5. BIOREDUCTION BASED REMEDIATION OF TANNERY EFFLUENTS USING...

71

5. BIOREDUCTION BASED REMEDIATION OF

TANNERY EFFLUENTS USING FUNGI

5.1 INTRODUCTION

Traditional methods for the clean up of pollutants usually involve the

removal of unwanted materials through sedimentation and filtration, and

subsequent chemical treatments such as flocculation, neutralization and electro-

dialysis before disposal. These processes may not guarantee adequate treatment

of the effluent. Moreover, they are often laborious and expensive, considering

the volume of wastes released during the industrial production process. The past

two decades have seen a tremendous upsurge in the search for cost-effective and

environmentally sound alternatives to the conventional methods for dealing with

wastes. The technologies that have emerged as most promising are those that

closely mimic the time tested, natural system that have restored environments to

their original status following undesirable perturbations. In fact the self-restoring

process in nature is what has actually given birth to the concept that the self-

cleansing ability of nature is infinite.

Many conventional processes are carried out to treat wastewater from

tannery industry such as biological process (Farabegoli et al., 2004), oxidation

process (Dogruel et al., 2004), and chemical process (Song et al., 2004).

Although the removal of toxic pollutants from industrial waste waters has been

practiced for several decades, the cost-effectiveness of the most common

physico-chemical processes such as oxidation and reduction, chemical

precipitation, filtration, electrochemical treatment, evaporation, ion-exchange

and reverse osmosis is limited. This calls for the development of new

72

technologies that emphasize the destruction of pollutants rather than the

conventional approach of disposal.

The search for alternative and innovative treatment techniques has

focused on the use of biological materials such as algae, fungi, yeast and bacteria

for the removal and recovery, and the technique has gained importance during

recent years because of their better performance and the low-cost of these

biological materials. Of all the technologies that have been investigated,

bioremediation has emerged as the most desirable approach for cleaning up

many environmental pollutants in effluents.

Bioremediation uses living systems especially microorganisms to catalyze

the degradation of wastes without disruption of the environment. Bioremediation

is the transformation or degradation of contaminants into non-hazardous or less

hazardous chemicals. Bacteria are generally used for bioremediation, but fungi,

algae and plants have also been used. Bioremediation is not a new technology.

There has been evidence that compost piles existed as far back as 6000 BC, and

in 1891 the first biological sewage treatment plant was created in Sussex, UK.

However, the word “bioremediation” did not appear in peer-reviewed scientific

literature until 1987.

In the recent era of environmental protection, the use of microorganisms

for the recovery of metals from waste streams, as well as the employment of

plants for landfill applications, has generated growing attention (Kotrba and

Ruml, 2000). There are a wide variety of microorganisms, encompassing

bacteria, fungi, yeast, and algae that can interact with metals and radionuclides

73

through several mechanisms to transform them (Volesky, 1994; Kapoor and

Viraraghavan, 1998). Biosorption, the chief process for microbial remediation

can be defined as metabolism independent adsorption of pollutants on microbial

biomass, based on partition process (Ringot et al., 2006).

Compared with the conventional chemical treatment, biological treatment

shows some advantages, such as low operation cost, steady effect, easy recovery

of some valuable metals (Rehman and Shakoori, 2001; Wang et al., 2001).

Today these waste products range from raw industrial effluents to nuclear waste.

In the past disposal of these wastes meant digging a hole, dumping the waste

material in, then filling it all in. But lately this method has become insufficient.

The toxic materials from these “dig and dump” sites have begun to leak into

water sources and into areas that sustain human life. This problem has led to the

development of biodegradation or modern-day bioremediation.

Bio-degradation refers to the biological transformation of an organic

chemical to another form. Though no extent is specified, scientists and

engineers usually mean that it can be mineralized. Bioaccumulation or

biosorption is the accumulation of the toxic compounds inside the cell without

any degradation of the toxic molecule. This method can be effective in aquatic

environments where the organisms can be removed after being loaded with the

toxic substance.

Bioremediation technologies offer a cost effective, permanent solution to

cleanup effluents from industries. By this novel technology we can remove the

pollutants and their toxicity from the effluents through the metabolic reaction

74

mediated by microorganisms. Bioremediation is an organic approach to the

reclamation of waste materials at the site. It is simply a new application of a very

old technology once primarily used in the waste water treatment. Bioremediation

through land farming is both simple and cost-effective to implement compared

with other treatment technologies (Pearce and Ollerman 1998).

In general, microbes in the environment play an important role in cycling

and destroying them through bio-degradation. Microbial populations have

amazing enzymatic and metabolic potential to degrade a variety of organic

compounds. Microbes regenerate quite rapidly in the environment and over a

period of time develop the genetic competence to synthesize enzymes and other

cell components that are necessary for the dissimilation of environmental

chemicals. Bacteria and fungi are the chief agents for biodegradation, while

yeast, algae, diatoms, some plants and animals also metabolize chemicals

(Alexander, 1981). Bacteria, fungi and other microorganisms, exhibit a number

of metabolism-dependent and independent processes for the uptake and

accumulation of pollutants from industrial waste waters.

The removal or reduction of such harmful substances from tannery

effluents by microbe-based technologies may provide an alternative or additional

means of waste recovery for economic reasons and environmental protection.

