so far and thus further studies are merited. The intermittent mode of supply, mainly characterized by water stagnation and flow interruption associated possibly with biofilm detachment, raised the possibility that the studied bacterial populations in such systems represented organisms coming from 2 different niches, the biofilm and the water column.     

Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide ore

The evolution of microbial populations involved in simulated-heap leaching of a polymetallic black schist sulfide ore (from the recently-commissioned Talvivaara mine, Finland) was monitored in aerated packed bed column reactors over a period of 40 weeks. The influence of ore particle size (2-6.5 mm and 6.5-12 mm) on changes in composition of the bioleaching microflora and mineral leaching dynamics in columns was investigated and compared to fine-grain (<2 microm) ore that was bioprocessed in shake flask cultures. Both column reactors and shake flasks were inoculated with 24 different species and strains of mineral-oxidizing and other acidophilic micro-organisms, and maintained at 37 degrees C. Mineral oxidation was most rapid in shake flask cultures, with about 80% of both manganese and nickel and 68% of zinc being leached within 6 weeks, though relatively little of the copper present in the ore was solubilised. The microbial consortium that emerged from the original inoculum was relatively simple in shake flasks, and was dominated by the iron-oxidizing autotroph Leptospirillum ferriphilum, with smaller numbers of Acidimicrobium ferrooxidans, Acidithiobacillus caldus and Leptospirillum ferrooxidans. Both metal recovery and (for the most part) total numbers of prokaryotes were greater in the column reactor containing the medium-grain than that containing the coarse-grain ore. The bioleaching communities in the columns displayed temporal changes in composition and differed radically from those in shake flask cultures. While iron-oxidizing chemoautotrophic bacteria were always the most numerically dominant bacteria in the medium-grain column bioreactor, there were major shifts in the most abundant species present, with the type strain of Acidithiobacillus ferrooxidans dominating in the early phase of the experiment and other bacteria (At. ferrooxidans NO37 and L. ferriphilum) dominating from week 4 to week 40. With the coarse-grain column bioreactor, similar transitions in populations of iron-oxidizing chemoautotrophs were observed, though heterotrophic acidophiles were often the most abundant bacteria found in mineral leach liquors. Four bacteria not included in the mixed culture used to inoculate the columns were detected by biomolecular techniques and three of these (all Alicyclobacillus-like Firmicutes) were isolated as pure cultures. The fourth bacterium, identified from a clone library, was related to the Gram-positive sulfate reducer Desulfotomaculum salinum. All four were considered to have been present as endospores on the dried ore, which was not sterilized in the column bioreactors. Two of the Alicyclobacillus-like isolates were found, transiently, in large numbers in mineral leachates. The data support the hypothesis that temporal and spatial heterogeneity in mineral heaps create conditions that favour different mineral-oxidizing microflora, and that it is therefore important that sufficient microbial diversity is present in heaps to optimize metal extraction.     

Culture confirmation of Listeria monocytogenes using tmRNA as a diagnostics target

16s ribosomal RNA (rRNA) is routinely used to identify bacteria in direct detection culture confirmation assays. In some instances rRNA cannot be used as a target to distinguish between phylogenetically closely related bacteria. Here we evaluate an alternative target, transfer messenger RNA (tmRNA), for the culture confirmation of Listeria monocytogenes.     

Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

Azo dyes are widely used in dye manufacturing, paper printing, textile industries, and as tattoo pigmentation. Since intestinal and skin bacteria can metabolize certain azo dyes to carcinogenic compounds, many researchers have studied the azoreductases of these bacteria. In this study, we used a microarray method to identify the intestinal bacterial species from cultured fecal samples in Brain Heart Infusion (BHI) broth with or without azo dyes that may be involved in azo dye reduction. The microarray was designed to identify 40 bacterial species that are reported in the literature to be predominant in human feces. Results from this study showed 26-30 species are present in the cultured fecal samples. The representative bacteria were then examined for the azo dye reduction activity.     

