Bioremediation of Remazol Black B by newly isolated Bacillus endophyticus LWIS strain
Munesh Kumari1, Maulin P Shah2* and Swaranjit Singh Cameotra1
1Institute of Microbial Technology, India
2Industrial Waste Water Research Lab, India
Submission: September 28, 2016; Published: November 18, 2016
*Corresponding author: Maulin P Shah, Industrial Waste Water Research Lab, Division of Applied & Environmental Microbiology, Enviro Technology Limited, Gujarat, India, Email: shahmp@beil.co.in
How to cite this article: Munesh K, Maulin P S, Swaranjit S C. Bioremediation of Remazol Black B by newly isolated Bacillus endophyticus LWIS strain
. Adv Biotech & Micro. 2016; 1(4): 555568. DOI: 10.19080/AIBM.2016.01.555568
Azo group containing most of the dyes are released into untreated wastewater lacking of any pretreatment and pollute water and soil environments. To thwart pollution of our prime resources, elimination of these dyes containing impurity is of enormous significance. For this purpose, wastewater samples were brought to gather from dye-contaminated sites of India. Total14 bacterial isolates were isolated through enrichment and then tested for their potential to remove Remazol Black-B azo dye in liquid medium. Isolate LWIS1, LWS2 & LWS3 having competence to degrade Remazol Black-B proficiently were screened on modified mineral salt medium. LWS1 was competent to absolutely remove the dye from the liquid medium in 10 h. The isolate LWIS 1 exhibited the most excellent performance at the dye concentration of 150 mg L-1 medium (pH 7.5) and at temperature 37°C. Similarly, yeast extract proved to be the best carbon source for decolorization purpose. The results imply that the isolate LWIS1 could be used for the removal of the reactive dyes from textile effluents.
Effluent discharged from the textile industries has variable characteristics in terms of pH, dissolved oxygen, organic, and inorganic chemical content, etc. Together with industrialization, awareness towards the environmental problems arising due to effluent discharge is of critical importance. Pollution caused by dye effluent is mainly due to durability of the dyes in waste water [1]. Existing effluent treatment procedures utilize pH neutralization, coagulation followed by biological treatment, but they are unable to remove recalcitrant dyes completely from effluents. This is because of the color fastness, stability, and resistance of dyes to degradation [2].
Bioremediation is the microbial clean up approach, microbes can acclimatize themselves to toxic wastes and new resistant strains develop naturally, which can transform various toxic chemicals to less harmful forms. Several reports have suggested that the degradation of complex organic substances can be brought about by bacterial enzymes [3-8]. Different dyes used in textile industry usually have a synthetic origin and complex aromatic molecular structures which make them more stable and more difficult to be biodegraded. Due to their ease of manufacturing methodology, azo dye accounts for almost 80% of annual production of commercial dyes all over the world. There are over 10, 0000 commercially available dyes with a production of over 7×105 tons per year [9]. Azo dyes, containing one or more azo bond (-N=N-), account for 60-70% of all textile dyestuffs used. It is estimated that about 10-15% of the total production of colorants is lost during their synthesis and dyeing Processes [10,11]. Whereas, in the case of reactive dyes almost 50% of the initial dye load is found in the dye bath effluents.
Colored industrial effluent is the most obvious indicator of water pollution and the discharge of highly colored synthetic dye effluents is aesthetically displeasing and cause considerable damage to the aquatic life. Although several physical-chemical methods have been used to eliminate the colored effluents in wastewater, they are generally expensive, produce large amounts of sludge. More often these conventional modes of treatment lead to the formation of some harmful side products. Interest is therefore now focused on the microbial biodegradation of dyes as a better alternative [12].
Some microorganisms, including bacteria, fungi and algae,
can degrade or absorb a wide range of dyes [13].The biological
mode of treatment of dye bath effluents offers distinct advantages
over the conventional modes of treatment. This method is
more economical and leads to less accumulation of relatively
harmless sludge. Most importantly, biological treatment of dye
bath effluents is eco friendly. It causes mineralization of dyes
to simpler inorganic compounds which are not lethal to life
forms. The basic step in the decolorization and degradation of
azo dyes is breakdown of azo bonds, leading to removal of color.
