Seawater carbonate chemistry, nitrogen concentration and macro community analyse, 2011

Rising anthropogenic CO2 emissions acidify the oceans, and cause changes to seawater carbon chemistry. Bacterial biofilm communities reflect environmental disturbances and may rapidly respond to ocean acidification. This study investigates community composition and activity responses to experimental ocean acidification in biofilms from the Australian Great Barrier Reef. Natural biofilms grown on glass slides were exposed for 11 d to four controlled pCO2 concentrations representing the following scenarios: A) pre-industrial (~300 ppm), B) present-day (~400 ppm), C) mid century (~560 ppm) and D) late century (~1140 ppm). Terminal restriction fragment length polymorphism and clone library analyses of 16S rRNA genes revealed CO2-correlated bacterial community shifts between treatments A, B and D. Observed bacterial community shifts were driven by decreases in the relative abundance of Alphaproteobacteria and increases of Flavobacteriales (Bacteroidetes) at increased CO2 concentrations, indicating pH sensitivity of specific bacterial groups. Elevated pCO2 (C + D) shifted biofilm algal communities and significantly increased C and N contents, yet O2 fluxes, measured using in light and dark incubations, remained unchanged. Our findings suggest that bacterial biofilm communities rapidly adapt and reorganize in response to high pCO2 to maintain activity such as oxygen production.

Bacterial diversity within the human subgingival crevice

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.

Phylogenetic and physiological characterization of a heterotrophic, chemolithoautotrophic Thiothrix strain isolated from activated sludge

The sheathed filamentous bacterium known as strain CT3, isolated by micromanipulation from an activated sludge treatment plant in Italy, is a member of the genus Thiothrix in the gamma-Proteobacteria according to 16S rDNA sequence analysis. The closest phylogenetic neighbours of strain CT3 are strains I and Q(T), which were also isolated from activated sludge and belong to the species Thiothrix fructosivorans. These strains have respectively 99.2 and 99.4 % similarity to CT3 by 16S rDNA sequence comparison. CT3 shows 63-67 % DNA-DNA hybridization with strain I, which is the only currently viable strain of T. fructosivorans. CT3 is the second strain in the genus Thiothrix that has been shown to be capable of growing autotrophically with reduced sulfur compounds as the sole energy source; autotrophy was also confirmed in strain I. The first reported chemolithoautotrophic isolate of this genus was a strain of 'Thiothrix ramosa' that was isolated from a hydrogen sulfide spring and is morphologically distinguishable from all other described strains of Thiothrix, including CT3. CT3 is an aerobic organism that is non-fermentative, not capable of denitrification and able to grow heterotrophically. Autotrophy in the genus Thiothrix should be investigated more fully to better define the taxonomy of this genus.

Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis

Aims: Isolation, identification and characterization of a highly efficient isomaltulose producer. Methods and Results: After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90-100 U mg(-1) DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41-66 U mg(-1) DW) or a reference strain of Erwinia rhapontici (0.3-0.9 U mg(-1) DW). Maximum yield of isomaltulose at 78-80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. Significance and Impact of Study: This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose.

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.

Systematics and evolution of ticks with a list of valid genus and species names

In recent years there has been much progress in our understanding of the phylogeny and evolution of ticks, in particular the hard ticks (Ixodidae). Indeed, a consensus about the phylogeny of the hard ticks has emerged which is quite different to the working hypothesis of 10 years ago. So that the classification reflects our knowledge of ticks, several changes to the nomenclature of ticks are imminent or have been made. One subfamily, the Hyalomminae, should be sunk, while another, the Bothriocrotoninae, has been created (Klompen, Dobson & Barker, 2002). Bothriocrotoninae, and its sole genus Bothriocroton, have been created to house an early-diverging ('basal') lineage of endemic Australian ticks that used to be in the genus Aponomma. The remaining species of the genus Aponomma have been moved to the genus Amblyomma. Thus, the name Aponomma is no longer a valid genus name. The genus Rhipicephalus is paraphyletic with respect to the genus Boophilus. Thus, the genus Boophilus has become a subgenus of the genus Rhipicephalus (Murrell & Barker, 2003). Knowledge of the phylogenetic relationships of ticks has also provided new insights into the evolution of ornateness and of their life cycles, and has allowed the historical zoogeography of ticks to be studied. Finally, we present a list of the 899 valid genus and species names of ticks as of February 2004.

