Chapter V

Designing of bioreactor for effective decolorization of

textile dyes

5. Introduction

Textile dyeing industry in India largely utilizes a considerable amount of water in

dyeing process that eventually results into wastewater with a large amount of dye

particulates/ molecules. This wastewater discarded to the water sources causes a drastic

decrease in oxygen concentration due to the presence of hydrosulfides in certain dyes that

can react with oxygen. It also blocks the passage of light to the water body by increasing

the turbidity, which is detrimental to water ecosystem (Liu et al., 2007). Microbes are the

only entities in the biosphere with an exceptional ability to exploit various organic/

inorganic compounds for their growth. They are empowered to inhabit various ecological

niches and pursue unusual metabolic and physiological activities (Reineke, 2001).

Decolorization of azo dye by sequential reactions involves the degradation of azo dye by

reduction or cleavage of azo bond by anaerobic digestion and ultimate biotransformation of

aromatic amines in aerobic conditions (Jirawat, 1998). Aerobic sludge granulation is a

novel wastewater treatment technology that decontaminates high-strength wastewater at an

acceptable rate. The aerobic granules produced have a dense and strong structure, good

settleability, high biomass retention, and high tolerance to medium toxicity (Oktem et al.,

2007). Biogranulation involves cell-to-cell interactions that include biological, physical and

chemical phenomena. Biogranulation can be classified as aerobic and anaerobic

granulation. Biogranules form through self immobilization of microorganisms. These

granules are dense microbial consortia packed with different bacterial species and typically

contain millions of organisms per gram of biomass (Wang et al., 2007). Formation of

anaerobic granules has been extensively studied and is probably best recognized in the up

flow anaerobic sludge blanket (UASB) reactor. Many wastewater treatment plants already

apply anaerobic granulation technology (Alves et al., 2000). Anaerobic granulation is

relatively well known, but research on aerobic granulation commenced only recently. Many

full-scale anaerobic granular sludge units have been operated worldwide, but no report

exists of similar units for aerobic granulation. Microbial granules play an important role in

the field of biological wastewater treatment due to their advantages over the conventional

sludge flocs, such as a denser and stronger aggregate structure, better settleability and

ensured solid-effluent separation, higher biomass concentration, and greater ability to

withstand shock loadings (Lin et al., 2005). The limited past investigations have shown that

azo dyes can be completely decolorized and some intermediates such as aromatic amines

with side groups (-SO3, - OH, -COOH, -Cl, N) containing metabolites were quantitatively

detected (O’Neill et al., 2000). Several microorganisms have been reported to transform

azo dyes into colorless metabolites corresponding to aromatic amines. Certain aromatic

amines from azo dyes are known to be toxic, mutagenic, and carcinogenic to various

organisms including animals (Ooi et al., 2007). The objective of the study was to develop a

bioreactor containing granules and to study its potential in decolorization and degradation

of textile dyes.

5.1. Materials and methods

5.1.1. Dyes and chemicals

The textile dyes, reactive blue 59 was a generous gift from local textile industry,

Ichalkaranji, Maharashtra, India. All the chemicals used were of the highest purity available

and of analytical grade.

5.1.2. Reactor setup, operation and media composition

A cylindrical glass reactor with a working volume of 3.0 L, a total height of 150 cm

and internal diameter of 5.5 cm was used to grow and cultivate aerobic granules. The

bioreactor was inoculated with different bacterial isolates that were isolated earlier in our

laboratory. Air was introduced with the help of fine bubble aerator located at the bottom of

the reactor at a superficial air pressure of 0.012 MPa/h. An airflow controller was used to

control the flow rate of air. The reactor was operated for about 60 days with constant

hydraulic retention time (HRT) of 24 h and at 30±2 °C throughout the study. The air flow

was stopped after 6 h for 5 min to allow the settling of the cell mass and the used media was

removed and fresh media was fed and again the operation is continued, this is repeated after

every 6 h. The reactor was fed with nutrient media containing (g l-1) peptone, 1.5; yeast

extract, 1.0; and sodium chloride, 1.5; as the sole carbon and nitrogen source. Pictures of

sludge appearance were taken once in a week with an Olympus CX-41 microscope.

Scanning electron microscopy (SEM) was performed to get an idea about the granulation

by fixing the sludge in a 2.5% glutaraldehyde solution, dehydrated in graded water-ethanol

solutions, then dried under vacuum conditions and sputter-coated with gold before SEM

pictures were taken with a JEOL JSM-500LV microscope.

