Nitrifying microbial community analysis of nitrite accumulating biofilm reactor by fluorescence in situ hybridization

Biological nitrogen removal via nitrite pathway in wastewater treatment is very important especially in the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulations were carried out in a biofilm reactor. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.2 kg N/m3/d. Nitrite accumulation was achieved by the selective inhibition of nitrite oxidizers by free ammonia and oxygen limitation. Nitrite oxidation activity was recovered as soon as the inhibition factor was removed. Fluorescence in situ hybridization studies of the nitrite accumulating biofilm system have shown that genus Nitrosomonas which is specifically hybridized with probe NSM 156 was the dominant nitrifying bacteria while Nitrospira was less abundant than those of normal nitrification systems. Further FISH analysis showed that the combinations of Nitrosomonas and Nitrospira cells were identified as important populations of nitrifying bacteria in an autotrophic nitrifying biofilm system.

Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity

Twenty-one strains of Bacillus (10 B. stearothermophilus, 3 B. cereus, and 8 B. licheniformis strains) were assayed for spore surface hydrophobicity on the basis of three measures: contact angle measurement (CAM), microbial adhesion to hydrocarbons (MATH), and hydrophobic interaction chromatography (HIC). On the basis of the spore surface characteristics obtained from these assays, along with data on the heat resistance of these spores in water, eight strains of Bacillus (three B. stearothermophilus, three B. cereus, and two B. licheniformis strains) either suspended in water or adhering to stainless steel were exposed to sublethal heat treatments at 90 to 110degreesC to determine heat resistance (D-value). Significant increases in heat resistance (ranging from 3 to 400%) were observed for the eight strains adhering to stainless steel. No significant correlation was found between these heat resistance increases and spore surface characteristics as determined by the three hydrophobicity assays. There was a significant positive correlation between the hydrophobicity data obtained by the MATH assay and those obtained by the HIC assay, but these data did not correlate with those obtained by the CAM assay.

Enrichment of nitrifying microbial communities from shrimp farms and commercial inocula

Nitrifying bacteria were selected from shrimp farm water and sediment (natural seed) in Thailand and from commercial seed cultures. The microbial consortia from each source giving the best ammonia removal during batch culture pre-enrichments were used as inocula for two sequencing batch reactors (SBRs). Nitrifiers were cultivated in the SBRs with 100 mg NH4-N/I and artificial wastewater containing 25 ppt salinity. The two SBRs were operated at a 7 d hydraulic retention time (HRT) for 77 d after which the HRT was reduced to 3.5 d. The amounts of ammonia removed from the influent by microorganisms sourced from the natural seed were 85% and 92% for the 7 d HIRT and the 3.5 d HRT, respectively. The ammonia removals of microbial consortia from the commercial seed were 71% and 83% for these HRTs respectively. The quantity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) was determined in the SBRs using the most probable number (MPN) technique. Both AOB and NOB increased in number over the long-term operation of both SBRs. According to quantitative fluorescence in situ hybridisation (FISH) probing, AOB from the natural seed and commercial seed comprised 21 +/- 2% and 30 +/- 2%, respectively of all bacteria. NOB could not be detected with currently-reported FISH probes, suggesting that novel NOB were enriched from both sources. Taken collectively, the results from this study provide an indication that the nitrifiers from shrimp farm sources are more effective at ammonia removal than those from commercial seed cultures.

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.

A dynamic mathematical model for sequential leach bed anaerobic digestion of organic fraction of municipal solid waste

A mathematical model that describes the operation of a sequential leach bed process for anaerobic digestion of organic fraction of municipal solid waste (MSW) is developed and validated. This model assumes that ultimate mineralisation of the organic component of the waste occurs in three steps, namely solubilisation of particulate matter, fermentation to volatile organic acids (modelled as acetic acid) along with liberation of carbon dioxide and hydrogen, and methanogenesis from acetate and hydrogen. The model incorporates the ionic equilibrium equations arising due to dissolution of carbon dioxide, generation of alkalinity from breakdown of solids and dissociation of acetic acid. Rather than a charge balance, a mass balance on the hydronium and hydroxide ions is used to calculate pH. The flow of liquid through the bed is modelled as occurring through two zones-a permeable zone with high flushing rates and the other more stagnant. Some of the kinetic parameters for the biological processes were obtained from batch MSW digestion experiments. The parameters for flow model were obtained from residence time distribution studies conducted using tritium as a tracer. The model was validated using data from leach bed digestion experiments in which a leachate volume equal to 10% of the fresh waste bed volume was sequenced. The model was then tested, without altering any kinetic or flow parameters, by varying volume of leachate that is sequenced between the beds. Simulations for sequencing/recirculating 5 and 30% of the bed volume are presented and compared with experimental results. (C) 2002 Elsevier Science B.V. All rights reserved.