The treatment of wastewater using microorganism is one of the most active

research fields in recent years (Leusch et al., 1995; Kaewsarn, 2002; Wu et al.,

1996). Compared with the conventional chemical treatment, biological

treatment shows some advantages, such as low operation cost, steady effect and

75

easy recovery of some valuable metals (Rehman and Shakoori, 2001;

Wang et al., 2001).

The fungi are unique among microorganisms in that they secrete a variety

of extracellular enzymes. The decomposition of lignocellulose is rated as the

most important degradative event in the carbon cycle of earth (Bennett and

Faison, 1997). Systems using Rhizopus arrhizus have been developed for

treating uranium and thorium. The ability of fungi to transform a wide variety of

hazardous chemicals has aroused interest in using them in bioremediation. The

white rot fungi are unique among eukaryotes for having evolved nonspecific

methods for the use of lignin as a carbon source for their growth (Kirk et al.,

1976). Many researchers studied the effectiveness of isolated fungi belonging to

the genus Aspergillus to degrade and remove pollutants from tannery waste

waters.

Fungal systems appear to be most appropriate in the treatment of coloured

and metallic effluents (Ezeronye and Okerentugba, 1999). Fungi can be easily

grown in substantial amounts using unsophisticated fermentation techniques and

inexpensive growth media (Kapoor et al., 1999). Biosorption carried out by

fungi could serve as an economical means of treating tannery effluents.

Among the fungal systems, Phanerochaete chrysosporium is emerging as

the model system for bioremediation. Phanerochaete chrysosporium has been

shown to degrade a number of toxic xenobiotics. The basidiomycetous fungus

Pleurobus ostreatus has been shown to produce an extracellular hydrogen

peroxide dependent lignolytic enzyme which removes the colour due to remozol

76

brilliant blue. Oxidative enzymes play a very major role in biodegradation.

Other fungi which can be used in bioremediation are obviously the members of

zygomycetes, e.g., the mycoraceous fungi and the arbuscular mycorrhizal fungi.

Aquatic fungi and anaerobic fungi are the other candidates for bioremediation.

Among other fungi used in bioremediation, the yeasts, e.g., Candida

tropicalis, Saccharomyces cerevisiae, S.carlbergensis and Candida utilis are

important in clearing industrial effluents of unwanted chemicals. Agaricus

bisporus and Lentinus oloides are important in lignocellulose decomposition.

Corius versicolor is important in clearing up pulp and paper mill wastes.

Consortia of fungi and bacteria (usually uncharacterised) are used in

composting, the most useful waste disposal practice. Phenolicazo dyes have

been shown to be oxidized by the enzyme laccase produced by Pyricularia

oryzae.

Fungi, in general, are well known for their metal biosorption (Tobin and

Roux, 1998; Venkobachor, 1990 and Pillichshammer et al., 1995). Biosorption

is a process that utilizes biological materials as adsorbent, and this method has

been studied by several researchers as an alternative technique to conventional

methods for heavy metal removal from waste water (Jean et al., 2001; Sag and

Kutsal, 2001). Two species of Aspergillus oryzae and Rhizopous oryzae, were

used for Cu+2 removal (Chihpin and Huang, 1998). Another investigation has

shown that Aspergillus niger can grow in tanning effluent (Sivaswamy, 1988). In

another report, the growth of Aspergillus niger and Aspergillus carbonaricus has

been studied (Marakis, 1995). Immobilized Aspergillus niger and Aspergillus

77

oryzae have been used from effluents for the removal of cadmium, lead, nickel

and chromium (Kapoor and Viraraghavan, 1998).

Fungi exhibit a number of metabolism-dependent and independent

processes for the uptake and accumulation of pollutants from industrial waste

waters. The removal or reduction of such harmful substances from tannery

effluents and waste waters by microbe-based technologies may provide an

alternative or additional means of waste recovery for economic reasons and

environmental protection. Due to its comparatively low cost and generally

benign environmental impact, bioremediation offers an attractive alternative

and/or supplement to more conventional clean-up technologies. Thus fungi

appear to be the best candidates, so far studied, in tannery effluent treatment in

situ. There is no doubt therefore, that if properly harnessed, fungi can be very

useful in the treatment and disposal of tannery and related effluents.

5.2 MATERIAL AND METHODS

5.2.1. Isolation of Fungi from Tannery Effluent

Tannery effluents were collected and used for the analysis of mycoflora.

The fungi were isolated using dilution plating method.

Dilution plating method

One ml of tannery effluent was dispersed thoroughly in 10 ml of

sterile distilled water, thus making the solution to the concentration of 1/10

dilution. From this sample solution, 1 ml was transferred to 9 ml of sterile

distilled water. The resulting solution was with the concentration of 1/100 or

10.2 dilution. From this diluted sample, 1 ml was pipetted into sterile

78

petridishes containing antibiotic amended agar medium (Potato Dextrose

Agar) and six replicates were maintained for each sample.

Culture Medium

Potato Dextrose Agar (PDA) was used as culture medium (Booth,

1971).

Potato dextrose agar (PDA) medium

Peeled potato extract 200 g

Dextrose 20 g

Agar 20 g

Distilled water 1000 ml

pH 6.8-7.0

Incubation

The petridishes containing the effluent sample in PDA were incubated at

room temperature 28 ± 2°C in a glass chamber for the isolation of fungi. After

incubation for a period of 4 to 5 days the growing colonies were counted and

identified upto species level with the aid of standard manuals. Wherever

necessary the colonies were sub cultured in Czapek's Dox Agar (CDA)

(Booth, 1971) for species identification. The composition of the medium is as

follows.

