INTRODUCTION

Chapter 1

1.1 INTRODUCTION

Society’s ever-expanding utilization of materials, energy and

space is accompanied by an increasing flux of anthropogenic organic

chemicals into the environment. The presence of man-made

hazardous compounds in the environment is a subject of intense

concern to many countries in the world .The different chemicals

discharged by the industries to the environment can upset the delicate

balance of the ecosystem. Minute quantities of these compounds

entering into the living organisms may accumulate in the various levels

of food chain. Studies about the degradation and transformation of

such compounds and the proper processing of the effluents containing

them are essential for the safe and clean environment.

Surfactants are the largest class of compounds present in raw

domestic wastewater. They are used in household and industrial

laundry and cleaning operations. The ever-increasing demand of

surfactant since the middle of this century is causing great concern

about its role in environmental pollution. Surfactants cause foam at

sewage treatment plants (1) and pollute underground water (2). Due to

their toxic nature, their presence endangers the aquatic flora and

fauna (3). Also, they create many health hazards like dermatitis (4).

They exert a solubilizing effect on many organic compounds and

increase the carcinogenic impact.(5) Having penetrated into water, the

surfactant changes its quality by causing it to have an unpleasant

smell and taste.

Surfactants basically are of three types- anionic, cationic and

non-ionic. Anionic and cationic detergents have a permanent negative

and positive charge respectively attached to nonpolar (hydrophobic)

2 Chapter 1

carbon- carbon chains. Non-ionic detergents do not have any

permanent charge. Surfactant can also be classified on the presence

of either sulphate or phosphate. Surfactant generally contains strong

lipophilic, hydrophobic group and strong, hydrophilic group.Therfore it

simultaneously attracted and repelled by water. Consequently, it tends

to its cleaning and foaming action. The hydrophobes in general use for

detergent formulation are aliphatic or alkyl aromatic hydrocarbon

radicals containing 12 to 20 carbon atoms. The hydrophobic groups

are generally anionic, such as sulfonates sulfates, ester carboxylates

or nonionic polyethers or polyalcohols (Amund et al, 1997).

Sodium dodecyl sulphate (SDS) is a widely applied anionic

surfactant that accounts for approximately 25% to 30% of the

world’s total synthetic surfactants. SDS is of high demand due to its less

cost and excellent foaming characteristics. SDS otherwise known as

sodium lauryl sulfate (SLS) is used in household products such as

toothpastes, shampoos, shaving foams and bubble baths, in laboratories

and in various other industries. The high prevalence and usage of this

detergent thus demands knowledge on its degradation aspects.

The molecular structure of SDS (C12H25NaO4 ) is composed of

two units namely, (1) A hydrocarbon chain (C1- C12), (2) A sulphate

group attached to the carbon (Schleheck et al, 2003). The molecule

has a tail of 12 carbon atoms, attached to a sulphate group giving the

molecule the amphiphilic properties required of a detergent.

Introduction 3

The pollution of soil and water with SDS is widespread in

the environment and is creating major health problems. High

concentrations of SDS in the environment cause may cause dermatitis

and inflammation. SDS is found to be toxic even in microgram

concentrations to aquatic flora and fauna. Moreover, reports

supporting and opposing the carcinogenicity of this component in high

concentrations do exist.

Degradation of SDS is a major challenge for bacteria because

this detergent solubilizes biological membrane and denatures proteins.

(Helenius et al., 1975). Several resistant mechanisms against anionic

detergents like diffusion barriers (Nikaido et al., 1985), multidrug efflux

pumps (Poole, 2004), or CIP proteases (Rajagopal et al, 2002) have

been described. All these resistance mechanisms require energy and

have been shown to protect cells which grow in the presence of

detergents Bacteria using detergents for growth face an additional

challenge. They have to invest part of their energy into protection

while taking an increased risk of damage because they have to take

up the toxic detergents to metabolize them. (Klebensberger et al.,

2006)

In relation to surfactant biodegradability, it is important to

distinguish between primary and ultimate biodegradation. The breakdown

of a substance as measured by a substance specific analytical method,

for example, the loss of the sulphate groups from surfactants, such as

Sodium dodecyl Sulphate would be a primary biodegradation step that

would lead to an immediate loss of surfactant properties. (Measured by

the methylene blue anionic surfactants (MBAS) reduction test. (Hayashi,

1975). However, ultimate biodegradation implies the complete

conversion of these surfactants into products such as CO2, H2O,

4 Chapter 1

inorganic salts and cellular products, and many more metabolic steps

would therefore be involved. nevertheless, confirmation of ultimate

biodegradation is important because primary biodegradation, although

leading to losses in surfactant properties. may still yield recalcitrant

and possible toxic metabolites. (Roig et al., 1998).

Several enzymes are involved in SDS degradation.

(Alkylsulphatases, Dehydrogenases etc.). Degradation of SDS is

started by alkylsulphatases which hydrolyses SDS to sulphate and 1-

dodecanol (Thomas and White, 1989). The liberated alcohols are

subsequently oxidized by the enzyme alcohol dehydrogenases

(Dodgson & White, 1983). Of these enzymes alkylsulphatases is of

great importance as high prevalence of this enzyme can serve as an

indicator of detergent contamination. Further utility of immobilized

forms of these enzymes in bioreactors may increase their usage

potential.

There is an urgent need to search for new possibilities of

enhanced degradation of surfactants. Bacterial degradation of

pollutions has proved to be very efficient especially in combination

with immobilization methods. Immobilization of bacterial cells offers

several advantages such as prevention of cell losses in continual

processes, and allows working with high cell densities. Furthermore,

immobilized organisms are more resistant to adverse effects occurring

during the degradation processes like changes of physico-chemical

parameters such as pH, temperature, fluctuations of substrate

concentration, presence of toxic substances etc.

To improve the biodegradation of surfactants at higher

concentration biotechnological approaches may be applied to bring

Introduction 5

out efficient solutions for biological cleanup of industrial wastewater. In

order to increase bacterial concentration in the bulk solution and to

enhance biodegradation rates, membrane bioreactors have been

successfully used as one of the alternatives. (Mortazavi et al.2008).

This work has been largely motivated by concerns over the

biodegradation of SDS like surfactant at high concentration using a

suitable bioprocess. The present work was addressed towards

biodegradation of high concentration of SDS using a novel bioreactor

for the effective bioremediation strategy. Attempts were also made to

study the metabolic products of SDS biodegradation to ensure that all

the degradative products are non toxic to the ecosystem.

1.1.1 Objectives

1. Isolation, screening, and identification of efficient Sodium

dodecyl sulphate degrading bacteria

2. Optimization of factors for efficient biodegradation

3. Analysis of metabolic byproducts of sodium dodecyl sulphate.

4. Extraction and purification of enzyme responsible for SDS

degradation.

5. Designing of a multistage reactor for detoxifying SDS polluted

wastewater.

