Microscale structure and function of anaerobic-aerobic granules containing glycogen accumulating organisms

The spatial arrangement and metabolic activity of 'Candidatus Competibacter phosphatis' was investigated in granular sludge from an anaerobic-aerobic sequencing batch reactor enriched for glycogen-accumulating organisms. In this process, the electron donor (acetate) and the electron acceptor (oxygen) were supplied sequentially in each phase. The organism, identified by fluorescence in situ hybridisation, was present throughout the granules; however, metabolic activity was limited to a 100-mum-thick layer immediately below the surface of the granules. To investigate the cause of this, oxygen microsensors and a novel microscale biosensor for volatile fatty acids were used in conjunction with chemical staining for intracellular storage polymers. It was found that the limited distribution of activity was caused by mass transport limitation of oxygen into the granules during the aerobic phase. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

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.

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.

Postsynthesis stabilization of free-standing mesoporous silica films

Mixed ammonia-water vapor postsynthesis treatment provides a simple and convenient method for stabilizing mesostructured silica films. X-ray diffraction, transmission electron microscopy, nitrogen adsorption/desorption, and solid-state NMR (C-13, Si-29) were applied to study the effects of mixed ammonia-water vapor at 90 degreesC on the mesostructure of the films. An increased cross-linking of the silica network was observed. Subsequent calcination of the silica films was seen to cause a bimodal pore-size distribution, with an accompanying increase in the volume and surface area ratios of the primary (d = 3 nm) to secondary (d = 5-30 nm) pores. Additionally, mixed ammonia-water treatment was observed to cause a narrowing of the primary pore-size distribution. These findings have implications for thin film based applications and devices, such as sensors, membranes, or surfaces for heterogeneous catalysis.

Effects of phosphorus on the growth and nitrogen fixation rates of Lyngbya majuscula: implications for management in Moreton Bay, Queensland

Significant acetylene reduction and therefore N-2 fixation was observed for Lyngbya majuscula only during dark periods, which suggests that oxygenic photosynthesis and N-2 fixation are incompatible processes for this species. Results from a series of batch and continuous-flow-culture reactor studies showed that the specific growth rate and N-2 fixation rate of L, majuscula increased with phosphate (P-PO4) concentration up to a maximum value and thereafter remained constant. The P-PO4 concentrations corresponding to the maximum N-2 fixation and maximum growth rates were -0.27 and -0.18 muM respectively and these values are denoted as the saturation values for N-2 fixation and growth respectively. Regular monitoring studies in Moreton Bay, Queensland, show that concentrations Of P-PO4 generally exceed these saturation values over a large portion of the Bay and therefore, the growth of the bloom-forming L, majuscula is potentially maximised throughout much of the Bay by the elevated P-PO4 concentrations. Results from other studies suggest that the elevated P-PO4 concentrations in the Bay can be largely attributed to discharges from waste-water treatment plants (WWTPs), and thus it is proposed that the control of the growth of L. majuscula in Moreton Bay will require a significant reduction in the P load from the WWTP discharges. If the current strategy of N load reduction for these discharges is maintained in the absence of substantial P load reduction, it is hypothesised that the growth of L, majuscula and other diazotrophs in Moreton Bay will increase in the future.

Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria

A bacterial culture collection of 104 strains was obtained from an activated sludge wastewater treatment plant to pursue studies into microbial flocculation. Characterisation of the culture collection using a polyphasic approach indicated seven isolates, phylogenetically affiliated with the deep-branching Xanthomonas group of the class Gammaproteobacteria, were unable to hybridise the GAM42a fluorescence in situ hybridisation (FISH) probe for Gammaproteobacteria. The sequence of the GAM42a probe target region in the 23S rRNA gene of these isolates was determined to have mismatches to GAM42a. Probes perfectly targeting the mismatches (GAM42a_TI038_G1031, and GAM42a_T1038 and GAM42a_A1041_A1040) were synthesised, and used in conjunction with GAM42a in FISH to,study the Gammaproteobacteria community structure in one full-scale activated sludge plant. Several bacteria in the activated sludge biomass bound the modified probes demonstrating their presence and the fact that these Gammaproteobacteria have been overlooked in community structure analyses of activated sludge. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

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.