Mass collection of magnetotactic bacteria from the permanently stratified ferruginous Lake Pavin,France

Obtaining high biomass yields of specific microorganisms for culture-independent approaches is a challenge faced by scientists studying organism's recalcitrant to laboratory conditions and culture. This difficulty is highly decreased when studying magnetotactic bacteria (MTB) since their unique behaviour allows their enrichment and purification from other microorganisms present in aquatic environments. Here, we use Lake Pavin, a permanently stratified lake in the French Massif Central, as a natural laboratory to optimize collection and concentration of MTB that thrive in the water column and sediments. A method is presented to separate MTB from highly abundant abiotic magnetic particles in the sediment of this crater lake. For the water column, different sampling approaches are compared such as in situ collection using a Niskin bottle and online pumping. By monitoring several physicochemical parameters of the water column, we identify the ecological niche where MTB live. Then, by focusing our sampling at the peak of MTB abundance, we show that the online pumping system is the most efficient for fast recovering of large volumes of water at a high spatial resolution, which is necessary considering the sharp physicochemical gradients observed in the water column. Taking advantage of aerotactic and magnetic MTB properties, we present an efficient method for MTB concentration from large volumes of water. Our methodology represents a first step for further multidisciplinary investigations of the diversity, metagenomic and ecology of MTB populations in Lake Pavin and elsewhere, as well as chemical and isotopic analyses of their magnetosomes.     

Investigations on pyrite oxidation in mine spoils of the Lusatian lignite mining district

The impact of organic waste material and fly ash on microbial and chemical pyrite oxidation was investigated in a field experiment, as well as in column tests under laboratory conditions. For the field experiment, pyritic mine spoil was ameliorated with fly ash and treated either with mineral fertiliser, with sewage sludge or with compost. Independent of treatment, during the 18 months following application, the pyrite-S contents decreased steadily in the top spoil (0–30 cm depth). However, high variations of the pyrite-S content were observed. Compared to other pyrite oxidation studies, the pyrite content of the mine spoil at the experimental site was low. Therefore, a model spoil with a higher pyrite content, derived from Tertiary strata of the overburden sequence in the same open-cast mine, was used for the column experiments. For the first column experiment, the model spoil was mixed with fly ash and mineral fertiliser, reflecting the common reclamation practice in the Lusatian open-cast lignite mining district. Columns with this spoil were either inoculated with different cell numbers of autochthonous acidophilic bacteria, isolated from the model spoil, or with a commercial strain of Thiobacillus ferrooxidans. The ratio of sulphate-S to total S was used as a measure for the degree of pyrite oxidation. The ratio of sulphate-S to total S increased within 28 days of incubation. The increase was related to the inoculated cell numbers of bacteria, but independent of the origin of the bacteria. It can be stated, that autochthonous bacteria from the model spoil oxidised pyrite at a similar rate as did the commercial T. ferrooxidans strain. For the second column test, mineral fertiliser, sewage sludge or compost were applied to the model spoil. The columns were inoculated with autochthonous bacteria, isolated from the model spoil. Application of sewage sludge and compost seemed to promote the weathering of pyrite, as the ratio of sulphate-S to total S increased more rapidly in these treatments compared to control or mineral fertiliser application. Both experiments showed an increase of cell numbers of inoculated bacteria, independent of the ratio of sulphate-S to total S. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date.     

BACLAB: a computer simulation of a medical bacteriology laboratory–an aid for teaching tertiary level microbiology

A program, BACLAB, is described, which has been devised to help students to learn the main biochemical tests and profiles that are used to identify medically important bacteria in a local hospital laboratory.     

Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column.

The vertical distribution of major and intermediate electron acceptors and donors was measured in a shallow stratified fjord. Peaks of zero valence sulfur, Mn(IV), and Fe(III) were observed in the chemocline separating oxic surface waters from sulfidic and anoxic bottom waters. The vertical fluxes of electron acceptors and donors (principally O2 and H2S) balanced within 5%; however, the zones of oxygen reduction and sulfide oxidation were clearly separated. The pathway of electron transfer between O2 and H2S was not apparent from the distribution of sulfur, nitrogen, or metal compounds investigated. The chemical zonation was related to bacterial populations as detected by ethidium bromide (EtBr) staining and by in situ hybridization with fluorescent oligonucleotide probes of increasing specificity. About half of all EtBr-stained cells were detectable with a general oligonucleotide probe for all eubacteria when digital image analysis algorithms were used to improve sensitivity. Both EtBr staining and hybridization indicated a surprisingly uniform distribution of bacteria throughout the water column. However, the average cell size and staining intensity as well as the abundance of different morphotypes changed markedly within the chemocline. The constant overall cell counts thus concealed pronounced population shifts within the water column. Cells stained with a delta 385 probe (presumably sulfate-reducing bacteria) were detected at the chemocline at about 5 x 10(4) cells per ml, and this concentration increased to 2 x 10(5) cells per ml beneath the chemocline. A long slim rod-shaped bacterium was found in large numbers in the oxic part of the chemocline, whereas large ellipsoid cells dominated at greater depth. Application of selective probes for known genera of sulfate-reducing bacteria gave only low cell counts, and thus it was not possible to identify the dominant morphotypes of the sulfate-reducing community.