scholarly journals Elucidation of the microbial community structure within a laboratory scale activated sludge process using molecular techniques

2006 ◽  
Author(s):  
◽  
Pamela Padayachee
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Denaturing Gradient Gel Electrophoresis ◽  
In Situ Hybridisation ◽  
Molecular Techniques ◽  
Laboratory Scale ◽  
Oligonucleotide Probes ◽  
Mass Balances ◽  
Nitrogen Levels ◽  
Gradient Gel Electrophoresis

The microbial community present in a laboratory-scale modified Ludzack-Ettinger activated sludge system was investigated using a combination of novel molecular techniques. The parent system was investigated for a duration of one year and samples were taken at regular intervals to determine the profile and structure of the microbial community present within the anoxic and aerobic zones of the MLE system. The combination of molecular techniques included fluorescent in situ hybridisation (FISH) and denaturing gradient gel electrophoresis (DGGE). FISH was performed using oligonucleotide probes, which were complementary to conserved regions of the rRNA for the alpha, beta and gamma subclasses of the gram negative family Proteobacteria as well as a group-specific HGC oligonucleotide probe as a representative of the gram positive actinomycetes branch. The total eubacteria present was determined using the EUB oligonucleotide probes, EUB388, EUB388-II and EUB388-III. The DGGE analysis of PCR-amplified 16S rDNA gene segments was used to examine the microbial community profile in the anoxic and aerobic zones. The profile for each of the zones revealed a number of consistent bands throughout the duration of the laboratory-scale process. However, the profiles obtained suggested that a diverse microbial community existed within the aerobic and anoxic zones. The bands also indicated the presence of dominant and less dominant species of bacteria. Hybridisations obtained from the FISH analyses indicated that the alpha and gamma subclasses were predominant within the anoxic zone and the aerobic zone showed a dominance of the beta subclass of Proteobacteria. The steady state behaviour of the MLE system was confirmed with the results obtained from COD, TKN, nitrates and OUR analytical tests. COD and nitrogen mass balances were conducted to confirm the acceptance of the results obtained for each batch as an indication of the system performance for the MLE model. Nitrogen mass balances indicated an upset in the nitrogen levels for batches two and seven.

Application of molecular methods to microbial community analysis of activated sludge

2000 ◽  
Vol 42 (3-4) ◽  
pp. 17-22 ◽  
Author(s):  
M. Onuki ◽  
H. Satoh ◽  
T. Mino ◽  
T. Matsuo
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Wastewater Treatment Plants ◽  
Community Analysis ◽  
Molecular Methods ◽  
Microbial Community Analysis ◽  
Community Structures ◽  
Microbial Community Structures ◽  
Gradient Gel Electrophoresis

In the last decade, molecular biology has made significant progress, and innovative molecular methods have become available to analyze microbial community structures. Among them, we applied the FISH (Fluorescent in situ Hybridization) method to analyze activated sludge in wastewater treatment plants (WWTPs). As a result, domain- or division-level community structures in activated sludge were determined successfully without cultivation. We also applied the PCR (polymerase chain reaction) -DGGE (Denaturing Gradient Gel Electrophoresis) method for laboratory nitrifying sludge in order to investigate more detailed microbial community structure. By this method, genus- or species-level community structures were characterized well. This method was also found to be powerful for monitoring the change of microbial community structures. For example, the behavior of Nitrosomonas group was successfully detected in the reactor with nitrification by the PCR-DGGE method.

Beginning a molecular analysis of the eukaryal community in activated sludge

1998 ◽  
Vol 37 (4-5) ◽  
pp. 455-460 ◽  
Author(s):  
Terence L. Marsh ◽  
Wen-Tso Liu ◽  
Larry J. Forney ◽  
Cheng Hans
Keyword(s):  
Activated Sludge ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Comparative Sequence Analysis ◽  
Small Subunit ◽  
Molecular Techniques ◽  
Initial Inoculum ◽  
Eukaryotic Community ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Total Community