6 Chapter 1

1.2 REVIEW OF LITERATURE

Surfactants are organic compounds that reduce surface tension

in water and other liquids (Kowalska et al; 2004). In the domestic

wastewater produced by the households, surfactants invariably exists

in significant amount due to detergents used for all kinds of washing.

Surfactants have also been widely used in textiles, fibers, paints,

polymers, cosmetics, pharmaceuticals, mining, oil recovery pulp and

paper (Sheng et al., 1999). These applications of the surfactant,

increasing its discharge in the wastewater, produce foam and enter

into the underground water resources and constituting an ecological

risk for aquatic organisms (Nasiruddin and Uzva, 2005).

Surfactant contains both strong hydrophobic and hydrophilic

moieties. According to the charge of their hydrophilic moiety,

surfactants can be classified into four categories: anionic, non-ionic,

cationic and amphoteric (Mozia et al., 2005). Anionic surfactants are

one of the most frequently employed surfactants and constitute

approximately two-third of these surfactants. Cationic surfactants

constitute less than 10% of the ionic surfactants and rest is anionic

surfactant. The predominant class of anionic surfactant is linear

alkylbenzene sulfonate and linear alkyl sulfate (Liwarska and Bizukojc

2006). As a result, their fate in the environment has been widely

studied. An example of linear alkyl sulfate is Sodium Dodecyl Sulfate

(SDS), which is a representative of anionic surfactant (Adac et al.,

2005).

The increasing releases of organic pollutions by industries

cause many health related problems. However increased awareness

of the harmful effects of environmental pollution has led to the dramatic

Review of Literature 7

increase in research on various strategies that may be employed to

clean up the environment. It is now realized that microbial metabolism

provides a safer, more efficient, and less expensive alternative to

physico- chemical methods for pollution abetment (Hebes et al.,

1987).

1.2.1 Ecological impact of SDS

Surfactants are widely used in everyday personal care and

household products as well as in a variety of industrial applications (Li,

2008). In fact, many surfactants and their degradation products have

been found worldwide in waste water discharges, sewage treatment,

plant effluents, natural water and sediments (Ying, 2006). Because

many surfactants are ubiquitous (Ying et al., 2002; Venhuis and

Mehrvar, 2004), the potential toxic effects of these chemicals have

attracted much research attention in the past several decades (Abel,

1974; Lewis and Suprenant, 1983; Lewis, 1991). Many different

mechanisms of toxicities exist for different types of surfactants and

one single surfactant can produce its toxicity through more than one

mechanism (Li, 2008).

SDS have ambiguous effect on the environment .Intensive

research has done to investigate the effects of surfactants on natural

communities (Abel, 1974; Malagrino et al., 1987; Augier, 1991; Lewis,

1992; Huang and Wang, 1994; Hansen et al., 1997; Rocha et al.,

2007; Rosety et al., 2007). The interaction of surfactants with cell

membrane lipids appears to disrupt membrane integrity, thus causing

toxic effects (Abel, 1974).The toxicity of surfactants is primarily

determined by their ability to be adsorbed and penetrating the cell

membrane (Rosen et al., 2001).

8 Chapter 1

Anionic surfactants such as SDS exert toxic and harmful effects

on higher organisms .Some of the reports are described below. SDS

may be damaging to the immune system, especially within the skin.

Skin layers may separate and inflame due to its protein denaturing

properties (CIR publication, 1983). Carcinogenic nitrosamines can form

in the manufacturing of sodium dodecyl sulfate or by its interaction

with other nitrogen bearing ingredients within a formulation utilizing

this ingredient (CIR publication, 1983). SDS can induce damage of the

skin barrier function (Léveque et al., 1993). SDS act against cell

membranes and can solubilize proteins causing their denaturation

(Cserhati et al., 2002).They can also modify the activity of an enzyme

by binding to it (Cserhati et al., 2002). The normal levels of blood

chemical parameters are altered due to topical exposure to SDS,

evidently indicate that SDS is capable of deep adsorption and

penetration through the skin barrier and can reach deeper internal

organs such as liver and kidneys to cause systemic effect.(Wadaan et

al., 2009). This poses the question whether it could be a serious

potential health threat from its use in shampoos, cleansers and

toothpastes (CIR publication, 1983).

Ducks have been observed to be at risk for hypothermia when

exposed to detergent-polluted waters in low temperatures (e.g., 0.07

Mm SDS at 0 degrees C) (Singer and Tjeerdema, 1993). Surfactants

such as SDS have the potential to enhance the penetration of water

into the birds’ feathers. This may decreases the feathers’ insulating

capacity, which decreases the bird’s ability to maintain body

temperature.

The SDS is nowadays widely spread in many different aquatic

environments, where it has an important pollution potential.

Review of Literature 9

(Ainsworth, 1992). According to literature data, anionic surfactants

give toxic effects to various aquatic organisms at concentrations as

low as 0.0025 mg l-1 (Petterson et al., 2000). Since the 1970’s decade,

investigations on the toxic effects of the SDS have been conducted

with aquatic organisms,as fish (Barbieri et al., 2002, Rocha et al.,

2007), crustaceans (Lewis & Suprenant 1983, Singh and Kumar,

2002) and molluscs (Marin et al., 1994, Da Ros et al. ,1995, Hansen et

al., 1997) ans Daphanides (PAN 2005).The main adverse effects

already reported are cellular, histological and physiological damages,

which comprise alterations in the fish gill tissues (Abel 1974)

lysosomal disturbances and enzymatic inhibition or stimulation (Drewa

1988, Da Ros et al. ,1995), growth reduction (Hansen et al. ,1997) and

alteration of the cardiac activity. Bromnage and Fuchs (1976) reported

the acute toxicity of SDS to gold fish. The LC50 values of SDS were

estimated to be 5.25 mg/l for 24 hour & 48 hour and 5.17 mg/l for 72

hr & 92 hr exposures. Madhyastha et al., (1979) reported the LC50

values of SDS for Rasbora daniencnius is 6.30 mg/l regarding

molluscs. The detrimental effects of this detergent on stimulation of

Chlorella growth and photosynthesis without affecting respiration

(Petrea,1979) decrease in salinity tolerance of water hyacinth exposed

to SDS (Muramato, 1988), alteration in growth pattern of Chlorella and

Microcystis (Lipnitskaya et al., 1989), inhibition of filter feeding habits

of bivalves (Ostroumov, 2003), marine invertebrate embryos and

larvae (Bellas et al., 2005), morphological and physiological damage

of surfactant polluted sea spray on Pinus pinea & Pinus halepensis

(Nicolotti et al., 2005) inhibition of mussel suspension feeding

(Ostromouv & Widdows, 2006), are some of the studies pointing out

the toxicity of SDS.