Czapak's Dox Agar medium (Booth, 1971)

Sodium nitrate 3.0 g

Dipotassium hydrogen phosphate 1.0 g

Magnesium sulphate 7H20 0.5 g

79

Potassium chloride 0.5 g

Ferrous sulphate 7H20 0.1 g

Sucrose 30.0 g

Agar 20.0 g


pH 6.5

Slide Preparation

Lactophenol and lactophenol with cotton blue (for hyaline moulds)

were used for preparing slides for examination and the slides were sealed with

DPX mountant for future use.

Preparation of lactophenol - cotton blue

Lactic acid 40 ml

Phenol 40 ml

Glycerol 40 ml

Cotton blue 0.5 mg


Mass Culture of Fungi

After identification of the fungi, mass culture was carried out in the

laboratory to be used for the biotreatment of the tannery effluent. Fungi such as

Aspergillus niger and Aspergillus terreus were cultured in mass, separately for

the treatment of tannery effluent. Apart from them, spores of Aspergillus niger

and Aspergillus terreus were mixed together in a ratio of 1:1 and grown

separately for mixed culture degradation of tannery effluent.

80

The cultures of Aspergillus niger, Aspergillus terreus and mixed culture

were carried out using the following medium:

Malt Extract Agar

Malt extract 20 g

D. Glucose 20 g


Isolation and Identification of Fungi

The fungi were mounted in lactophenol cotton blue and observed through

light microscope. They were identified using standard manuals (Gilmann, 1967;

Subramanian, 1971; Ellis, 1971; Barnett and Hunter, 1972).

Presentation of data

The results obtained were presented as (colony forming units) “cfu/ml”

and “percent contribution”.

No. of cfu/ml of Average No of colonies/plate tannery effluent = ––––––––––––––––––––––––– x Dilution factor (102) Volume of the effluent

The term “percent contribution” refers to the contribution of individual

species to the total, and is calculated as follows:

No. of cfu/ml of No of cfu/ml of an individual species An individual species = ––––––––––––––––––––––––––––––––– x 100 Total No of cfu/ml of all species

81

5.2.2. Biodreduction of Tannery Effluent using Fungi

Mycelial mat of fungi grown in liquid culture was recovered, washed in

sterile distilled water. Approximately 10 g (fresh weight) of mycelia of

Aspergillus niger and Aspergillus terreu, were transferred to experimental jars

containing 500 ml of different concentrations (25%, 50%, 75% and 100%) of

tannery effluent. They were kept in an orbit shaker for 72 hours and maintained

at 28 ± 2°C. Parameters such as pH, BOD, COD, TSS and TDS were estimated

before and after 72 hours (3 days) to check the degradation process. Similar

procedure was adopted for the bioreduction of tannery effluent using mixed

fungi.

5.2.3. Statistical analysis

The data obtained from the various experiments was analyzed and

expressed as mean and standard deviation (Zar, 1974). Percentage change was

calculated between the control and experimental for the various experiments

using the formula

Control - Experimental % change = ––––––––––––––––––––––– x 100 Control

For statistical analysis SPSS/PC+ was used in the present study to

calculate mean, standard deviation and F test.

Index of dominance (c) was done to find out the dominance of fungus

(Simpson, 1949).

82

c = E (ni/N)2

Where

ni = Importance value for each species

N = Total of importance values.

Shannon and Weiner Index of general diversity was calculated to find

the diversity of fungal population during the course of study (Shannon and

Weiner, 1963) (H).

H = -E Pi log Pi

Where,

ni = Importance value for each species

N = Total of importance values

Pi = Importance probability for each species = ni/N I

5.3 RESULTS

5.3.1. Microbial Analysis

5.3.1.1. Isolation and Identification of fungi

Isolation and identification of fungal population present in the tannery

effluent during the month of January 2008 to December 2008 is shown in Table

5.1.

10 species were isolated and identified during the study period, i.e, from

January 2008 to December 2008 (Plate 5.1). Among the species, one belonged to

Zygomycotina, and another, to Basidiomycotina and the remaining 8 species

belong to Deuteromycotina.

83

Maximum number of six fungal species was isolated and identified during

the month of October 2008 and minimum of two species belonging to the same

genus Aspergillus was noticed during March and December 2008.

Five species of fungi were identified in the months of January, May and

August 2008. The maximum percentage was contributed by Aspergillus terreus

(98.1%) with 17.3 x 102 cfu/ml during March 2008. The minimum percentage

was contributed by three species (0.63% with 0.17 x 102 cfu/ml) namely

Paecilomyces variotii during January and Monilia sitophila and Curvularia

lunata during October 2008 (Table 5.1).

5.3.2. Ecological Indices of Isolated Fungi

During the course of study, index of dominance (c) was maximum during

the months of March 2008 and October 2008. During the month of March 2008,

only two species belonging to the genus Aspergillus was recorded. In the month

of October 2008 five species were recorded. Minimum was recorded during the

month of May 2008 and five species of fungi was reported during this month.