As a means to furthering our understanding of the eukaryal community in activated sludge, we have applied contemporary molecular techniques to an activated sludge community maintained in a laboratory-scale bioreactor. The initial inoculum was derived from a local wastewater treatment facility in East Lansing, MI and maintained with continuous aeration and a daily feeding regime that included glucose and peptone. Samples were taken on a weekly basis for 5 weeks and community DNA was extracted from 2-5 ml of activated sludge. Using a variety of oligonucleotide primers specific to eukaryotic small subunit ribosomal DNA, we PCR amplified rDNA from the total community DNA. PCR products were analyzed by three techniques, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of rDNA clones. Over the course of 5 weeks, analysis with DGGE revealed dramatic changes in the eukaryotic community based on differences in denaturation profiles. However, the analysis is limited to 5-6 bands corresponding to 5-6 different “ribotypes”. Analysis with T-RFLP also suggests changes to the eukaryotic community over time. Both increases and decreases in population size can be detected as a function of time. Up to 15 different terminal restriction fragments can be detected with T-RFLP indicating that this technique is considerably more sensitive than DGGE. Phylogenetic analysis of partial sequences from 11 cloned rDNAs indicate that all 11 clones are from the Ciliophora phylum.

The comparison of the diversity of activated sludge plants

1998 ◽  
Vol 37 (4-5) ◽  
pp. 71-78 ◽  
Author(s):  
Thomas P. Curtis ◽  
Noel G. Craine
Keyword(s):  
Cluster Analysis ◽  
Activated Sludge ◽  
Treatment Process ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Optimal Sampling ◽  
Bacterial Populations ◽  
Large Numbers ◽  
Probable Presence ◽  
Gradient Gel Electrophoresis ◽  
Sampling Regime

The explicit engineering of bacterial populations requires that we know which organisms perform which tasks. The comparison of the bacterial diversity of activated sludge plants may give important information about the functions of different bacteria. This difficult task may be made easier by the use of technologies based on 16S rRNA based techniques. In this study we have used denaturing gradient gel electrophoresis (DGGE) to determine the optimal sampling regime for comparative studies and used cluster analysis to show how plants may be quantitatively compared. We sought evidence of spatial, diurnal and intrasample variation in a number of sites. No evidence for variation was found in the plants studied and we concluded that a single sample of an activated sludge plant was sufficient for a plant to plant comparison. The cluster analysis was able to distinguish between plants, though further work is required to find the most appropriate basis for such comparisons. We found organisms from raw sewage in the mixed liquor samples, these organisms may have no functional significance in the treatment process and thus complicate plant to plant comparisons as will the probable presence of heteroduplex rDNA products. Nevertheless we believe that these drawbacks do not outweigh the advantages of being able to take and compare relatively large numbers of samples.

scholarly journals Diversity of methanogens in ruminants in Queensland

2008 ◽  
Vol 48 (7) ◽  
pp. 722 ◽  
Author(s):  
D. Ouwerkerk ◽  
A. F. Turner ◽  
A. V. Klieve
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Rumen Fluid ◽  
Bos Indicus ◽  
Methane Emissions ◽  
Molecular Techniques ◽  
Feed Conversion ◽  
Methanogen Community ◽  
Gradient Gel Electrophoresis ◽  
Reduce Emissions ◽  
Cattle And Sheep

Methane emissions from ruminant livestock represent a loss of carbon during feed conversion, which has implications for both animal productivity and the environment because this gas is considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in different breeds of cattle and sheep, as well as in response to different diets, is required. A study was undertaken using the molecular techniques denaturing gradient gel electrophoresis, DNA cloning and DNA sequence analysis to define the extent of diversity among methanogens in ruminants, particularly Bos indicus cross cattle, on differing forages in Queensland. It was found that the diversity of methanogens in forage-fed cattle in Queensland was greater than in grain-fed cattle but there was little variability in methanogen community composition between cattle fed different forages. The species that dominate the rumen microbial communities of B. indicus cross cattle are from the genus Methanobrevibacter, although rumen-fluid inoculated digestors fed Leucaena leucocephala leaf were populated with Methanosphaera-like strains, with the Methanobrevibacter-like strains displaced. If ruminant methane emissions are to be reduced, then antimethanogen bioactives that target both broad groups of ruminant methanogens are most likely to be needed, and as a part of an integrated suite of approaches that redirect rumen fermentation towards other more useful end products.

Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene

2001 ◽  
Vol 43 (1) ◽  
pp. 77-82 ◽  
Author(s):  
O.-C. Chan ◽  
W.-T. Liu ◽  
H. H. Fang
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Granular Sludge ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Rrna Gene ◽  
Related Sequence ◽  
Gradient Gel Electrophoresis

The microbial community structure of granular sludge from an upflow anaerobic sludge blanket (UASB) reactor treating brewery effluent was studied by denaturing gradient gel electrophoresis (DGGE). Twelve major bands were observed in the DGGE fingerprint for the Bacteria domain and four bands for the Archaea domain. Of the bacterial bands observed, six were successfully purified and sequenced. Among them, three were related to the gram-positive low G+C group, one to the Delta subclass of the Proteobacteria, one to the Gamma subclass, and one to the Cytophaga group with no close related sequence. The 16S rRNA sequences of the four archaeal bands were closely associated with Methanosaeta concilii and Methanobacterium formicum.

