International Journal of Advanced Research and Publications

ISSN: 2456-9992

Application Of Biotechnology In Environment, In

Case Of Biodegradation Potential Of Bacterial
Isolates From Tannery Effluent With Special
Reference To Hexavalent Chromium
Buzayehu Desisa
Ethiopian Environment and Forest Research Institute, Wood Technology Research Center, Addis Ababa, Ethiopia
buzayehudesisa@gmail.com

ABSTRACT: Chromium is important metal due to its high corrosion resistance and hardness. The trivalent chromium (Cr (III)) is
required in trace amounts for sugar and lipid metabolism in humans and its deficiency causes disease. But, due to environmental pollution
caused as a result of hexavalent chromium (Cr(VI)) is a highly toxic metal pollutant that affects the environment and is abundantly available
in the environment and has toxicity and harmful effect on living system and its cleanup is highly essential such as contaminants contained
hexavalent chromium (Cr(VI). To alleviate such problems by applying essential microorganisms were easily degradable by naturally
occurring bacteria and the isolated one. Considering its potential for hazardous toxicity and exposure, Cr(VI) has been designated as a
priority pollutant in many countries. Chromium (VI) is toxic and harmful to human health, mainly for the people who are working in
industries where Cr(VI) is widely used. Therefore, this review article was initiated to study the application of isolated bacterial culture as
biological tool for hexavalent chromium removal from tannery industry waste water and investigated to degrade Chromium (VI) and clean
up the environments.

Keywords: biodegradation, tannery effluent, chromium, heavy metals

1. INTRODUCTION components of waste products. Environmental engineers

Biotechnology, broadly defined as any technique that uses
living organisms to make or modify a product, improve
plants or animals, or develop micro-organisms for specific
use and applicable for the processes of the protection and
restoration of the quality of our environment. And used as
the environmentally sound technology (EST) of choice in
many applications, particularly pollution clean-up [1].
Biotechnological processes to protect the environment
have been used for almost a century now, even longer than
the term ‘biotechnology’ exists. Municipal sewage
treatment plants and filters to purify town gas were
developed around the turn of the century. They proved
very effective although at the time, little was known about
the biological/ principles underlying their function. Since
that time our knowledge base has increased enormously.
However, modern biotechnology, based on the use of new
tissue culture methods, and recombinant-DNA
technology, or genetic engineering, is an exciting science
and rich in potential and new applications are expected to
include water treatment, treatment of solid wastes
(including biodegradable plastics) [2]. Biotechnological
techniques to treat waste before or after it has been
brought into the environment are described and
exemplified in the section on bioremediation.
Biotechnology can also be used to develop products and
processes that generate less waste and use less non-
renewable resources and energy [1]. Environmental
biotechnology is the used in waste treatment and pollution
prevention. Additionally, environmental biotechnology
can more efficiently clean up many wastes than
conventional methods and greatly reduce our dependence
on methods for land-based disposal. Every organism
ingests nutrients to live and produces by-products as a
result of different organism need different types of
nutrients. Some bacteria thrive on the chemical
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use bioremediation, the broadest application of
environmental biotechnology, in two basic ways. They
introduce nutrients to stimulate the activity of bacteria
already present in the soil at a waste site, or add new
bacteria to the soil. The bacteria digest the waste at the
site and turn it into harmless by products. This process of
bioremediation is an area of increasing interest. Through
application of biotechnical methods, enzyme bioreactors
are being developed that will pretreat some industrial
waste and food waste components and allow their removal
through the sewage system rather than through solid waste
disposal mechanisms. Waste can also be converted to
biofuel to run generators. Microbes can be induced to
produce enzymes needed to convert plant and vegetable
materials into building blocks for biodegradable plastics
[3]. In some cases, the byproducts of the pollution-
fighting microorganisms are themselves useful. For
example, methane can be derived from a form of bacteria
that degrades sulfur liquor, a waste product of paper
manufacturing. This methane can then be used as a fuel or
in other industrial processes. The role of microbiology in
environmental biotechnology the restoration, maintenance
and protection of the environment with the help of
biological agents, which includes both the living
organisms and their components. Environmental
remediation, pollution protection, detection and
monitoring are evaluated considering the achievements, as
well as the perspectives in the development of
biotechnology. Waste water treatment, soil waste
treatment, solid waste treatment, and waste gas treatment,
dealing with both microbial and process engineering
aspects. The distinct role of environmental biotechnology
in the future is emphasized considering the opportunities
to contribute with new solutions and directions in
remediation of contaminated environments, minimizing
future waste release and creating pollution prevention
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ISSN: 2456-9992
alternatives [3]. To take advantage of these opportunities,
innovative new strategies which advance use of molecular
biological methods and genetic engineering technology
are examined. These methods would improve the
understanding of existing biological process in order to
increase their efficiency, productivity and flexibility. In
case of waste water and solid industrial effluents micro-
organisms in sewage treatment plants remove the more
common pollutants from waste water before it is
discharged into rivers or the sea. Increasing industrial and
agricultural pollution has led to a greater need for
processes that remove specific pollutants such as nitrogen
and phosphorus compounds, heavy metals and chlorinated
compounds. New methods include aerobic, anaerobic and
Physico-chemical processes in fixed-bed filters and in
bioreactors in which the materials and microbes are held
in suspension. Extensive application of heavy metals like
chromium in industries particularly leather tanning
industries leads to the formation of chromium-
contaminated soil and ground water which pose a serious
threat to living biota particularly to human health.
