Microbial quantification in activated sludge: the hits and misses

Since the implementation of the activated sludge process for treating wastewater, there has been a reliance on chemical and physical parameters to monitor the system. However, in biological nutrient removal (BNR) processes, the microorganisms responsible for some of the transformations should be used to monitor the processes with the overall goal to achieve better treatment performance. The development of in situ identification and rapid quantification techniques for key microorganisms involved in BNR are required to achieve this goal. This study explored the quantification of Nitrospira, a key organism in the oxidation of nitrite to nitrate in BNR. Two molecular genetic microbial quantification techniques were evaluated: real-time polymerase chain reaction (PCR) and fluorescence in situ hybridisation (FISH) followed by digital image analysis. A correlation between the Nitrospira quantitative data and the nitrate production rate, determined in batch tests, was attempted. The disadvantages and advantages of both methods will be discussed.

Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community

The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 1 6S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16SrDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like 'Dehalococcoides ethenogenes' or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56(.)6%) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18(.)5%), the Chloroflexi phylum (16(.)4%), the Bacteroidetes phylum (6(.)3%), the Synergistes genus (11(.)1%) and a lineage that could not be affiliated with existing phyla (11(.)1%). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured microorganisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNAand differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.

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.

The treatment of domestic wastewater using small-scale vermicompost filter beds

The aim of this study is to quantity the effect of filter bed depth and solid waste inputs on the performance of small-scale vermicompost filter beds that treat the soluble contaminants within domestic wastewater. The study also aims to identify environmental conditions within the filters by quantifying the oxygen content and pH of wastewater held within it. Vermicompost is being utilised within commercially available on-site domestic waste treatment systems however, there are few reported studies that have examined this medium for the purpose of wastewater treatment. Three replicate small-scale reactors were designed to enable wastewater sampling at five reactor depths in 10-cm intervals. The surface of each reactor received household solid organic waste and 1301 m(-2) per day of raw domestic wastewater. The solid waste at the filter bed surface leached oxygen demand into the wastewater flowing through it. The oxygen demand was subsequently removed in lower reactor sections. Both nitrification and denitrification occurred in the bed. The extent of denitrification was a function of BOD leached from the solid waste. The environmental conditions measured within the bed were found to be suitable for earthworms living within them. The study identified factors that will affect the performance and application of the vermicompost filtration technology. (C) 2004 Elsevier B.V. All rights reserved.

The missing ingredient:inter-municipal cooperation and central-local relations in the UK