Azo dyes are known to undergo reductive cleavage whereas
the resultant aromatic amines are metabolized under aerobic
conditions [14]. So for complete mineralization of azo dyes the
microbial population forming part of treatment system should
be able to work efficiently. In view of these problems the most
potent bacterial culture was selected in this study for maximum
decolorization of Remazol Black B.
Ankleshwar, Gujarat, India is one of the most industrialized
cities in India. It is ideal for waste water sample collection.
The samples were collected from the activated sludge of the
common effluent treatment plant of Ankleshwar, Gujarat,
India. Samples were collected from the aeration tank of
common effluent treatment plant. The Temperature and pH
was analyzed at the site. The temperature was analyzed using
laboratory grade thermometer and pH was analyzed by using
pH meter (Hanna digital pH meter). The sample was transferred
to laboratory at 4 °C as per the standard methods [24]. The
physicochemical parameters such as Color, Biological Oxidation
Demand, Chemical Oxygen Demand, Total Suspended Solids,
and Total Dissolved Solids were analyzed as soon as the sample
was brought to the laboratory (Table 1). Sample color was
analyzed by spectrophotometer (SHIMADZU UV-1700). BOD
was determined by employing evaporation method by dissolve
oxygen meter while chemical oxygen demand was measured by
instrument directly.
Table 1: Effect of formulation parameters on the particle size, polydispersity index, drug loading and encapsulation efficiency of CS-MIAA coated magnetic nanoparticles.
The bacterial strains were isolated from waste water
containing activated sludge. Inoculums developed from the
isolate LWS1 was first enriched using a modified MSM medium
with Remazol Black-B dye as the source of C and N [20].
Remazol Black B dye was added to a concentration of 150 mg
L-1. The cultures containing 200 ml MSM with a dye broth in
500 ml Erlenmeyer flasks were inoculated with 10 ml volume
of activated sludge. The flasks were incubated at 37°C for 5 days
in static conditions. After incubation, the cell suspensions from
each vial were plated on MSM agar medium and incubated at 37
°C for 24 h. Bacterial colonies that appeared on the agar medium
were picked, washed gently with sterile water and re suspended
in flasks containing fresh MSM broth enriched with the dye.
Approximately 14 colonies actively growing were selected for
purification.
The selected isolates were purified by streaking on agar
containing MSM medium at 20 g L-1 concentration. Streaking
was done thrice in Zig-zag manner. The purified cultures were
preserved in a refrigerator for subsequent study.
Using modified MSM medium, primary screening was
done to obtain proficient bacterial strains able to decolorize
the azo dye. For this purpose, 14 isolates gained ability to
decolorize Remazol Black-B from all samples was selected. The
decolorization ability of each isolate was tested in liquid medium.
Remazol Black B amended media were incubated at 37 °C for 24
h with respective bacterial inoculum. After 24 h, harvesting of
the cells was done by centrifugation at 10,000 rpm (REMI R-23,
India) for 10 minutes. Then decolorization was measured with
that facilitate of spectrophotometer (SHIMADZU-1700, Japan) at
597 nm. Uninoculated media were run as blanks to check abiotic
decolorization. The three most efficient bacterial isolates (LWIS
1, 2 & 3) from the concluding screening were again tested for
their decolorization potentials in test tubes at different time
intervals. 0.5% yeast extract as a co-substrate were added in
ten milliliters sterilized MSM broth containing azo dye (Remazol
Black B) at 150 mg L-1 concentration.
Uniform cell density of the inocula were added with respective
bacterial strains at (OD: 0.6) 597 nm. Azo dye containing MSM
medium with yeast extract in uninoculated test tubes were
incubated under parallel environment to make sure for abiotic
decolorization of dye. Decolorization was measured after every
6 hrs interval up to 24 hrs at 597 nm by spectrophotometer as
described by Khalid et al. [20].