Chromium is a potent pollutant which is mutagenic,
carcinogenic and teratogenic in humans [4]- [6]. It is
found to be toxic in plants also [7]. Among different forms
of chromium, chromium VI is known to cause serious
health hazard effects. It can cause allergic reactions, nose
irritation and nose bleeds. It creates respiratory problems
and weaken-immune system. It causes renal tubular
necrosis and produce cytotoxic and genotoxic effects [8].
Hexavalent chromium toxicity appears to be due to its
rapid permeability through biological and subsequent
interaction of chromium with intracellular proteins and
nucleic acids. Usually the tanning industry uses chrome
liquor in the tanning process. A large number of tanning
industries discharge their effluents into the environment
containing chrome salts exceeding the maximum
permissible limit. As a result, they provide a natural
environment for enrichment of chromium-resistant
bacteria and consequently these bacteria may be employed
for removal of hexavalent chromium from tannery waste.
Cr (VI) containing tannery effluents are commonly treated
by chemical means. These methods may be a source of
potential heavy metal pollution from the resultant metal-
containing chemical sludge. The commonly employed
methods include excavation, to pump and treat, in situ
vitrification and chemical treatment with a reductant [3].
The conventional method to detoxify and remove Cr (VI)
from the environment involve chemical reduction
followed by precipitation, ion exchange and absorption on
coal, activated carbon, alum and kaolinite [9], [10].
Several reports have indicated biological reduction of Cr
(VI) by aerobic and anaerobic bacteria as biological
reduction of Cr (VI) usually occurs at a neutral pH range.
It generates an insignificant quantity of chemical sludge. It
offers potentially cost-effective remediation strategy [11].
It does not require high energy input or toxic chemical
reagents and finally it offers an economical as well as eco
friendly option of metal detoxification and
bioremediation. The processes by which microorganisms
interact with toxic metals enabling their removal/and
recovery are bioaccumulation, biosorption and enzymatic
reduction [12]. Recent studies have shown that certain
species of bacteria are capable of transforming Cr (VI)
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into the much less toxic and less mobile Cr (III) [13a],
[14].
2.Pollution of Water
Pollution of water by industrial effluents of process
industries is a serious problem in most countries [15].
Industrial waste consists of both organic and inorganic
substances. Organic wastes include pesticide residues,
various hydrocarbons, solvents, cleaning fluids, dissolved
residue from fruit and vegetables, and lignin from pulp
and paper. Effluents can also contain inorganic wastes
such as brine salts and metals. The increased industrial
activities have reduced the availability of good quality
water by producing a large amount of effluents to the
rivers. Industrial effluents often contain various toxic
metals, harmful gases, and several organic and inorganic
compounds [15]. Due to discharge of toxic effluents long-
term consequence of exposure can cause cancer, delayed
nervous damage, malformation in urban children,
mutagenic changes, neurological disorders [16]. Phenol
and chromium are the major contaminant present in the
effluent discharged from the various industrial processes
such as wood preserving, metal finishing, petroleum
refining, leather tanning and finishing, paint and ink
formulation, pulp and paper industry, Textile Industry
Pharmaceutical industry and manufacturing of automobile
parts industries [17].
