The rehabilitation of degraded soils, such as those associated with post-mining sites, requires knowledge of the soil ecosystem and its physical, chemical, and biological composition in order for rehabilitation efforts to fulfil the long-term goal of reconstructing a stable ecosystem for rehabilitated mine soil. This study addresses the need for appropriate assessment criteria to determine the progress of rehabilitation and subsequently the success of management practices. Significant contributions made by this investigation included the establishment of minimum and maximum values for microbial community measurements from two case studies of rehabilitated coal discard sites. Furthermore, it was shown that there was no relationship between changes in microbial community function and structure and the rehabilitation age of the sites. Following this, the considerable impact of management practices on microbial communities was illustrated. The first part of the study investigated the temporal changes in microbial community function and structure in a chronosequence of rehabilitated coal discard sites aged 1 to 11 years. The most important observation made during the investigation of the microbial communities in the different aged soil covers of the rehabilitated coal discard sites, was that there was no relationship between rehabilitation age and microbial activity or abundance of certain microbial groups. What was responsible for a clear differentiation between sites and a shift in microbial community attributes was the management practices applied. A comparison of two chronosequences of rehabilitated coal discard sites was achieved by an application of the 'space-for-time' hypothesis. Sites of different ages and at separate locations ('space') were identified to obtain a chronosequence of ages ('time'). The two chronosequences included sites aged 1 to 11 years (chronosequence A) and 6 to 17 years (chronosequence B), respectively. Sites in the same chronosequence were managed identically, while there was a distinct difference in management practices applied to each chronosequence. The long-term effect of the different management regimes on the soil microbial community function and structure was investigated. Again, there was no relationship between rehabilitation age and microbial community measurements. Fluctuations of selected microbial properties occurred in both chronosequences and similar temporal trends existed over the rehabilitation periods. However, the less intensively managed chronosequence (8) seemed more stable (less fluctuation occurred) over the rehabilitation period than the more intensively managed chronosequence (A). It was therefore concluded that the microbial communities in the less managed sites maintained their functional and structural integrity within bounds in the absence of management inputs or disturbance. While there was similarity in the trends over time for individual microbial community measurements, the seemingly more stable conditions in chronosequence 6 are important in terms of the goal of rehabilitation. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007

A review of the literature indicates that the understanding of the mixing phenomena in anaerobic digesters is limited and the ability to measure digester mixing characteristics is lacking. Moreover, rheological characteristics of the sludge are largely ignored. The need for a more thorough understanding of fundamental mixing relationships and the ability to measure these relationships in the anaerobic digester is recognized. To this end, investigations using experimental and numerical methods to visualize flow patterns and quantify mixing that impact biogas yields is reported. Results from this research identifies optimum mixing regimes for digesters depending on their sludge rheology, operational regimes, digester dimensions and mixing systems, and also produces an industrial guide to improve digester design. The results show that in order to accurately predict the optimum mixing regime for a particular anaerobic digester, it is necessary to determine to what extent biogas output is dependent upon, and can be influenced by flow patterns in an anaerobic digester. These flows are in turn determined by physical characteristics of the digester, the feed regime, the mixing system and the rheological properties of the sludge. Mixing regimes are tailored to produce flow patterns that could potentially increase biogas production and/or reduce mixing energy input of the digester. An all-inclusive anaerobic digester model is developed and validated using advanced experimental techniques that can be used to optimize the digestion process in terms of physical and biological parameters. In either case, the model provides an ongoing management of anaerobic digester process to improve overall energy yield from the digester and reduce its environmental impact.

The new field of ‘omics’ has spawned the development of metaproteomics, an approach that has the ability to identify and decipher the metabolic functions of a proteome derived from a microbial community that is largely uncultivable. With the development and availabilities of high throughput proteomics, high performance liquid chromatography coupled to mass spectrometry (MS) has been leading the field for metaproteomics. MS-based metaproteomics has been successful in its’ investigations of complex microbial communities from soils to the human body. Like the environment, the human body is host to a multitude of microorganisms that reside within the skin, oral cavity, vagina, and gastrointestinal tract, referred to as the human microbiome. The human microbiome is made up of trillions of bacteria that outnumber human genes by several orders of magnitude. These microbes are essential for human survival with a significant dependence on the microbes to encode and carryout metabolic functions that humans have not evolved on their own. Recently, metaproteomics has emerged as the primary technology to understand the metabolic functional signature of the human microbiome. Using a newly developed integrated approach that combines metagenomics and metaproteomics, we attempted to address the following questions: i) do humans share a core functional microbiome and ii) how do microbial communities change in response to disease. This resulted in a comprehensive identification and characterization of the metaproteome from two healthy human gut microbiomes. These analyses have resulted in an extended application to characterize how Crohn’s disease affects the functional signature of the microbiota. Contrary to measuring highly complex and representative gut metaproteomes is a less complex, controlled human-derived microbial community present in the gut of gnotobiotic mice. This human gut model system enhanced the capability to directly monitor fundamental interactions between two dominant phyla, Bacteroides and Firmicutes, in gut microbiomes colonized with two or more phylotypes. These analyses revealed membership abundance and functional differences between phylotypes when present in either a binary or 12-member consortia. This dissertation aims to characterize host microbial interactions and develop MS-based methods that can provide a better understanding of the human gut microbiota composition and function using both approaches.