Distinctive manner of parameters like substrate
concentration, temperature and pH were optimized during the performing tests for distinct carbon sources (Mannitol, Yeast
extract, glucose, maltose) at 5 g L-1 concentration were explored
as co- substrate in the dye removal development. Optimization
studies have involved the selection of the concentration of the
dye (50, 75, 100, 125, 150, 200 and 250 mg L-1), the temperatures
(25, 30, 35, 40, 45oC) and pH (5, 6, 7, 8, 9). Isolated strains LWIS
1, 2 & 3 have been with in order to optimize their color removal
competence. As culture conditions were the unchanged as used in
color removal experiment i.e., 100 mg L-1 of Remazol Black-B azo
dye with MSM was used. Uninoculated blanks were run to make
sure the abiotic decolorization throughout the experimentation.
To scrutinize the color potency depth of the azo dye Remazol
Black-B decolorizing activity by an effectual bacterial isolates,
liquid medium was used. Based on the relative aptitude of
the decolorization of different isolates, three most excellent
performing bacterial isolates (LWIS1, 2 & 3) with more than 90%
of decolorizing activity were preferred for more experiments
(Data not shown).
Microbial decolorization of Remazol Black-B by the most
promising selected bacterial isolates (LWIS1, 2 & 3) was
corroborated by performing one more experiment in liquid
medium at distinctive time frame (Figure 1). It was surveyed
that distinctive bacterial isolates had patchy aptitude to
eliminate Remazol Black-B in actively growing cultures. The
most competent bacterial isolate to decolorize Remazol black-B
colorless was by isolate LWIS1 with 98% color degrading
efficiency in 10 h incubation interlude while left over isolates exhibited utmost decolorization in 16h. Isolate 2 was the
second largely skilled bacterial isolate and it removes the color
of Remazol Black-B up to 94% in 16 h. Similarly, isolate 3 had
decolorization potential of 86%.
Potential of selected isolates (LWIS1, 2, and 3) was
further explored for the optimization of assorted incubation/
environmental circumstances for removal of the azo dye in liquid medium. It was apparent (Figure 2) that Remazol Black-B
azo dye decolorization piercingly improved up to 100 mg L-1 of
substrate concentration and utmost removal was observed at
100 mg L-1 of substrate concentration. Then, there was a slow
but sure decline in the azo dye decolorization. Isolate LWIS1was
the most proficient azo dye removal strain with more or less
complete removal of the color i.e., 100% decolorization at 100
mg L-1 and minimum decolorization was recorded at 50 mg L-1
while after 100 mg L-1 substrate concentration, again LWIS1
showed a decreasing trend. Isolate 2 was the second at the rank
with 90% decolorization at 100 mg L-1. But, isolate 3 showed
different trends from the other isolates; it indicated enhanced
decolorization up to 200 mg L-1(82%).
Sound effects of distinctive carbon sources such as maltose,
mannitol, glucose and yeast extract were assessed on Remazol
Black-B decolorization by bacterial isolates (Figure 3). It was
noted that utmost decolorization take place with 5% yeast
extract in all selected strains (85 to 98%) that was pursued by
glucose in which decolorization takes place in the range of 20 to
25%. However, slightest decolorization was seen in the case of
mannitol (10 to 15%and maltose (up to 18%).
For pH optimization study, pH ranging from 5 to 9 were
exploited and all selected isolates were inoculated at these
levels (Figure 4). To begin with increase in pH from 5 to 7,
decolorization improved and utmost decolorization takes
place at 7.5 pH. Likewise, auxiliary raise in pH from 7 to 9 had
pessimistic outcome on decolorization aptitude of a mixture of
isolates. The peak decolorization was observed with the isolate
LWIS1 (98%) at pH 7.5 though least decolorization take place
at pH 9. Comparable tendency in lingering isolates 2 and 3 were
examined at pH 7.5. Overall, it was noted that all the bacterial
isolates demonstrated most favorable decolorization from pH 5
to 7.5.