They are Aspergillus niger, A.terreus, P.variotii and Absida corymbifera

(Table 5.2)

Index of general diversity (H) was recorded maximum during the months

of May 2008 (sp 5) followed by November 2008 (sp 4) and January (sp 5) and

minimum diversity was observed in the month of October 2008 represented by

six fungi namely Aspergillus flavus, A. terreus, A. niger, A. tamarii, M. sitophila

and C. lunata.

84

5.3.3. Bioreduction Based Remediation of Tannery Effluent (72 Hours) Of

Different Concentrations Using Fungi and Mixed Culture

Results of the tannery effluent treated with A. niger, A. terreus and mixed

culture at different concentrations (25%, 50, 75% and 100%) for 72 hrs is shown

in Plate 5.1

5.3.3.1. pH of tannery effluent after biodegradation

pH of tannery effluent in control and after bioreduction (72 hours) by

individual fungus and mixed culture is shown in Table 5.4 and plate 5.2 to 5.4.

In 100% concentration of tannery effluent Aspergillus terreus showed the

maximum percentage change (+18.60%) followed by mixed culture (+16.55%).

In 75% concentration of tannery effluent, Aspergillus terreus showed the

maximum amount of percentage increase (+17.44%) followed by mixed culture

(+14.4%) and the least was found with A. niger (+11.52%). In 50%

concentration of tannery effluent A. terreus evoked a maximum percentage

change (+7.48%) followed by the mixed culture (+6.41%). In 25% concentration

of tannery effluent maximum percentage increase was observed in A.terreus

bottles (+7.56%) followed by mixed culture (+5.93%) %) and the percentage

change was least in A. niger (+4.31%). The increase in pH from acidic to neutral

was statistically significant (P<0.001).

5.3.3.2. BOD of Tannery effluent after biodegradation

The BOD of tannery effluent in control and after bioreduction (72 hr) by

individual fungus and mixed culture is shown in Table 5.5. In 100%

concentration of tannery effluent, Aspergillus terreus reduce the BOD to a

maximum extent (-58.89%), followed by A. niger (-37.26%) and the mixed

85

culture (-27.75%). In 75% concentration of tannery effluent, Aspergillus terreus

was found to reduce BOD to a maximum extent (-42.96%), followed by the

mixed culture (-28 %) and A. niger (-23.98%). In 50% concentration of tannery

effluent, the mixed culture was found to reduce BOD to a maximum extent

(-63.45%), followed by A.terreus (62.18%) and A. niger (56.51%), while in

25% concentration of tannery effluent, maximum percentage change was

observed with mixed culture(-65.91%), followed by A.terreus (-53%) and the

minimum with A.niger (-50.91%). The decrease in BOD value in the biotreated

effluents was statistically significant at (<0.001).

5.3.3.3. COD of tannery effluent after biodegradation

The COD of tannery effluent in control and after bioreduction (72 hours)

by individual fungus and mixed culture is shown in Table 5.6. In 100%

concentration of tannery effluent, Aspergillus terreus reduced the COD to a

maximum extent (-56.17%), followed by the mixed culture (-52.03%) and

A.niger (-41.53%). In 75% concentration of tannery effluent, the maximum

percentage reduction was observed with Aspergillus terreus (-57.04%) followed

by mixed culture (-52.24 %) and A. niger (-45.95%). In 50% concentration of

tannery effluent, the maximum percentage reduction (-70.90%) was brought

about by A.terreus followed by the mixed culture (-64.59%) and A.niger

(56.31%). In 25% concentration of tannery effluent the maximum percentage

change (-62.5%) was observed with A.terreus followed by the mixed culture

(-58.83%) and A. niger (-49.17%). The reduction in COD value in the biotreated

effluents was statistically significant (P<0.001).

86

5.3.3.4. TSS of tannery effluent after biodegradation

The Total Suspended Solids of tannery effluent in control and after

bioreduction (72 hours) by individual fungus and mixed culture is shown in

Table 5.7 and Plates 5.2 to 5.4. In 100% concentration of tannery

effluent Aspergillus terreus reduced the TSS to a maximum extent (-55.29%)

followed by the mixed culture (-47.97%) and least percentage change (-43.66%)

was observed with A.niger. In 75% concentration of tannery effluent the

maximum percentage reduction (-29.60%) was observed with Aspergillus

terreus followed by mixed culture (-27.51 %) and the least percentage reduction

was found with A. niger (-19.65%). In 50% concentration of tannery effluent

A.terreus (-44.56%) showed maximum percentage change followed by the

mixed culture (-31.76%) and the least percentage reduction was found with

A.niger (-28.95%). In 25% concentration of tannery effluent A.terreus (-46.77%)

showed maximum percentage change followed by the mixed culture (-44.19%),

and the minimum percentage change was observed with A.niger (-18.39%). The

decrease in TSS in the biotreated effluents was statistically significant

(P<0.001).

5.3.3.5. TDS of tannery effluent after biodegradation

The Total Dissolved Solids of tannery effluent before control and after

biodegradation (72 hours) by individual fungus and mixed culture is shown in

Table 5.8. In 100% concentration of tannery effluent, the mixed culture

(-14.68%) reduced TDS to a maximum extent followed by A. terreus (-12.5%),

and A. niger (-9.7%). In 75% concentration of tannery effluent maximum

percentage change was observed in mixed culture (-31.45%) followed by

A. terreus (-20.33%) and A.niger (-12.5%). In 50% concentration of tannery

87

effluent, the maximum percentage change was observed in mixed culture

(-32.32%) followed by A. terreus (-20.72%) and least percentage change was

observed with A. niger (-14.37%). In 25% concentration of tannery effluent, the

maximum percentage of reduction was observed in mixed culture (-35.37%)

followed by A. terreus (-21.85%) and the least percentage reduction was

observed for A.niger (-16.63%). Decrease in TDS in the biotreated effluents was

statistically significant (P<0.001).