Dynamics of a temperature-related coral disease outbreak

During the austral summer of 2001/2002, a coral epizootic occurred almost simultaneously with a bleaching event on the fringing reefs of Magnetic Island (Great Barrier Reef region), Australia. This resulted in a 3- to 4-fold increase in the mean percentage of partial mortality rate in a population of the hard coral Montipora aequituberculata. The putative disease state, ‘atramentous necrosis’, was observed on both bleached and normally-pigmented M. aequituberculata, and presented blackened lesions that spread within days across the colony surface and throughout the population. Diseased portions of the corals were only visible for 3 to 4 wk, with diseased tissues becoming covered in sediment and algae, which rapidly obscured evidence of the outbreak. Diseased colonies were again observed in the summer of 2002/2003 after being absent over the 2002 winter. Analysis of when diseased and bleached corals were first observed, and when and where the mortality occurred on individual colonies, indicated virtually all the mortality over the summer could be attributed to the disease and not to the bleaching. Fluorescence in situ hybridisation (FISH) techniques and cloning, and analysis of the 16S rRNA genes from diseased coral tissue, identified a mixed microbial assemblage in the diseased tissues particularly within the Alphaproteobacteria, Firmicutes and Bacteroidetes. While it is not possible in this study to distinguish between a disease-causing microbial community versus secondary invaders, the bacterial 16S rDNA sequences identified within the blackened lesions demonstrated high similarity to sequences from black band disease and white plague infected corals, suggesting either common aetiological agents or development of a bacterial community that is specific to degrading coral tissues. Temperature-induced coral disease outbreaks, with the potential for elevated levels of mortality, may represent an added problem for corals during the warmer summer months and an added dimension to predicted increases in water temperature from climate change.

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer

Aims: Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. Methods and Results: Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced L-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. Conclusions: Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. Significance and Impact of the Study: Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.

The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract

Seventy-two lactic acid producing bacterial isolates (excluding streptococci) were cultured from the gastrointestinal tract of six horses. Two of the horses were orally dosed with raftilose to induce lactic acidosis and laminitis while the remaining four were maintained on a roughage diet. Near complete 16S rDNA was amplified by PCR from the genomic DNA of each isolate. Following RFLP analysis with the restriction enzymes MboI, HhaI and HinfI, the PCR products from the IS isolates that produced L- and/or D-lactate were subsequently cloned and sequenced. DNA sequence analysis indicated that the majority of the isolates were closely related to species within the genus Lactobacillus, including Lactobacillus salivarius, Lactobacillus mucosae and Lactobacillus delbrueckii. Four isolates were closely related to Mitsuokella jalaludinii. Lactic acid producing bacteria (LAB) from the equine gastrointestinal tract was dominated by representatives from the genus Lactobacillus, but also included D-lactate-producing bacteria closely related to M. jalaludinii. Identification and characterization of LAB from the equine gastrointestinal tract should contribute to our understanding and management of fermentative acidosis, ulceration of the stomach and laminitis. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates Arthrobacter sp 2AC and Comamonas sp 4BC

