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Chapter 5

Biodegradation of benzenesulfonates in aqueous

environment

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146

5.1 Introduction

Environmental conditions have tremendous effect on the living

organisms especially microorganisms. They adapt to the climatic

conditions very fast. Their versatility makes them useful for various

biotechnological applications. They are widely used for biodegradation or

biosorption for removal of pollutants from the environmental matrices.

Here their nutritional source is exploited for biodegradation of

environmental pollutants. In this process it is observed that certain

microorganisms metabolize toxic pollutants by obtaining energy from

organic substances. Finally, the chemicals are transformed into harmless

compounds such as carbon dioxide and water. Organic pollutants are

generally degraded aerobically or anaerobically. It has also been observed

that biosorption has a great potential for the removal of xenobiotic

compounds from industrial effluents. The surface properties of bacteria,

yeasts, fungi and algae enable them to adsorb different kinds of pollutants

from aqueous solutions allowing the recovery and/or environmentally

acceptable disposal of the pollutants.

Biodegradation is the process by which organic substances are

broken down by the enzymes produced by living organisms. Later it

undergoes mineralization thereby converting organic matter to minerals.

Biodegradable matter is generally organic material such as plant, animal

and other substances originating from living organisms. Some

microorganisms have the astonishing, naturally occurring, microbial

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catabolic diversity to degrade, transform or accumulate a huge range of

compounds. Microbes (bacteria and fungi) found in natural waters and

soils have a very broad ability to utilize (catabolise) virtually all naturally

occurring compounds as their sources of carbon and energy, thus

recycling the fixed organic carbon back into harmless biomass and carbon

dioxide. This capability of microbes has evolved over 3 billion years of the

planets history and is responsible for the balance between photosynthesis

(by plants and algae), fixing carbon dioxide into biomass, and respiration

(by animals and bacteria), converting organic compounds back to carbon

dioxide by oxidation.

The advent of modern chemical industry has resulted in the release

of huge amounts of novel organic compounds, as industrial by-products,

pesticides, and other agrochemicals etc. into the environment. Bacteria

appear to adapt their pre-existing catabolic breadth to enable attack and

degrade many of the novel xenobiotic compounds. This has led to the

possibility of using consortia of bacteria or even cultures of single

organisms to either clean-up polluted environments or to degrade

potential pollutants at source and before their release into the

environment. This has led to an entire new industry, that of

bioremediation, whose role is to optimize conditions for natural bacteria to

degrade. However some compounds with complex structures are highly

recalcitrant to biodegradation, and some sites have become so polluted

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with toxic mixtures of both organic and inorganic compounds that nothing

can live there. In these instances, chemical or physical processes for

clean-up still have to be utilized.

Natural degradation is slow due to various reasons:

1. Bio availability

2. Physical, chemical factors unsatisfactory

3. pH, oxygen, moisture, redox potential, electron acceptors

4. Concentration of pollutants 5. Recalcitrance

6. Xenobiotic nature of pollutant.

7. Toxic intermediates produced

Microbial degradation or transformation of organic compounds may

involve either of the processes of aerobic (oxygen dependant) or anaerobic

situation, while in some cases it may need both the conditions to detoxify

some of the xenobiotic compounds.

5.1.1 Aerobic biodegradation of pollutants

In the conventional aerobic system, the substrate is used as a

source of carbon and energy. It serves as an electron donor resulting in

bacterial growth. The extent of degradation is correlated with the rate of

O2 consumption, as also previous acclimatization of the organism in the

same substrate. Two enzymes primarily involved in the process are mono-

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and di -oxygenase. The later enzymes can act on both aromatic and

aliphatic compounds, while for the former, only aromatic compounds can

act as substrates. Another class of enzymes involved in aerobic condition

is peroxidase, which is receiving attention for their ability to degrade

lignin.

5.1.2 Anaerobic biodegradation of pollutants

Anaerobic biodegradation is the breakdown of organic contaminants

by microorganisms when oxygen is not present. Some anaerobic bacteria

use nitrate, sulfate, iron and manganese as their electron acceptors, and

break down organic chemicals into smaller compounds often producing

carbon dioxide and methane as the final products. This general

mechanism of anaerobic microorganisms is an example of anaerobic

respiration. Alternatively some anaerobic microorganisms can break down

organic contaminants by fermentation. Fermentation is where the organic

chemical acts as an electron acceptor. Anaerobic biodegradation is an

important component of the natural attenuation of contaminants at many

hazardous waste sites.

The ability of microorganisms to metabolize, or use nutrients

depends on the chemical composition of the environment, and the

different microorganisms have evolved to the advantage of varying

conditions. In most organisms, including bacteria, the metabolic process

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requires the exchange of oxygen and carbon. In the absence of oxygen,

microorganisms use contaminants as their primary food supply.

