Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3-.N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO3-.N mg of MLVSS-1 h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [C-13] methanol to biomark the DNA of the denitrifiers. The extracted [C-13]DNA and [C-12]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [C-13]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [C-12]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [C-14] methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

Determination of external and internal mass transfer limitation in nitrifying microbial aggregates

In this article we present a study of the effects of external and internal mass transfer limitation of oxygen in a nitrifying system. The oxygen uptake rates (OUR) were measured on both a macro-scale with a respirometric reactor using off-gas analysis (Titrimetric and Off-Gas Analysis (TOGA) sensor) and on a micro-scale with microsensors. These two methods provide independent, accurate measurements of the reaction rates and concentration profiles around and in the granules. The TOGA sensor and micro-sensor measurements showed a significant external mass transfer effect at low dissolved oxygen (DO) concentrations in the bulk liquid while it was insignificant at higher DO concentrations. The oxygen distribution with anaerobic or anoxic conditions in the center clearly shows major mass transfer limitation in the aggregate interior. The large drop in DO concentration of 22 - 80% between the bulk liquid and aggregate surface demonstrates that the external mass transfer resistance is also highly important. The maximum OUR even for floccular biomass was only attained at much higher DO concentrations ( approximate to 8 mg/L) than typically used in such systems. For granules, the DO required for maximal activity was estimated to be > 20mg/L, clearly indicating the effects of the major external and internal mass transfer limitations on the overall biomass activity. Smaller aggregates had a larger volumetric OUR indicating that the granules may have a lower activity in the interior part of the aggregate. (C) 2004 Wiley Periodicals, Inc.

An in vitro study of the anti-microbial efficacy of a 1% silver sulphadiazine and 0.2% chlorhexidine digluconate cream Silvazine (TM), 1% silver sulphadiazine cream Flamazine (TM) and a silver coated dressing Acticoat (TM)

Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical anti-microbial agents has helped improve the survival in these patients. There are a number of anti-microbials available, one of which, Silvazine(TM) (1% silver sulphadiazine (SSD) and 0.2% chlorhexidine digluconate), is used only in Australasia. No study, in vitro or clinical, had compared Silvazine(TM) with the new dressing Acticoat(TM). This study compared the anti-microbial activity of Silvazine(TM), Acticoa(TM) and 1% silver sulphadiazine (Flamazine(TM)) against eight common burn wound pathogens. Methods: Each organism was prepared as a suspension. A 10 mul inoculum of the chosen bacterial isolate (representing approximately between 104 and 105 total bacteria) was added to each of four vials, followed by samples of each dressing and a control. The broths were then incubated and 10 mul loops removed at specified intervals and transferred onto Horse Blood Agar. These plates were then incubated for 18 hours and a colony count was performed. Results: The data demonstrates that the combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) results in the most effective killing of all bacteria. SSD and Acticoat(TM) had similar efficacies against a number of isolates, but Acticoat(TM) seemed only bacteriostatic against E. faecalis and methicillin-resistant Staphylococcus aureus. Viable quantities of Enterobacter cloacae and Proteus mirabilis rei named at 24 h. Conclusion: The combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) is a more effective anti-microbial against a number of burn wound pathogens in this in vitro study. A clinical study of its in vivo anti-microbial efficacy is required. (C) 2003 Elsevier Ltd and ISBI. All rights reserved.

Geochemistry of late Archaean stromatolites from Zimbabwe: evidence for microbial life in restricted epicontinental seas

