Title: Microbial Diversity in a Dye Treating SBR
1Microbial Diversityin a Dye Treating SBR
by Dr. Naeem ud din Islamia College Peshawar
2Biotreatment Different modes
B isolated organisms
A using mixed culture
C isolated enzymes
Dyes are hard to degrade, and often result in
harmful intermediates
3(No Transcript)
4- A Specially Designed Airlift BR from a previous
Experiment for SND achieving was used - The Nitrogen Removing Process was well
established in that Reactor - 93 of Ammonia and COD at an HRT of 12 hrs.
5Table 1. Physical and Operational Conditions of the SBR Table 1. Physical and Operational Conditions of the SBR
Parameter Value
Working volume (L) Temperature (oC)Dissolved oxygen (mg/l) pH of bioreactor Aeration No aeration(minutes) 3.5 25 - 30 0.05 - 2.0 6.5-8.6 30 120
6The NITRIFYING MEDIUM
Constituents Quantity
NH4Cl (mg N L-1 ) 120
NaCl (mg L-1 ) 1000
C6H12O6 (mg/L) 1000
FeSO4 (mg L-1 ) 55.00
K2HPO4 (mg L-1 ) 140.00
CaCO3(g L-1 ) 2.00
Trace metal solution(ml/L) 2
Yeast Extract(mg L-1 ) 10
pH 7.8
g/l MgSO47H2O 5, FeCl24H2O 6, COCl2 0.88, H3BO3 0.1, ZnSO47H2O 0.1, CuSO4 0.05, NiSO4 1, MnCl2 5, (NH4)6MO7O244H2O, 0.64 and CaCl22H2O 5. g/l MgSO47H2O 5, FeCl24H2O 6, COCl2 0.88, H3BO3 0.1, ZnSO47H2O 0.1, CuSO4 0.05, NiSO4 1, MnCl2 5, (NH4)6MO7O244H2O, 0.64 and CaCl22H2O 5.
7MG dye-
- textile industry, biological stain and
antifungal. - phytotoxic, a respiratory poison, and teratogen
8- This SBR was subjected to gradually increasing
dye concentration - Optimization was achieved at a dye concentration
of 25 mg/l and increased HRT of 36 hrs - In this experiment we used the activated sludge
as a renewable biological resource to adsorb the
usual environmental concentrations of the MG dye.
9Synthetic DYE CONTAINING wastewater composition
Constituents Quantity
NH4Cl (mg N L-1 ) 120
NaCl (mg L-1 ) 1000
C6H12O6 (mg/L) 1000
FeSO4 (mg L-1 ) 55.00
K2HPO4 (mg L-1 ) 140.00
CaCO3(g L-1 ) 2.00
Trace metal solution(ml/L) 2
Yeast Extract(mg L-1 ) 10
MG (mg L-1 ) 25
pH 7.8
g/l MgSO47H2O 5, FeCl24H2O 6, COCl2 0.88, H3BO3 0.1, ZnSO47H2O 0.1, CuSO4 0.05, NiSO4 1, MnCl2 5, (NH4)6MO7O244H2O, 0.64 and CaCl22H2O 5. g/l MgSO47H2O 5, FeCl24H2O 6, COCl2 0.88, H3BO3 0.1, ZnSO47H2O 0.1, CuSO4 0.05, NiSO4 1, MnCl2 5, (NH4)6MO7O244H2O, 0.64 and CaCl22H2O 5.
10- In that optimized state
- The Color and COD removal was 80
- ammonia removal declined to 70 .
- Biomass, 4 0.7 to 6 0.5 gm/l, SVI was in the
range of 30 to 65 ml/gm
11COD Color removal
12?max 618 nm
UV-Vis spectrophotometric scan of the
biodecolorization of malachite green.
13Correlation between ammonia, biomass, dye
concentration and OUR
14- Knowledge about the microbial community in a dye
treating reactor would be useful in association
with operational conditions, to eliminate the
pollutants efficiently. - likely to cause the domination of certain
groups of bacteria - This aspect inspired our interest to know the
microbial community evolved under the selective
pressure of the Dye in the SBR.