This process is of widespread occurrence and relies on the metabolic

versatility of mixed microbial populations present in soils or sediments

when O2 supply is limited. Growth yield of anaerobic bacteria is extremely

low due to low energy yields. It has drawn attention these years due to the

possibility of decomposition of extremely recalcitrant xenobiotics through

this process.

Anaerobic microbial mineralization of recalcitrant organic pollutants

is of great environmental significance and involves intriguing novel

biochemical reactions. This form of degradation, under anaerobic

conditions (Fig. 5.1a), depends not only upon the compound, but the

temperature, pH and salinity of the subsurface. The overall process of

anaerobic degradation of sulphonates is shown in Fig. 5.1.

Fig. 5.1 Anaerobic degradation.

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Fig. 5.1a Schematic representation of aerobic and anaerobic

biodegradation of sulphonates.

5.1.3 Cometabolism

Some substances can be degraded by microorganisms only when

they are associated with other utilizable substrates. The metabolism

where cell growth can take place only in the presence of another

substance that is utilizable (co-substrate) is called cometabolism or co-

oxidation. The basics for cometabolism is the supply of energy, cofactors

or metabolites at various levels, from the transformation of one substrate,

to processes such as substrate transport, energy biosynthesis or

functioning involved in the transformation of second substrate. It has

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recently emerged as an important for the biotreatment of many xenobiotic

compounds. Due to the lack of specificity of existing enzymes and co-

factors, cometabolism is the fortuitous biotransformation of a non-growth-

supporting compound by microorganisms that are growing on metabolism

of growth substrate or by resting cells.

Such transformations have considerable environmental and

ecological significance since large number of organic compounds are

subjected to cometabolism. However, the toxicity of non-growth substrate

or its transformation products may result in the injury of some cellular

components and therefore inactive the cells. The presence of non-growth

substrate can inhibit the metabolism of the natural growth substrate,

because of its toxicity or recalcitrance, thereby decreasing cell growth and

retarding biodegradation. Cometabolism can be exploited, for example, by

purification of industrial effluents that contain degradation-resistant

synthetics together with domestic sewage water in a common wastewater

treatment plant. In some cases the mechanism is not yet obvious. In

cometabolism chemical substances are metabolically converted to non-

toxic products by one of several different types of reactions:

1. degradation of complex substrate into simple products

2. detoxication of chemical substances to a nontoxic compound.

3. conjugation of chemical substances with other compounds or cell

metabolites.

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5.1.4 Microbial degradation of aromatic sulphonates

Aromatic sulfonates are produced on a large scale in the chemical

industry and eventually large quantities of it are released into the

environment. The main source comprises of pollutants such as the

anionic detergents and dyestuffs, byproducts and their intermediates.

Benzene and naphthalene sulfonates are used in chemical industry as

intermediates for manufacturing of pharmaceuticals, dyes and tanning

agents. Sulfonated naphthalene-formaldehyde condensates are important

commercial plasticizers for concrete, dispersants and tanning agents.

Sulfonated azo dyes are extensively applied in the textile industry to

colour natural fibers, inks and pigments. Stilbenesulfonates are applied in

the paper industry as optical brighters. Alkanesulfonates and linear

alkylbenzene sulfonates (LASs) are frequently used anionic surfactants in

detergents and laundry.

Dyes are water-soluble dispersible synthetic aromatic organic

compounds, which are normally used for coloration of various substances

and extensively used for dyeing and printing in various industries. Due to

their chemical structure, dyes are resistant to fading on exposure to light,

water and many chemicals. Many dyes especially azo dyes are difficult to

decolorize due to their complex structure and synthetic origin. They are

recalcitrant to microbial degradation. Among dyes, azo dyes are the largest

and most versatile class of dyes considered to be toxic to the aquatic biota

and considered to be carcinogenic to humans. Because of their aromatic

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molecular structures and the strong electron-withdrawing property, the

azo group is thought to protect them against attack by oxygenases thereby

the conventional aerobic wastewater treatment processes cannot

efficiently decolorize azo dye-contaminated effluents. Hence these dyes are

first reduced under anaerobic conditions to the corresponding aromatic

amines, which though resisting further anaerobic degradation, were

reported to be well amenable for aerobic degradation. Aromatic amines

can be mineralized by means of aerobic treatment by non-specific enzymes

through hydroxylation and ring-fission of aromatic compounds.

Aromatic sulfonates are highly acidic and strongly hydrophilic in nature.

The persistency of aromatic sulfonates to microbial degradation is distinct.