10 Chapter 1

The toxic effects of detergents were reported by Abdel-Hamid

(1986) on phytoplankton, on bacteria and soil fungi.The effects of SDS

to algae have been studied by Singer and Tjeerdema,1993.At low

levels, growth of some algae species was stimulated, but at higher

levels, growth was inhibited. SDS also inhibited nitrogen fixation of a

cyanobacterium Gloeocapsa at 50 ppm, corresponding to 1.73 ×10-4

mol L-1. (Tozum-Calgan et al., 1994). These results emphasized the

need to control concentrations of surfactants entering the soil system.

Improper and/or excessive use of SDS could adversely affect

the survival and function of soil organisms, including earthworms,

bacteria, algae, and protozoa. SDS misuse or spills could also result

in the damage and even death of areas of organic crops given its non-

selective herbicidal properties.Nandlal et al., (2003) reported the effect

of synthetic detergent (surf excel) on germination parameters,

seedling growth and photosynthetic pigments in Mungbean.

Mungbean seeds failed to germinate at detergent levels beyond

0.17%; it showed marked decrease in rate and vigour index

SDS showed sensitivity to the microorganisms with symptoms

including acute toxicity (Mariani et al., 2006; Tozum Calgan et al.,

1994). The sewage sludge isolates A.johnsonii and Oligotropha

carboxidovorans showed nearer 59% and 20 % viability during the

treatment with 0.2 and 2 mg mgl-1 of SDS.Toxicity of anionic and

cationic surfactant to Acinetobacter Junii, (Phosphate accumulating

bacterium) in pure culture was studied by Mariani et al., 2006;

Hrenvoic et al., 2007. Results showed a 100 % inhibition of both CFU

and P-utake rate at a concentration of 10-3 molL-1 and higher .The

decreased P-accumulation as a result of surfactants represents one

important mechanism of surfactant toxicity against P-accumulating

Review of Literature 11

bacteria. In another study consisting of various taxa ,the EC 50 value

obtained for the bacterium Vibrio fischeri was 2.6 mgL-1 of SDS

,corresponding to 9.02 ×10-6 mol L -1 . (Tozum Calgan et al.,1994).

A reduction in counts of bacteria, fungi, actinomycetes,

phosphate solubilizers and nitrogen fixers were observed on exposure

to anionic surfactants. The dehydrogenase activity of the detergent

exposed soil was significantly decreased with increased

concentrations of detergents. On exposure to detergent germination

percentage of paddy was found to be decreasing. When the treatment

concentration increased from 1mg/l to 10 mg/l the germination

percentage drops below 50% (Asok et al., 2009).

Most cleaning products are used in water solutions that are

typically disposed down-the-drain, where they combine with other

wastes. These compounds can act on biological wastewater treatment

processes and cause problems in sewage aeration and treatment

facilities due to their high foaming, lower oxygenation potentials and

making death of waterborne organisms (Eichnorn et al., 2002).

Generally the presence of surfactants helps in the degradation

of polycyclic hydrocarbons. But the degradation of PAH was inhibited

by sodium dodecyl sulfate because this surfactant was preferred as a

growth substrate (A Tiehm, 1994). This point out the presence of this

detergent in the water bodies will indirectly lead to bioaccumulation of

other hydrocarbons. Similar inhibitory effects were seen when effect of

sodium dodecyl sulfate (SDS) and biosurfactants from Pseudomonas

aeruginosa UG2 was assessed during biodegradation of 13 of the 16

USEPA priority polycyclic aromatic hydrocarbons (PAH) in a wood-

preserving soil contaminated with creosote and pentacholorophenol

12 Chapter 1

for a period of at least 20 years (Deschênes et al., 1996). Assimilation

of sulfur from alkyl- and arylsulfonates by Clostridium sp. was

analyzed by Denger and Cook (1997).

1.2.2 SDS Degradation

Microbial degradation is the most efficient way of SDS

degradation (White et al., 1990). One of the several sets of

transformations that remove organic compounds from the environment

is that group of reactions mediated by organisms.Many

microorganisms were responsible for the degradation of organic

compounds.

Payne and co workers (1963a, 1963b, 1965) and Hsu (1965)

have done extensive work with the genus Pseudomonas aeruginosa

(C12 and C12B). These isolates were obtained from enriched soils

and cultured on media containing detergent compounds as sole

sources of carbon (Payne and Feisal, 1963b). Both isolate destroyed

the foaming capacity of dodecyl sulfate; but C12B could only grow on

dodecyl benzene sulfonate (DBS) and destroy the foaming capacity of

this surfactant. Sigoillot et al., 1988 isolated and characterized 26

surfactant degrading bacteria from marine environment.A laboratory

system was developed for modeling the colonization of slide-discs by

reverine epilithic biofilm capable of the bio-degradation of anionic

surfactants. Initial experiments using batch or fed-batch systems

produced biofilms yielding at least 10 times fewer viable bacteria and

slower surfactant bio degradation rates than occur in indigenous bio

film formed on slate-discs.

Review of Literature 13

Many investigators pointed out that continued growth and

biomass accumulation of the bacteria were coincidental. This indicates

that the bacteria are actually utilizing SDS as their sole carbon source.

(Di Cocia et al., 1994; Jimenez et al., 1991 and Sigillot et al., 1990).

Mechesi et al., 1994, identified the SDS-degrading bacterial

attachment to reverine sediment in response to the surfactant primary

degradation and identified as Pseudomonas sp

Past experience have demonstrated anionic surfactants

biodegradation are exclusively conducted by bacteria (Cain et al.,1981

and Juker et al., 1994). In another work, a surfactant-degrading

Klebsiella sp. is isolated from a lagoon contaminated with surfactant

(Amund et al., 1997). Other surfactant-degrading bacteria that have

been reported are Pseudomonas sp. strain C12B (Payne and Feisal,

1963; Thomas and White, 1989) Pseudomonas sp., strain DES1 (Hales

et al., 1982) Nocardia amarae (Bhatia and Singh, 1996), Comamonas

terrigena (Roig et al., 1998), Bacillus cereus (Singh et al., 1998),

Citrobacter braakii (Dhouib et al., 2003), and other bacterial genus such

as Acinetobacter, Pantoea, Vibrio, Flavobacterium, Pseudomonas,

Enterobacter, Actinomyces, Eschericia, Shigella, Proteus, Anaebena,

Corynebacterium and Staphylococcus (Gledhill, 1974; Abboud et al.,

2007; Ogbulie et al., 2008). Table1 gives a list of various micro

organisms capable of degrading SDS.

Singh et al., 1998 isolated Bacillus cereus, capable of degrading

SDS. B.cereus was isolated from a detergent polluted pond. This strain

showed growth with exceedingly high concentration of both anionic and

nonionic detergents. Increased aeration, and the continuous-feeding of

the microcosm with nutrient amended discharge from the sewage

14 Chapter 1

treatment plant separately gave rise to increased eplithic bacterial cell

numbers and SDS biodegradation rates (Lee et al., 1996).