5.4 DISCUSSION

Biological cleaning procedures make use of the fact that most organic

chemicals are subject to enzymatic attack of living organisms. These activities

are summarized under the term biodegradation (Alexander, 1994).

Bioremediation refers to the productive use of microorganisms to remove or

detoxify pollutants that otherwise threaten public health. Microorganisms have

been used to remove organic matter and toxic chemicals from domestic and

industrial waste discharged for many years (Gupta and Mukerji, 2001)

Acidic pH, excessive hardness, high TSS, TDS, BOD, COD of the

tannery effluent of Tiruchirappalli district revealed its highly polluted nature

and the need for its treatment, but earlier reports indicate that even the similarly

treated effluent from tanneries elsewhere do not satisfy the limits prescribed by

of the CPCB (1995). Hence it is imperative that additional or alternative

technologies be adopted to reduce / degrade the tannery effluent. According to

Mc Eldowney et al. (1993), the most appropriate method for pollution control

depends on various factors linked to the nature and type of pollution, with

environmental statutes, cost benefits analysis and commodity acceptance. Since

88

no single technology can satisfy all these requirements, combined treatment

strategies could be a wise and prudent option. Microbes in the environment play

an important role in the cycling and fate of organic chemicals, and can destroy

them through biodegradation. Microbial populations have amazing enzymatic

and metabolic potential to degrade a variety of organic compounds. Micro-

organisms regenerate quite rapidly in the environment and over periods of time,

develop the genetic competence to synthesize enzymes and other cell

components that are necessary for the dissimilation of environmental chemicals.

Bacteria and fungi are the chief agents for the biodegradation of organic

compounds. Yeast, algae, and diatoms as well as some higher plants and animals

metabolize a variety of chemicals (Ninnekar, 1992).

Researchers have clearly indicated that aquatic fungi play a key role in

the productivity of streams, estuaries, and oceans (Johnson and Sparrow, 1961;

Jones, 1976; Wicklow and Carroll, 1981). The spectacular array of fungal

populations adorning aquatic habitats has recently been reviewed by Wadhwani

et al. (1992) and thus prompted the present analysis of the native microbial

population of tannery effluent of Tiruchirappalli, and to use it for biodegradation

instead of introducing exogeneous microbes. The analysis of tannery effluent

revealed the occurrence of 12 species of mycoflora. This finding could help to

pave way for three major areas of research, namely;

• Isolation and identification of new fungal isolates from tannery effluent.

• Provide clues on the ability of the microbes to survive adapt and colonize

in the polluted environment.

• Assessment the biodegradation potential of the mycoflora present in the

effluent.

89

Tannery effluent is rich in organic and inorganic nutrients which would

support the growth of a variety of microbial population. Govindan and Uma

(1991) identified 36 species of mycoflora from waste stabilisation ponds

containing sewage, and attributed the high TDS concentration in the sewage to

favour the growth of the mycofloral population. Panneerselvam (1998), Prabakar

(1999) and Jamal Mohamed (2002) also reported the occurrence of various fungi

in sago, tannery and sugar mill and distillery effluents containing high TDS.

The presence of 12 species of fungi in tannery effluent prompted to study

the Index of dominance and index of general diversity. The study revealed that

not all fungi in the community were equally important in determining the nature

and function of the whole community. Out of the several species of fungi that

were present, a few where relatively important by virtue of their numerical

abundance in the effluent. They might have had synergistic rather than

competitive relationship. Within these fungi, certain species might strongly

control or affect the environment of the other species and hence could be

considered as ecological dominants. The Index of dominance (Simpson, 1949)

indicated that degree of dominance might be centered in one or many species. Of

the total number of species, relatively small percent was abundant. The few

which are abundant determined the dominance. (Shanon and Weiner, 1949).

Dominance is largely concentrated in one or few species when "c" (Index

of dominance) is greater than 0.6. In the present study greater diversity was

observed in March, April, July, August, October and December 2008. The

dominance and diversity pattern did follow any seasonal variation as the samples

were anthropogenic. It appeared that the relative abundance of the fungal

90

population was directly related to the physico-chemical characteristics of the

effluent. Polluted environment or other stress factors are known to reduce

species diversity, such that the number of rare species is reduced and the

concentration of dominance is in one or few common species that are tolerant to

such levels of pollution (Odum, 1971; Rao and Rao, 2000).

Inspite of moderate dominance, diversity was high in May 2008 and

November 2008. Maximum number of species was observed in October 2008

(sp 6) followed by January 2008, May 2008 and August 2008 (sp 5 each) each

and November 2008 (4 species). Maximum number of cfu for A. terreus was

observed in December (n=379) followed by January, October and March 2008.