Compounds containing a sulfonic acid group are highly soluble in the

aqueous environment and consequently more abundant there; they do not

accumulate in the sediment. Since the aromatic sulfonic acids are

xenobiotics, they are mainly found in the wastewater discharges from the

industries producing processing these compounds e.g. chemical, leather,

printing, paper, textile and pharmaceutical industries. These chemicals

are released in to the environment through industrial wastes via

wastewater treatment plant effluent discharges.

Aromatic sulfonates can be divided into two main groups. The first

group comprises the linear alkylbenzenesulfonates. Their fate when

released in the environment has been extensively studied and reviewed.

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The second group of aromatic sulfonic acid compounds comprises the

aromatic sulfonates.

This chapter focuses on the use of isolated bacteria for cometabolic

degradation of benzenesulfonic acids in aqueous environment.

5.2 Literature review

Generally aromatic sulfonic acids are not easily biodegradable. The

ability of aerobic bacteria to mineralize aromatic sulfonic acid compounds

was observed for the first time in the seventies. As a result of this

biodegradation, the sulfur moiety of the sulfonic acid group can enter in

the sulfur cycle. Today, our knowledge of the biodegradation of aromatic

sulfonic acids compounds is still rather limited.

Presence of sulfonic acid group on aromatic ring not only confers the

xenobiotic character but also recalcitrant nature to these compounds, as

not many aromatic sulfonic acids are known among natural compounds

[1]. Further the polar nature of sulfonic acid group requires highly specific

transport enzymes for their entry into the cell, thus rendering these

compounds resistant to biodegradation by unadapted activated sludge

and bacterial species utilizing normal aromatics [2,3]. However, some

mixed cultures as well as few pure bacterial strains which can utilize

amino-aromatic sulfonic acids as sole carbon and energy sources have

been isolated [4-11] Junker et al. [12] showed the transformation of 2-

aminobenzenesulfonic acid to 2-hydroxymucoic acid by the enzymes from

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Alcaligenous species O-1. Activated sludge bioreactors were used by Tan

et al {13} for degradation of positional isomers of aminobenzenesulfonic

acids. Different species of isolated Pseudomanas were used to transform

amino- and hydroxynaphthalene-sulfonic acids into various degradation

products [14-18]. Where as Nortemann et al have used mixed organisms

for degradation of 6-amino-2-naphthalenesulfonic acids (6A2NS). It was

initially converted into 5-aminosalicylate (5AS) by Pseudomonas sp. BN6,

and later was completely degraded by Pseudomonas sp. BN9 [16]. The

degradation pathways of 5-aminonaphthalene-2-sulfonic acid (5A2NS) by

Pseudomonas sp. BN6 were further investigated. It was found that 5A2NS

converted into a dead-end product, 5-Hydroxyquinoline-2-carboxylic acid

[17].

Ercole et al [19] used mixed culture (Co 27) along with a single strain

(RMNT) [19] to degrade 2-naphthalenesulfonic acid into β-naphthol.

Contzen et al., [20] have used mixed bacterial culture (RW2) for

degradation of benzene-1,3-disulfonic acid. They also found it suitable to

degrade the benzene-1,3-disulfonic acid to catechol-4-sulfonic acid in the

presence of 4-nitrocatechol. Catechol-4-sulfonic acid was further

metabolized into 3-sulfomuconate and 4-carboxymethyl-4-sulfobut-2-en-

4-olide. Ruff et al., [21] have used Pseudomonas Putida S-313 for

degradation of many of the sulfonic acids, viz, 4-chlorobenzenesulfonic

acid, 2-nitrobenzenesulfonic acid, 3-nitrobenzenesulfonic acid, 4-

nitrobenzenesulfonic acid, 4-nitrotoluene-2-sulfonic acid, 5-amino-2-

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chlorotoluene-4-sulfonic acid, 1,5-naphthalenedisulfonic acid, 1,6-

naphthalenedisulfonic acid, 2,6-naphthalenedisulfonic acid, 2,7-

naphthalenedisulfonic acid, 8-amino-1,5-naphthalene-disulfonic acid, 3-

amino-1,5-naphthalenedisulfonic acid, 6-amino-1,3-naphthalene-

disulfonic acid and 3-amino-2,7-naphthalenedisulfonic acid and found

that all test compounds were desulfonated except 6-amino-1,3-

naphthalenedisulfonic acid.

Chien [22] used aromatic sulfonic acids as a source of sulfur for

Clostridium Pasteurianum DSM 12136 and found that benzenesulfonic

acid, 4-toluenesulfonic acid, 4-xylene-2-sulfonic acid, 4-

aminobenzenesulfonic acid, 4-sulfobenzoic acid, 1,3-benzene-disulfonic

acid were successful. Similarly, Song et al [23] have isolated two bacterial

strains Arthrobacter sp.2AC and Comamonas sp.4BC and found that both

were capable of utilizing naphthalene-2-sulfonic acid. Most xenobiotic

aromatic sulfonates, which were examined as carbon or sulfur sources for

the growth of aerobic bacteria [24]. Kertesz et al [25] and Cook et al [26]

conducted a detailed study and reviewed the varying stages at which the

mechanism of microbial desulfonation of aromatic sulfonates occurs viz.,

(a) before (b) during or (c) after ring cleavage. Fig. 5.2 shows the probable

path ways of desulphonatons.