5.1.3. Analytical methods

Different parameters of the granules are studied such as dry weight of granules,

chemical oxygen demand (COD), settling velocity (SV), total suspended solids (TSS) and

settling velocity index (SVI) measured according to Standard Methods (APHA, 1998). The

decolorization of textile dye Reactive blue 59 was monitored by using UV-Vis

spectrophotometer.

5.1.4. Granular decolorization test

The granules were collected in 500 ml Erlenmeyer conical flask, washed with

water, and kept at room temperature 30±2°C and 37±2°C. To study the ability of the

granules to remove the azo dyes from the aqueous solution, reactive blue 59 were

inoculated with different concentrations range from 0.5-5 g l-1 at static anoxic conditions.

After addition of dye and after decolorization (clear filtrate) the COD was measured. The

optical density was measured by using spectrophotometer.

5.1.5. Enzyme extraction and activity determination

The granules were incubated in nutrient media at room temperature (30±2 °C) and

dye was amended in the concentration 1 g l-1. After complete decolorization the granules

were separated and suspended in 0.1 M sodium phosphate buffer (pH 7.4). The collected

granules were homogenized in a tissue homogenizer at 4°C. The suspension was sonicated

on ultrasonicator and centrifuged at 10,000 rpm at 4°C for 20 min and supernatant was used

as enzyme. The cytosolic cytochrome P-450 was measured by the method of Omura and

Sato, (1962). NADH-dichloro phenol indophenol (NADH–DCIP) reductase was

determined by method of Wakeyama et al. (1983). Azoreductase activity was assayed as

per Zimmerman et al. (1982). Proteins were quantified using the Bradford assay with BSA

as standard (Bradford 1976).

5.1.6. Extraction and isolation of metabolites

The granules were weighed and 10% was added to nutrient media and was amended

with the Reactive Blue 59 at a concentration 1 g l-1 and decolorization was measured at

different time intervals on an UV-Visible spectrophotometer. After complete decolorization

the supernatant is collected by decantation and extracted with dichloromethane and dried

over anhydrous sodium sulfate. The solvent was evaporated and the samples were used for

genotoxic and cytotoxic studies.

5.1.7. Comet assay for genotoxicity

In vitro genotoxicity assessment was carried out by using comet assay for the dye

and degradation products on the earthworm coelomocytes as described in earlier chapter.

5.1.8. MTT assay for cytotoxicity

The cytotoxicity analysis was carried out by MTT assay as described in earlier

chapter for the dye and granular mediated degradation products.

5.1.9. PCR amplification of 16S rRNA gene sequences and library construction

The community analysis of the granules was carried out by extraction of the DNA

from the unexposed granules (control) and granules (test) that were used for biodegradation

of dye. These samples were centrifuged for 10 min at 10,000 × g. The pellet was

resuspended in phosphate buffered saline (PBS, pH 7.6), lysozyme 1 mg/ml, Proteinase K 1

mg/ml, and incubated at 55°C for 12 h. Total DNA was then purified from this solution

using phenol-chloroform extraction procedure. Partial 16S rRNA genes were amplified

from the extracted genomic DNA by PCR using a thermal cycler (Eppendorf). The variable

V3 region of the 16S rRNA gene from members of the domain Bacteria was amplified

using the forward (5′- CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA

CGG GGG GCC TAC GGG AGG CAG CAG -3’) and the reverse primer (5′- ATT ACC

GCG GCT GCT GG -3’) with a GC clamp. The final 50 µl reaction mixture contained 1×

PCR buffer (NEB, England), 1 nmol of dNTPs,1 pmol of forward and reverse primers, 1

unit of Taq DNA polymerase (NEB, England), and ∼10 ng of template DNA. The PCR

protocol included initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 sec, 55°C

for 30 sec, and 72°C for 45 sec, followed by 10 min at 72°C and incubation at 20°C until

further process. All PCR reactions were carried out in triplicates and the product were

collected into 50 µl autoclaved distilled water. The denaturating gradient gel electrophoresis

