Microbial Diversity in a Dye Treating SBR

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Microbial Diversityin a Dye Treating SBR
by Dr. Naeem ud din Islamia College Peshawar
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Biotreatment Different modes
B isolated organisms
A using mixed culture
C isolated enzymes
Dyes are hard to degrade, and often result in
harmful intermediates
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(No Transcript)
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• 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.

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Table 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
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The 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.
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MG dye-

• textile industry, biological stain and
  antifungal.
• phytotoxic, a respiratory poison, and teratogen

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• 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.
  

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Synthetic 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.
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• 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

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COD Color removal
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?max 618 nm
UV-Vis spectrophotometric scan of the
biodecolorization of malachite green.
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Correlation between ammonia, biomass, dye
concentration and OUR
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• 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.

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Microbial community structure in the Dye Treating
SBR Sludge

• 16S rRNA gene Library
• Phylogenetic Analysis

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• PCR-amplification, clone library construction and
  sequencing
• Bacterial universal primers
• 27F (3'-AGAGTTTGATCATGGCTCAG-5') and
• 1492R (3'-TACGGYTACCTTGTTACGACTT-5') were used
  for amplification.

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BLAST Analysis of the OTUs (culture-
independent)
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• 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.

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• 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).

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Culture-Independent Phylogenetic tree of the
clones from the dye treating SBR,
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Phylogenetic distribution profile of microbial
community (Culture Independent) in the SBR.
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• 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.

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Similarity of 16S rRNA gene sequences of the
isolates
Is No
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Phylogenetic tree of isolates from the dye
treating SBR
The isolates Identified with a- g-
Proteobacteria
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Phylogenetic distribution, as illustrated by
isolates in the SBR involved in the biotreatment
MG.
All these groups well represented in the
polluted environments
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• 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.

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inferences

• 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.

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inferences

• 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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