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a) Desulphonation before ring cleavage

SO3-

R1 R1

OHSO3- OH

OH

R1

OH

OH

O2NADH

H+ HSO3-

b) Desulphonation during ring cleavage

NH2

SO3- SO3-OH

OH

SO3-O

COO-

OH COO- OH

COO-NH3

O2NADHH

+

H+

O2

HSO3-

OH2

c) Desulphonation after ring cleavage

SO3-

NH2

SO3-

OHOH

COO-COO-

SO3-

O

SO3-COO-

OCOO-

OCOO-

HSO3-

OH2

Fig. 5.2 Different path ways of aerobic desulfonation of aromatic sulfonic

acids.

However, only a few strains were found to be suitable for

degradation of nitro-substituted aromatic sulfonic acids [5,27]. It was

found that aromatic pollutants containing multiple nitro and azo groups

were resistant to biodegradation by aerobic bacteria. Hence these

compounds were first subjected to anaerobic consortia to form aromatic

amines. They were not mineralized. The aromatic amines produced by this

process were transformed into highly reactive electrophiles in mammals

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which in turn could form covalent adducts with DNA and pose health

risks [28-33].

Reports on cometabolism of environmentally hazardous compounds

with bacterial cells, grown exclusively in a conventional carbon source

such as glucose are limited [34, 35]. No reports are available on

degradation of subsitiuted benzenesulfonic acids viz.,

metaphenylenediamine-4-sulfonic acid (MPDSA), 3-Aminoacetanilide-4-

sulfonic acid (AASA), paranitrotoluene-orthosulfonic acid (PNTSA) and 2,4-

dinitrobenzenesulfonic acid (DNBSA) in the literature.

This chapter describes the biodegradation studies of substituted

benzenesulfonic acids viz., metaphenylenediamine-4-sulfonic acid

(MPDSA), 3-Aminoacetanilide-4-sulfonic acid (AASA), paranitrotoluene-

orthosulfonic acid (PNTSA) and 2,4-dinitrobenzenesulfonic acid (DNBSA)

in aqueous media by Arthrobactor species in presence of various

carbohydrates viz., glucose, fructose and dextrose etc., as growth

substrates.

5.3 Aims and objectives

� Isolation of the bacterial strain, Arthrobactor species suitable for

removing substituted benzenesulfonic acids from aqueous media.

� Optimization of growth parameters of isolated bacteria Arthrobactor

species.

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� Degradation studies of the substituted benzenesulfonic acids using the

bacterial Arthrobactor species.

� Application to industrial effluents containing the tested benzenesulfonic

acids.

5.4 Materials and methods

5.4.1 Reagents and test compounds

All the chemicals used in the present study were of highest purity

(>99%). The sulfonic acids, metaphenylenediaminesulfonic acid (MPDSA),

3-aminoacetanilide-4-sulfonic acid (AASA), paranitrotoluenesulfonic acid

(PNTSA) and 2,4-dinitrobenzenesulfonic acid (DNBSA) were kind gift from

M/S. Orchem industries Pvt. Ltd. (Hyderabad, India). Glucose, sucrose,

fructose, arabinose, galactose, maltose, xylose, soluble starch and potato

starch (M/s High Media, Mumbai, India), potassium dihydrogen

orthophosphate, dipotassium hydrogen orthophosphate, calcium chloride,

magnesium sulphate, ammonium nitrate, ferric chloride and ammonium

chloride (M/s Ranbaxy Fine Chemicals Ltd, S.A.S. Nagar, India) were

used.

5.4.2 Isolation of microorganisms

Bacterial strains were isolated using the spread plate method. The

aerobic sludge from the common effluent treatment plant located in

Hyderabad, India (Jeedimetla effluent treatment plant) was diluted 100

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times and spread on the nutrient agar media supplemented with 0.05 mM

each of MPDSA, AASA, PNTSA and DNBSA. Petri dishes were

subsequently incubated at 250C. The bacterial strains were isolated as

soon as the colonies were appeared. As soon as the colonies were

appeared, different bacterial strains were sub-cultured by spreading

separately on the nutrient agar media supplemented with the 0.05 mM of

each test compound.