Five levels (25, 30, 35, 40 and 45°C) of the temperature
used for the assessment of optimum biodecolorization Remazol
Black - B by selected bacterial isolates. It is apparent (Figure 5),
when the temperature is raised from 25 to 35°C there was an
inconsistent trend in decolorization due to different isolates.
Isolate LWIS 1and showed gradual increase in decolorization,
while isolate3 indicated maximum decolorization at 25oC. Other
two bacterial isolates (LWIS1 and 2) with a gradual increase
from 25 to 35°C showed maximum decolorization at 35 C. As
the temperature is further raised from 35oC to 45°C, there was sharp decline in decolorization capacity in all isolates
increased. Utmost decolorization was detected with the isolate
1 (98%) at 35°C and is followed by isolate 2 (94%) at the similar
temperature. Least decolorization was observed at 45oC in all
selected isolates.
Among three selected bacterial isolates LWIS1exhibited
maximum decolorizing potential against azo dyes, so it was
identified by 16s rRNA gene sequencing approach. Sequence
analysis of 16s rRNAgene showed that isolate LWIS1 had 98.89%
similarity with the species Bacillus endophyticus. Phylogeny tree
(Figure 6) based on Mega 6 indicated that isolate LWIS was a
Bacillus endophyticus strain. The 16s rRNA gene sequence
submitted in Gene Bank with accession number KT945244.
Industrial effluent is very much unstable and it fluctuates
repeatedly in an ample assortment depending upon the
course practiced. Most of the south Asian countries have
rigorous environmental trouble due to hasty industrialization
development. This observable fact is very widespread where
the polluting industries like paper, pulp, textile, dyeing, leather,
tanning processing, sugar manufacturing flourish as clusters.
Together with these, the textile industries are huge industrial
patrons of waters as well as producers of wastewater. The
effluent discharged by this industry headed to grim pollution
of groundwater and soils and eventually has an effect on the
livelihood of the poor [21].
During the dying process a significant amount of dyes and
other chemicals lost in the wastewater. It is estimated that the
dye loses between 10 - 15 % [22]. Dye is generally non-toxic to
the environment, but the color waters there may impede high
penetration influenced by the aquatic life and the usage limit [23].
Decolorization of industrial effluent has been a foremost anxiety
in waste water that instigates from textile and dye stuff plant
with a unremitting discharge of immense quantity of residual
dyes to the environment. A well-organized management of the
effluent is an environmental friendly approach for medication
of textile effluent.
The microbial degradation of dye molecules in the
environment is likely to be slow, which signifies that it is
promising for high intensity of dye to keep on and probably
accumulate. Because of the poor biodegradability of dyes,
unadventurous process of biological treatments is unproductive
in the management of dye containing wastewater. Biological
decolorization is used in both aerobic and anaerobic
environments. A number of reports discourage decolorization
of the azo dye by microorganism under anaerobic conditions
since it leads to the formation of the corresponding aromatic
amines. The efficiency of microbial discolorization depends on
the adaptive capacity and activity of selected microorganisms. In
recent decades, many microorganisms are capable of degrading
azo dyes, including bacteria, fungi and yeast. The discharge of
waste water color in the river by the textile industry represents
a serious environmental problem and a public health problem.
The main part of the wastewater contains azo dyes which
are increasingly used in industries because of their ease and
cost effective in the synthesis compared with natural dyes.
Relative effectiveness of bacteria isolated for decolorization
Remazol Black- B clearly implies that these can be effectively
used for removing Remazol Black- B from industrial wastewater
contaminated. Azoreductase is reported to be the key enzyme
expressed in bacterial azo dyes degrading and catalyses the
reductive cleavage of the azo bond [24, 20]. Azoreductase
activity had been identified in a number of bacterial species
recently, such as Staphylococcus aureus, Shewanella putrefaciens,
Shewanella and Pseudomonas spp. [24- 26].
