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Microbial Analysis Report Microbial Analysis Submitted to XXXXXXXXXXXX December 7, 2015 Submitted by Trevor Ghylin, P.E., PhD www.microbedetectives.com [email protected] Executive Summary The DNA data revealed the following: - Two samples were very similar even though one came from a system with an anaerobic zone and one did not - Traditional AOB and NOB were not detected at all indicating potential for SND (simultaneous nitrification denitrification) by non-traditional AOB/NOB - PAOs were detected in somewhat low abundance but relatives of known PAOs were also detected - A small amount of iron oxidizing bacteria were detected in Basin 7 but not basin 6 - Filaments and foaming were not abundant - Some nitrogen fixing bacteria were detected indicating potential for nitrogen limitation - Sulfate reducing bacteria were detected. Some have been associated with high alkalinity and thermophilic conditions. This is unusual for municipal wastewater - Photosynthetic bacteria were detected indicating potential growth in clarifiers or other sun-exposed locations Background 2 samples were received by Microbe Detectives on 11/13/15 labelled: AerationBasin6_S2 AerationBasin7_S3 DNA results and analysis of this sample are presented below. Results DNA data are displayed in Table 1 and the DNAmyWastewater™ tables. The data in these tables is a subset of the total data, focusing on bacteria that are typically important in wastewater systems. The full data is also provided in a spreadsheet. Data presented are bacterial relative abundance (% of total bacteria in the sample). For example 0.12% Actinobacteria means that 0.12% of bacteria in the sample are Actinobacteria. The two samples had very similar bacterial communities even though they were apparently collected from different systems where one system includes an anaerobic environment and the other does not. The similarity of these samples suggests that either the sludge from the two locations is mixed or the anaerobic environment in the one system is not anaerobic enough to have an impact on the microbial population. Phylum Figures 1-2 show the phylum distribution for the samples. The predominant phyla were Proteobacteria and Bacteroidetes which contain many common environmental bacteria.. Fermentation/Anaerobic Decay The data shows only a small amount of fermenters (Actinobacteria and Firmicutes) in the sample. The fermenting bacteria produce volatile fatty acids necessary for biological phosphorus removal. Fermentation can also occur upstream in sewer systems that have extended residence times. Phosphorus Dechloromonas and Rhodocyclus were detected at somewhat low concentrations (~0.5% each). Both of these bacteria are associated with biological phosphorus removal. Additionally, several bacteria related to Dechloromonas and Rhodocyclus were detected including Azonexus, Zoogloea and Thauera. These bacteria are closely related to PAOs but have not been shown to perform enhanced biological uptake of phosphorus at this point. The science may change on this in the future. Nitrogen The common ammonia and nitrite oxidizing bacteria Nitrosomonas and Nitrospira were not detected. This is somewhat unusual. It is likely that ammonia removal is being performed by other bacteria, possibly Dechloromonas, Thauera, or others that have been shown to perform simultaneous nitrification-denitrification in low DO environments. A relatively high amount of nitrogen fixing bacteria (Azonexus, Azohydromonas) were found in the samples, indicating that the wastewater may be somewhat nitrogen limited. Filaments/Foaming Actinomycetales and Acidimicrobiales were found only in low abundance. Filaments and foaming do not appear to be an issue. Zoogloea was also found in relatively low abundance. This bacteria causes slime bulking and is associated with nutrient deficiency, high f:m, high soluble organic loading or low pH. Iron/Sulfur Gallionella were detected in Aeration Basin 7 S3 but not in Aeration basin 6 S2. These bacteria oxidize iron for energy. Sulfur associated bacteria were also detected in lower quantities (<0.5%), indicating the presence of reduced sulfur and also sulfate. Desulfonatronospira, Thermodesulforhabdus, Thiorhodospira were detected. Desulfonatronospira has been associated with alkaline high pH conditions. Thermodesulforhabdus has been associated with thermophilic conditions and Thiorhodospira is a strictly anaerobic, photosynthetic purple sulfur bacteria. The presence of sulfur can hinder ammonia removal and can also cause filamentous issues, although the primary sulfur filament (Thiothrix, aka 021N) was not observed. Photosynthesis A cyanobacteria (Aphanothece) and a photosynthetic purple sulfur bacteria (Thiorhodospira) were detected. This is unusual for wastewater. This may indicate some