5.4.3 Growth and maintenance of the bacteria

Stock cultures of Arthrobactor species were maintained by periodic sub-

transfer on nutrient agar slants for every 15 days. The stock solutions of

bacterial culture were prepared using slants, which were stored at 40C in a

refrigerator. All the batch cultures were grown in 250 mL cotton plugged

erlenmayer flasks containing 100 mL of mineral medium consisting of 1.0 g/L

of potassium dihydrogen orthophosphate, 1.0 g/L of dipotassium hydrogen

orthophosphate, 0.02 g/L of calcium chloride, 0.2 g/L of magnesium sulfate,

1.0 g/L of ammonium nitrate, 0.05 g/L of ferric chloride and 2.5 g/L of

ammonium chloride. Glucose (1% w/v) was used as a primary growth

substrate unless otherwise mentioned. The test compounds used in the study

were added to the medium before it was autoclaved. This was because of the

fact that the compounds were quite stable at high temperatures. The

autoclaved (at 1210C, 1.5 kgf/cm2 for 20 min.) flasks were inoculated with

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bacterial culture and placed on a rotary platform incubator shaker at 200

rpm and 300C.

5.4.4 Adaptation of bacteria to the test substances

Preliminary experiments involved the adaptation of the isolated

bacteria to an increased concentration of the test substances. The isolated

strains were initially grown in basal medium containing 0.05 mM each of test

compounds and 1% glucose as a co-substrate. Then bacterial medium was

gradually exposed to the increased concentrations of the test compounds

through successive transfer into fresh medium containing 0.1, 0.15, and 0.2

mM of the test compounds and 1% glucose. Finally the exposed culture was

used for all the experiments.

5.4.5 Preliminary experiments with bacterial culture

0.1 mM of the test compounds (MPDSA-1.88 mg/L, AASA-2.3 mg/L,

PNTSA-2.18 mg/L and DNBSA-2.47 mg/L) was chosen to conduct

preliminary studies. Four sets of experiments were carried out in parallel. In

one set, growth of bacteria was initiated in sterile media containing the test

compounds, in the second set 1% glucose and the chosen test compounds. In

the third set, the growth of bacteria was initiated in presence of glucose

without test compounds. The fourth set was run in the absence of inoculum

as a control. Aliquots of the spent growth media were withdrawn after 5 days

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to analyze the residual contents of test compounds as well as measured the

growth of the bacteria respectively.

5.4.6 Optimization of growth parameters

5.4.6.1 Effect of different carbon sources

To study the effect of different carbon sources on the degradation of

test compounds by bacteria, the growth of the bacteria was initiated in a

medium containing 0.1 mM of the test compounds and 1% of each of

glucose, fructose, sucrose, maltose, galactose, arabinose, xylose, lactose,

soluble starch and potato starch separately used as carbon sources.

Aliquots of the spent growth media were withdrawn after 5 days to analyze

the residual contents of test compounds as well as measured the growth

of the bacteria respectively.

5.4.6.2 Effect of initial concentration of carbon source

In order to study the effect of the initial concentration of glucose on

the degradation of test compounds by bacteria, it was varied from 0.25 to

2.0% in the medium containing 0.1 mM of the test compounds. Aliquots of

the spent growth media were withdrawn after 5 days to analyze the

residual contents of test compounds as well as measured the growth of

the bacteria respectively.

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164

5.4.6.3 Effect of pH of the medium

pH of the medium is one of the important parameters used to study the

degradation of the test compounds by bacteria. Experiments were

conducted in the medium containing 0.1 mM of test compounds, 1%

glucose and the pH varied from 4.0 to 11.0. The pH was adjusted using 2N

HCl/NaOH. Aliquots of the spent growth media were withdrawn after 5

days to analyze the residual contents of test compounds. Growth of the

bacteria was also measured. Separate set of experiments was carried out

in parallel in the absence of innoculum to know the stability of the test

compounds at different pH conditions.

5.4.7 Removal of test compounds at optimized conditions

Two sets of experiments were conducted to find out the degradation

of the test compounds and bacterial growth pattern at optimized

conditions. In the first set, growth of bacteria was initiated in sterile

medium containing the test compounds and 1% glucose as a carbon

source and the second set with out test compounds. Aliquots of the spent

growth medium were withdrawn at regular intervals and analyzed by

HPLC.

5.4.8 Application to industrial effluents

The effluents of MPDSA, AASA, PNTSA and DNBSA collected from

M/s M/S. Orchem industries Pvt. Ltd, Hyderabad were filtered through

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165

Whatman No. 41 filter paper and subjected to the treatment with bacteria

in the medium containing 1% glucose.