Zhang et al., 1998 reported the aerobic biodegradation kinetics

of four anionic and nonionic surfactants of sub and supra-critical

micelle concentration. (CMCs). Primary degradation at supra- CMCs

level ( 2500 mg/l for SDS) and ultimate biodegradation at both sub and

supra CMCs were best described by first order kinetics. Increasing

surfactant concentration from sub to supra-CMC significantly

decreased primary biodegradation, ultimate biodegradation and foam

degradation.

Many researchers used activated sludge in order to isolate

bacteria that are able to degrade anionic surfactants (Schleheck et al.,

2000). Activated sludge provides elevated nutritional carbon and other

factors necessary for growth of a wide variety of microorganisms;

therefore it is an ideal source for isolation of degrading strains. (Cain,

1981).

Schleheck et al., (2000) isolated Proteobacterium strain DS-1

that could utilize commercial SDS in aerobic culture.The influence of

natural organic matter (NOM) on the absorption of SDS and bacteria

to riverine sediment surfaces and the effects of these interactions on

the rates of SDS biodegradation in riverine environments were

investigated by Marshall et al., 2000.

Pure culture degradation studies revealed that 66% of SDS

was degraded (Payne, 2004). Enrichment cultures from river

sediments yielded several nonfermentative dentrifying bacteria,

capable of anaerobic respiratory growth on the surfactant SDS as a

Review of Literature 15

sole source of carbon and energy. Four selected isolates grew

aerobically or anaerobically (with added nitrate) on primary alkyl

sulfates of chain length C6-C12 but not on the shorter homologues

(C2-C5), or DL- octan-2-yl sulfate, or sodium triethoxy sulphate.

Hosseini et al., 2007 studied the biodegradation of anionic

surfactants by isolated bacteria from activated sludge. In this survey, 2

different bacteria, Pseudomonas beteli and Acinetobacter Johnsoni

were isolated from Tehran municipal activated sludge. Cell aggregation

of Pseudomonas aeruginosa strain PAO1 was found as an energy

dependent stress response during growth with sodium dodecyl sulphate

(Klebensberger et al., 2006). The growth started with the formation of

macroscopic cell aggregates which consists of respiring cells

embedded in an exracellular matrix composed of acidic polysaccharides

and DNA. Damaged cells accumulated in these aggregates compared

to those cells that remained suspended.These isolates were able to

degrade 97.27 & 96.4% of the SDS after 10 days of growth

respectively. The highest peak of SDS degradation occurred during the

log phase of bacterial growth.

Ojo et al., 2009 studied the biodegradation of synthetic

detergents in waste water. The heterotrophic bacterial count from the

76 randomly collected effluent samples was 42.9x106 cfu/ml, while the

mean bacterial detergent-degrader population was 20.94x106 cfu/ml.

Klebsiella oxytoca was able to degrade approximately 80% of 2.0 gl-1

SDS after 4 days of incubation concomitant with increase in cellular

growth. (Shukor et al., 2009).

Kramer et al., 1980 have described the growth of Enterobacter

cloacae in 25% SDS. The bacteria appeared to tolerate SDS rather

16 Chapter 1

than metabolize it. The process was energy dependent, and cell lysis

occurred during stationary phase. Extreme detergent resistance may

be characteristic of the Genus Enterobacter.

Another survey of riverine bacteria by White et al., (1985)

showed that the ability to grow in the presence of the surfactant

sodium dodecyl sulfate (SDS) was widespread but the proportion of

SDS-resistant isolates was significantly greater at a polluted site, as

compared with clean source water

The capacities of epilithic and planktonic river bacterial

populations to degrade sodium dodecyl sulfate (SDS) in samples

taken at two times during 1987 from one clean and four polluted sites

in a South Wales river were estimated in die-away tests under

simulated environmental conditions (Anderson et al., 1990). There

was a relatively slow disappearance of SDS in die-away tests for both

planktonic and epilithic populations taken from the clean source site,

as compared with those taken from the downstream polluted sites, for

which the rate of biodegradation was accelerated, sometimes after an

apparent initial lag period.

Anderson et al., 1989 investigated the die-away kinetic analysis

of the capacity of epilithic and planktonic bacteria from clean and

polluted river water to biodegraded SDS.

Review of Literature 17

Table 1. Microorganisms in the biodegradation of SDS

Sl.No Microorganism Reference

1 Comamonas terrigena Matcham et al ., 1977

2 Pseudomonas spp.DESI and C12B Hales et al., 1985

3 Coryneform Bia White et al., 1987

4 Pseudomonas strain Stavskia et al .,1989

5 Pseudomonas BPS – D Marchesi et al .,1991

6 Pseudomonas strain Marchesi et al .,1994,

7 Vibrio, klebsiella, flavobacterium, Pseudomonas, E-coli, shigella, citrobacter

Amund et al., 1997

8 Bacillus cereus Sing et al., 1998

9 Pseudomonas C12B Jerabkova et al., 1999

10 Proteobacterium strains DS-1 Schleheck et al., 2000

11 Pseudomonas AE-A, AE-B, AE-D Ellis et al., 2001

12 Citrobacter braakii Dhouib et al., 2002

13 Rhodococcus ruber DSM 44541 Pogoreve et al., 2003

14 Pseudomonas strain Ellis et al.,2004

15 Pseudomonas aeruginosa strain PA01

Klebensberger et at., 2006

16 Klebsiella oxytoca,Acinetobacter calcoacetiacus,Serratia odorifera

Khleifat et al., 2006

17 Pseudomonas beteli, Acinetobacter Johnsoni

Hosseini et al., 2007

18 Acinetobacter calcoaceticus and Pantoea agglomerans

Abound et al., 2007

19 Enterobacter cloacae Khleifat et al., 2008

20 Stenotrophomonas maltophIlia Farzaneh et al., 2009

21 Enterobacter liquifasciens, klebsiella liquifasciens, klebsiella aerogenes,

Escherichia coli

Ojo et al., 2009

18 Chapter 1

1.2.3 Molecular studies based on SDS degradation

Loss of function at a rate higher than expected rate of mutation

are generally accepted properties that provide genetic evidence for

the presence of plasmid involved in conferring a particular function to

a cell. (Williams, 1978). Pseudomonas C12B, an SDS degrading

strain, harbors a plasmid coding for degradation of medium chain

length n-alkanes. (Kostal et al., 1998). The plasmid was designated as

pDEC.Its size was estimated at several hundred kb according to the

mobility in agarose gels. (Payne and Feisal, 1963; White et al., 1985).

Genes coding for degradation of halogenated aromatic compounds

are often plasmid-borne (Chakrabarty, 1972). Degradation of salicylic

acid or n-alkanes (C6, C18) which are so far reported to be carried on

one or more well characterized plasmids such as the SAL and OCT

plasmid respectively (Chakrabarty 1976).