Densification of tailings slurries to mature fine tailings (MFT) is important in the oil sands industry for tailings inventory reduction, pore water recovery and tailings reclamation. The cause of methane release from the tailings pond of Shell Albian Sands (Albian) and the effects this process has on densification of Albian tailings was investigated. Citrate, added to tailings with polyacrylamide and hydrocarbon-diluent, was identified as the methanogenic substrate. Bacterial and Archaeal 16S rRNA gene sequences in Albian MFT were dominated by matches to Rhodoferax, some Clostridia and sulfate-reducing bacteria, and acetoclastic methanogens. Citrate-, diluent-, and polyacrylamide-amendments to Albian MFT did not cause a microbial shift over a 10-month laboratory incubation period. A potential pathway for microbial methane production in Albian MFT is proposed. Methane production and release from citrate-amended Albian MFT correlated to accelerated densification. Though diluent and polyacrylamide did not affect methanogenesis, they potentially affect gas bubble formation and release. / Microbiology and Biotechnology

Contaminated aquatic sediments pose health risks to fish, wildlife, and humans and can limit recreational and economic uses of surface waters. Technical and cost effective in situ approaches for sediment management and remediation have been identified as a research need. Subaqueous in situ capping is a promising remedial approach; however, little is known regarding its impact on underlying sedimentary processes and the feasibility of bioaugmented caps at sites subject to contaminated groundwater seepage. This work specifically addresses (1) the impact of capping on biogeochemical processes at the sediment-water interface, (2) the ability and degree to which indigenous sediment microorganisms colonize an overlying cap, (3) the effect of advective flow direction on redox conditions within a cap, (4) natural contaminant bioattenuation processes within capped sediment, and (5) limitations toward a functional bioreactive in situ cap. Laboratory-scale experiments with capped sediment columns demonstrated that emplacement of a sand-based in situ cap induced an upward, vertical shift of terminal electron accepting processes into the overlying cap while simultaneously conserving redox stratification. Upflow conditions simulating a groundwater seep compressed anaerobic processes towards the cap-water interface. Microorganisms indigenous to the underlying sediment colonized cap material and spatial population differences generally reflected redox stratification. Downflow of oxic surface water through the cap, simulating tidally-induced recharge, created fully oxic conditions within the cap, demonstrating that flow direction strongly contributes to redox conditions. Experiments simulating capped sediment subject to contaminated groundwater seepage revealed a reduction of natural bioattenuation processes with time, stemming from the elimination of labile organic matter deposition to the sediment and a subsequent lack of electron donor. Thus, parent contaminants within groundwater seeps will be subject to minimal biotransformations within the sediment before entering a reducing cap. A bioreactive cap, inoculated with microorganisms capable of reductive dehalogenation, was established to reductively dechlorinate tetrachloroethene present in the groundwater; however electron donor amendments to sediment effluent were required to achieve complete dechlorination of tetrachloroethene to non-toxic ethene. Results from this work improve understanding of biogeochemical and bioattenuation processes within capped aquatic sediments and should aid in the development of active capping technologies.