2.1. Chromium
Chromium is a chemical element discovered in 1797 by
Louis Nicolas Vauquelin which has the symbol Cr. Its
atomic number is 24. It is a hard metal of steely gray
colour and also it has a high melting point of 1907°C. It is
odourless and tasteless metal. Many of its compounds are
intensely coloured. Chromium is important metal due to
its high corrosion resistance and hardness. The trivalent
chromium (Cr(III)) is required in trace amounts for sugar
and lipid metabolism in humans and its deficiency causes
disease. Hexavalent chromium (Cr(VI)) is a highly toxic
metal pollutant that affects the environment and is
abundantly available in the environment. Due to its
toxicity and harmful effect on living system its cleanup is
highly essential [18].
2.1.1. Uses of chromium
Chromium metal is found in the effluents of industries,
such as in metal finishing, petroleum refining, iron and
steel industries, textile manufacturing, electroplating,
leather tanning metal plating [18], [19]. Effluents of these
industries contain large quantities of chromium-laden in
their wastewater [20]. Hexavalent chromium Cr(VI)
compounds are considered to be highly toxic,
carcinogenic, and mutagenic to living organisms [21].
Considering its potential for hazardous toxicity and
exposure, Cr(VI) has been designated as a priority
pollutant in many countries.
2.1.2. Toxicity of chromium
Considering its potential for hazardous toxicity and
exposure, Cr(VI) has been designated as a priority
pollutant in many countries. Chromium(VI) is toxic and
harmful to human health, mainly for the people who are
working in industries where Cr(VI) is widely used.
Chromium (VI) causes various health problems to human
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beings as reported by [22b]. It has been reported by
various authors that hexavalent chromium causes lung
cancer, ulcer, severe damage to the liver and kidneys,
perforation of nasal septum, leukocytosis, Skin rashes in
humans [20]. There is sufficient evidence for
carcinogenicity of Cr(VI) in animals for the hexavalent
chromium compounds like calcium chromate, chromium
trioxide, lead chromate, strontium chromate and zinc
chromate stated by [23]. IARC and ACGIH has also
classified chromium metal and trivalent chromium
compounds as not human carcinogen. According to
central pollution control board the minimum permissible
limits of Cr(VI) at 0.05-0.1 ppm and 2 ppm for total
chromium is sited [19].
2.2. Different Physical and chemical Methods for
chromium degradation and their draw backs.
Traditionally, by the use of various expensive chemical
and physical processes high concentrations of both Cr(VI)
and phenol are reduced from the industrial wastewater.
Those methods include ozonization, adsorption, ion
exchange, membrane filtration, chemical oxidation [19],
[20]. But, these processes are high energy consuming,
non- economic and release effluents and waste waters
which requires further treatment and thus are alarming for
the environment. Also, complete removal of the
pollutants cannot be possible by the use of physical and
chemical processes [14].
2.2.1. Ozonization
Chemical oxidation with ozone can be used to treat
organic pollutants or act as disinfectant agents. Ozone is a
powerful oxidant that can oxidize a great number of
organic and inorganic materials. Ozone based
technologies research is also being focused on the
catalytic ozonation where the presence of catalyst
significantly improved the oxidation rate of organic
compounds compared to non- catalytic ionization. The
ozonization processes are possibly one of the most
effective methods for treatment of wastewater containing
organic products such as effluents from chemical and
agrochemical industries, textile industry, paints, [21].
However, the characteristics of the wastewater such as
pH, temperature and concentration of organic pollutant
play an important role in organic degradation. Similarly
[22]. have studied the treatment of industrial effluent by
treatment with ozone initial pH, ozone dosages and
dosages of catalyst on effluent treatment. Then, organic
matter remained was removed by BAF (Biological aerated
filter), and the effect of hydraulic retention time (HRT) of
BAF was also considered. Their result showed that under
optimum pH and dose of H2O2 was about 30% COD could
be removed by ozonization alone. The use of high
oxidation potential like ozonization has recently received
much attention in wastewater treatment studies. Ozonizer
in which the oxygen molecules in the gas were dissociated
to form ozone was used in many cases [23]. The
disadvantages associated with the process are high
operating cost. The cost of the equipment is very high and
also it requires high voltage and electricity for its
operation [24].
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2.2. 2. Ion exchange
Ion exchange means the removal of an ion from an
aqueous solution by replacing another ionic species. There
are natural and synthetic materials available which are
specially designed to enable ion exchange operations at
high levels. So, ion exchangers are used to perform this
ion exchange for removal of organic and inorganic
pollutants along with other heavy metals for purification
and decontamination of industrial effluent. Synthetic and
industrially produced ion exchange resins are mainly
made up of polystyrene and polyacrylate are in the form
of small and porous beads. Also, there are some naturally
occurring minerals which have ion exchange properties.
