Obligate Anaerobes in Granules

The representatives of phylogenetic groups in aerobically grown granules were determined and then hypothetically inferred physiological groups

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3 200000

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Distance along radius of granule, |m

Fig. 7.4. Distribution of enterobacteria in aerobically grown granule.

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Distance along radius of granule, |m

Fig. 7.4. Distribution of enterobacteria in aerobically grown granule.

of microbial community in granules have been used for analysis of microbial physiological diversity in the granules. One group includes obligate anaerobic bacteria from genus Bacteroides. Eleven clone sequence types were assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) group in clone library. A maximum likelihood tree generated by fast-DNAml program (Olsen et al., 1994) is shown in Fig. 7.5.

Clone 051 was affiliated with Bacteroides fragilis. A sequence of 06 clone was identified as belonging to Bacteroides spp. by its position on 16S rDNA phylogenetic tree (Fig. 7.5). It was closed to B. distasonis and B. merdae. Cloning of 16S rDNA and its sequencing demonstrated the presence of obligate anaerobe Bacteroides spp. in aerobically grown microbial granules.

These anaerobic bacteria were selected to detect the boundary of anaerobic microzone in the granules. Cells of Bacteroides spp., detected by FISH and CLSM, were concentrated in a layer with a thickness about 100 |xm. This layer was on the depth approximately 800 |xm from the surface of the big granules. Cells of Bacteroides spp. were concentrated on inner surface of the walls of the big granule and were almost absent in the small granules.

Cells of Bacteroides spp., which were stained after the incubation in hybridization buffer with Bacto1080 probe (Dore et al., 1998;

Bacteroides splanchnicus

100 I- Prevotella buccalis

Prevotella bivia

Bacteroides eggerthii Bacteroides uniformis

Prevotella zoogleoformans Prevotella heparinolytica Bacteroides ovatus Bacteroides acidofaciens

Bacteroides fragilis Bacteroides thetaiotaomicron str. E50

Bacteroides caccae _AG clone 06

Uncultured bacterium mlel-2 Bacteroides ASF519 str. ASF 519

- Bacteroides distasonis

Bacteroides merdae

- Porphyromonas macacae

Porphyromonas endodontalis Porphyromonas asaccharolytica

Bacteroides forsythus - Flavobacterium succinicans

Fig. 7.5. 16S rRNA phylogenetic tree for cloned sequence 06. The numbers at the branch nodes are bootstrap values based on 100 resamplings for maximum likelihood. Only bootstrap values greater than 50% are shown. Scale bar = 1% nucleotide divergence.

Sghir et al., 2000) for FISH and washed in the washing buffer, were also clearly distinguished by flow cytometry on the dot plots showing red fluorescence (FL3) versus forward light scatter (FSC) (Fig. 7.6). The stained (hybridized) cells were concentrated in the region R2 and non-stained cells were appeared in the region R1 (Fig. 7.6b). These non-stained cells in the region R1 were determined in the control where the cells were treated as in the experiment but the Bacto1080 probe for detection of Bacteroides spp. was not added (Fig. 7.6a).

The ratio of the cells of Bacteroides spp. cells was determined as the ratio of the number of the events in the region R2 to the number of the events in the region R1. It was 0.56% for the cells from big granules and 0.22% for the cells from small granules. It is not absolute but a conventional ratio, because some events detected in the region R1 are not the microbial cells but the particles produced during the disintegration of the granules. FSC for the stained cells was higher for the non-stained cells.

Fig. 7.6. Dot plot of red fluorescence (FL3) and forward light scatter (FSC) of the cells, which were not incubated with the probe (a, control) and incubated with Bacto1080 probe (b, experiment). The cells were produced by the disintegration of big granules. The region R1 is corresponding to non-labeled cells and particles and R2 region shows the cells, which were hybridized with Bacto1080 probe.

Fig. 7.6. Dot plot of red fluorescence (FL3) and forward light scatter (FSC) of the cells, which were not incubated with the probe (a, control) and incubated with Bacto1080 probe (b, experiment). The cells were produced by the disintegration of big granules. The region R1 is corresponding to non-labeled cells and particles and R2 region shows the cells, which were hybridized with Bacto1080 probe.

Flow cytometry, additionally to SCLM data, proves the presence of Bac-teroides spp. in the granules and the bigger share of obligate anaerobic bacteria in big granules rather than in small granules.

All methods confirmed that aerobically grown microbial granules contain obligate anaerobe Bacteroides spp. It is well known that these bacteria usually dominate in human feces. Probably, the source of the initial inoculation of aerobic microbial flocs in wastewater treatment plant by Bacteroides spp. was the raw sewage or the effluent from anaerobic digester. Ecological consequence from the detection of obligate anaerobic bacteria in aerobically grown granule is that the boundary of anaerobic microzone in microbial matrix of the granule or thick biofilm can be detected by the visualization of the layer of Bacteroides spp. or other anaerobic microorganisms by FISH with specific fluorescence-labeled oligonucleotide probe. This method reliably shows the place of anaerobic layer, which is depending on average oxygen gradient during the microbial granule growth and activity. If the place of anaerobic zone is detected by the microelectrodes, it could be depended on the variable oxygen gradient in the granule during the measurement. Applied significance of the detection of obligate anaerobes in aerobically grown granule is that there must be optimal size of such granules to ensure optimal balance of aerobic and anaerobic biodegradation of organic matter in the granule.

Fig. 7.7. Floating of the granules after settling. (a) to (e) correspond to 2, 24, 25, 28, and 32 min after settling of the granules.

The produced gases of fermentation can destroy the granule if the size of the granule is big and, correspondingly, the number of anaerobic bacteria inside the granule will be high.

Another negative effect of anaerobic bacteria on the wastewater treatment process is the occurrence of floating granules, which could occur if anaerobic bacteria, and especially denitrifying bacteria, are allowed to incubate in medium with carbon source after biomass settling (Fig. 7.7). This potential floating of the microbial granules in case of high organic or nitrate load, leading to the production of gases in anaerobic zone of the granule, can deteriorate wastewater treatment.

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