(DGGE) was performed on a Universal Mutation Detection System (Bio-Rad). Samples

containing approximately equal amounts of PCR amplicons were loaded onto 8% (w/v)

polyacrylamide gels (37.5:1, acrylamide: bisacrylamide) in 1X Tris-acetate-EDTA (TAE)

buffer with a denaturing gradient ranging from 40 to 60% denaturant (100% denaturant

contains 7 M urea and 40% (v/v) and formamide in 1× TAE buffer). Electrophoresis was

performed at 60°C, at 80 V (20 h). After electrophoresis, the gel was stained with ethidium

bromide (Sigma), and visualized under UV transilluminator. The most prominent bands

from the DGGE gel were excised by puncturing the gel using sterile pipette tips. The tips

were placed in a 1.5 ml microcentrifuge tube containing 20 µl of sterile deionized water and

incubated overnight at 4°C. A 2-5 µl aliquot of this solution was used as template for PCR

re-amplification. The purity of the re-amplified PCR product was confirmed by agarose gel

electrophoresis (1.5%). Selected products were then purified by PEG-NaCl precipitation

(Sambrook et al., 1989).

5.1.10. Construction of clone libraries for 16S rRNA and functional genes

The PCR products were purified using Qiagen PCR purification kit (Qiagen, USA),

cloned into the pGEMT easy vector (Promega, USA) and then transformed into E. coli

JM109 (Promega, USA) following the manufacturer’s instructions. One hundred clones

(from each 16S rRNA gene library) and 50 clones (from each functional gene library) were

picked for direct colony PCR with M13F/M13R primers targeting the flanking vector

sequences. The PCR products were run on agarose gels with DNA ladder to confirm the

correct size of the cloned inserts and subsequently purified by PEG-NaCl precipitation

(Sambrook et al., 1989) and sequenced with forward- 5’-CC TAC GGG AGG CAG CAG -

3’ and reverse 5′- ATT ACC GCG GCT GCT GG -3’ primers.

5.1.11. DNA sequencing and phylogenetic analysis

The DNA sequencing was performed on a 3730 DNA analyzer (Applied

Biosystems, USA) using the ABI Big-Dye version 3.1 sequencing kit as per the

manufacturer’s instructions with both M13F and M13R primers for all functional gene

library based PCR products and with only M13F for 16S rRNA gene library based products

(partial sequencing). The generated sequences were analyzed using ChromasPro software

(http://www.technelysium.com.au/ChromasPro.html) and compared to the current database

of nucleotide sequences at GenBank and Ribosomal Database Project (RDP). Reference

sequences were chosen based on BLASTN similarities. All 16S rRNA gene sequences were

checked for possible chimeric artifacts by using the Pintail program (Ashelford et al., 2006)

in conjunction with Bellerophon (Huber et al., 2004). Functional gene sequences were

inspected for chimeras by BLASTN analysis. Multiple sequence alignments of 16S rRNA

genes were performed with MEGA 4 software with neighbour algorithm p-distance

(Tamura et al., 2007).

5.2. Results and discussions

5.2.1. Granulation

The continuous aeration and feeding lead to increase in bacterial cell mass and

granulation was initiated at around 20 days and reached to mature granules in 60 days of

reactor operation. The sizes of the small granules were measured using phase contrast

microscopy (Olympus CX 41). High hydraulic selection pressure exerts in systems will

retain denser, heavier, more compact and smooth granules, as well as improved metabolic

activity (Chen et al., 2007). No granular sludge was observed with low superficial up flow

air velocity less than 1.2 cm/s (Tay et al., 2001b). The morphology of mature granules in

the reactor is shown in (Fig. 1) and it can be seen that granules in the reactor were different

in color and appearance (Liu et al., 2007).

Fig. 1. Images analysis of aerobic granular in the three reactors at steady state. a) cultures at

initial stage of granulation (b) Initial stage of accumulation of cultures (c) 40 days granules

(d) 60 days of mature granules

In nature, selection pressures for aerobic granulation are triggering forces that play a

crucial role in granulation process and further influence the granular characteristics and

reactor performance (Qin et al., 2004; Wang et al., 2007). In this study, dense and good

settling ability aerobic granules were developed with increasing hydraulic shear force.

5.2.2. Microstructure of aerobic granules

The microstructure of aerobic granules was examined using SEM and is shown in

(Fig. 2) It can be clearly seen that the cell mass before granulation process in the reactor

had a fluffy loose structure (Fig. 2a) and later on after 20 days it showed filament-dominant

outer surface (Fig. 2b).