15Microbial community structure in the Dye Treating
SBR Sludge
- 16S rRNA gene Library
- Phylogenetic Analysis
16- PCR-amplification, clone library construction and
sequencing - Bacterial universal primers
- 27F (3'-AGAGTTTGATCATGGCTCAG-5') and
- 1492R (3'-TACGGYTACCTTGTTACGACTT-5') were used
for amplification.
17 BLAST Analysis of the OTUs (culture-
independent)
18- Phylogenetic analysis
- The obtained sequences were edited and
aligned using the BioEdit software and CLUSTAL_W
program (Thompson, 199724). - The sequences were compared to the known GenBank
sequences using Basic Local Alignment Search Tool
(BLAST). - Phylogenetic trees were constructed by
neighbor-joining method with the MEGA package .
Identical sequences were recognized by
phylogenetic tree analysis.
19- Phylo-genetic analysis
- If the sequences similarity was more than 97 ,
they were considered as identical and used for
further phylogenetic analysis as an operational
taxonomic unit (OTU).
20Culture-Independent Phylogenetic tree of the
clones from the dye treating SBR,
21Phylogenetic distribution profile of microbial
community (Culture Independent) in the SBR.
22- Culture-Dependent Method
- Nineteen isolates were selected from the SBR and
their 16S rRNA genes were sequenced, and compared
with similar sequences of the reference organisms
BLAST search. Figure 6 shows the phylogenetic
tree based on the culture dependent isolates
identified with sequences of the NCBI BLAST. - Some of the clones identified with the well-known
biodegraders, the notable being Dokdonella
koreensis, Rhodobactor, Shingomonas and
Paracoccus species.
23Similarity of 16S rRNA gene sequences of the
isolates
Is No
24Phylogenetic tree of isolates from the dye
treating SBR
The isolates Identified with a- g-
Proteobacteria
25Phylogenetic distribution, as illustrated by
isolates in the SBR involved in the biotreatment
MG.
All these groups well represented in the
polluted environments
26- Table 2 shows the phylogenic affiliation and
abundance of the clones. The sequences
identifying with 5 divisions of Proteoabacteria
i.e ?-, ß-, ?- -proteobacteria and
Verrucomicrobia groups were obtained. The ß-, and
?-proteobacteria were in high abundance, valuing
24 and 45 of the total clones. The other
small groups, consisting of ?-,-proteobacteria
and Verrumicrobia groups, were 4 , 9 , and 2
respectively. A moderate amount of clones, about
9 , ranked with the uncultured bacterial strains
with sequenced data in the NCBI. - The similarity of six culture independent
clones(HT-69, HT-47, HT-51, HT-66, HT-38, HT-7,
HT-72), to the known sequences in the GenBank was
lower than 95. Due to difficulty in translating
16S rRNA gene sequence similarity values into
nomenclature, it is assumed that similarity
values to the known sequences below 95 may be
regarded as evidence of the discovery of novel
species(3). Thus there is ample possibility of
unidentified bacteria in the SBR used in the
present study.
27inferences
- SBR with good SVI, effectively removed MG, COD
and nitrogen up to 25 mg/L dye, above which a
strong inhibition of these processes was
observed. - The autotrophic nitrifying bacteria were not
detected at high dye concentration, acting as
bio-indicators for the MG toxicity. The ammonia
removal pathway was, however, present, an
indication of the microbial redundancy.
28inferences
- Majority of the sequences identified with the ß-
and ?-Proteobacteria. pollutant degrading
bacteria, like rhodobacterales, sphingomonadales
were in plenty. - The first time that MG treated in a nitrifying
BR, with its inhibitory effects, and microbial
community monitored. - Both culture-dependent and Culture independent
methods must be used to have a true picuture of
microbial diversity.
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31Thanks