Similarly antibiotic resistance is a common plasmid encoded

character. Plasmid encoded character often plays significant role in

bacterial adaptation to xenobiotics in the environment. (Kado and Liu,

1981). There have been reports on the involvement of bulky plasmids

in the biodegradation of e-caprolactam and related compounds

(Boronin et al., 1984; Esikova et al., 1990, 1993; Grishchenkov et al.,

1993). Ojo et al., 2009 isolated plasmid DNA from synthetic detergent

degraders in waste water from a tropical environment. The size of the

plasmid was 14-15 kbp.It was evident that the genetic information for

detergent-hydrocarbon utilization was not plasmid mediated since the

cured isolates grew on detergent supplemented medium after plasmid

was removed. Two alkane utilizing plasmid encoding genes have been

described, CAM-OCT and a plasmid in a strain of P.maltophilia (Lee et

Review of Literature 19

al., 1996). Pseudomonas putida MCM B-408 capable of utilizing e-

caprolactam (monomer of nylon-6) as the sole source of carbon and

nitrogen was found to harbor a single 32-kb plasmid (Kulkarni et al.,

1998). Analysis of alkyl sulphatase in parent and cured strain

confirmed that both enzymes are encoded by the chromosome. The

ability of Pseudomonas C12 B to utilize alkyl benzene sulphonate also

appears to be coded by the chromosome (Kostal et al.,

1998).Deshpande et al., 2001 reported the Plasmid-mediated

dimethoate degradation in Pseudomonas aeruginosa MCMB-427 and

located in 6.6 kbp plasmid.

Curing agents used by Deshpande et al., (2001) were

plumbagin (50 µg /ml), acridine orange (50 µg /ml)and ethidium

bromide (500 µg /ml).Curing of the plasmid by plumbagin or ethidium

bromide resulted in the loss of ability of MCMB-427 to degrade

dimethoate. Mitomycin C is the only mutagen that has been reported

to cure caprolactam degradative plasmids from Pseudomonas species

(Boronin et al., 1984). SDS at 0.1 g ml was not cured by Mitomycin C

(Kulkarni et al., 1998). Pseudomonas C12B was also cured by

mitomycin C resulting in the loss of pDEC plasmid. (Kostal et

al.,1998). Other Pseudomonas plasmids such as CAM, OT, SAL, NAH

and TOL were removed by treatment with Mitomycin C. (Williams,

1978).

1.2.4 Factors affecting the SDS biodegrdation

There were many reports to examine the impact of

environmental factors on the process of surfactant biodegradation in

seawater, namely: temperature, pH, aeration, concentration and the

presence of other sources. (Sales et al., 1998).

20 Chapter 1

a) Temperature

The dependence on temperature on the rate of the degradation

process is related to the metabolism of the micro-organisms

responsible for degradation. Many other mesophilic SDS degrading

bacteria reported in the literature required 300C for optimum SDS

degradation.Citrobacter braakii,Delftia acidovorans strain SPB1,

Pseudomonas strain C12B, Acinetobacter calcoaceticus and Pantoea

agglomerans required 30oC for optimal SDS degradation (Payne and

Feisal, 1963; Schulz et al., 2000; Dhouib et al., 2003; Abboud et al.,

2007) . Comamonas terrigena strain N3H showed optimum growth at

28O C for optimum SDS degradation (Roig et al., 1998). Marchesi et al.

(1997) also reported a lower temperature (25oC.) for the degradation

of SDS by Pseudomonas sp.

Shukor et al., 2009 have isolated an SDS-degrading Klebsiella

oxytoca from an SDS-polluted water sample from Malaysia. The effect

of temperature on the cellular growth of strain DRY14 on 2.0 g l -1 SDS

was studied at temperatures ranging from 10 to 50oC. Cellular growth

was increased as the temperature was increased from 10oC reaching

an optimum at 37oC before a dramatic decline in growth was seen at

higher temperatures.

Abboud et al., 2007 observed the 300C or 370C incubation

temperature for optimum SDS and LAS biodegradation by mixed cultures

of Acinetobacter calcoaceticus and Pantoea agglomerans. Temperature at

42 0C did not produce more than 10% of the SDS degradation where as in

the case of LAS 50% degradation observed at 420C. The optimum SDS

degradation temperature for Pseudomonas betelli and Acinetobacter

sp.was also 30oC (Hosseini et al., 2007).

Review of Literature 21

b) pH

The effect of pH on SDS degradation was studied by many

investigators. The preference to neutrality or near neutrality in terms of

optimal growth on SDS is shared by many other SDS-degrading

bacteria such as Pseudomonas strain C12B at pH 7.5 to 8.0 (Payne

and Feisal, 1963), Citrobacter braakii at pH 7.0 (Dhouib et al., 2003),

Comamonas terrigena strain at pH 7.4 (Roig et al., 1998) and,

Klebsiella oxytoca at pH 7.25 (Shukor et al., 2009). Other pHs like 7.5

and 6.5 gave intermediate effect on the degradation ability of LAS and

SDS while pH 5.5 produced the lowest extent of degradation (Shukor

et al., 2009). In another report, growth on SDS by a novel consortium

of Acinetobacter calcoaceticus and Pantoea agglomerans required pH

8.5 for efficient degradation. Growth was dramatically reduced at lower

or higher pHs than the optimum (Abboud et al., 2007).

In bioremediation works, a cheap source of pH controlling

chemical such as calcium carbonate can be added to soil during

bioremediation to achieve near neutrality in order to optimize

remediation .pH was studied using an overlapping buffer system

consisting of phosphate and carbonate (50 Mm) spanning the pH

range from 6.5 to 8.5. (Abboud et al., 2007).

c) SDS concentration

Many studies show that there is an inverse relationship

between SDS concentration and degradation. The growth of

Enterobacter cloalace in 25% SDS is described by Kramer et al.,

(1980).The bacteria appeared to tolerate SDS rather than metabolize

it. The process was energy dependent, and lysis occurred during

stationary phase. Klebsiella oxytoca degraded almost 80% of 2.0 g l-1

22 Chapter 1

SDS was after 4 days of incubation concomitant with increase in

cellular growth. Ten days of incubation were needed to degrade SDS

completely. Growth then enters plateau on 8 th days onwards; possibly

due to substrate depletion. Disappearance of bubbles during

incubation period also indicates the absence of SDS due to

degradation of this surfactant from the culture. (Shukor et al., 2009)

Khleifal et al., 2006 studied the biodegradation of sodium lauryl

ether sulfate by 2 bacterial isolates (Acinetobacter and klebsiella).

