P. C. Burrell

Identification, Detection, and Spatial Resolution of Clostridium Populations Responsible for Cellulose Degradation in a Methanogenic Landfill Leachate Bioreactor

ABSTRACT An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.

Identification of bacteria responsible for ammonia oxidation in freshwater aquaria.

ABSTRACT Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to theNitrosomonas marina cluster, theNitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the β subdivision of the class Proteobacteria. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established.Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.

Microbial ecology of the equine hindgut during oligofructose-induced laminitis

Alimentary carbohydrate overload is a significant cause of laminitis in horses and is correlated with drastic shifts in the composition of hindgut microbiota. Equine hindgut streptococcal species (EHSS), predominantly Streptococcus lutetiensis, have been shown to be the most common microorganisms culturable from the equine caecum prior to the onset of laminitis. However, the inherent biases of culture-based methods are estimated to preclude up to 70% of the normal caecal microbiota. The objective of this study was to evaluate bacterial population shifts occurring in the equine caecum throughout the course of oligofructose-induced laminitis using several culture-independent techniques and to correlate these with caecal lactate, volatile fatty acid and degrees of polymerization 3–7 fructo-oligosaccharide concentrations. Our data conclusively show that of the total microbiota present in the equine hindgut, the EHSS S. lutetiensis is the predominant microorganism that proliferates prior to the onset of laminitis, utilizing oligofructose to produce large quantities of lactate. Population shifts in lactobacilli and Escherichia coli subpopulations occur secondarily to the EHSS population shifts, thus confirming that lactobacilli and coliforms have no role in laminitis. A large, curved, Gram-negative rod previously observed during the early phases of laminitis induction was most closely related to the Anaerovibrio genus and most likely represents a new, yet to be cultured, genus and species. Correlation of fluorescence in situ hybridization and quantitative real-time PCR results provide evidence supporting the hypothesis that laminitis is associated with the death en masse and rapid cell lysis of EHSS. If EHSS are lysed, liberated cellular components may initiate laminitis.

The use of 16S rDNA clone libraries to describe the microbial diversity of activated sludge communities

Clone libraries were prepared from polymerase chain reaction amplified 16S rDNAs from activated sludge community DNAs. Eight different libraries from a range of samples were prepared. From each library, up to 100 clones were examined. In some libraries, the clone inserts were grouped into operational taxonomic units (OTUs) by restriction enzyme analysis (REA). Then, either the clones or representatives of OTUs were partially sequenced using either 27f or 530f conserved bacterial primers. The sequence data was phylogenetically analysed to group the clones and the method currently gives the best insight into the activated sludge microbial community biodiversity. The method for clone library production is described and the pros and cons of the procedure are outlined. In summary, the use of clone libraries has resulted in the discovery of unimagined biodiversity in activated sludge. The abundance of some unpredicted bacterial groups (e.g. beta subclass Proteobacteria) and the paucity of expected ones (e.g. Acinetobacter ) highlights the inadequacy of traditional culture dependent methods that rely on sample dilution and spread plate inoculation.

Characterisation of the bacterial consortium involved in nitrite oxidation in activated sludge

A sequencing batch reactor (SBR) was operated to selectively grow a nitrite oxidising microbial community and was called the nitrite oxidising SBR (NOSBR). The nitrite oxidising characteristics of the reactor biomass were studied as well as the microbial composition. Molecular biological methods of clone libraries were used to evaluate the microorganisms in both the seed sludge and in the NOSBR sludge. We have found that the nitrite oxidation in the NOSBR was due the presence of bacteria from the Nitrospira phylum and not because of the presence of Nitrobacter which were in very low numbers in the NOSBR and not detected in the seed sludge. We hypothesize that the unknown nitrite oxidising bacteria in wastewater treatment plants are a range of species related to Nitrospira moscoviensis. A suite of primers were developed from the clone sequence data and used in a diagnostic polymerase chain reaction to prove the presence of these novel nitrite oxidisers in a range of full scale and laboratory scale activated sludge plants.

