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Characterization of microbial communities in a
fluidized-pellet-bed bioreactor by DGGE analysis
Introduction
As an extension of the fluidized pellet bed operation used for high rate solid-liquid separation of high concentration suspensions, the authors developed a new
method to promote sludge granulation by rational utilization of inorganic coagulant and organic polymer, continuous supply of dissolved oxygen (DO), and
moderate mechanical agitation in an up-flow reactor for domestic wastewater treatment. In this study, the authors used the DGGE method based on 16S
rRNA gene and paid attention to the microbial diversity and community succession of pellets in the FPB bioreactor. For an overall grasp of the amount and
distribution of aerobic microorganisms, bacteria enumeration was also conducted.
Results & Discussion
Aerobic condition and total counts of aerobic bacteria
in the FPB bioreactor
Fig. 1 showed the result of DO consumption for biodegradation. It can be
seen from the DO profile that the liquid was with a DO > 1.0 mg/L as h <
40 cm, a DO between 0.23 and 1.0 mg/L as 40 < h < 110 cm, and an
almost constant DO about 0.23 mg/L as h > 110 cm. Therefore, the FPB
bioreactor is generally under an aerobic condition especially in its bottom
section.
180
160
Height of fluidized bed (cm)
140
120
Microbial diversity analysis through DGGE
Fig. 4 shows the finger printing obtained by DGGE for all the samples.
According to the principle of DGGE analysis, each electrophoresis strip
represents an independent distinct fragment, and each fragment
represents a microbial species. Larger number of strips is an indication of
variety of microbial species, and stronger signal indicates a greater
amount of organisms. It is recognized from Fig. 4 that there are 23
microbial species (17 common + 6 specific) in the bottom (h=10cm), 21
(17 common + 4 specific) in the middle (h=60cm) and 20 (17 common + 3
specific) in the top sections of the FPB bioreactor. The comparability of the
microbial communities can thus be evaluated as 83.1%, indicating a very
stable microbial structure throughout the FPB bioreactor. Contrarily, the
succession of microbial communities in the vertical direction of the FPB
bioreactor is not obvious.
100
80
(a)
60
40
20
(b)
0
0
1
2
3
4
DO (mg/L)
Fig. 1 DO profile along the FPB height
Aerobic bacteria count (1/g)
(c)
1.0E+09
9.5E+07
1.0E+08
6.9E+07
Fig. 3 Agarose gel
electrophoregrams of the genome
DNA, PCR products and purified
PCR products. (1, 2, 3: samples of
granular sludge from the FPB at 10,
60, 110 cm, respectively)
4.2E+07
1.0E+07
1.0E+06
1.0E+05
10cm
60cm
110cm
Fig. 4 DGGE finger printing and
cluster analysis Results (1, 2, 3:
samples of granular sludge from the
F P B a t 1 0 c m , 6 0 c m , 11 0 c m ,
respectively; "○": specific strip;
" Δ " : c om m o n s tr i p . )
Phylogenetic analysis based on 16S rRNA
sequence similarity
Height of fluidized bed
Fig. 2 Total counts of aerobic bacteria along
the height of FPB bioreactor
Uncultured bacterium(AY976349)
Uncultured bacterium(AY982499)
Band 1-g
S.maxima(X76650)
Uncultured bacterium(AF371837)
Bacillaceae bacterium(AF513473)
Cytophaga sp.(X85210)
Uncultured bacterium(AY212722)
Band 1-j
Flavobacterium columnare(AB023660)
Flavobacterium sp.(AM110999)
Clostridium disporicum(Y18176)
S.ventriculi(X76649)
Bacillus thuringiensis(DQ286359)
Band 3-c
Sarcina ventriculi(AF110272)
Band 1-f
Bacillus sp.(DQ314542)
Bacillus sp.(AY124766)
Bacillus cereus(DQ314542)
Bacillus sp.(AJ746155)
Fig. 2 shows the results of enumeration of total heterotrophic bacteria in
per unit weight of sludge in the FPB. At the bottom section, i.e. h=10cm,
the total aerobic bacteria count was as high as 9.5×107 count/g, and it
kept the same order in the upward direction though there were slight
decreases to 6.9×107 at h=60cm, and to 4.2×107 at h=110cm. The
difference of DO concentration along the vertical direction did not affect
the growth of aerobic bacteria so much.
DNA extraction and PCR amplification
From each of the granular sludge samples collected from the FPB
bioreactor, the genome DNA was extracted by chemical cleavage method
and then amplified by PCR with universal primers of Eubacteria and
Archaebacteria (530F and 1490R) to 16s rRNA gene. As can be seen in
Fig. 3a, the genome DNA extracted from all the samples were at a level
larger than 14 kb, indicating that the genome DNA is unabridged. After
PCR amplification, 16s rRNA fragments with size of 1 kb were obtained
from each of the samples (Fig. 3b). Although certain non-special products
appeared in the electrophoregrams possibly due to interference from the
universal primers and low purity of DNA samples, they disappeared after
gel recovery purification (Fig. 3c).
277.5
250
200
150
100
50
0
Fig. 5 Phylogenetic tree of the OTUs and their relatives among
Actinobacteria.
(Sequences are aligned with Clustal W, and the distance and number
represent the nucleotide substitutions. Numbers in parentheses represent
the sequences accession number in GenBank).
As a result of phylogenitic analysis based on 16S rRNA sequence
similarity, the 18 OTUs are all found to belong to Eubacteria of which 11 or
61 % are Proteobacteria, 3 or 17 % are Actinobacteria, 2 or 11% are low
G+C gram-positive bacteria and the remaining 2 or 11% belong to other
bacteria branches.
Conclusions
This paper illustrated the characteristics of microbial communities in the FPB bioreactor through DGGE analysis and aerobic bacteria enumeration. As a
result, 17 common microbial species were identified from the granular sludge sampled from the bottom, middle and top sections of the FPB. The
comparability of the microbial communities in the three samples was 83.1%. The 16S rRNA sequence analysis results revealed that the 18 OTUs obtained in
the DGGE finger printing all belong to the domain of Eubacteria.
Acknowledgement: This study is supported by the National Natural Science Foundation of China (Grant No. 50578132)