ability for photosynthetic organisms to grow in the secondary clarifiers. Table 1. Most Abundant Wastewater Bacteria (% of Total Bacteria in Sample) AerationBasin6_S AerationBasin7_S Treatment Function Bacteria Identity (Genus) 2 3 Nitrogen Fixation; Related to PAOs Azonexus 3.41% 3.12% Cyanobacteria/Blue-green algae (photosynthetic) Aphanothece 1.25% 1.30% PAO (phosphorus) Rhodocyclus 0.68% 0.53% Slime forming - related to PAO Zoogloea 0.63% 0.51% Filament Haliscomenobacter 0.57% 0.65% PAO (phosphorus) Dechloromonas 0.42% 0.46% Sulfate reduction Desulfonatronospira 0.41% 0.33% Sulfate reduction Thermodesulforhabdus 0.30% 0.16% Sulfur oxidation Thiorhodospira 0.22% 0.19% Denitrification - related to PAO Thauera 0.20% 0.15% Nitrite removal Nitrospira 0.00% 0.18% Nitrite removal Nitrobacter 0.00% 0.00% Sulfate reduction Desulfofaba 0.00% 0.00% Ammonia oxidation Nitrosomonas 0.00% 0.00% Note: Only bacteria that are well known and relevant to wastewater treatment are included in this table. Other bacteria can be found in the excel data. Figure 1. Phylum Analysis Armatimonadetes, Verrucomicrobia, 0.80% AerationBasin6_S2 0.80% Actinobacteria, Cyanobacteria, 1.30% Other, 2.00% 0.70% Chloroflexi, 1.40% Firmicutes, 1.50% Proteobacteria Bacteroidetes Firmicutes Bacteroidetes, 21.00% Chloroflexi Cyanobacteria Verrucomicrobia Proteobacteria, 70.50% Armatimonadetes Actinobacteria Other Figure 2. Phylum Analysis Armatimonadetes, 0.40% AerationBasin7_S3 Verrucomicrobia, 0.40%Actinobacteria, 0.50% Cyanobacteria, 1.30% Other, 3.00% Chloroflexi, 0.80% Firmicutes, 1.10% Proteobacteria Bacteroidetes Firmicutes Bacteroidetes, 20.30% Chloroflexi Cyanobacteria Verrucomicrobia Proteobacteria, 72.20% Armatimonadetes Actinobacteria Other Microbe Detectives DNAmyWasteWater™ Copyright© Microbe Detectives™ All Rights Reserved Bacterial Relative Abundance (% of Total Bacteria in Sample) Bolded or highlighted text indicates bacteria that were found greater than 0.2% of total bacteria. Bacteria Name AerationBasin6_S2 AerationBasin7_S3 Notes Phylum Proteobacteria 70.50% 72.20% Bacteroidetes 21.00% 20.30% Firmicutes 1.50% 1.10% Chloroflexi 1.40% 0.80% Cyanobacteria 1.30% 1.30% Verrucomicrobia 0.80% 0.40% Armatimonadetes 0.80% 0.40% Actinobacteria 0.70% 0.50% Other 2.00% 3.00% Fermenters AerationBasin6_S2 AerationBasin7_S3 Actinobacteria 0.70% 0.50% Firmicutes 1.50% 1.10% Lactobacillus 0.00% 0.00% Propionivibrio 0.00% 0.00% Tetrasphaera 0.00% 0.00% Propionicimonas 0.00% 0.00% Streptococcus 0.00% 0.00% Lactococcus 0.00% 0.00% Phosphorus AerationBasin6_S2 AerationBasin7_S3 Dechloromonas 0.42% 0.46% PAO (Phosphorus Accumulating Organism) Rhodocyclus 0.68% 0.53% PAO (Phosphorus Accumulating Organism) Accumulibacter 0.00% 0.00% PAO (Phosphorus Accumulating Organism) Tetrasphaera 0.00% 0.00% PAO (Phosphorus Accumulating Organism) CandidatusCompetibacter 0.00% 0.00% GAO (Glycogen Accumulating Organisms) Defluviicoccus 0.00% 0.00% GAO (Glycogen Accumulating Organisms) Related to Dechloromonas and Accumulibacter; Rhizobiales (Order) 8.70% 6.50% Potential PAO Nitrogen AerationBasin6_S2 AerationBasin7_S3 Notes Rhodococcus 0.00% 0.00% SND (Simultaneous Nitrification/Denitrification) Pseudomonas 0.20% 0.00% SND (Simultaneous Nitrification/Denitrification) Paracoccus 0.00% 0.00% SND (Simultaneous Nitrification/Denitrification) Thauera 0.20% 0.15% SND (Simultaneous Nitrification/Denitrification) Dechloromonas 0.42% 0.46% SND (Simultaneous Nitrification/Denitrification) Aromatoleum 0.00% 0.00% SND (Simultaneous Nitrification/Denitrification) Nitrosomonas 0.00% 0.00% AOB (Ammonia Oxidizing Bacteria) Nitrosococcus 0.00% 0.00% AOB (Ammonia Oxidizing Bacteria) Chromatiales 0.00% 0.00% AOB (Ammonia Oxidizing Bacteria) Nitrosovibrio 0.00% 0.00% AOB (Ammonia Oxidizing Bacteria) Nitrosopumilus 0.00% 0.00% AOA (Ammonia Oxidizing Archaea) Nitrososphaera 0.00% 0.00% AOA (Ammonia Oxidizing Archaea) Nitrosocaldus 0.00% 0.00% AOA (Ammonia Oxidizing Archaea) Candidatus_Nitrosoarchaeum 0.00% 0.00% AOA (Ammonia Oxidizing Archaea) Nitrobacter 0.00% 0.00% NOB (Nitrite Oxidizing Bacteria) Nitrobacter-related 0.00% 0.00% NOB (Nitrite Oxidizing Bacteria) Nitrospira 0.00% 0.18% NOB (Nitrite Oxidizing Bacteria) Nitrospina 0.00% 0.00% NOB (Nitrite Oxidizing Bacteria) Bradyrhizobiaceae 0.00% 0.00% Nitrogen Fixation Thauera 0.20% 0.15% NO3 Reduction Nitratireductor 0.00% 0.00% NO3 Reduction Nitratiruptor 0.00% 0.00% NO3 Reduction Brocadia 0.00% 0.00% Anammox Jettenia 0.00% 0.00% Anammox Scalindua 0.00% 0.00% Anammox Kueninia 0.00% 0.00% Anammox Anammoxoglobus 0.00% 0.00% Anammox Nitriliruptor 0.00% 0.00% Nitrile Oxidation Foaming AerationBasin6_S2 AerationBasin7_S3 Notes Acidimicrobiales (order) 0.30% 0.30% Related to Microthrix Actinomycetales (order) 0.28% 0.22% Mycolic Acid Producing Bacteria
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