Streptococcus henryi sp. nov. and Streptococcus caballi sp. nov., isolated from the hindgut of horses with oligofructose-induced laminitis

Four Gram-positive, catalase-negative, coccoid-shaped isolates were obtained from the caecum and rectum of horses with oligofructose-induced equine laminitis. Phenotypic and phylogenetic studies were performed on these isolates. Initial biochemical profiling assigned two of the isolates to Streptococcus bovis. The other two isolates, however, could not be assigned conclusively on the basis of their biochemical profiles. Gene sequence analysis demonstrated that the four new isolates were related most closely to Streptococcus suis based on the 16S rRNA gene and to Streptococcus orisratti based on the manganese-dependent superoxide dismutase gene (sodA). Sequence divergence values from recognized Streptococcus species based on these two genes were >3 and >13%, respectively, for all four isolates. Phylogenetic and phenotypic analyses demonstrated that the four isolates formed two distinct clonal groups that are suggested to represent two novel species of the genus Streptococcus. The names proposed for these organisms are Streptococcus henryi sp. nov. (type strain 126(T) =ATCC BAA-1484(T) =DSM 19005(T)) and Streptococcus caballi sp. nov. (type strain 151(T) =ATCC BAA-1485(T) =DSM 19004(T)).

Phylogenetic analysis of Porphyromonas species isolated from the oral cavity of Australian marsupials

Porphyromonas species are frequently isolated from the oral cavity and are associated with periodontal disease in both animals and humans. Black, pigmented Porphyromonas spp. isolated from the gingival margins of selected wild and captive Australian marsupials with varying degrees of periodontal disease (brushtail possums, koalas and macropods) were compared phylogenetically to Porphyromonas strains from non-marsupials (bear, wolf, coyote, cats and dogs) and Porphyromonas gingivalis strains from humans using 16S rRNA gene sequence analysis. The results of the phylogenetic analysis identified three distinct groups of strains. A monophyletic P. gingivalis group (Group 1) contained only strains isolated from humans and a Porphyromonas gulae group (Group 2) was divided into three distinct subclades, each containing both marsupial and non-marsupial strains. Group 3, which contained only marsupial strains, including all six strains isolated from captive koalas, was genetically distinct from P. gulae and may constitute a new Porphyromonas species.

Application of flowcell technology for monitoring biofilm development and cellulose degradation in leachate and rumen systems.

In this study, a flat plate flowcell was modified to provide a reactor system that could maintain anaerobic, cellulolytic biofilms while providing the data needed to carry out a chemical oxygen demand mass balance to determine the cellulose digestion rates. The results showed that biofilms could be observed to grow and develop on cellulose particle surfaces from both anaerobic digester leachate and rumen fluid inocula. The observations suggest that the architecture of rumen and leachate derived biofilms may be significantly different with rumen derived organisms forming stable, dense biofilms while the leachate derived organisms formed less tenacious surface attachments. This experiment has indicated the utility of flowcells in the study of anaerobic biofilms.

A survey of the relative abundance of specific groups of cellulose degrading bacteria in anaerobic environments using fluorescence in situ hybridization

Aims:  The utility of fluorescence in situ hybridization (FISH) for detecting uncultured micro‐organisms in environmental samples has been shown in numerous habitats. In this study a suite of three FISH probes for cellulolytic bacteria is described and their efficacy is demonstrated by quantifying the relative abundance of the target micro‐organisms in a range of industrial biomass samples.

The microbiology of nitrogen removal in activated sludge systems

Eutrophication can be defined as a deterioration in the aesthetic and life-supporting qualities of lakes, estuaries and streams, caused by the continued input of nutrients like nitrogen, phosphorus and organic growth factors, which exceed the concentrations limiting the growth of photosynthetic cyanobacteria, eukaryotic algae and/or macrophytes (Parker, 1984; Blanc et al., 1986; Meganck and Faup, 1988; Argaman, 1991; Cole, 1993). In a healthy water body, stable microbial communities exist as components of balanced food chains (Winkler, 1984), and this stability or homeostasis is destroyed when the supply of these nutrients exceeds the metabolic requirements of these communities (Winkler, 1984). Our understanding of what these acceptable levels are is often poor and not always based on extensive scientific data. They will also vary of course with the receiving water body and its intended use and, because of political reasons, differ between countries. However, it is generally believed that phosphorus enrichment is more influential, especially since cyanobacteria can fix atmospheric nitrogen (Kortstee et al., 1994). Much has been written on the environmental effects of eutrophication, now a world-wide problem, on a water body and its ecosystem (Vinconneau et al., 1985; Meganck and Faup, 1988; Argaman, 1991), but the most obvious visual change is the development of a bloom of these photosynthetic organisms Fig. 8.1, which not only decreases the aesthetic appeal but places an increased demand on the utilization of oxygen present in that water. When these blooms eventually die, breakdown of the biomass by aerobic chemoheterotrophic bacteria leads to oxygen consumption and depletion, often followed by death of the fauna (Argaman, 1991). Proliferation of phytoplankton increases water turbidity (Mackenthun, 1973) further threatening the existence of other photosynthetic organisms requiring light energy, and consequently fish species, due to a decrease in 02 production.