5.4.9 Analytical methods

5.4.9.1 Assay of sulfonic acids

The concentrations of MPDSA, AASA, PNTSA and DNBSA were

measured by HPLC system composed of two LC-10AT vp pumps, an SPD-

M10 A vp photodiode array detector, an SIL-10AD vp auto injector, a

DGU-12A degasser and an SCL-10A vp system controller (all from

Shimadzu, Kyoto, Japan). A reversed-phase Inertsil ODS-3V C18 column

(250 x 4.6 mm i.d., 5 µm particle size) (GL Sciences Ltd, Tokyo, Japan)

was used for separation. The mobile phase, consisting of methanol-0.01 M

ammonium acetate, was initially programmed to elute 100% 0.01 M

ammonium acetate up to 4 min, followed by a linear gradient of 60%

methanol within 20 min. and back to 100% ammonium acetate within 25

min. and maintained the same up to 30 min. The mobile phase was

filtered through a 0.45 m, PTFE filter and degassed using a vacuum

before delivering into the system. The analysis was carried out at ambient

temperature with a flow rate of 1.0 ml/min. Chromatograms were

recorded using an SPD-M10A vp photodiode array detector at 254 nm.

The chromatographic and integrated data were recorded using a HP-

Vectra (Hewlett Packard, Waldbronn, Germany) computer system.

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166

5.4.9.2 Measurement of bacterial growth

The cell growth was determined by weighting the cells retained after

filtering the samples by Whatman GF/F (0.7 μm) filters.

5.4.9.3 Assay of glucose

Glucose concentration was determined by dinitrosalicylic acid (DNS)

method. DNS reagent was prepared by adding 10 g of 2,4-dinitrosalicylic

acid, 0.5 g of sodium sulfite and 10 g of sodium hydroxide in to 1 L

distilled water. 40 g of Potassium sodium tartrate was dissolved in 100 mL

DI water to prepare 40% solution. Added 3 mL of DNS reagent to 3 mL of

sample in a test tube. Heated the test tube at 90oC for 5-15 minutes to

develop the red-brown color. Added 1 mL of potassium sodium tartrate

solution to stabilize the color. After cooling to room temperature recorded

the absorbance with a spectrophotometer at 575 nm.

5.5 Results and discussion

In the present investigation, the bacterial strain Arthrobactor species

were isolated from a combined effluent collected from the treatment plant

in Hyderabad, India. The isolated strains were tested for degradation of

benzenesulfonic acids. Screening, characterization of fungi and bacteria

and the effects of different growth parameters were studied.

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167

5.5.1 Screening and characterization of microorganisms

Combined effluent of a treatment plant at Hyderabd, India was

taken for study. Strains, which have shown tolerance to high

concentrations of test compounds, were screened and isolated from the

combined effluent taken. In the preliminary screening three bacterial

strains (B1, B2, B3) were isolated which have shown tolerance to test

compounds. Here nutrient agar spread plate method was used. It was

found that, B2, of the three bacterial strains, suitable for the removal of

high concentration of test compounds. The isolated bacterial strain (B2)

was sent to at Institute of Microbial Technology, Chandigarh, India for

identification. According to the morphology and physiochemical

characteristics determined, the isolated strain was identified as as

Arthrobactor species (MTCC No. 7254). The bacterial colonies on nutrient

agar plate are shown in Fig. 5.3. The characteristic parameters of

Arthrobactor species are recorded in Tables 5.1.

5.5.2 Biodegradation experiments with Arthrobactor species

Preliminary experiments were conducted in the medium containing

0.1 mM of test compounds in presence and absence of glucose. In the

absence of glucose, growth of the bacteria as well as removal of the test

compounds was negligible. In the presence of glucose, the growth patterns

of bacteria were found to be similar for all the four test compounds viz.,

MPDSA, AASA, PNTSA and DNBSA, but differ in their degradation. Within

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the 5 days inoculation period, 64% of MPDSA, 37% of AASA and 22% of

PNTSA were converted into products whereas the concentration of DNBSA

remained intact. The control experiments conducted in the absence of

bacterial culture has ruled out the possibility of degradation due to abiotic

mechanism. Further it was noticed that the growth of bacteria was found

to be similar in absence and presence of test compounds. It suggests that

in presence of glucose, the bacteria was able to grow in the medium

containing test compounds and also able to degrade the test compounds.

Further these species were adapted to higher concentrations of the test

compounds for improved effectiveness. The adapted species were used for

further studies. The parameters viz., different carbon sources,

concentration of carbon source and pH were optimized.

Table 5.1 Characteristics of Arthrobactor species

S.No. Parameter Observation

1. Cell morphology Round, mucoid, translucent

and creamish yellow,

2. Gram’s staining positive

3. Cell shape Rods and coccus

4. Arrangement Single and in groups

5. Motility positive

6. Growth temperature 15-370C

7. Growth pH 4-10

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Fig. 5.3 Colonies of Arthrobactor species on a nutrient agar plate.

