3. Materials and Methods
4.2 Station 84 clone library
To get a picture of the sediment microbial communities at station 84, a 16S rDNA clone library was constructed. 351 clones were sequenced, and FastGroupII was
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Figure V-2. Water column temperature and oxygen concentrations.
Water column temperatures (black) and oxygen concentrations (white) measured in August 2006. The western basin stations 357, 974, 969 are shown in the top panel and central basin stations 84 and 961 as well as station 958 (Cleveland) are shown in the bottom panel.
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Table V-1. Concentrations of PAHs in Lake Erie sediments.
Concentrations are average mg/kg ± standard deviation, n=3. - =Not detected, LMW PAH = low molecular weight PAHs (3 rings or less), HMW PAH = high molecular weight PAH (more than 3 rings).
Western basin Central basin Cleve-land Fluoranthene 0.31 ±
0.02
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used for rarefaction analysis (Yu et al. 2006). Groups were determined for 80, 85, 90, 95, and 97% sequence identity, which grouped the sequences into 61, 96, 149, 207, and 240 phylotype groups, respectively; rarefaction curves are shown in Figure V-3. The predicted Chao1 values indicate that there is a great deal of diversity in these sediment communities, and this library reflects only a portion of it: at 95% sequence identity, the observed phylotypes (207) make up only 38% of predicted diversity (Chao1 = 549).
The phylogenetic affiliations of the clones sequenced are shown in Figure V-4.
Major divisions of bacteria common to sediment and aquatic microbial communities were represented, including Proteobacteria, Actinobacteria, Acidobacteria, Nitrospira, Firmicutes, Bacterioidetes, Planctomycetes, and
Cyanobacteria. Phylogenies were determined using RDP (Cole et al. 2007) with a confidence threshold of 80%.
A neighbor-joining phylogenetic tree of the major divisions recovered in the clone library is shown in Figure V-5. Over half (51%) of the clones in the library were proteobacteria. Within the proteobacteria, 30.3% were β-proteobacteria. Some were similar to Rhodocycales, Burkholderiales, and Commamonas, however many fell into clades which were closely related to sequences from other
environmental clone libraries which had no cultured representatives (Figure V-6).
27.5% of the proteobacteria clones were γ-proteobacteria, falling into orders Methyococcales, Xanthomonadales, Chromatiales, and Legionellales, also with a large number of unclassified γ-proteobacteria matching only other environmental (uncultured) sequences in the databases (Figure V-7). 10.7 % of the
proteobacteria sequences were α-proteobacteria, closely related to Sphingomonas, Methylocycstis, and Rhodobacter (Figure V-8). 15.2% were δ-proteobacteria, falling into two distinct clades: one group was closely related to Geobacter and Desulfobacter, while the other group was more closely related to Bacteriovorax and Desulfovibrio (Figure V-8). 16.3% were unclassified proteobacteria.
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Number of sequences
0 100 200 300
Number of ribotypes
0 50 100 150 200 250
97% identity (Chao1 = 764) 95% identity (Chao1 = 549) 90% identity (Chao1 = 345) 85% identity (Chao1 = 171) 80% identity (Chao1 = 118)
Figure V-3. Rarefaction analysis of Lake Erie 16S clone library.
Curves were calculated for 80, 85, 90, 95, and 97% sequence identity. Chao1 values are listed for each.
F
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Figure V-5. Neighbor-joining phylogenetic tree of all 351 16S rDNA clones from sediment microbial community at station 84.
Phylogenetic classifications based on 80% confidence level. A euryarchaeota was used as the outgroup. Size of the wedge represents the number of clones
recovered within that phylogenetic classification. “Unclassified” groups could not be assigned to a particular phylogenetic group. Clones not falling into a distinguishable clade are represented by their clone name (e.g. ES6-16).
aG Proteobacteria (unclassified) Proteobacteria (unclassified)
19-8 Unclassified bacteria
eV
Cyanobacteria
Unclassified bacteria
Unclassified bacteria
Cloroflexi
Firmicutes (Clostridium) ES6-16
Unclassified bacteria Unclassified bacteria
ES13-11
Bacteroidetes
Unclassified bacteria ES11-2
Nitrospira
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Figure V-6. Neighbor-joining phylogenetic tree of proteobacteria 16S sequences from Lake Erie sediments, highlighting the β subdivision.