We report comprehensive trace element and Sr-isotope data for microbial carbonates from the Archaean Mushandike limestone, Masvingo Greenstone Belt, Zimbabwe. The stromatolites have very coherent REE + Y patterns and share the essential shale-normalised characteristics of other Archaean marine precipitates (positive La and Gd anomalies, absence of a negative Cc anomaly and a strongly superchondritic Y/Ho ratio). Mixing models constrain the maximum amount of shale contamination to 0.25-1% and calculated detritus-free carbonate REE + Y systematics require precipitation from seawater. In terms of light-REE over heavy-REE depletion, however, the studied samples are very different from all other known Archaean marine precipitates. In shale-normalised plots, the Mushandike samples yield a negative slope. A very restricted, regional input source of the dissolved load is indicated because normalisation with locally occurring tonalite gneiss REE + Y data yields a pattern closely resembling typical shale-normalised Archaean marine chemical sediments. The disappearance of a negative Eu anomaly when patterns are normalised with local tonalite gneiss strengthens this interpretation. Sr-isotope ratios are strongly correlated with trace element contents and ratios, which explains the modest scatter in Sr-isotope ratios as representing (minor) clastic contamination. Importantly, even the least contaminated samples have very radiogenic initial Sr-87/Sr-86 ratios (0.7184) implying Sr input from an ancient high Rb/Sr source, such as the early Archaean gneisses of south-central Zimbabwe. A local ancient (3.5-3.8 Ga) source is also indicated by previously published Pb-isotope datasets for the Mushandike stromatolites. This is entirely compatible with the occurrence of 3.7-3.8 Ga zircons in quartzites and metapelites from comparably old greenstone belts within less than 150 km of the studied locality. Comparison of the Pb-isotope ratios of the Mushandike stromatolites with 2.7 and 2.6 Ga old stromatolites from the neighbouring, Belingwe Greenstone Belt demonstrates differences in initial isotope composition that relate to the extent of exchange with the open ocean. The development of numerous basins on old continental crust, with water masses variably restricted from the open ocean. suggests a lack of strong vertical topography on this late Archaean craton. (C) 2004 Elsevier B.V. All rights reserved.

Anomalous magnetic susceptibility values and traces of subsurface microbial activity in carbonate banks on San Salvador Island, Bahamas

Pure limestones beneath the paleosols on San Salvador Island, Bahamas, contain strong positive magnetic susceptibility anomalies, although the iron content is generally very low. These magnetic phenomena differ from those associated with disconformities, which are marked by accumulation of paramagnetic airborne dust deposits with relatively high iron content. The strength and characters of the magnetic response in these subsurface zones correspond to the presence of magnetite, particularly small single-domain magnetite crystals of microbial origin. These crystals are not present elsewhere in the intergranular rock pores or microvugs. They are preferentially concentrated in capillary microborings, which developed concurrently with formation of calcite cements that have soil-related C and O isotope compositions. These magnetic zones occur several meters below the overlying soil horizons. Very thin and long linear microborings may be attributable to cyanobacterial microborers. The single-domain magnetites in these micrometer-size tunnels plugged by calcite appear to result from later occupation of these tiny holes by magnetotactic bacteria. Inorganic origin of the magnetite seems unlikely. Numerous traces that suggest subsurface microbial activity provide evidence that may be used to develop possible scenarios for subsequent biological studies of the precise bacteria involved.

A soil-based microbial biofilm exposed to 2,4-D: bacterial community development and establishment of conjugative plasmid pJP4

A soil suspension was used as a source to initiate the development of microbial communities in flow cells irrigated with 2,4-dichlorophenoxyacetic acid (2,4-D) (25 mu g ml(-1)). Culturable bacterial members of the community were identified by 16S rRNA gene sequencing and found to be members of the genera Pseudomonas, Burkholderia, Collimonas and Rhodococcus. A 2,4-D degrading donor strain, Pseudomonas putida SM 1443 (pJP4::gfp), was inoculated into flow cell chambers containing 2-day old biofilm communities. Transfer of pJP4::gfp from the donor to the bacterial community was detectable as GFP fluorescing cells and images were captured using confocal scanning laser microscopy (GFP fluorescence was repressed in the donor due to the presence of a chromosomally located lacl(q) repressor gene). Approximately 5-10 transconjugant microcolonies, 20-40 mu m in diameter, could be seen to develop in each chamber. A 2,4-D degrading transconjugant strain was isolated from the flow cell system belonging to the genus Burkholderia.

Microbial hyaluronic acid production

Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.