The impact of market forces on the sustainability of the biotechnology industry

his paper contains a warning for investors, executives, analysts and scientists about the sustainability of the biotechnology industry. The study upon which the paper is based examines the impact of market forces on the biotechnology industry and argues that the short-term focus of market driven policies and practices impacts on the sustainability of firms operating in the industry. The market is represented by the National Association of Securities Dealers, Automated Quotations Market (NASDAQ), considered to be one of the vehicles of the promotion of ''new economy'' companies and principles. Through the application of bibliometric data (using both refereed and non-refereed papers), matched with the long term tracking of the NASDAQ Biotechnology Index, the authors provide a clear indication that the short-term investment thinking is leading an industry that is characterised by long R&D cycles. There is an incompatibility between the shorter-term investment considerations and the long-term scientific developments the biotechnology industry is attempting to achieve. Graphs and illustrations are provided to portray the comparative data.

Towards definition of the global biotechnology value chain using cases from Australian biotechnology SMEs

The generic pharmaceutical value chain model has been employed to describe both the global pharmaceutical and biotechnology industries till now. This research investigates the organisational value chain in Australian biotechnology companies in order to assess the appropriateness of the pharmaceutical value chain to small-and medium-sized biotechnology companies. The main theme of the research is: Can a generic model of the organisational value chain be defined for the biotechnology industry? Emanating from the literature, two research propositions were developed. RP1: there are eight major definable elements/activities of the organisational value chain for the biotechnology industry. RP2: Coverage of the elements in the biotechnology value chain ranges from focused to broad. A multiple case study methodology was used to explore these propositions. To develop a number of case studies, data was collected from senior managers of small and medium Australian biotechnology companies using an interview instrument, as well as from publicly available documentation and through observation. The results were analysed using cross-case comparisons. The results showed that an aggregation of the value chains of each organisation can be reduced to these eight definable elements that constitute the biotechnology value chain: basic research, applied research, development, verification and validation, prototype development, clinical trials, manufacturing and marketing. However, the findings also indicate that these major elements of the value chain need to be further reduced into sub-activities or sub-tasks to cater for the unique differences between biotechnology companies. Generally, the findings were consistent with the literature. However, a wider sampling, including international biotechnology organisations should be studied. The major contribution of this research is in the development of a value chain model, including general sub-tasks, for the Australian biotechnology industry.

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.

The 'parallel bridge' and its relevance to biotechnology new ventures

A firm's competitive strategy and innovation processes are strongly influenced by, and must be responsive to, its competitive environment. This is nowhere more strongly evident than in the high technology industries. In the present work, case studies of biotechnology new ventures are presented. These studies illustrate how an initial market entry strategy of parallel competition (through creative imitation) has enabled several biotechnology start-ups to reduce their mortality risk. We coin the term ''parallel bridge'' to describe this strategy. The parallel bridge provides early cash flows which support research and development and provide time for new ventures to develop core competencies, including a capacity to produce second and third horizon products that will sustain longer term competitiveness.