5.5.2.1 Effect of different carbon sources

The results of preliminary experiments indicated that the presence

of additional carbon source plays an important role in the growth of the

bacteria and degradation of test compounds. The following is the weight of

bacteria grown in the corresponding carbon sourse:

Table 5.2: Bacterial growth with different carbon source

Weight of bacteria (g) Carbon

source

Weight of bacteria (g) Carbon source

0.688 Glucose 1.220 Galactose

0.661 Fructose 0.827 Xylose

0.702 Sucrose 0.932 Lactose

0.716 Maltose 0.171 Soluble starch

1.097 Arabinose 0.314 Potato starch

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The pattern of degradation with different carbon sources is shown in

Fig.5.4.

Fig. 5.4 Effect of different carbon sources on degradation of MPDSA,

AASA, PNTSA and DNBSA (pH-6.8, 200 rpm and 300C).

Degradation of test compound (%) and growth of bacteria

(g/L).

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171

It could be seen from Fig.5.4 that the maximum of the test compounds

were removed with glucose, sucrose, maltose followed by fructose, soluble

starch, potato starch, lactose, arabinose, galactose and xylose. It was also

observed that the maximum growth was in the presence of galactose

followed by arabinose, lactose, xylose, maltose, sucrose, glucose, fructose,

potato starch and soluble starch. Inspite of less bacterial growth complete

degradation of MPDSA and AASA was observed in presence of glucose,

sucrose and maltose when compared to galactose, arabinose, lactose and

xylose. This infers that there was sufficient production of required

enzymes for degradation of test compounds in presence of glucose,

sucrose and maltose. Based on these results, glucose was selected as

carbon source for further studies.

5.5.2.2 Effect of concentration of glucose

Fig. 5.5 shows the effect of different concentrations of glucose on

degradation of MPDSA, AASA, PNTSA and DNBSA. As the initial

concentration of glucose was increased from 0.25% to 0.5%, an increase

in the removal of test compounds was observed. Almost similar results

were obtained from 0.5% to 1.5% where complete degradation of MPDSA

and AASA, 58% degradation of PNTSA and 2% degradation of DNBSA were

observed. These results clearly indicate the positive influence of primary

growth substrate on removal of the test compounds. Thus 1% glucose was

used as carbon source for further experiments.

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5.5.2.3 Effect of pH

Growth of bacteria and degradation of test compounds was studied

different pH.

Table 5.3 Bacterial growth at different pH

Weight of bacteria (g)

pH taken

0.345 4.0

0.403 5.0

0.704 6.0

0.804 6.7

1.747 8.0

0.615 9.0

0.432 10.0

0.173 11.0

From the above data it was evident that maximum growth of

bacteria is with the pH 6-8. Fig. 5.6 shows the effect of pH on degradation

of MPDSA, AASA, PNTSA and DNBSA. The degradation was found to be

maximum in the pH range 6-8. At this pH range complete degradation of

MPDSA and AASA, 36% of PNTSA was observed, whereas only 2% of

DNBSA was degraded. There was significance decrease in the growth of

bacteria at pH > 8.0. It indicated that there was a direct relation between

the growth of bacteria and degradation of test compounds in relation to its

pH. So pH 6.8 was selected for experiments through out the study.

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173

Fig. 5.5 Effect of concentration of glucose on degradation of MPDSA,

AASA, PNTSA and DNBSA (pH-6.8, 200 rpm and 300C).

Degradation of test compound (%), consumption of glucose

(%) and growth of bacteria (g/L).

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174

Fig. 5.6 Effect of pH on degradation of MPDSA, AASA, PNTSA and DNBSA.

(Concentration of glucose 1%, 200 rpm and 300C). Degradation

of test compound (%) and growth of bacteria (g/L).

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175

Fig. 5.7 Effect of initial concentrations of the test substances (0.5% glucose,

pH 6.7, 200 rpm and 30°C).

5.5.2.4 Degradation of test compounds at optimized conditions

Fig. 5.8 shows the degradation patterns of the test compounds,

consumption of glucose and growth patterns of bacteria under optimized

conditions. MPDSA, containing two amino groups and AASA with one

amino and one acetamido group were degraded completely.

Chapter 5

176

Fig. 5.8 Degradation of test compounds and growth of the bacteria under

optimized conditions. (Concentration of test compounds 0.1 mM,

concentration of glucose-1%, pH – 6.8, 200 rpm and 300C).

Concentration of test compound (%), concentration of

glucose and growth of bacteria (g/L).

PNTSA with one methyl and one nitro group degraded partially whereas

degradation of DNSDA with two nitro groups was negligible. It could be

seen clearly from the Fig. 5.8 that MPDSA and AASA were completely

Chapter 5

177

degraded within 18 h and 24 h respectively. PNTSA was degraded partially

(56%) within 36 h later the degradation was stopped whereas only 2%

degradation of DNBSA was observed within five days when the initial

concentration was 0.1 mM. The degradation of the test compounds was

stopped after 36 h. The growth pattern of the bacteria in presence of all

the four test compounds was comparable with each other with maximum

weight of 0.88-0.90 g/L. The same growth of the organism in presence and

absence of the test compounds suggests that the bacterial species were

able to tolerate the test compounds and complete their growth pattern.