ES9-6
Acidovorax facilis AF078765 ES16-6
Leptothrix mobilis X97071 Variovorax paradoxus AJ420329 ES13-8
Polaromonas naphthalenivorans AY166684 ES18-9
ES2-21 ES10-19 ES15-14
Comamonas testosteroni M11224 ES3-16 Azoarcus evansii X77679
Dechloromonas denitrificans AJ318917 ES5-3
ES14-2
Burkholderia glathei Y17052 Alcaligenes faecalis AJ242986
Ralstonia syzygii U28237 Oxalobacter formigenes U49757 Limnobacter thiooxidans AJ289885
ES5-1
Sterolibacterium denitrificans AJ306683 ES8-18
ES11-19 ES3-10 ES11-11 ES14-4
Methylophilus methylotrophus L15475 ES8-22
Nitrosomonas oligotropha AJ298736 ES12-7
Gamma proteobacteria (Xanthomonads)
Gamma proteobacteria
Delta proteobacteria
Alpha proteobacteria
Delta proteobacteria (Geobacter)
Unclassified proteobacteria
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Figure V-7. Neighbor-joining phylogenetic tree of proteobacteria 16S sequences from Lake Erie sediments, highlighting the γ subdivision.
Beta proteobacteria
Xanthomonas pisi Y10758 ES18-14
Legionella oakridgensis X73397 ES13-5
ES19-2 ES2-23
Methylococcus capsulatus X72770 ES5-17
ES12-12
Pseudomonas aeruginosa X06684 Pseudomonas putida D37923
Pseudoalteromonas denitrificans X82138 Shewanella denitrificans AJ311964
ES3-24
Delta proteobacteria (Geobacter)
Unclassified proteobacteria
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Figure V-8. Neighbor-joining phylogenetic tree of proteobacteria 16S sequences from Lake Erie sediments, highlighting the α and δ subdivisions.
Beta proteobacteria
Gamma proteobacteria (Xanthomonads)
Gamma proteobacteria
Syntrophobacter wolinii X70905
Bacteriovorax litoralis AF084859 ES4-18
ES15-1 ES9-5
ES19-12
Desulfovibrio aespoeensis X95230 ES17-11
Roseococcus thiosulfatophilus X72908 ES6-11
ES14-5
Rhodospirillum photometricum AJ222662 ES13-6
Caulobacter fusiformis AJ227759 Rhizobium leguminosarum AY509899
ES3-8
Rhodobacter sphaeroides X53853 ES6-20
Hyphomicrobium denitrificans Y14308 ES15-17
ES11-16
Sphingomonas paucimobilis U20776 ES18-18
ES20-3 ES8-20
ES17-7
Rhodopseudomonas palustris D25312 Beijerinckia indica subsp. indica M59060 ES6-5
ES5-12
Methylocystis echinoides AJ458473
Alpha proteobacteria
ES4-3 ES13-10
ES2-10 ES16-1
Geobacter sulfurreducens U13928 ES13-9
Desulfuromonas thiophila Y11560 ES20-2
ES15-15 ES17-3 ES16-18
ES9-18
Desulfobacter curvatus M34413 ES5-15
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Twenty sequences were recovered that fell into the phylum and class
Actinobacteria (Figure V-9). Of these, 10 were in the order Rubrobacterales, which included Rubrobacterineae and Conexibacter. Seven were Actinomycetes, including Actinosynnemataceae, Mycobacteriacea and Frankineae. The two sequences most closely related to Mycobacterium had 98% sequence identity to other fast-growing, environmental Mycobacterium spp. Verrucomicrobia was represented by 18 sequences, falling into subdivision 3, Opitutus,
Xiphinematobacteriaceae and Verrucomicrobiaceae (Figure V-10). The clone that was most closely related to a cultured representative (ES4-7) had 97% sequence identity to Opitutus sp. VeSm13.
Other sequences of interest include clones most closely related to candidate divisions WS3 and OP10, monophyletic groups that currently have no cultured representatives. Chloroplast sequences were also recovered: eleven clones had 98-99% identity to Aulacoseira and Thalassiosira spp. chloroplast sequences.