Effect of SLES concentration on the rate of biodegradation was

studied and observed that maximum SLES degradation occurs within

a concentration range of 1000-3000 ppm in 96 hours, whereas higher

concentration of SLES (5000, 7000 ppm) were degraded over a longer

incubation time (120-144 hours). This could be interpreted as the

higher concentration of SLES possibly leading to a decrease in

substrate bioavailability, which slows down its removal

Effect of substance mass on surfactants biodegradation was

observed by Abboud et al., 2007. A much higher degradation of SDS

at 4.0 g l-1 has been reported using a consortium of Acinetobacter

calcoaceticus and Pantoea agglomerans with complete degradation

occurring after approximately 5 days. The higher ranges of SDS

concentration (3000–8000 ppm) were degraded by the same mixed

culture but still an inverse relationship existed between the increase in

mass and the extent of degradation. Many scientists investigate the

toxicity effects of high surfactant amounts on bacterial growth. The

mixed culture showed better resistance to high surfactant mass toxicity

than either bacterial strain alone. Khleifal et al., 2006 studied the toxic

effect of SDS at mass range of 4000–8000 ppm to mixed bacterial

consortium (Acinetobacter calcoacetiacus, klebsiella oxytocan (A-K)

Review of Literature 23

and Serratia odorifera and Acinetobacter calcoacetiacus (A-S)) growing

in broth medium. The growth rate achieved for A-K and A-S were 0.26

h-1 and 0.21h-1 respectively. A mixed culture of activated sludge from a

municipal waste water treatment plant grown on higher SDS

concentrations (500-2500 mg/l) increased the microbial specific growth

rate in comparison to cultures, incubated with lower SDS

concentrations( in the range of 0.379 to 0.0567h-1) (zhang et al ., 1999).

The growth expressed as colony forming unit (CFU), was found

to be proportionally suppressed with increasing the amounts of

surfactant present in culture. Such mass intolerance was more

pronounced on the Acinetobacter than on Pantoea strain particularly,

when elevated amounts of SDS were encountered. (Abboud et al.,

2007).

The effect of three different rates of shaking (aeration) on

surfactants biodegradation was investigated. High and medium

shaking rates were also substantially effective on SDS breakdown

giving 100% degradation while the low shaking rates gave less than

10% degradation of this surfactant. (Abboud et al., 2007)

The effect of various carbon sources including glucose,

sucrose, maltose, mannitol and succinic acid were added separately

at a fixed concentration of 0.2% to broth medium before the

biodegradation. The addition of same carbon sources caused a slow

down in the degradation process of SDS (140 h). (Abboud et al.,

2007).

24 Chapter 1

1.2.5 Analysis of metabolic byproducts

Gas chromatography (GC) provided the convincing evidence

of synthetic degradation in sewage treatment plant.The detection of

unusual peaks in the GC profiles provided the scientific evidence of

inclusion of certain hydrocarbons in detergent formulation outside that

of industry specifications.The unusual peaks are attributable to

inclusion of certain chemical optical brightners.(Ojo et al., 2009 ).

Sawyer et al., 1956 have shown that gas chromatography provides a

very effective analytical method for determining intermediate in and

time required for, degradation in such system.

The biodegradation of SDS is initiated by sulfate release

catalyzed by alkyl sulfatase enzymes, to produce an alcohol which is

readily assimilated by the central metabolic pathways found in bacteria

(Anderson et al., 1999). Sulfate ester detergents like sodium dodecyl

sulphate are considered as readily biodegradable .Several SDS

degradation is started by an alkyl sulfatase which hydrolyses SDS to

sulfate and 1-dodecenol. This primary alcohol is oxidized to lauric acid

and further degraded by oxidation to acetyl COA residues. (Thomas

et al., 1989).

Studies with four different naturally occurring organisms have

shown that the three possible routes for primary biodegradation of

alkyl ethoxy sulphate surfactant (etherase, sulphatase, ω and β

oxidation). Thomas et al., 1989 studied the metabolic pathway for the

biodegradation of sodium dodecyl sulphate. Analysis of the extractable

lipids established the sequential production from SDS to 1- dodecanol,

dodecanal and dodecanoic acid.

Review of Literature 25

Metabolic pathway of alkyl sulphate is studied by Lijmbach and

Brinkuis, 1973. They observed that alkyl sulphatase hydrolyse

inorganic sulphate from its ester linkage with its alcohol, the later

being readily assimilated through metabolic pathway. Biodegradation

of alkyl polyethoxy sulphate, by microorganism is intiated by the

breakdown of related nonionic alkyl ethoxy alcohol surfactants by

cleavage of the alkyl ether bond. (Patterson et al., 1970; Tobin et al.,

1976; Williams et al., 1966). The liberated polyethylene glycol moieties

are subsequently degrade more slowly.

A number of different thin layer chromatographic systems on

silica gel and cellulose plates were employed to separate and

identify35 S labeled metabolites of SDTES. Metabolites have been

identified as mono-di and tri ethylene glycol monosulphate (major

metabolites) and acetic acid -2-ethoxy sulphate and acetic acid

diethoxy sulphate. Major metabolites were produced by rupture of one

or other ether linkage present in the surfactant molecule by a single

etherase enzyme. Acetic acid -2-ethoxy sulphate and acetic acid

dietoxy sulphate were formed by the oxidation of the free alcohol

groups of di and triethylene glycol monosulphates respectively and

increased in amount during the stationary phase of growth. Inorganic35

S-sulphate also appeared from the parent surfactant by the action of

sulphatase enzyme. (Hales et al., 1982). Primary degradation of

SDTES by Pseudomonas sp.DES1 involved in the action of one or

more sulfatase and one or more ether cleaving enzymes. By analogy

with alkyl sulphatase of Pseudomonas sp.C12B (Dodgson, 1981), the

sulphatase may expected to liberate the free alcohol and inorganic

sulphate. In the case of SDTES the products of etherase are alcohol,

aldehyde or carboxylic acid.

26 Chapter 1

Pathway of SDTES was identified and the intermediate could

have produced by the subterminal oxidation and cleavage between

C-11, C-10 or C-9 and C-8 of the dodecyl chain followed by beta

oxidation (Hales et al., 1986). Normally beta oxidation route appears

to be used only when other mechanisms are unavailable. Example for

beta oxidation were the bacterial degradation of alkyl benzene

sulphonate (Cain 1981) and mammalanian degradation of alkyl

sulphate (Denner et al, 1969; Maggs et al., 1982) alkyl ethoxy

sulphates (Taylor et al., 1978). Steber and Wierich (1985) observed

the combination of omega /beta oxidation and ether cleavage during

the degradation of nonionic alkyl ethoxylate by mixed sewage culture.

In order to obtain a detailed insight into the environmental fate

of complex surfactant mixtures a sensitive and specific analytical

technique is required to enable the detection and identification of

individual components and metabolites. Eichhorn and Knepper., 2000

used LC-MS for separating and identifying the components.

Extensive investigation of anionic surfactant,LAS with respect

to its degradation behaviour and metabolic pathway under aerobic

conditions that occur in sewage treatment plants was carried out by

Schoberl.,1989.Sulfophenyl carboxylates (SPCs) have been identified

in the environment as primary degradation intermediates of LAS.

(Dicorcia and Samperi., 1994).

The intial step in the aerobic metabolism of a Cn–LAS is the

omega oxidation of the terminal methyl group of the alkyl chain,

releasing a Cn SPC.This metabolite was further breakdown by beta

oxidation leading to the short chain of SPC. (Eichhorn and Knepper.,

2000). Analytical detection of SPC with non specific detectors was

Review of Literature 27

difficult due to the lack of commercial available standard. Using mass

selective detector allowing identifying the compounds via its molecular

ion, and additionally by one or more characteristic fragments.