The thrust of the argument presented in this chapter is that inter-municipal cooperation (IMC) in the United Kingdom reflects local government's constitutional position and its exposure to the exigencies of Westminster (elected central government) and Whitehall (centre of the professional civil service that services central government). For the most part councils are without general powers of competence and are restricted in what they can do by Parliament. This suggests that the capacity for locally driven IMC is restricted and operates principally within a framework constructed by central government's policy objectives and legislation and the political expediencies of the governing political party. In practice, however, recent examples of IMC demonstrate that the practices are more complex than this initial analysis suggests. Central government may exert top-down pressures and impose hierarchical directives, but there are important countervailing forces. Constitutional changes in Scotland and Wales have shifted the locus of central- local relations away from Westminster and Whitehall. In England, the seeding of English government regional offices in 1994 has evolved into an important structural arrangement that encourages councils to work together. Within the local government community there is now widespread acknowledgement that to achieve the ambitious targets set by central government, councils are, by necessity, bound to cooperate and work with other agencies. In recent years, the fragmentation of public service delivery has affected the scope of IMC. Elected local government in the UK is now only one piece of a complex jigsaw of agencies that provides services to the public; whether it is with non-elected bodies, such as health authorities, public protection authorities (police and fire), voluntary nonprofit organisations or for-profit bodies, councils are expected to cooperate widely with agencies in their localities. Indeed, for projects such as regeneration and community renewal, councils may act as the coordinating agency but the success of such projects is measured by collaboration and partnership working (Davies 2002). To place these developments in context, IMC is an example of how, in spite of the fragmentation of traditional forms of government, councils work with other public service agencies and other councils through the medium of interagency partnerships, collaboration between organisations and a mixed economy of service providers. Such an analysis suggests that, following changes to the system of local government, contemporary forms of IMC are less dependent on vertical arrangements (top-down direction from central government) as they are replaced by horizontal modes (expansion of networks and partnership arrangements). Evidence suggests, however that central government continues to steer local authorities through the agency of inspectorates and regulatory bodies, and through policy initiatives, such as local strategic partnerships and local area agreements (Kelly 2006), thus questioning whether, in the case of UK local government, the shift from hierarchy to network and market solutions is less differentiated and transformation less complete than some literature suggests. Vertical or horizontal pressures may promote IMC, yet similar drivers may deter collaboration between local authorities. An example of negative vertical pressure was central government's change of the systems of local taxation during the 1980s. The new taxation regime replaced a tax on property with a tax on individual residency. Although the community charge lasted only a few years, it was a highpoint of the then Conservative government policy that encouraged councils to compete with each other on the basis of the level of local taxation. In practice, however, the complexity of local government funding in the UK rendered worthless any meaningful ambition of councils competing with each other, especially as central government granting to local authorities is predicated (however imperfectly) on at least notional equalisation between those areas with lower tax yields and the more prosperous locations. Horizontal pressures comprise factors such as planning decisions. Over the last quarter century, councils have competed on the granting of permission to out-of-town retail and leisure complexes, now recognised as detrimental to neighbouring authorities because economic forces prevail and local, independent shops are unable to compete with multiple companies. These examples illustrate tensions at the core of the UK polity of whether IMC is feasible when competition between local authorities heightened by local differences reduces opportunities for collaboration. An alternative perspective on IMC is to explore whether specific purposes or functions promote or restrict it. Whether in the principle areas of local government responsibilities relating to social welfare, development and maintenance of the local infrastructure or environmental matters, there are examples of IMC. But opportunities have diminished considerably as councils lost responsibility for services provision as a result of privatisation and transfer of powers to new government agencies or to central government. Over the last twenty years councils have lost their role in the provision of further-or higher-education, public transport and water/sewage. Councils have commissioning power but only a limited presence in providing housing needs, social care and waste management. In other words, as a result of central government policy, there are, in practice, currently far fewer opportunities for councils to cooperate. Since 1997, the New Labour government has promoted IMC through vertical drivers and the development; the operation of these policy initiatives is discussed following the framework of the editors. Current examples of IMC are notable for being driven by higher tiers of government, working with subordinate authorities in principal-agent relations. Collaboration between local authorities and intra-interand cross-sectoral partnerships are initiated by central government. In other words, IMC is shaped by hierarchical drivers from higher levels of government but, in practice, is locally varied and determined less by formula than by necessity and function. © 2007 Springer.

High temperature gas cleaning for advanced syngas utilization:Paper # 95

Biofuels and chemicals from biomass mean the gasification of biogenic feedstocks and the synthesis via methanol, dimethylester (DME) or Fischer-Tropsch products. To prevent the sensitive synthesis catalysts from poisoning the syngas must be free of tar and particulates. The trace concentrations of S-, C1-, N-species, alkali and heavy metals must be of the order of a few ppb. Moreover maximum conversion efficiency will be achieved performing the gas cleaning above the synthesis conditions. The concept of an innovative dry HTHP syngas cleaning is presented. Based on the HT particle filtration and suitable sorption and catalysis processes for the relevant contaminants a total concept will be derived, which leads to a syngas quality required for synthesis catalysts in only 2 combined stages. The experimental setup for the HT gas cleaning behind the 60 kWtherm entrained flow gasifier REGA of the institute is described. Results from HT filter experiments in pilot scale are presented. The performance of 2 natural minerals for HC1 and H2S sorption is discussed with respect to the parameters temperature, surface and residence time. Results from lab scale investigations on low temperature tar catalysts' performance (commercial and proprietary development) are discussed finally.