Fig.2. SEM observation of the granules (a) cultures at initial stage of granulation= 1µm (b)

Initial stage of accumulation of cultures = 1µm (c) 40 days granule = 100µm (d) 60 days

mature granule = 200µm

However, after 40 days the granules in the reactor showed a very compact structure

(Fig. 2c), and after 60 days, rod-like shape were found to be predominant and some cavities

were also found to be present (Fig. 2d). These cavities would be favorable to enhance

substrate and oxygen transfer from the bulk to the interior of granules (Wang et al., 2007).

5.2.3. Reactor performance and cultivated granules

The testing performance of the reactor was monitored for over the period of 60

days. The granule sizes in the reactor after 60 days of operation were 1-2 mm in size. The

volatile suspended solid of the aerobic granular bioreactor was 6.5 g l−1. Mixed volatile

suspended solids (MVSS) was slightly reduced during initial granulation phase because the

cell mass has low settling velocity and washed out (Adav et al., 2008). Granules were

assessed after every 20 days for their MVSS and found to be significantly increased. Since

aerobic granulation represents a form of self immobilization of cells, the autoaggregation

abilities of the isolates were also examined. Generally, strains with autoaggregation ability

can contribute to the structural stability of cell aggregates. Autoaggregation took place

when cultures were cultivated in a reactor with constant shear force of air. Cell aggregates

possessed good settleability, as these aggregates settled immediately to the bottom of the

cylindrical reactor. The settling velocity (SV) was 13.3 ml-1 min in the initial stage of

operation of reactor which was significantly increased up to 46.6 ml -1 min after 60 days of

granulation (Fig. 3).

0 2 4 6 8 10 12 14 16200

300

400

500

600

700

800

900

1000

Set

tling

vol

. in

ml

Time in min

0 Days 20 Days 40 Days 60 Days

Fig. 3. Time profiles of settling velocity (SV) during the granular maturation.

0 2 4 6 8 10 12 14 16

300

400

500

600

700

800

900

Set

tling

vol

. in

ml

Time in min

Without exposed to dye Exposed to dye

Fig. 4. Effects of OLR applied 5 kg m-3 of the dye RB59 on settling velocity (SV) of

granules

The settling velocity of granules was found to be decreased by 20% after the

exposure of 5 kg m-3 of dye (Fig. 4). The total suspended solids (TSS) value was 1.4-1.5 g l-

1 in the initial stage of granulation, whereas it was significantly increased up to 6.05-6.25 g

l-1 after 60 days (Fig. 5). The average SVI values were reduced from 550 ml g-1 at initial

stage to 46-59 ml g-1 after formation of matured granules at 60 days (Fig. 6). There are

many evidences showing that a high shear force favors the formation of aerobic granules

and granular stability (Shin et al., 1992; Tay et al., 2001a; Liu and Tay, 2004; Wang et al.,

2004).

0 20 40 60

1

2

3

4

5

6

7

TSS

in g

m/L

Time in days

Fig. 5. Time profiles in days of granular bioreactor on volatile suspension solids (TSS)

0 20 40 600

100

200

300

400

500

600

SV

I in

ml g

-1

Time in days

Fig. 6. Time profiles in days of granular bioreactor on settling volume index (SVI)

5.2.4. Decolorization of Reactive Blue 59 dye

The decolorization of textile dye (reactive blue 59) was carried out at 30±2 and

37±2°C. The decolorization efficiency of bacterial granules was examined for the dye,

reactive blue 59, under static anoxic conditions at 37 °C in the increasing concentration in

range of 0.5-5 kg m-3 of the dye. The efficiency of the granules from bioreactor was

assessed at an organic loading rate of 5 kg m−3 per day of the dye RB59 and complete

decolorization of the dye was obtained within 24 h. The COD removal efficiency of

bacterial granule was 30-40% immediately after decolorization and it was found to be

increased upto 50-60% after incubation for another 24 h (Fig. 7).

1 2 3 4 50

10

20

30

40

50

60

70

80

90

100

CO

D (g

/l)

Concentration of dye (g/l)

Before degradation Immediately after degradation After 24h of incubation

Fig. 7. Change in COD after exposure to the dye RB59 at 37°C

Similarly anaerobic/aerobic sequential process showed decolorization of the dye reactive

black 5. The COD (59%) and color removal (85%) efficiencies was obtained (Sponza and

Isik, 2002). Anaerobic or static conditions were necessary for bacterial decolorization for

initial reduction of azo bond. Under aerobic conditions azo dyes are generally resistant to

attack by bacteria (Hu, 1998). Degradation samples were analyzed by UV–Vis

spectroscopy to confirm that decolorization was due to biodegradation and not merely

decolorization. Abiotic control without granules and with reactive blue 59 was incubated

and scanned from 200 to 800 nm that showed the maximum absorbance at 585 nm and no

abiotic decolorization. Samples from the inoculated broth were also scanned similarly,

which showed a significant decrease in the absorption at 585 nm. Disappearance of peak

indicates decolorization (Fig. 8).