Molecular investigation of the microbial populations of the pink sugarcane mealybug, Saccharicoccus sacchari

In an attempt to better understand the microbial diversity and endosymbiotic microbiota of the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homoptera: Pseudococcidae), culture-independent approaches, namely PCR, a 16S rDNA clone library, and temperature gradient gel electrophoresis (TGGE) were used. Previous work has indicated that the acetic acid bacteria Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens represent only a small proportion of the microbial community of the PSMB. These findings were supported in this study by TGGE, where no bands representing G. sacchari, G. diazotrophicus, and G. liquefaciens on the acrylamide gel could be observed following electrophoresis, and by a 16S rDNA clone library study, where no clones with the sequence of an acetic acid bacterium were found. Instead, TGGE revealed that the mealybug microbial community was dominated by beta- and gamma-Proteobacteria. The dominant band in TGGE gels found in a majority of the mealybug samples was most similar, according to BLAST analysis, to the beta-symbiont of the craw mealybug Antonina crawii and to Candidatus Tremblaya princeps, an endosymbiont from the mealybug Paracoccus nothofagicola. The sequences of other dominant bands were identified as gamma-Proteobacteria, and were most closely related to uncultured bacterial clones obtained from soil samples. Mealybugs collected from different areas in Queensland, Australia, were found to produce similar TGGE profiles, although there were a few exceptions. A 16S rDNA clone library based on DNA extracted from a mealybug collected from sugarcane in the Burdekin region in Queensland, Australia, indicated very low levels of diversity among mealybug microbial populations. All sequenced clones were most closely related to the same members of the gamma-Proteobacteria, according to BLAST analysis.

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [C-13]acetate was used in SIP to label the DNA of the denitrifiers. The [C-13]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the C-13 library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking Up [C-14] acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the waste-water industry to enhance denitrification.

Challenges for simultaneous nitrification, denitrification, and phosphorus removal in microbial aggregates: mass transfer limitation and nitrous oxide production

The microbial community composition and activity was investigated in aggregates from a lab-scale bioreactor, in which nitrification, denitrification and phosphorus removal occurred simultaneously. The biomass was highly enriched for polyphosphate accumulating organisms facilitating complete removal of phosphorus from the bulk liquid; however, some inorganic nitrogen still remained at the end of the reactor cycle. This was ascribed to incomplete coupling of nitrification and denitrification causing NO3- accumulation. After 2 h of aeration, denitrification was dependent on the activity of nitrifying bacteria facilitating the formation of anoxic zones in the aggregates; hence, denitrification could not occur without simultaneous nitrification towards the end of the reactor cycle. Nitrous oxide was identified as a product of denitrification, when based on stored PHA as carbon source. This observation is of critical importance to the outlook of applying PHA-driven denitrification in activated sludge processes. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus)

The ornate tropical rock lobster, Panulirus ornatus has substantial potential as an aquaculture species though disease outbreaks during the animal's extended larval lifecycle are major constraints for success. In order to effectively address such disease-related issues, an improved understanding of the composition and dynamics of the microbial communities in the larval rearing tanks is required. This study used flow cytometry and molecular microbial techniques (clone libraries and denaturing gradient gel electrophoresis (DGGE)) to quantify and characterise the microbial community of the water column in the early stages (developmental stage I-II) of a P. ornatus larval rearing system. DGGE analysis of a 5000 L larval rearing trial demonstrated a dynamic microbial community with distinct changes in the community structure after initial stocking (day I to day 2) and from day 4 to day 5, after which the structure was relatively stable. Flow cytometry analysis of water samples taken over the duration of the trial demonstrated a major increase in bacterial load leading up to and peaking on the first day of the initial larval moult (day 7), before markedly decreasing prior to when > 50% of larvae moulted (day 9). A clone library of a day 10 water sample taken following a mass larval mortality event reflected high microbial diversity confirmed by statistical analysis indices. Sequences retrieved from both clone library and DGGE analyses were dominated by gamma- and alpha-Proteobacteria affiliated organisms with additional sequences affiliated with beta- and epsilon-Proteobacteria, Bacteroidetes, Cytophagales and Chlamydiales groups. Vibrio affiliated species were commonly retrieved in the clone library, though absent from DGGE analysis.

Microbial Fuel Cells: Methodology and Technology

Microbial fuel cell (MFC) research is a rapidly evolving field that lacks established terminology and methods for the analysis of system performance. This makes it difficult for researchers to compare devices on an equivalent basis. The construction and analysis of MFCs requires knowledge of different scientific and engineering fields, ranging from microbiology and electrochemistry to materials and environmental engineering. DescribingMFCsystems therefore involves an understanding of these different scientific and engineering principles. In this paper, we provide a review of the different materials and methods used to construct MFCs, techniques used to analyze system performance, and recommendations on what information to include in MFC studies and the most useful ways to present results.