On the average, Aspergillus terreus formed the dominant (77.5%) fungus

followed by A. niger (15.14%) in the tannery effluent. Rao and Rao (2000) have

shown that the fungal diversity is higher in polluted water bodies, compared to

non-polluted sources. Baldi et al. (1990) isolated chromium resistant yeast from

sewage treatment plant receiving tannery. Turick et al. (1996) isolated chromium

reducing anaerobes from contaminated and non-contaminated environments.

The common mycoflora present in the tannery effluent: A. terreus,

A.tamarrii, P. variotii and A. niger have the potential to be used as biosorbents

for the removal of pollutants from industrial wastewaters. They may also be

utilized as biological indicators (Rao and Rao, 2000). Further, as pointed out by

Radha (1995), the presence of native microbes in tannery effluent can

successfully be exploited to remove the pollutants, through biosorption

technique which are more economically and industrially effective.

91

Use of biological materials for heavy metal removal or recovery has

gained importance in recent years due to their good performance and low cost

(Volesky, 1987; Gadd, 1990; Mattuschka and Straube 1993). Work of Akthar

and Mohan (1995) shows that 95% of chromium can be removed through

biosorptions by Aspergillus niger from electroplating effluents.

Samudo et al. (2005) reported the bioremediation of textile effluent using

Phanerochaete chrysosporium. Accumulation of chromium by P. ambigua,

algae such as Chlorella vulgaris, Anabaena doliolum (Mallick and Rai, 1994)

and fungi such as Saccharomyces carlsbergensis (Kumpalainen and

Koivisloinen, 1978) have also been reported. Reports on the recovery of

chromium from effluents using cyanobacteria (Verma et al., 1995) and

P. mendocia (Dhakaphalkar et al., 1996) are also available. Chromium removal

from tannery effluent using biomass of Aspergillus oryzae was reported by

Nasseri et al. (2002). More et al. (2001) suggested that bioremediation will be a

cleaner way to treat the effluents as they operate under milder conditions with

minimum generation of by-products. Only a limited number of attempts have

been done in this direction in the past.

Based upon the previous reports, present study was planned to study the

biodegrading capacity of the native fungi present in tannery effluent,

individually and also as mixed cultures. Results showed the effectiveness of this

technique in bringing about favourable reductions in the levels of physico-

chemical parameters like, pH, BOD, COD, TDS and TSS. The study indicated

that the pH of diluted effluent changed from acidic to near neutral / neutral, on

92

biodegration by A. niger and A. terreus and by mixed cultures. Further it could

be suggested that microbes might degrade the organic matter thus releasing CO2

and water, diluting still further to arrive at CPCB limit for the discharge of

effluent in inland surface water or for irrigation. Greater change of pH was

observed in lower concentrations and this observation was in agreement with the

work of Sujatha et al. (1995). Their study suggest that lower the pH of effluent

favours the uptake of chromium in which case chromium can be recovered at

lower pH and simultaneously the effluent may be degraded by the microbes. It

may be noticed that A. terreus and A. niger had the ability to change the pH from

acidic to neutral, than the other microbes indicating their efficiency and because

of this trait, these microbes had been recommended widely for biodegradation

earlier (Kirk and Farrell, 1987). Complete change of pH was achieved in 25%

tannery effluent with A. terreus and A. niger. Non uniformity in the change of

pH at different concentrations of effluents could be due to other factors like

release of enzymes by the fungus to change the pH.

Biodegrading abilities of fungi in terms of the percentage removal of

BOD have been reported by Gaddad and Rodgi (1985), but literature concerning

a comparative BOD removal efficiency of native microbes in tannery effluent is

wanting. Results on undiluted effluent revealed that A. terreus reduced

comparatively larger percentage of BOD followed by A. niger and then by the

mixed culture of A. niger and A. terreus. Jamal Mohamed (2002) also confirmed

the good degradating capacity of A. niger. But according to Gaddad and Rodgi

(1985) BOD removal efficiencies of filamentous A. niger is poor, thus

contradicting with the present observation. This difference may be due to

difference in the source of effluents or even due to physiological variations in

93

the strains of microbes. Gaddad and Rodgi (1985) were also of the opinion that

mixed culture, where fungi in combination would show better BOD removal

efficiency than individual microbes. Such efficient reduction in BOD was

discernible with mixed fungal cultures, in the present study where the effluents

were diluted to 50% or below (Table 5.5).

More et al., (2001) suggested the use of alkalo-tolerant / alkalo-philic

Actinomycetes NCIM 5080 and NCIM 5142 strains for effective reduction of

COD of tannery effluent. In present study, COD was reduced effectively by

A. terreus and A. niger, and also by mixed culture. A. terreus showed higher

reduction of COD. More et al. (2001) opine that this reduction of COD may

depend on the growth medium, because about 60% of COD was removed by

immobilized Actinomycetes in their study. Saravanan et al. (1999) showed that

Flavobacterium sp. EK1 can be used to reduce COD of tannery effluent by about

93%. According to them, immobilized cells were more effective than the free

cells in the bioremediation of tannery effluent, and they also suggested that the

medium of growth was also responsible for percentage reduction of COD.

TSS in tannery effluent was found to be degraded more by A. terreus

followed by A. niger, mixed culture of A. terreus and A. niger (Table 5.5). About

55% degradation of TSS was achieved by A. terreus within 72 hours of

incubation, which would have paved way for the reduction of BOD, in the

present study. 25% concentration of tannery effluent showed maximum

reduction of TDS (35%) in the mixed culture, when compared to those by

individual microbes (Table 5.8). In general, mixed culture showed gradual

94

increase in the reduction of TDS with decrease in concentration of tannery

effluent.