It was denoted that enhance in substrate concentration from
its best possible level had pessimistic effect on decolorization
capacity of isolated isolates. Explorations with distinctive dye
concentrations in additional experiments also reported higher
net color removal efficiencies at lower dye concentrations
[27-29]. Dwindle in color removal ability at high substrate
concentration might be due to the toxicity of the dye and co
contaminants [30]. On aromatic ring of azo dye structure one or
more sulphonic-acid groups are generally endorse, which might
be active as detergents to hold back the growth of microbes [30].
One more explanation of the toxicity at elevated concentration
may be due to the presence of heavy metals or metal complex
dyes and/or the occurrence of non hydrolyzed reactive groups
which may impede the bacterial growth [29]. In the same way,
diminution in color removal at stumpy concentration of the
substrate might be due to the shrink in enzyme capability to be
acquainted with the substrate proficiently.
While in case of diverse carbon sources experimented yeast
extract corroborated to be the most excellent amongst tested
carbon source. Our results were in accordance with the research
performed by Guo et al. [31] in which the bacterial strains grew
well and absolutely decolorized K-2BP where either peptone or yeast extract was contemporary in the medium; however,
sucrose, glycerol, glucose, starch and lactose resulted in poorer
rates of growth and decolorization of these dyes. Supplementary
studies also accounted the utmost color removal of azo dyes in
the presence of yeast extract by bacteria [32]. In case of variable
pH, decolorization was on upper side at pH 7.5.
Whereas at alkaline pH conditions declined the decolorization
efficiency of all the tested isolates were observed. So, from
this investigation, it could be bring to a close that neutral pH
sustained bacterial movement to decolorize Remazol Black-B
in liquid medium [33-34]. Temperature is one more incredibly
significant parameter for anaerobic treatment of wastewater.
Selected isolates were mesophilic bacteria because they all
showed better decolorization in the temperature range of 25
to 35 °C. Similar results were also reported by Guo et al. [31].
The mesophilic range is traditionally used [35-37] since it is
generally thought that maintaining high temperature would be
uneconomical, while degradation within the psychrophilic range
is too slow. Overall, one of the selected isolate [1] of bacteria
was able to completely remove color of the dye in 18 h. However,
these isolates should be tested at large scale treatment system
to examine their potential for bioremediation of dye-polluted
wastewaters.
The current study divulges that the selected three isolates
can be exploited lucratively for decolorizing Reactive Black B
dye. The cultures exhibited maximum decolorization ability at
pH between 7.5 for all the three isolates and 35 °C for 1, 2 & 25 °C
for 3. Moreover, 5 g/L yeast extract was found to be optimum for
decolorization. In conclusion, isolate LWS1 was found as one of
the most efficient among others for bioremediation of Remazol
Black B dye, which can be studied further for bioremediation of
dye-polluted waters including rate of degradation of azo dyes
other than the selected Black dye through an application of
Bioaugmentation.
The risk factors for autoimmune dermatologic diseases are
genetics, sex, other immunologic disorders, infectious diseases
and drugs. About 60% of dogs is now purebred and pure breeding
always involves inbreeding. This brings genetic complexity which
result development and regulation of autoimmune disease. Intact
females have the highest incidence of immune diseases and
intact males have the lowest. Infections can trigger allergies and
the formation of auto antibodies. If autoimmune dermatologic
diseases are treated with the appropriate glucocorticoids the
prognosis for both of these ailments is fair to good and if not it
shifts poor to guard.
Since intact females have the highest incidence of
immune diseases owners should be informed for spaying
of their bitches because ovariohysterectoy reduces the
incidence of these ailments.
Whenever the ailment is encountered patients should
be treated by appropriate glucocorticoids so that clinician
should always remember the drugs are more likely to kill the
patient than the disease.
Veterinarians, universities, hospitals and animal wale
fare opponents should undertake research on immune
mediated dermatologic diseases of dogs in Ethiopia.
The susceptibility to autoimmune diseases is should be
controlled by the interplay of environmental and heritable
factors so that cross breeding shall be adapted.