1.2.6 Role of enzymes on SDS degradation

Microorganisms use a range of sulfatases of widely varying

substrate specificities to use sulfate esters as carbon and/or sulfur

source. At least three mechanically distinct groups of sulfatases exist:

arylsulfatases, the best studied group (hereby assigned to group I),

are predominantly eukaryotic. They are characterized by an active-site

serine or cysteine post translational modified to formylglycine that

mediates the cleavage of the CO-S bond of sulfate esters, producing

inorganic sulfate and the corresponding alcohol The Fe(II)

α-ketoglutarate-dependent dioxygenase super family of enzymes

constitutes a second group (group II) of sulfatases. These enzymes

oxidatively cleave sulfate esters into inorganic sulfate and the

corresponding aldehyde and require α-ketoglutarate as a cosubstrate

.Third group of alkyl sulphatase is microbial origin.

Alkyl sulphates are among most environmentaly acceptable

synthetic surfactants because they are readilly biodegraded by

environmental and sewage baceria.Biodegradation is intiated by alkyl

sulphatase enzymes. Sulphate esters make up a large propotition of

sulfur that is found in aerobic soils, and so it is not surprising that many

soil microorganisms have evolved enzymes that catalyse the hydrolysis

of theses compounds ,either to release sulfate moitey as a sulfur source

for growth. or as the first step in their mineralization. (Dodgson et al.,

1982; Dodgson and White,1983; White et al .,1985 ).

28 Chapter 1

Sulphatase catalyse hydrolytic cleavage of the sulphate ester

bond by liberating inorganic sulphate and corresponding alcohol.They

are present in wide variety of species ,ranging from bacteria to

humans. (Kertesz., 1999; Roy., 1971). In contrast to the role of human

sulphatase, which are involved in the sulphation of sulfated

glycolipids, the primary role of bacterial alkyl sulfatase is the

assimilation of sulfur(Denger et al., 1997; Dodgson et al.,1982) or in

the provision of carbon and energy sources for cell growth. (Fitzgerald

et al., 1977).

Strains that are able to grow with alkyl sulphatases such as

SDS as the source of carbon are widespread in the environment,even

in samples isolated from uncontaminated sites. (White et al., 1983)

Alkyl sulphatases have mostly been found in gram negative

bacteria; the only exceptions to date are strains of Bacillus cereus

(singh et al., 1998) and coryneform strain BI a (white et al., 1987).

Alkyl sulphatase activity has also been found in Psedomonas

aeruginosa, Aerobacter aerogenes, Comamonas terrigena and

Pseudomonas putida .Most of the work on alkyl sulfatases was carried

out with gram negative bacteria, Pseudomonas sp strain C12B

(NC1MB 11753 = ATCC 43648) and Comamonas terrigena (NCIMB

8193) (cloves et al., 1980.

White et al., 1992 studied the biodegradation of short-chain

alkyl sulphates by coryneform species .Growth of the organism was

accompanied by disappearance of butyl-1-sulphate. Non-denaturing

polyacrylamide gel electrophoresis of extracts of cells grown on butyl-

1-sulphate, followed by incubation of gels in butyl-1-sulphate and

Review of Literature 29

precipitation of liberated SO4-2 as BaSO4 revealed a single white band

of alkylsulphatase activity.

A strain of Bacillus cereus isolated from a detergent polluted

pond rapidly degraded SDS to dodecan-1-ol by the enzyme

alkylsulfatase (Singh et al., 1998). Marches et al., 1994 studied the

SDS-degrading bacteria attached to reverine sediment in response to

the surfactant or its primary biodegradation. Three of the Pseudomonas

strains were chosen for their known ability to express alkylsulphatse

enzymes capable of hydrolyzing SDS. The alkylsulphatase phenotypes

were confirmed by gel zymography of all extracts.

Kahnert and Kertesz (2000) characterized a sulfur-regulated

oxygenative alkylsulfatase from Pseudomonas putida S-313. Ellis et al.,

2002 isolated novel alkylsulphatase capable of degrading branched

primary alkylsulphate surfactant 2-butyloctyl sulphate.

Pogorevc and Faber (2003) isolated and purified

alkylsulphatases from the whole cells of Rhodococcus ruber DSM

44541 capable of hydrolyzing ()-2-octyl sulphate in a stereo- and

enantiospecific fashion .When grown on complex medium, the cells

produced two sec- alkylsulphatases and (at least) one primary

alkylsulphatase in the absence of an inducer, such as sec- alkyl

sulphate or a sec- alcohol.

The alkylsuphatases of Pseudomonas C12 B appears to have

a periplasmic localization in the cell (Fitzgerald et al., 1975). Bacteria

containing the alkylsulfatases that initiate SDS degradation were also

widespread. The polluted site yielded significantly more alkylsulfatase-

containing strains than the clean site, and this difference was due to a

30 Chapter 1

greater number of strains at the polluted site bearing constitutive

rather than inducible enzymes. The bacteria involved in surfactant

biodegradation are often capable of producing a multiplicity of

alkylsulphatases.

Thomas et al. (1989) studied the location of long chain alkyl

sulphatase in the bacterial cell. A location for long-chain

alkylsulphatases in the outer cell wall is rational because it eliminates

the threat to the cell while still allowing assimilation of the carbon as

the long-chain (non-surfactant) alcohol. There was no activity inside

the cells. In contrast, the short-chain (C1-C4) alkylsulphatase in a

coryneform isolated for its ability to grow on butyl-1-sulphate was

located entirely in the cytoplasm. The butyl sulphatase was apparently

associated with granules of poly-P-hydroxybutyric acid. The different

locations for the long- and short-chain alkylsulphatases may be related

to the relative potential toxicities of their ester substrates.

Bacillus cereus metabolized SDS into 1-dodecanol alkyl

sulphatase within 8 hours of incuation.The appearance of enzyme

activity in the SDS supplemented medium started after 1 hour of

incubation and last up to 8 hours. Therefore the enzyme sulphatase is

inducible in nature because the appearance of enzyme activity was

not instant. Rather it appeared after 1 h of incubation in SDS-

supplemented medium and thereafter there was abrupt and linear

increase lasting up to 8h. Addition of chloramphenicol (25 mg/ml) to

SDS-supplemented cells did not show alkylsulphatase activity

suggesting that synthesis of enzyme upon SDS addition is required to

synthesis of enzyme upon SDS addition is required to manifest

activity. (Singh et al., 1998).

Review of Literature 31

Pseudomonas ATCC 19151 and Pseudomonas BPS-D contain

inducible alkylsulphatase enzyme, it converts SDS into1-dodecanol

which is responsible for the attachment of bacteria to the riverine

sediment.The period of maximum attachment coincided with that of

maximum production of dodecanol .The alcohol was further

metabolized and its concentration declined, the bacteria detached

from the sediment surface. (Marchesi et al., 1994).