The most common one is aluminium silicate minerals
which are also called zeolites [25]. There are different
zeolites available made up of various ionic materials
which have affinity towards some particular metals. The
main features of the ionic resins include material
properties such as like adsorption capacity, porosity,
density [26]. The main disadvantages associated with ion
exchange method are the high cost of the ion exchange
resins and each resin must be selectively removes one type
of contaminant only. Further, complete removal of the
contaminant is not possible. Besides, it can be used for
limited cycles only as by passing concentrated metal
solution the matrix gets easily owned out by organics and
other solids in the wastewater after several uses.
Moreover, ion exchange is also highly sensitive to pH of
the solution [27], [28].
2.2.3. Adsorption
Adsorption is a widely used method for the treatment of
industrial wastewater containing colour, heavy metals and
other inorganic and organic impurities stated by [29], [30].
Adsorbent materials are basically derived from low-cost
agricultural wastes, activated carbon prepared from
various raw materials such as sawdust, nut shells, coconut
shells [31]. These adsorbents are basically used for the
effective removal and recovery of organic and metal
pollutants from wastewater streams [32]. It is a complex
process affected by several factors. Mechanisms involved
in the adsorption process mainly focus on the selection of
the adsorbent material like their particle size surface area
and porosity [33]. This method suffers from low
adsorption capacity and in some cases complete removal
is not possible and high cost of the adsorbent. After use
the disposal of adsorbants creates problems.
2.2.4. Membrane filtration
Membrane filtration technique has received a significant
attention for the wastewater treatment. It considers the
application of hydraulic pressure to bring about the
desired separation through the semi permeable membrane
[34]. Membranes are of different pore size and it is
necessary to select membranes of appropriate pore size for
specific purpose so that effluent and wastewater could be
purified and permeate could be recycled a number of
times. Mainly three types of membrane filtration are there.
They are Ultra-filtration, Nano filtration and Reverse
osmosis reported by [31], [35]. Various other types of
membranes such as inorganic, polymeric, and liquid
membranes can be used in this process. The main problem
associated with this process is incomplete removal of
contaminants, high energy requirement, high cost of the
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membrane and longevity of the membrane. After long
term use the membrane get clogged with the contaminants
present in the waste water and is damaged due to extra
pressure on the membrane.
2.2.5. Chemical oxidation
In this process the waste materials from the industrial
waste water are removed by the help of chemical
oxidation by the use of various chemicals mainly
hydrogen peroxide is widely used for this purpose as
reported [36a]. There are many disadvantages associated
with this process like the high cost of the chemicals,
emission of various harmful by products, it creates
hazardous constituent like secondary effluent problem
along with the production of harmful gases.
2.3. Biodegradation
Biodegradation is the process of decaying or reduction of
different organic materials and toxic metals to their non-
toxic form with the help of microorganisms. In this
process complete mineralization of the starting compound
to simpler ones like CO2, H2O, NO3 and other inorganic
compounds takes place [38]. In the mixed culture of
microorganism’s phenol degrading organisms utilizes
phenol as sole source of carbon and produce energy,
metabolites, electron donor which is used by the
chromium degrading organisms to reduce chromium [39],
[40]. Biodegradation is a microbial process in which
nutrients and physical conditions plays important role.
Temperature and pH are the important physical variables
and carbon, nitrogen, oxygen, phosphorus, sulfur,
calcium, magnesium, and several metals are the
micronutrients that also shows a significant impact on
degradation behavior is reported [41].
2.3.1. Advantages of biodegradation
There are various advantages associated with
biodegradation such as the process is a simple process. It
is an eco-friendly and cost-effective process that requires
low capital and operating cost. Being environmentally
friendly process, it produces no harmful end products
2.3.2. Microorganisms involved in biodegradation of
chromium
Chromium degradation can be possible with the help of
variety of bacteria and fungal groups like Arthrobacter sp.
and Bacillus sp. [42], E. coli ATCC 33456[43].
Pseudomonas aeruginosa [44]. 4), Brevebacterium casei
[19], Acinetobacter sp and some unidentified species like
Pseudomonas fluorescens, Pseudomonas synxantha,
Alcaligenes eutrophus. A number of bacterias Bacillus
spp., Shewanella alga BrY-MT and a few unidentified
strains have also been shown to reduce Cr 6+ [2].
