Environmental Engineering Reference
In-Depth Information
favorable for the degradation of a certain compound (Mueller 1992 ). Presence of
toxic materials, type and amount of organic material (carbon) present, pH, electron
acceptor and inorganic nutrients such as nitrogen and phosphorus are the most
important abiotic factors limiting bacterial population from the perspective of
bioremediation (Baker and Herson 1994 ).
4 Involvement of Oxidoreductive Enzymes in Degradation
Process
Various bacterial strains reduce azo dyes under anaerobic condition. The most
accepted hypothesis for this phenomenon is that many bacterial strains possess
rather unspeci
c cytoplasmic enzymes which act as
azo reductases
. It was earlier
suggested that under anaerobic condition, unspeci
c cytoplasmic azo reductases act
via the intermediate formation of free reduced
avin (Walker 1970 ). Previous
reports on azo dye degradation by bacteria focused on a certain enzyme which is the
azo reductase responsible for the reduction of azo dye. According to Russ et al.
( 2000 ), enzymatic reduction can occur both intracellularly and extracellularly. Ra
i
et al. ( 1990 ) isolated anaerobic bacteria that produced azo reductases constitutively
and released extracellularly. An azo reductase was puri
ed form E. faecalis by
hydrophobic, anion exchange and af
nity chromatography (Punj and John 2008 ).
Maier et al. ( 2004 ) have shown that a NADPH-dependent azoreductase is
responsible for azo dye reduction by Bacillus strain SF. In a study of the anaerobic
reduction by whole cells, cell extracts and cell membranes of Sphingomonas sp.
strain BN6, enzymatic azo dye reduction activity was found to be located in the
cytoplasm as well as in the membrane fraction. It was also suggested that azo dye
reduction by whole cells is mainly related to the membrane fraction (Kudlich et al.
1997 ).
5 Mechanism and Pathways of Degradation
In our study, we conducted test to determine if the azo dye degrading enzyme is
located intracellularly or extracellularly. To test if the enzyme is located extracel-
lularly, we used the decolorized culture broth of monocultures and centrifuged at
10,000 rpm for 10 min at 5
°
C. The supernatant was collected and then
lter
sterilized using a 2
µ
m sterile syringe
lter. The sterile supernatant was incubated at
°
30
C with Congo red and decolorization was observed. To test for the intracellular
location of the enzyme, we prepared the cell free extract by growing whole cells in
nutrient broth up to its logarithmic phase based on the growth curve. Samples were
taken and then centrifuged at 10,000 rpm and the pellet was suspended in 30 ml
phosphate buffer. The pellet suspension was transferred to a 50 ml ask containing
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