The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass

The development of a strong, active granular sludge bed is necessary for optimal operation of upflow anaerobic sludge blanket reactors. The microbial and mechanical structure of the granules may have a strong influence on desirable properties such as growth rate, settling velocity and shear strength. Theories have been proposed for granule microbial structure based on the relative kinetics of substrate degradation, but contradict some observations from both modelling and microscopic studies. In this paper, the structures of four granule types were examined from full-scale UASB reactors, treating wastewater from a cannery, a slaughterhouse, and two breweries. Microbial structure was determined using fluorescence in situ hybridisation probing with 16S rRNA-directed oligonucleotide probes, and superficial structure and microbial density (volume occupied by cells and microbial debris) assessed using scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The granules were also modelled using a distributed parameter biofilm model, with a previously published biochemical model structure, biofilm modelling approach, and model parameters. The model results reflected the trophic structures observed, indicating that the structures were possibly determined by kinetics. Of particular interest were results from simulations of the protein grown granules, which were predicted to have slow growth rates, low microbial density, and no trophic layers, the last two of which were reflected by microscopic observations. The primary cause of this structure, as assessed by modelling, was the particulate nature of the wastewater, and the slow rate of particulate hydrolysis, rather than the presence of proteins in the wastewater. Because solids hydrolysis was rate limiting, soluble substrate concentrations were very low (below Monod half saturation concentration), which caused low growth rates. (C) 2003 Elsevier Ltd. All rights reserved.

Enhanced biological phosphorus removal in a sequencing batch reactor using propionate as the sole carbon source

An enhanced biological phosphorus removal (EBPR) system was developed in a sequencing batch reactor (SBR) using propionate as the sole carbon source. The microbial community was followed using fluorescence in situ hybridization (FISH) techniques and Candidatus 'Accumulibacter phosphatis' were quantified from the start up of the reactor until steady state. A series of SBR cycle studies was performed when 55% of the SBR biomass was Accumulibacter, a confirmed polyphosphate accumulating organism (PAO) and when Candidatus 'Competibacter phosphatis,' a confirmed glycogen-accumulating organism (GAO), was essentially undetectable. These experiments evaluated two different carbon sources (propionate and acetate), and in every case, two different P-release rates were detected. The highest rate took place while there was volatile fatty acid (VFA) in the mixed liquor, and after the VFA was depleted a second P-release rate was observed. This second rate was very similar to the one detected in experiments performed without added VFA. A kinetic and stoichiometric model developed as a modification of Activated Sludge Model 2 (ASM2) including glycogen economy, was fitted to the experimental profiles. The validation and calibration of this model was carried out with the cycle study experiments performed using both VFAs. The effect of pH from 6.5 to 8.0 on anaerobic P-release and VFA-uptake and aerobic P-uptake was also studied using propionate. The optimal overall working pH was around 7.5. This is the first study of the microbial community involved in EBPR developed with propionate as a sole carbon source along with detailed process performance investigations of the propionate-utilizing PAOs. (C) 2004 Wiley Periodicals, Inc.

An in vitro study of the anti-microbial efficacy of a 1% silver sulphadiazine and 0.2% chlorhexidine digluconate cream Silvazine (TM), 1% silver sulphadiazine cream Flamazine (TM) and a silver coated dressing Acticoat (TM)

Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical anti-microbial agents has helped improve the survival in these patients. There are a number of anti-microbials available, one of which, Silvazine(TM) (1% silver sulphadiazine (SSD) and 0.2% chlorhexidine digluconate), is used only in Australasia. No study, in vitro or clinical, had compared Silvazine(TM) with the new dressing Acticoat(TM). This study compared the anti-microbial activity of Silvazine(TM), Acticoa(TM) and 1% silver sulphadiazine (Flamazine(TM)) against eight common burn wound pathogens. Methods: Each organism was prepared as a suspension. A 10 mul inoculum of the chosen bacterial isolate (representing approximately between 104 and 105 total bacteria) was added to each of four vials, followed by samples of each dressing and a control. The broths were then incubated and 10 mul loops removed at specified intervals and transferred onto Horse Blood Agar. These plates were then incubated for 18 hours and a colony count was performed. Results: The data demonstrates that the combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) results in the most effective killing of all bacteria. SSD and Acticoat(TM) had similar efficacies against a number of isolates, but Acticoat(TM) seemed only bacteriostatic against E. faecalis and methicillin-resistant Staphylococcus aureus. Viable quantities of Enterobacter cloacae and Proteus mirabilis rei named at 24 h. Conclusion: The combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) is a more effective anti-microbial against a number of burn wound pathogens in this in vitro study. A clinical study of its in vivo anti-microbial efficacy is required. (C) 2003 Elsevier Ltd and ISBI. All rights reserved.