TDS is a major concern of all the tanneries. Though the conventional

treatment is able to reduce the TDS considerably (Thabaraj et al., 1965), still it

exceeds the permissible limits set by CPCB (1995). Numerous physical and

chemical methods have been employed for the disposal of wastes (Tomlinson,

1968; Chaurasia and Rai, 1990; Dansal and Bajpai, 1993). The most reliable way

seems to be the biological treatment in which micro-organisms serve as efficient

detoxifiers. It is cost-effective and therefore highly suitable for reducing the

pollution load of effluents. Among the varied concentrations of tannery effluent,

25% concentration of effluent favoured the degradation of all the parameters at

72 hours of incubation, by the mixed cultures. Individual culture of A. terreus

showed significant of reduction. A niger seems also a promising candidate for

the bioremediation of tannery effluent.

Whatever be the fungal species, biodegradation was not very effective at

higher concentrations of effluent which could be due to the excessively high

organic and inorganic load in the tannery effluent. In the present study 25%

concentration of tannery effluent was found to be suitable for biodegradation. To

achieve 100% degradation, amendments to the concentration of inoculum and

the period of treatment needs to be standardized, and this deserves further

investigation. During the biodegradation process, the toxic organic chemicals are

converted to useful by-products as in the production of methane through

anaerobic fermentation, or they are completely destroyed through biodegradation

95

by the heterotrophic micro-organisms, which utilize the organic waste as a food

source.

Sanglard et al. (1986) and Bumpus (1989) recommended the use

P. chrysosporium for the biodegradation of various pollutants. For tannery

effluent treatment A. niger was recommended by Gadd and Rodgi (1985) and

Kapoor and Viraraghavan (1998); Aspergillus oryzae by Nasseri et al., (2002),

and Staphylococus cohnii by Saxena et al. (2000). Studies have recommended

the immobilization of the microbes which will efficiently help in biodegradation

of the effluents where as immobilized fungal technique can be utilized

(Ninnekar, 1992).

From the results, it became evident that a beneficial association between

the fungal groups prevailed in the tannery effluent and if amended in proper

composition, reduction of BOD, COD and TDS can be achieved to the

permissible standards, or even far below it.

Effluent treatment is one the important steps in pollution abatement.

Recycling / reuse of the biotreated effluent for various purposes offer an

attractive option of ecofriendly use. Based on the present observations and the

results of similar studies elsewhere, it is suggested that instead of using

monoculture of fungus, co-culture/mixed cultures with immobilization will be

advantageous for the biodegradation and purification of effluents.

Table 5.1. Fungi isolated from the tannery effluent during January 2008 to December 2008

Months SI. No.

Fungi Number

of Colonies

Average cfu/ml x 102

% Contribution

Jan 2008

1. Aspergillus niger 3 0.5 1.91

2. Aspergillus flavus 5 0.83 3.18

3. Aspergillus terreus 145 24.16 92.4

4. Aspergillus versicolor 2 0.33 1.27

5. Paecilomyces variotii 1 0.17 0.63

Feb 2008




March 2008



April 2008


2. Aspergillus fumigatus 1 0.17 1.04


May 2008





5. Absidia corymbifera 1 0.17 3.44

June 2008


2 Aspergillus terreus 9 1.5 69.23


July 2008



3. Phanerochaete chrysosporium 18 3.0 15.4

Months SI. No.

Fungi Number

of Colonies

Average cfu/ml x 102

% Contribution

August 2008

1. Monilia sitophila 3 0.5 2.8


3. Curvularia lunata 1 0.17 0.94

4. Aspergillus versicolor 1 0.17 0.94


Sep 2008

1. Aspergillus tamarii 2 0.33 66.7


Oct 2008




4. Aspergillus tamarii 3 0.5 1.9

5. Monilia sitophila 1 0.17 0.63


Nov 2008



3. Aspergillus terreus 7 1.17 50

4. Scopulariopsis brevicaulis 1 0.17 7.14

Dec 2008



Table 5.2. Index of dominance (c) in relation to Index of general diversity (H) of fungi present in the tannery effluent from January 2008 to December 2008

Months

2008 Index of dominance (c)

Index of general diversity

(H)

January 0.48 1.063

February 0.41 0.973

March 0.96 0.093

April 0.67 0.553

May 0.30 1.386

June 0.54 0.790

July 0.73 0.477

August 0.86 0.358

September 0.56 0.637

October 0.97 0.079

November 0.36 1.171

December 0.73 0.436

Table 5.3. Fungal Colonies isolated from the tannery effluent from Jan 2008 to Dec 2008

Fungi

Months

January 2008 to December 2008 Total

Jan Feb March April May June July Aug Sep Oct Nov Dec

Zygomycotina

Absidia corymbifera

1 1

Basidoimycotina

Phanerochaete chrysosporium

18 18

Deuteromycotina

Aspergillus flavus

5 2 11 1 2 2 23

A.fumigatus 1 1

A.niger 3 5 5 3 98 5 4 71 194

A.tamarii 2 3 5

A.terreus 145 19 104 76 10 9 99 145 7 379 993

A.versicolor 2 1 3

Curvularia lunata

1 1 1 3

Paecilomyces variotti

1 11 19 1 1 2 35

Monilia sitophila

3 1 4

Scopulariopsis brevicaulis

1 1

TOTAL 156 35 106 96 28 13 117 106 3 157 14 450 1281

Table 5.4. pH of tannery effluent before (control) and after bioremediation (72 hrs) using fungi