200 300 400 500 600 700 800

0

1

2

3

4

Abs

Wavelength in nm

0 h 4 h 8 h 12 h

Fig. 8. UV visible spectral analysis of RB59 decolorization

5.2.5. Ecotoxicological analysis of degradation products formed after degradation

MTT (cytotoxicity) and comet (genotocity) assays were performed in order to

determine the toxicity of azo dye reactive blue 59 and its degraded products. The

cytotoxicity was measured in terms of % cell viability of L-929 cell lines. Toxicity tests

showed that the product formed after granular mediated degradation did not exhibit

cytotoxic response toward L-929 cells, whereas the original dye found to induce cytotoxic

response toward L-929 cells, the viability of cells was decreased by 25% of that of the

control (Fig. 9).

Control Before degradation After degradation0

20

40

60

80

100

120

140

160

% V

iabi

lity

Fig. 9. Cytotoxic analysis of before degradation and after degradation of RB59

The genotoxic effect of reactive blue 59 on earthworm coelomocytes was increased

with increasing concentration of dye. The degradation product did not exhibit the genotoxic

effect even when high concentration of dye was used for decolorization studies. This

toxicity tests showed that the reactive blue 59 was cytotoxic and genotoxic, whereas

granular decolorization of dye (reactive blue 59) generated metabolites were not toxic to L-

929 cell line and earthworm coelomocytes, indicating that the products causing no toxicity

to the environment (Fig. 10).

0 1 2 3 4 5 60

40

80

120

160

200

240

280

320

360

400

Control 1µg/ml 2µg/ml 3µg/ml 4µg/ml H

2O

2 (70.4µM)

degradation product

Arb

itrar

y un

its

Fig. 10. Genotoxic analysis of RB59 at different concentrations and product formed after

degradation

Similar studies were carried out for the cytotoxicity studies revealed that

biodegradation of the dye Direct Black-38, by the isolated culture resulted in detoxification

of the dye and no significant total cell death in product formed after decolorization (Kumar

et al., 2006). A yeast-based bioassay was used to compare the potential cytotoxicity of the

Sudan Orange G with its enzymatic biotransformation products and found that the dye was

cytotoxic to the yeast cells, whereas laccase enzymatic biotransformed products were

significantly less toxic to the yeast cell population (Pereira et al., 2009).

5.2.6. Role of enzyme on the decolorization

A major mechanism of biodegradation in living cells is because of the ability of the

cytosolic enzymes to mineralize synthetic dyes. The major contributions of azoreductase,

NADH-DCIP reductase and cytochrome P450 system mediated both the reduction and the

N-demethylation reactions (Cha et al., 2001). The bacterial granules were exposed to the

dye (reactive blue 59) and the induction in the enzyme system viz. cytochrome P450,

azoreductase and DCIP reductase was observed (Table 1).

Table 1. Enzyme activities in control and induced state Enzyme Before dye degradation After dye degradation aAzoreductase 2.23 ± 0.46 3.60 ± 0.23* bCytochrome P-450 0.012 ± 0.001 3.031 ± 0.18***

Values are mean of three experiments ± SEM. Significantly different from control cells at *P < 0.05, ***P < 0.0001 by One way analysis of variance (ANOVA) with Tukey–Kramer comparison test. a nmol min−1 mg−1 protein b nmol mg−1 protein

The increase in content of cytochrome P450 and azoreductase activity after

exposure to dye represents that there is induction in the enzyme system for the degradation

of dyes as compared to unexposed granules. This study demonstrates that bacterial granules

and its enzyme systems are involved in efficiently dye decolorization. Similarly it has been

previously reported that the strain Achromobacter sp. DBTN3, a heterotrophic denitrifying

bacterium, exhibits nitrous oxide reductase activity and was identified as an abundant and

dominant denitrifying community in granular mediated wastewater treatment (Zhang et al.,

2008; Juang et al., 2009).