Two bacterial strains, 2AC and 4BC, both capable of utilizing naphthalene-2-sulfonic acid (2-NSA) as a sole source of carbon, were isolated from activated sludges previously exposed to tannery wastewater. Enrichments were carried out in mineral salt medium (MSM) with 2-NSA as the sole carbon source. 16S rDNA sequencing analysis indicated that 2AC is an Arthrobacter sp. and 4BC is a Comamonas sp. Within 33 h, both isolates degraded 100% of 2-NSA in MSM and also 2-NSA in non-sterile tannery wastewater. The yield coefficient was 0.33 g biomass dry weight per gram of 2-NSA. A conceptual model, which describes the aerobic transformation of organic matter, was used for interpreting the biodegradation kinetics of 2-NSA. The half-lives for 2-NSA, at initial concentrations of 100 and 500 mg/l in MSM, ranged from 20 h (2AC) to 26 h (4BC) with lag-phases of 8 h (2AC) and 12 h (4BC). The carbon balance indicates that 75-90% of the initial TOC (total organic carbon) was mineralized, 5-20% remained as DOC (dissolved organic carbon) and 3-10% was biomass carbon. The principal metabolite of 2-NSA biodegradation (in both MSM and tannery wastewater) produced by Comamonas sp. 4BC had a MW of 174 and accounted for the residual DOC (7.0-19.0% of the initial TOC and 66% of the remaining TOC). Three to ten percent of the initial TOC (33% of the remaining TOC) was associated with biomass. The metabolite was not detected when Arthrobacter sp. 2AC was used, and a lower residual DOC and biomass carbon were recorded. This suggests that the two strains may use different catabolic pathways for 2-NSA degradation. The rapid biodegradation of 2-NSA (100 mg/l) added to non-sterile tannery wastewater (total 2-NSA, 105 mg/l) when inoculated with either Arthrobacter 2AC or Comamonas 4BC showed that both strains were able to compete with the indigenous microorganisms and degrade 2-NSA even in the presence of alternate carbon sources (DOC in tannery wastewater = 91 mg/l). The results provide information useful for the rational design of bioreactors for tannery wastewater treatment.

Archaeal diversity in two thermophilic chalcopyrite bioleaching reactors

This study used a culture-independent molecular approach to investigate the archaeal community composition of thermophilic bioleaching reactors. Two culture samples, MTC-A and MTC-B, grown with different concentrations of chalcopyrite (CuFeS2), a copper sulfidic ore, at a temperature of 78 degrees C and pH 1.6 were studied. Phylogenetic analysis of the 16S rRNA genes revealed that both cultures consisted of Archaea belonging to the Sulfolobales. The 16S rRNA gene clone library of MTC-A grown with 4% (w/v) chalcopyrite was dominated by a unique phylotype related to Sulfolobus shibatae (69% of total clones). The remaining clones were affiliated with Stygiolobus azoricus (11%), Metallosphaera sp. J1 (8%), Acidianus infernus (2%), and a novel phylotype related to Sulfurisphaera ohwakuensis (10%). In contrast, the clones from MTC-B grown with 12% (w/v) chalcopyrite did not appear to contain Sulfolobus shibatae-like organisms. Instead the bioleaching consortium was dominated by clones related to Sulfurisphaera ohwakuensis (73.9% of total clones). The remaining microorganisms detected were similar to those found in MTC-A.

Biodegradation of the cyanobacterial toxin microcystin LR in natural water and biologically active slow sand filters

A bacterium (MJ-PV) previously demonstrated to degrade the cyanobacterial toxin microcystin LR, was investigated for bioremediation applications in natural water microcosms and biologically active slow sand filters. Enhanced degradation of microcystin LR was observed with inoculated (1 x 10(6) cell/mL) treatments of river water dosed with microcystin LR (> 80% degradation within 2 days) compared to uninoculated controls. Inoculation of MJ-PV at lower concentrations (1 x 10(2)-1 x 10(5)cells/mL) also demonstrated enhanced microcystin LR degradation over control treatments. Polymerase chain reactions (PCR) specifically targeting amplification of 16S rDNA of MJ-PV and the gene responsible for initial degradation of microcystin LR (mlrA) were successfully applied to monitor the presence of the bacterium in experimental trials. No amplified products indicative of an endemic MJ-PV population were observed in uninoculated treatments indicating other bacterial strains were active in degradation of microcystin LR, Pilot scale biologically active slow sand filters demonstrated degradation of microcystin LR irrespective of MJ-PV bacterial inoculation. PCR analysis detected the MJ-PV population at all locations within the sand filters where microcystin degradation was measured. Despite not observing enhanced degradation of microcystin LR in inoculated columns compared to uninoculated column, these studies demonstrate the effectiveness of a low-technology water treatment system like biologically active slow sand filters for removal of microcystins from reticulated water supplies. Crown Copyright (c) 2006 Published by Elsevier Ltd. 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.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.