Microbial Community Analysis of Underground Drinking Water Using Denaturing Gradient Gel Electrophoresis and Flow Cytometry Technologies

2014 ◽  
Vol 700 ◽  
pp. 519-524 ◽  
Author(s):  
Jie Liu ◽  
Yan Li Ding ◽  
Mark Bartlam ◽  
Ying Ying Wang
Keyword(s):  
Drinking Water ◽  
Microbial Community ◽  
Rural Areas ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Poyang Lake ◽  
Removal Rate ◽  
Underground Water ◽  
Water Sources ◽  
Gradient Gel Electrophoresis ◽  
Bacteria And Fungi

Underground water is directly used as drinking water in most rural areas of developing countries due to limitations in infrastructure. As an important indicator of drinking water quality, however, microbial quality has been largely ignored for a long time. Microbial quality poses a great threat to the safety of underground drinking water, especially in rural areas. The current study compared microbial abundance and community structure of three different water sources, i.e. underground water, Poyang Lake and Hai River, combined with flow cytometry (FCM) and denaturing gradient gel electrophoresis (DGGE). FCM results showed that the bacterial concentration of underground water is the lowest (1.037×106cell/ml) of the three water sources, but still approximates that of the Poyang Lake. The removal rate of bacteria after filtration through a 0.45μm-pore-size filter is 98.16% in underground water. The removal rate for Poyang Lake and Hai River is much lower (i.e. 66.57% and 74.17% respectively). DGGE profiles demonstrated that the microbial community structure in underground water shares higher similarity to Poyang Lake (51.0% and 53.1% similarity for bacteria and fungi respectively) than Hai River. The microbial diversity index (i.e. Shannon-Weaver index) for bacteria and fungi are 2.906 and 2.847 respectively in underground water, which is lower than in Poyang Lake. The evenness (i.e. Simpson index) of groundwater was lowest among the three water sources tested. The results suggested that groundwater has a complex microbial community and hence it is critical to apply necessary hygienic barriers to remove microbes for the safety of underground drinking water.

Integration of nitrification and denitrification by combining anoxic and aerobic conditions in a membrane bioreactor

2010 ◽  
Vol 62 (11) ◽  
pp. 2590-2598 ◽  
Author(s):  
Jianfeng Li ◽  
Fenglin Yang ◽  
Dieudonné-Guy Ohandja ◽  
Fook-Sin Wong
Keyword(s):  
Activated Sludge ◽  
Nitrogen Removal ◽  
Membrane Bioreactor ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Biomass Concentration ◽  
Morphological Properties ◽  
Utilization Rate ◽  
Gradient Gel Electrophoresis ◽  
Microbial Characterization ◽  
Nitrification And Denitrification

A membrane bioreactor (MBR) was developed to achieve nitrogen removal by combining nitrification and denitrification conditions in one reactor. The activated sludge was alternated between aerobic and anoxic conditions using peristaltic pump. The biomass concentration and floc morphological properties were observed to be similar in anoxic and aerobic compartments. However, the homogeneous properties of the activated sludge did not lead to the failure of oxygen gradient formation in the reactor. Due to the position of the air diffuser, an anoxic compartment at the bottom and an aerobic compartment in the upper part of the reactor were formed after 40 days. The average total nitrogen (TN) removal efficiency was then observed to increase to 77%. The microbial characterization using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, as well as the specific nitrogen utilization rate measurements, indicated that the nitrogen removal in the reactor occurred via nitrification and denitrification processes.

scholarly journals Bioaugmentation of Activated Sludge by an Indigenous 3-Chloroaniline-Degrading Comamonas testosteroni Strain, I2gfp

2000 ◽  
Vol 66 (7) ◽  
pp. 2906-2913 ◽  
Author(s):  
Nico Boon ◽  
Johan Goris ◽  
Paul De Vos ◽  
Willy Verstraete ◽  
Eva M. Top
Keyword(s):  
Community Structure ◽  
Microbial Community ◽  
Activated Sludge ◽  
Microbial Community Structure ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Dynamic Change ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Adaptation Period ◽  
Comamonas Testosteroni

ABSTRACT A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distalmeta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.

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