Plant residues added to soil are a source of nutrients for plants and soil organisms and increase soil organic matter which has an important role in improving soil structure and fertility, hence maintaining soil quality for sustainable agriculture. In order to utilize plant residues for increasing soil organic matter more effectively, it is necessary to understand the mechanisms of plant residue decomposition. Soil organic matter decomposition is influenced by several factors such as plant residue quality, temperature, water availability, soil structure and soil texture, particularly clay content. The interaction of clay and decomposition of organic matter has been studied in the past. Nevertheless, many studies investigated this interaction in natural soil or under field conditions over long periods of time. Variation in environmental factors may influence the interaction of clay and decomposition of organic matter, thus in most previous studies their effect cannot be separated from the direct effect of clay on decomposition. To study the direct effect of clay on organic matter decomposition, four experiments with different objectives were carried out using isolated natural clay, under controlled conditions (e.g. temperature and organic matter input) and a short incubation period (approximately one month). All experiments were carried out using a sand matrix to which different clay types, clay fractions (natural or with iron oxide partially removed) or clay concentrations were added together with mature wheat straw (C/N 122 in most experiments, except Experiment 2 where the wheat straw had a C/N of 18) and a microbial inoculum. To investigate the effect of clay type, two clay types were added. They were isolated from Wiesenboden (W) and Red Brown Earth (RBE) soil. Clay types from both soils contained kaolinite and illite, but smectite only occurred in W clay. Iron oxide is thought to be important for the binding of organic matter to clay, therefore two clay fractions were used, the clay with native iron oxide (natural clay) and clay from which iron oxide was partially removed by citrate-dithionite-bicarbonate treatment (citrate-dithionite clay, CD clay). The following parameters were measured: pH, water loss, respiration rate, microbial community structure using phospholipid fatty acid analysis and, in some experiments, particulate organic matter. In all experiments, the water content of the substrate mixes was adjusted only at the start; water loss was greatest in the control and decreased with increasing clay content. The aim of the first experiment was to study the effect of the concentration of W clay on decomposition of wheat residues. Respiration (i.e. decomposition of the wheat straw) was affected by clay in two ways (i) decreased decomposition, thus protection of organic matter, in the initial phase at all concentrations (5, 10, 20 and 40%) and throughout the incubation period at ≤ 20% clay, and (ii) greater water retention at higher clay concentration particularly 40% clay that allowed maintenance of higher respiration rates towards the end of incubation. Generally, clay concentration had an effect on microbial community structure but not on microbial biomass. The effect of clay concentration was also investigated in the second experiment, but using RBE clay and a narrower range of concentrations (0, 2.5, 5, 10 and 20% clay) than in the first experiment with W clay. The wheat residue used in this experiment had a lower C/N ratio compared to the other three experiments (C/N 18 compared to 122). In contrast to the first experiment, cumulative respiration of the clay treatments was greater than that of control throughout the incubation, thus clay increased rather than decreased decomposition. This may be due to the properties of the wheat residue used in this experiment which contained more water-soluble compounds, the diffusion of which would be enhanced in treatments with clay compared to the control due to their higher water availability. However, considering only the treatments with added clay, cumulative respiration followed the same pattern as in the first experiment, with highest cumulative respiration at 20% clay. In general, microbial community structure, microbial biomass and microbial groups (i.e. bacterial and fungal fatty acids) were affected by the presence of clay and sampling time, but there was no clear relationship between these factors and the richness and diversity of the microbial community. The aim of the third experiment was to determine the effect of clay concentration (5 and 40% of W clay) and fraction (natural or citrate-dithionite clay) on decomposition of wheat straw and microbial community structure. Clay fraction and concentration strongly affected the respiration rate and microbial community structure as well as microbial biomass but not the concentration of particulate organic matter (POM). Compared to the control, partial removal of iron oxide strongly increased decomposition at both concentrations whereas clay with iron oxides reduced the decomposition. Microbial community structure was affected by clay fractions, particularly at 40% clay. The aim of the fourth experiment was to determine the effect of clay fraction (natural and citrate-dithionite clay) and clay type (W clay or RBE clay) at 5% clay on decomposition of wheat straw and microbial community structure. Clay type and the partial removal of iron oxide had a significant effect on the decomposition rate but did not affect POM concentration. As in the third experiment, partial removal of iron oxide increased respiration rate, the effect was less pronounced in RBE clay than in W clay. Clay type and fraction strongly affected microbial community structure. In conclusion, the experiments showed that native clay generally reduces organic matter decomposition by binding and occlusion. The importance of iron oxide for the protective effect of clay on organic matter decomposition was shown by the fact that partial removal of iron oxide strongly increased decomposition rate compared to the native clay. The two clay types differed in their effect. The W clay containing smectite protects organic matter to a greater extent than RBE clay with predominantly illite and kaolinite due to its higher surface area and CEC that lead to binding and or occlusion. The results also showed that although clay reduces organic matter decomposition under optimal water availability, this effect can be reversed as the substrates dry out because the greater water retention of substrates with clay concentrations > 10% compared to the pure sand matrix allows maintenance of a greater microbial activity. Clay type, fraction and concentration affected microbial community structure via their effect on organic matter and water availability. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1521949 / Thesis (M.Ag.Sc.) -- University of Adelaide, School of Earth and Environment Science, 2010