2.3.3. Mechanism of biodegradation
The biological mechanism E. coli can directly transform
the highly toxic Cr(VI) is to less toxic Cr(III) with the
help of various membrane associated, NADH dependent
soluble chromate reductase enzymes ([2], [43] and [45].
[46] suggest the effect of different electron donors on
Cr(VI) reduction using Ganoderm lucidum and
Brevibacterium casei respectively. Among several
electron donors and nitrogen sources screened they
concluded dextrose was the optimum electron donor and
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peptone showed maximum Cr(VI) reduction. Similarly,
maximum degradation of Cr(VI) evidenced in the
presence of dextrose by [43] by using E. Coli ATCC
33456. The optimum Cr(VI) reduction by E. coli ATCC
33456 within a pH range of (3-8) and the optimum pH
reported was 7. Similarly, Cr(VI) reduction in E. coli
ATCC 33456 was evaluated within a pH range of (3–8) by
Shen and Wang, (1994) and the result showed maximum
initial specific rate of Cr(VI) reduction at pH 7. Also, they
have reported that effect of pH on Cr(VI) reduction were
similar for both aerobic and anaerobic cultures. [19] used
Brevibacterium casei for degradation of chromium. They
observed the reduction of Cr(VI) using wide range of pH
ranging from pH 4 to pH 9 and the maximum degradation
was reported at pH 7. The above studies conformed that
pH has a significant influence on phenol as well as
chromium degradation. [43] studied Cr(VI) reduction
using E. coli ATCC 33456 in the temperature range of
400C to 600C and found optimum value at 37°C.
Temperature range of 100C to 450C was studied by [2].
and the temperature of 360C was found to be optimum.
Thus, results suggested that temperature has a great
influence on both phenol and chromium degradation. The
temperature is a very important factor effecting the
degradation as it is an enzymatic process where the
enzymes present in the microorganisms show maximum
functionality at a certain temperature.
3. Sampling Method
Effluent samples were collected from a tannery industry at
Kolkata, West Bengal. Samples were collected in
sterilized glass bottles aseptically and transported to the
laboratory in an ice bucket. Samples were analyzed within
6h of collection.
3.1. Isolation of chromium-resistant bacteria
Chromium-resistant bacteria were isolated from untreated
tannery effluent on agar plates under anoxic conditions.
The medium contains (per litre): casein 20g, dextrose 10g,
sodium thioglycollate 2g, sodium chloride 5g, sodium
formaldehyde sulphoxylate 1g, methylene blue 2mg, agar
20g, pH 7.2±0.2. These agar plates were amended with
200 μg ml-1 and 400 μg ml-1 Cr (VI) by standard plate
method [47], [48]. The inoculated agar plates were
incubated in BBL Gas Pak anaerobic Jars at 25°C for
5days. After incubation clones representing different
colony types were purified on agar plates under anoxic
condition. All the bacterial cultures were kept at -20°C in
agar stabs.
3.2. Determination of metal tolerance activity
The anoxic bacteria were examined for their resistance to
chromium by agar dilution method [49]. In this method,
freshly prepared agar plates amended with Cr (VI) as
dichromate at various Concentrations (50-800 μg ml-1)
were inoculated with overnight grown cultures under
anoxic condition. All the plates were incubated at room
temperature (25°C) for 48h. The minimal concentration of
metal in the Petri plate showing complete inhibition of
growth was considered as minimal inhibitory
concentration (MIC). Tolerance to Cr (VI) was confirmed
by broth-dilution method [50]. Tolerance to Zn, Cd and Pb
was determined by agar dilution method. The salts used
were zinc chloride, cadmium chloride and lead nitrate.
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3.4. Evaluation of Cr (VI) reduction
The test bacteria were inoculated in diluted peptone water
amended with Cr (VI) at the rate of 20μg ml-1 and
incubated at 25±2°C for up to 72h. The decrease in Cr
(VI) concentration in the experimental flasks was
measured after appropriate time intervals by 1, 5-diphenyl
carbazide (50μg ml-1) method [48]. The test samples were
acidified (pH 1-2) and 50μg ml-1 1, 5-diphenyl carbazide
was added and Cr (VI) concentration was detected by
spectrophotometer (CECIL CE 7200) at 540 nm. To
estimate the total chromium, samples were digested with
hydrochloric acid and perchloric acid mixture (6:1). Metal
concentration was determined by atomic absorption
spectrophotometer.
3.5. Characterization of bacterial isolates
Bacterial isolates from tannery effluents were
characterized according to Bergey’s Manual of Systematic
Bacteriology [51].
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