5.2.7. Taxonomic diversity of dye-degrading granules

The DGGE analysis of the variable region 3 of 16S rDNA sequences obtained from

the aerobic granules without exposure to dye (control) and exposed to dye (sample) was

performed. The control band represents the bacterial granules that were not exposed to the

dye (reactive blue 59) and sample represents the granules undergone continuous exposure

to dye (Fig. 11). The band pattern clearly reveals the granules which was exposed to the dye

showed the predominate species which are involved in the effective decolorization at higher

concentrations (5 kg m-3). The community profile of both the sample and control was

compared by observing the banding pattern in DGGE. The community profile of control

aerobic granules showed microbial diversity which was evident from 23 different bands in

the DGGE. The microbial diversity in the aerobic granules treated with dye was not much

diverse as compared with control and showed only 8 predominant bands. This discrepancy

can be attributed to the sensitivity of the bacterial species to the dye. The DGGE analysis

revealed that the bacterial community in the sample was similar with control as observed by

the banding pattern. All the bands present in the sample were also present in the control.

For example, in sample band number S1, S2, S3, S4, S5 and S6 were observed in both

control and sample whereas C2, C4, C7, C9, C13, C14, C16, C21 and C23 bands

disappeared, these species could not survive in the presence of dye. The excised bands were

cloned and sequenced and few of them showed homology with proteobacteria in the range

of 98-100% homology (Table 2). The phylogenic analysis of both control and sample

showed the nearer homology with each other, this evidence showed that these species are

from same origin and adapted in the presence of dye (Fig. 12). Although the microbial

diversity was stable in control and sample, such an occurrence suggests a better long-term

operational stability for granular bioreactor (Juang et al., 2009). The studies of community

analysis (DGGE) revealed the wide range of microbial species involved in the granulation.

The band patterns for control community were enriched after exposure of textile dye which

can be observed with few similarities with control.

Fig. 11. An ethidium bromidestained polyacrylamide denaturing gradient gel (35–70%)

with DGGE profiles of 16 S rDNA gene fragments for samples from bioreactor, The

samples were labeled as the top, control: granules without exposure to dye and sample:

granules with exposure to dye

c3f

s1f

c6f

s2f

c7f

s3f

c9f

C2F

C22F

c8f

c10f

s5f

c1f

C14F

s4f

C23F

s8f

s6f

s7f

c12f

0.05

Fig. 12. Phylogenetic tree of control and sample cloned sequences

Table 2. DGGE bands and their close relatives

Band no.

Close relative Accession no.

% Similarity

Phylogenetic affiliation

C1 Uncultured Acidobacteria HM062028 99 Acidobacterium C2 Uncultured Thauera sp. GU980064 100 Betaproteobacteria C3 Uncultured bacterium EU426931 98 Bacteria C6 Uncultured bacterium GQ252618 100 Bacteria C7 Estrogen-degrading bacterium DQ066432 99 Bacteria C8 Uncultured beta proteobacterium FM252746 100 Betaproteobacterium C9 Uncultured bacterium FJ612209 94 Bacteria C10 Thermomonas haemolytica

strain GU195191 98 Gammaproteobacteria

C14 Uncultured bacterium FM253000 93 Gammaproteobacteria C22 Uncultured bacterium GU980064 99 Proteobacteria C23 Uncultured bacterium FM253000 100 Proteobacteria S1 Uncultured bacterium EU426931 98 Bacteria S2 Uncultured bacterium GQ252618 100 Bacteria S3 Estrogen-degrading bacterium DQ066432 98 Bacteria S4 Uncultured bacterium FM252974 100 AlphaProteobacteria S5 Uncultured bacterium FM252746 100 BetaProteobacteria S6 Uncultured bacterium FM253000 100 AlphaProteobacteria S7 Uncultured bacterium FM252847 100 AlphaProteobacteria S8 Uncultured bacterium FM252920 100 AlphaProteobacteria

5.3. Conclusion

Maturation of granules was proved with SEM and images. The aerobic granules have

potential for the degradation RB59 up to 5 kg m-3 with in 12 h with significant decrease in

COD after the degradation. The cytotoxic and genotoxic analysis of degradation product

has proved that the products or intermediates are not toxic. Community analysis (DGGE)

indicates the wide microbial species involved in the granulation. Granulation technology

can be used to remove the dyes from industrial effluents, because of its reusability and

without any technical complications.