Microbial communities associated with gastrointestinal tract of animals play a critical role in gut development, digestion and resistance to disease, thus the prospect of altering these communities beneficially by using probiotics is attractive. In terrestrial animals, the gut provides a stable, moist habitat in an otherwise moisture-limited environment, thus microbial communities tend to be very stable. In contrast, farmed aquatic animals reside within an environment that can support microbes in high densities, and as many marine animals drink continuously for osmoregulation, they are subjected to potential re-inoculation. Consequently, little is known of the stability of gut microbial communities in marine shrimp or whether it is possible to establish beneficial bacteria in the gut. The aims of this thesis were therefore to examine the midgut microbial community associated with farmed black tiger shrimp, Penaeus monodon, and to investigate whether the introduction of potentially probiotic Bacillus could alter the species diversity or abundance of the present microbes. Using culture methods it was found that B. pumilus was able to transfer between animals via the water column and persisted in the midgut for at least 7 days, while B. subtilis was only recovered from animals directly fed the bacteria and persisted for less than 24 h in the midgut. V. parahaemolyticus, a known shrimp pathogen,remained in the tanks it was originally found in, and did not transfer via the water column to other tanks and is therefore tightly associated with its host. A bacterium with apparent probiotic qualities was isolated from control animals in the above study and identified as a strain of B. pumilus. Its safety for food animal use was confirmed due to the absence of B. cereus toxin genes, and the isolate’s pH and salt tolerances were investigated. Moreover, the isolate was highly inhibitory to crustacean pathogens in the family Vibrionaceae. Methods to investigate the gut microbiota using the full cycle 16S rRNA methodology were optimized. Fluorescence in situ hybridization (FISH) probes designed specifically targeting B. pumilus, B. subtilis and B. licheniformis, commercially available probiotics, were validated for specificity and optimal hybridization conditions. For FISH analysis of bacteria in situ in histological sections of shrimp midgut trunks, fixation times in 4 % paraformaldehyde wereoptimizedfor bacterial RNA retention whilst maintaining tissue integrity. Due to the broad range of autofluorescence in the shrimp tissue, spectral imaging is required to adequately differentiate between host tissue and multiple bacterial probes. The richness and diversity of the midgut microbiota of animals treated with the novel strain of B. pumiluswere analyzed using 16S rRNA gene clone libraries and FISH analysis of histological sections. It was confirmed that B. pumilus can enter the midgut via top-coated feed and through water inoculation. In the tanks that were treated with B. pumilus the proportion of Vibrio sp. in the microbial community decreased, however, only in the systems in which B. pumilus was recovered from the shrimp midgut did the proportion of pathogenic Vibrio species decrease. The application of the B. pumilus caused a shift in the shrimp midgut microbiota, but the community returned to its initial diversity over time. The midgut microbiota of P. monodon is relatively stable but can be adjusted using probiotics. The transience or residence of the probiotics is strain-specific and should be tested for any new strains before determining optimum application protocols. The methods designed in this study are applicable to future research in this field.