Parameter Concentration of the effluent

Mean +SD

& % Change

Control Aspergillus

niger Aspergillus

terreus Mixed

culture

pH

100% Mean +SD

5.86 ± 0.03

6.85 ± 0.04

6.95 ± 0.04

6.83 ± 0.03

% Change +16.89 +18.60 +16.55

75% Mean +SD

6.25 ± 0.03

6.97 ± 0.02

7.34 ± 0.01

7.15 ± 0.002

% Change +11.52 +17.44 +14.4

50% Mean +SD

6.55 ± 0.03

6.86 ± 0.03

7.04 ± 0.03

6.9 ± 0.02

% Change +4.73 +7.48 +6.41

25% Mean +SD

6.75 ± 0.03

7.04 ± 0.03

7.26 ± 0.02

7.15 ± 0.02

% Change +4.31 +7.56 +5.93

Table 5.5. BOD of tannery effluent before (control) and after bioremediation (72hrs) using fungi


Mean +SD

& % Change

Control Aspergillus

niger Aspergillus

terreus Mixed

culture

BOD

100% Mean +SD

883 ± 39.62

554 ± 11.40

363 ± 11.95

638 ± 10.39

% Change 37.26 58.89 27.75

75% Mean +SD

575 ± 14.40

440 ± 15.81

328 ± 10.37

414 ± 12.94

% Change 23.48 42.96 28

50% Mean +SD

476 ± 10.84

207 ± 11.51

180 ± 12.15

174 ± 18.88

% Change 56.51 62.18 63.45

25% Mean +SD

220 ± 12.75

108 ± 9.08

99 ± 8.22

75 ± 7.91

% Change 50.91 53 65.91

Table 5.6. COD of tannery effluent before (control) and after bioremediation (72 hrs) using fungi


Mean +SD

& % Change

Control Aspergillus

niger Aspergillus

terreus Mixed

culture

COD

100% Mean +SD

2439 ± 41.77

1426 ± 19.87

1069 ± 28.14

1170 ± 19.38

% Change 41.53 56.17 52.03

75% Mean +SD

2146 ± 83.17

1160 ± 19.38

922 ± 25.18

1025 ± 28.10

% Change 45.95 57.04 52.24

50% Mean +SD

1426 ± 79.87

623 ± 21.68

415 ± 15.38

505 ± 15.81

% Change 56.31 70.90 64.59

25% Mean +SD

600 ± 15.81

305 ± 12.76

225 ± 19.36

247 ± 11.51

% Change 49.17 62.5 58.83

Table 5.7. TSS of tannery effluent before (control) and after bioremediation (72hrs) using fungi


Mean +SD

& % Change

Control Aspergillus

niger Aspergillus

terreus Mixed

culture

TSS

100% Mean +SD

1720 ± 42.70

969 ± 8.37

770 ± 8.39

895 ± 11.18

% Change -43.66 55.29 47.97

75% Mean +SD

865 ± 8.47

695 ± 11.18

609 ± 7.42

627 ± 8.37

% Change 19.65 29.60 27.51

50% Mean +SD

570 ± 15.81

405 ± 11.18

316 ± 10.84

389 ± 8.56

% Change 28.95 44.56 31.76

25% Mean +SD

310 ± 10.84

253 ± 12.04

165 ± 10.0

173 ± 9.75

% Change 18.39 46.77 44.19

Table 5.8. TDS of tannery effluent before (control) and after bioremediation

(72hrs) using fungi


Mean +SD

& % Change

Control Aspergillus

niger Aspergillus

terreus Mixed

culture

TSS

100% Mean +SD

3100 ± 101

2800 ± 43.78

2712 ± 12.5

2645 ± 21.59

% Change 9.7 12.5 14.68

75% Mean +SD

2076 ± 20.99

1815 ± 10.80

1654 ± 14.36

1423 ± 32.34

% Change 12.5 20.33 31.45

50% Mean +SD

1482 ± 5.50

1269 ± 5.24

1175 ± 7.63

1003 ± 6.55

% Change 14.37 20.72 32.32

25% Mean +SD

998 ± 6.78

829 ± 8.72

780 ± 5.11

645 ± 8.61

% Change 16.63 21.85 35.37

Plate 5.1. Fungi isolated and identified from the tannery effluent (January

2008 to December 2008)

Aspergillus niger

Aspergillus tamarii

Aspergillus terreus

Aspergillus versicolor

Curvularia lunata

Fusarium sp

Paecilomyces variotii

Scopulariopsis brevicaulis

Plate 5.2. Biotreatment of tannery effluent using Aspergillus terreus

Plate 5.3. Biotreatment of tannery effluent using Aspergillus niger

Plate 5.4. Biotreatment of tannery effluent using mixed culture

5. BIOREDUCTION BASED REMEDIATION OF TANNERY EFFLUENTS USING...

Documents

Transcript of 5. BIOREDUCTION BASED REMEDIATION OF TANNERY EFFLUENTS USING...