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Microbial Diversity in a Dye Treating SBR. by Dr. Naeem ud din Islamia College Peshawar. 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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microbial diversity in a dye treating sbr

Microbial Diversityin a Dye Treating SBR


Dr. Naeemuddin

Islamia College Peshawar

biotreatment different modes
Biotreatment Different modes

B: isolatedorganisms

A: using mixed culture

C: isolated enzymes

Dyes are hard to degrade, and often result in harmful intermediates


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.

mg dye
MG dye-
  • textile industry, biological stain and antifungal.
  • phytotoxic, a respiratory poison, and teratogen

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.


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


λmax 618 nm

UV-Vis spectrophotometric scan of the biodecolorization of malachite green.


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.

microbial community structure in the dye treating sbr sludge
Microbial community structure in the Dye Treating SBR Sludge
  • 16S rRNA gene Library
  • Phylogenetic Analysis

PCR-amplification, clone library construction and sequencing

Bacterial universal primers


1492R (3′-TACGGYTACCTTGTTACGACTT-5′) were used for amplification.


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.


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).

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 Dokdonellakoreensis, Rhodobactor, ShingomonasandParacoccus species.
phylogenetic tree of isolates from the dye treating sbr
Phylogenetic tree of isolates from the dye treating SBR

The isolates Identified with a- & g-Proteobacteria

phylogenetic distribution as illustrated by isolates in the sbr involved in the biotreatment mg
Phylogenetic distribution, as illustrated by isolates in the SBR involved in the biotreatment MG.

All these groups well represented

in the polluted environments

Table 2 shows the phylogenic affiliation and abundance of the clones. The sequences identifying with 5 divisions of Proteoabacteriai.eά-, β-, γ- §-proteobacteria and Verrucomicrobia groups were obtained. The β-, and γ-proteobacteria were in high abundance, valuing 24 % and 45 % of the total clones. The other small groups, consisting of ά-,§-proteobacteriaandVerrumicrobia 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.
  • 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 microbialredundancy.
  • Majority of the sequences identified with the β- and γ-Proteobacteria. pollutant degrading bacteria, like rhodobacterales, sphingomonadaleswere 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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