Proper management of soil organic matter (SOM) contributes to increasing plant productivity and reducing dependency on mineral fertilizers. Organic matter is widely regarded as a vital component of a healthy soil as it plays an important role in soil physical, chemical and biological fertility. Plant residues are the primary source of SOM. Therefore, proper SOM management requires a better understanding of plant residue decomposition kinetics in order to synchronize nutrient release during decomposition and plant uptake and prevent nutrient losses. In natural and managed ecosystems, residues are added frequently to soil, in the form of dead roots and litter fall of plant species with different C/N ratios. However, in most studies on residue decomposition, residues with different C/N ratios are added once and the effect of the presence of plants on residue decomposition is rarely investigated. In this project, four experiments were carried out with different objectives in order to close these knowledge gaps. The aim of the first experiment was to investigate the effect of frequent wheat residue addition on C mineralization and N dynamics. The experiment consisted of five treatments with different frequency of residue addition (2% w/w of wheat residues in total): once (100%W), every 16 days (25%), every 8 days (12.5%) or every 4 days (6.25%) and noresidue addition (control) with four replicates. The results showed that increasing frequency of low-N wheat residue addition increased C mineralization. Compared to 100%W, cumulative respiration per g residue at the end of the incubation (day 80) was increased by 57, 82 and 92% at 25%W, 12.5%W and 6.25%W, respectively. Despite large increases in cumulative respiration, frequent residue addition did not affect inorganic N or available N concentrations, microbial biomass C and N or soil pH. It is concluded that experiments with single residue additions may underestimate residue decomposition rates in the field because with several additions, soil microbes respire more of the added C (and possibly native soil C) per unit biomass but that this does not change their N requirements or the microbial community composition. In the second experiment, the effect of mixing of high and low C/N residues at different times during incubation was investigated. There were 4 addition times; 25% of a total of 2% (w/w) residue was added either as wheat (high C/N) or lupin (low C/N) residue. Wheat residue was added to lupin residues on days 16 (LW-16), 32 (LW-32) or 48 (LW-48). Additional treatments were 100%L (added 25% of lupin residues on days 0, 16, 32 and 48) and 100%W (added 25% of wheat residues on days 0, 16, 32 and 48) and 0% (the control) with four replicates. Adding high C/N residues into decomposing low C/N ratio residue strongly decreased the respiration rate compared to the addition of low C/N residues, and lowered the availability of inorganic N, but significantly increased soil pH and altered microbial community composition. By the end of the incubation on day 64, the cumulative respiration of LW-16, LW-32 and LW-48 was similar and approximately 30% lower than in the treatment with only lupin residue addition. The third experiment studied the effect of spatial separation of high and low C/N residues on decomposition and N mineralization. Each microcosm consisted of two PVC caps of 70 mm diameter and 20 mm height with the open end facing each other separated by a 30μm mesh. The caps were filled with soil mixed with either low or high C/N residue with three replicates. Contact of high and low C/N residues led to an increase in the decomposition rate of the high C/N residues at the interface whereas it decreased it in the low C/N residues. The results showed that N and soluble C compounds moved from the easily decomposable residues into the surrounding soil, thereby enhancing microbial activity, increasing inorganic N and significantly changing soil pH in the layer 0-5 mm from the interface compared to the 5-10 mm layer of the high C/N residues, whereas the movement of soluble C and N to high C/N residues decreased the decomposition of the low C/N residues. The final experiment investigated the effect of living plants on decomposition of high and low C/N residues. Wheat was grown in pots with a 30 μm mesh at the bottom. After a root mat had formed (>50% root coverage), a PVC cap with soil with high and low C/N residues (2% w/w) was placed against the mesh. The presence of plant roots significantly increased the respiration rate, N immobilization and increased the soil pH in the 0-5 mm layer in the first 4 days compared to the 5-10 mm layer. This enhanced microbial activity (and probably microbial biomass) can be explained by root exudates. The microbial community composition of plant treatments differed significantly from treatments without plants and the effect was greater in the immediate vicinity of the roots. / Thesis (M.Ag.Sc.) -- University of Adelaide, School of Earth and Environmental Sciences, 2009

Les biofilms constituent un mode de vie microbien extrêmement répandu, aussi bien en milieu naturel que dans les environnements anthropisés. Dans ce dernier cas, les structures morphologique et microbiologique du biofilm vont conditionner son impact sur le système, que cet impact lui soit bénéfique ou préjudiciable. L'objectif de cette thèse est d'approfondir notre connaissance des phénomènes de structuration du biofilm afin, à terme, d'optimiser les performances de procédé. Afin de représenter au mieux les conditions industrielles, des régimes d'écoulement turbulents et des consortia microbiens complexes ont été utilisés. Une première partie se focalise sur l'impact des forces de cisaillement sur l'adhésion microbienne. Les résultats démontrent un changement progressif de la flore bactérienne fixée et de sa distribution spatiale. Dans un second temps, le projet s'est intéressé aux étapes de développement du biofilm et ont permis d'identifier un effet mémoire du biofilm mature. Il s'agit d'une conservation des structures morphologique et microbiologique au cours du temps en dépit d'un changement de régime hydrodynamique. Enfin la dernière partie a consisté en la mise au point d'une méthode de quantification des prédateurs mobiles dans les biofilms. Ces prédateurs participent à la structuration du biofilm et leur quantification peut s'avérer utile dans le contexte de l'épuration des eaux. / Biofilms are a biological mode of life widely spread in both natural and engineered environments. In the last case, whether the biofilm is beneficial or detrimental for the process under consideration, both morphology and microbial community of the biofilm determine its impact. The objective of this thesis is to deepen our knowledge of biofilm structuring and, as a further goal, optimize a given process. Turbulent flows and multi-species consortia were used in order to better mimic industrial conditions. The first part of the project focused on the impact of shear stress on microbial adhesion. Results have demonstrated a gradual shift in bacterial communities with shear and a change in the spatial distribution of adhered microorganisms. Secondly, the work dealt with biofilm development. A memory effect, defined as the conservation of initial morphological and microbiological features despite a change in the environmental conditions, has been observed. Finally, a method for quantification of moving predators in mature biofilms has been developed. These predators actively shape the biofilm and their quantification is valuable, especially for wastewater treatment.

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