Adaptations of strangler figs to life in the rainforest canopy

Figs are rainforest keystone species. Non-strangler figs establish on the forest floor; strangler figs establish epiphytically, followed by a dramatic transition from epiphyte to free-standing tree that kills its hosts. Free-standing figs display vigorous growth and resource demand suggesting that epiphytic strangler figs require special adaptations to deal with resource limitations imposed by the epiphytic environment. We studied epiphytic and free-standing strangler figs, and non-strangler figs in tropical rainforest and in cultivation, as well as strangler figs in controlled conditions. We investigated whether the transition from epiphyte to free-standing tree is characterised by morphological and physiological plasticity. Epiphyte substrate had higher levels of plant-available ammonium and phosphate, and similar levels of nitrate compared with rainforest soil, suggesting that N and P are initially not limiting resources. A relationship was found between taxonomic groups and plant N physiology; strangler figs, all members of subgenus Urostigma, had mostly low foliar nitrate assimilation rates whereas non-strangler figs, in subgenera Pharmacocycea, Sycidium, Sycomorus or Synoecia, had moderate to high rates. Nitrate is an energetically expensive N source, and low nitrate use may be an adaptation of strangler figs for conserving energy during epiphytic growth. Interestingly, significant amounts of nitrate were stored in fleshy taproot tubers of epiphytic stranglers. Supporting the concept of plasticity, leaves of epiphytic Ficus benjamina L. had lower N and C content per unit leaf area, lower stomatal density and 80% greater specific leaf area than leaves of conspecific free-standing trees. Similarly, glasshouse-grown stranglers strongly increased biomass allocation to roots under water limitation. Epiphytic and free-standing F. benjamina had similar average foliar delta C-13, but epiphytes had more extreme values; this indicates that both groups of plants use the C-3 pathway of CO2 fixation but that water availability is highly variable for epiphytes. We hypothesise that epiphytic figs use fleshy stem tubers to avoid water stress, and that nitrate acts as an osmotic compound in tubers. We conclude that strangler figs are a unique experimental system for studying the transition from rainforest epiphyte to tree, and the genetic and environmental triggers involved.

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation' Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO2- uptake than NH4 uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO2- from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal. (c) 2006 Elsevier Inc. All rights reserved.

Construction and analysis of a metagenomic library from an enhanced biological phosphorus removal biomass

The enhanced biological phosphorus removal (EBPR) process is regularly used for the treatment of wastewater, but suffers from erratic performance. Successful EBPR relies on the growth of bacteria called polyphosphate-accumulating organisms (PAOs), which store phosphorus intracellularly as polyphosphate, thus removing it from wastewater. Metabolic models have been proposed which describe the measured chemical transformations, however genetic evidence is lacking to confirm these hypotheses. The aim of this research was to generate a metagenomic library from biomass enriched in PAOs as determined by phenotypic data and fluorescence in situ hybridisation (FISH) using probes specific for the only described PAO to date, Candidatus Accumulibacter phosphatis. DNA extraction methods were optimised and two fosmid libraries were constructed which contained 93 million base pairs of metagenomic data. Initial screening of the library for 16S rRNA genes revealed fosmids originating from a range of non-pure-cultured wastewater bacteria. The metagenomic libraries constructed will provide the ability to link phylogenetic and metabolic data for bacteria involved in nutrient removal from wastewater. Keywords DNA extraction; EBPR; metagenomic library; 16S rRNA gene.

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass)center dot h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass)center dot h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD center dot h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO(2)(-)-N/L, independent of the presence or absence of CO2. (c) 2006 Wiley Periodicals, Inc.

Stoichiometric and kinetic characterisation of Nitrosomonas sp in mixed culture by decoupling the growth and energy generation processes

A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosolnonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO, were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14 - 0.16 mgN mgCOD(biomass)(-1) h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH7. (c) 2006 Elsevier B.V. All rights reserved.