These results indicate that the bacterial species were able to grow in

presence of the three test compounds, but differ in rates of degradation.

The differences in the removal were due to substituents present on the

benzene rings.

HPLC chromatograms of MPDSA, AASA, PNTSA and DNSDA are

shown in Fig. 5.9. It could be clearly seen from the HPLC chromatograms

that the MPDSA with two amino- groups and AASA with one amino- and

one acetamido group were completely converted into their products.

PNTSA with one methyl and one nitro group was partially converted in to

products whereas the degradation of DNBSA with no amino but two nitro

groups was found be negligible. This could be due to the electron releasing

character of the amino- group, which facilitates the electrophilic attack of

the extra cellular enzymes, produced by bacterial biomass whereas it was

retarded by the electron withdrawing nitro groups. Further it was

Chapter 5

178

observed that the degradation products of MPDSA and AASA were

completely disappeared within 72 h, whereas some products of PNTSA

remained intact till 120 h.

Figs. 5.9 HPLC chromatograms of I) MPDSA, II) AASA, III) PNTSA and IV)

DNBSA; a: before degradation; b: after 5days of degradation.

5.5.2.5 Biodegradation of industrial effluents

It was clear from above studies that the arthrobactor species were able

degrade to MPDSA, AASA and PNTSA, but not DNBSA. Thus the present

method was successfully adapted to the degradation of test compounds

Chapter 5

179

from the industrial effluents of MPDSA, AASA and PNTSA collected from

the manufacturing units in Hyderabad, India. The contents of the test

compounds before and after the biotreatment of three different effluents of

MPDSA, AASA, DNBSA and PNTSA are recorded in Table 5.3. It is clear

from Table 5.3 that 22.4 mg/L of metaphenylenediamine-4-sulfonic acid

(MPDSA) was completely degraded in effluent of MPDSA within five days.

24.7 mg/L of 3-Aminoacetanilide-4-sulfonic acid (AASA) and 19.3 mg/L

metaphenylenediamine-4-sulfonic acid (MPDSA) were completely

degraded. Whereas PNTSA was partially degraded and converted into its

products. 25.6 mg/L of PNTSA was degraded to 11.7 mg/L and its

products were remaining intact within 5 days of incubation period. It

could be clearly seen from the results that the Arthrobacor species was

capable of degrading MPDSA, AASA and PNTSA. The HPLC

chromatograms of effluents of MPDSA, AASA and PNTSA are shown

Fig.5.9.

Table 5.3 Contents of the organic pollutants before and after biotreatment

S.No. Effluent/ Concentration (mg/L)

compound Before biotreatment After 5 days of biotreatment

1. MPDSA 22.4 --

2. PNTSA 25.6 11.7

3. AASA 24.7 19.3

4. DNBSA 23.9 --

Chapter 5

180

5.6 Conclusions

Arhtrobactor species were isolated from the sludge of industrial effluent

treatment plant and used for degradation of MPDSA, AASA, PNTSA and

DNBSA from aqueous environment. The growth parameters for the

bacteria and degradation patterns of the test compounds were optimized.

The effect of different conventional carbon sources, concentration of

glucose and initial pH of the medium were tested. In absence of

conventional carbon source as growth substrate, the degradation of test

compounds as well as the growth of the bacteria was found to be

negligible. Maximum growth of the bacteria was observed in presence the

of galactose, arabinose, lactose and xylose whereas the maximum

degradation of the test compounds was observed in the presence of

glucose, sucrose and maltose. Thus glucose was selected as a primary

growth substrate. The degradation of the test compounds increased as the

concentration of glucose increases from 0.25-1.0 g/L and steady upto 1.5

g/L and then slightly decreased. Medium pH 6.0-8.0 was found to be

effective in the degradation of tested sulfonic acids. The isolated bacteria

have a great potential for complete degradation of MPDSA and AASA and

degradation products of MPDSA, partial degradation of PNTSA, but it was

not successful in the degradation of DNBSA and degradation products of

AASA and PNTSA. At optimized conditions a complete degradation of

MPDSA, AASA and 56% degradation of PNTSA were observed at 0.1 mM

sulfonic acid concentration whereas the concentration of DNBSA was

Chapter 5

181

remained intact (only 2% was degraded). The degradation products of

MPDSA were disappeared completely within 36 h whereas some products

of AASA and PNTSA were still remaining up to 5 days of incubation period.

Chapter 5

182

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