The maximum rate of SDS hydrolysis and dodecanol

accumulation correspond to the point at which bacterial surface has

become most hydrophobic and which the residual SDS concentration

had reached zero, the surface hydrophobic index returned to

zero.Increase in hydrophobicity is due to the conversion of SDS to

dodecanol close to the bacterial cell surface by primary alkyl

sulphatase, which is located in the periplasm. (Thomas et al., 1986)

In addition, the incidence of strains containing multiple

alkylsulfatases was much higher at the polluted than at the clean site.

(White et al., 1985). The bacteria involved in the biodegradation of

SDS are often capable of producing a multiplicity of alkylsulphatases.

Thus Pseudomonas C12B can produce five such enzymes (Dodgson

et al., 1974 and Pseudomonas DESI four (Hales, 1981). Of the five

alkylsulphatases produced by Pseudomonas C12B, two (designated

P1 and P2) are active towards primary alkyl sulphates, whereas the

other three (SI, S2 and S3) act on secondary alkyl sulphates

(Matcham et al., 1977a; Dodgson & White, 1983). P1 is constitutive

and P2 is Inducible in nature. The latter enzymes exhibit positional and

stereo-specificity, SI being active towards D-alk-2-yl sulphates, S2

towards L-alk-2-yl sulphates and S3 towards symmetrical and near

symmetrical secondary alkyl sulphates.All three enzymes surprisingly

32 Chapter 1

operate by rupture of the C-O bond of the ester sulphate linkage, and

this is accompanied by inversion of configuration (Bartholomew et al.,

1978; Shaw et al., 1980).

On some occasions, gels incubated with SDTES and Bacl2

contained a faint, transient band of precipitated BaSO4 in a position

corresponding to the P2 enzyme. On other occasions, the band did not

appear at all even though P2 was present, suggesting that the later

enzyme may not responsible for induction of the sulphatase activity.

Cell extracts from aerobic or anaerobic cultures of the most sulfatase

active towards octylsulphate but inactive towards propyl, butyl and

pentenyl sulfates and seconday alkyl sulphates (Dodgson et al., 1984)

The most striking difference between the two Pseudomonas

C12B enzymes is that relating to their modes of action. The P1

enzyme ruptures the O-S bond of the C-0-S linkage and hence would

be regarded as exhibiting the normal behaviour for a hydrolytic

enzyme active towards esters of the type alcohol--acid. (Bateman et

al., 1986). Dodgson et al. (1982) have previously drawn attention to

the fact that of six other known classes of hydrolytic enzymes active

towards this type of ester, all operate by scission of the 0-acid bond. In

sharp contrast with the P1 enzyme, the P2 enzyme attacks the C

(alcohol)-O bond, in common with the mechanisms of action of the

secondary alkylsulphatases of Pseudomonas C12B that have so far

been studied in detail (the SI and S3 enzymes). These C-0-bond

splitters are thus emerging as atypical of sulphatases in particular and

hydrolytic enzymes in general.

Alkyl sulphates phenotypes were confirmed by gel zymography.

Marchesi, 1994 studied the location of alkyl sulphatase by gel

Review of Literature 33

zymography by the generation of white bands of insoluble dodecanol in

the gel. The same method was used by Hales et al (1985) to study the

alkyl sulphatase activity .Sulphatase activity towards SDTES was

detected by incubating the gels at 300C in a solution containing 10 m M

–SDTES and 20Mm BaCl2 in 20 Mm tris /HCl, pH 7.8. Although the

liberated alcohol was not sufficiently insoluble to form a visible

precipitate, white bands were formed because the presence of Ba 2+

ions led to the precipitation of liberated SO4 2-

Extracts of Pseudomonas sp. strain C12 B cells grown on SDS

supplemented basal salt medium in batch culture produced P1 and P2

bands when gels were incubated with SDS.When the same cell

extracts were separated on gels and gels were with 2- butyl octyl

sulphate instead of SDS, no alkyl sulphatase bands were

observed.(Ellis et al ., 2001)

Stoichiometric liberation of butanol and SO42- from butyl

1-sulphate confirmed the true hydrolytic nature of reaction catalysed.

(White and Russel., 1992). Stoichiometric incorporation of 18 O from

H218O into SO4

2- during the degradation of butyl -1- sulphate by pure

enzyme showed that fission of the C-O-S ester linkage occurred at the

O-S bond.The O-S mechanism is common in all aryl sulphatase

(Dodgson et al., 1982)c,the P1 alkylsulphatase of Pseudomonas C12

B (Cloves et al., 1988) the 2,4 dichloro phenoxy ethyl sulphate in

P.putida (Lillis et al.,1983) and the D-lactate -2-sulphate sulphatase in

P. syringae (Crescenzi et al., 1984).

Primary alkylsulphohydrolase of the soil bacterium Pseudomonas

C12 B was purified to homogeneity by column chromatography on

DEAE- cellulose Sephadex G-100 and butyl agarose (Cloves et al.,

34 Chapter 1

1980a). The results showed the presence of a hydrophobic site on the

enzyme capable of accommodating an alkyl chain of considerable

length.

Matts et al., 1994 described the purification and characteristics

of the novel short chain alkyl sulphatase from strain Corenybacterium

B1a and compared with its properties with other alkyl sulphatase.

Butyl –sulphate was routinely employed as a substrate for the assay of

alkyl sulphatase activity.Alkyl sulphatase for short chain alkyl suphate

was measured by BaCl2 /gelatin method. (Dodgson, 1961). Purification

was done with streptomycin sulphate and DEAE cellulose column

chromatography and hydrophobic column chromatography.Purification

factor achieved from crude extract to final preparation was in the

range 65 to 75 fold. SDS/E of denatured protein yielded a single band

corresponding to 77.6 kDa.

Pogorev (2003) was also tried to purify secondary alkyl

sulphatase from Rhodococcus rubber by using DEAE –cellulose

batch, phenyl sepharose Q6, Blue sepharose and superdex 200.

These purification sequence allowed to purify alkyl sulphatase RS2

119 fold from crude cell extract in reproducible way.SDS/PAGE

revealed the presence of a single band at about 43 KDa for RS2.

Highly enantioselective stereo-inverting sec- alkylsulphatase

activity of hyperthermophilic Archae was noted by Wallner et al., 2005.

Wallner et al., 2005 also noted similar highly enantioselective sec-alkyl

sulfatase activity of the marine planctomycete Rhodospirulla baltica.

Gadler and Faber (2007) have reviewed the various scopes

and dimensions of alkylsulphatases, which can be employed for the

Review of Literature 35

enantio-convergent transformation of racemic sulfate esters into a

single stereoisomeric secondary alcohol, with a theoretical yield of

100%.

1.2.7 Effect of starvation on SDS biodegradation

One of the major environmental factors influencing microbial

physiology and cell growth is nutrient availability. Most natural

environments are characterized by low bioavailability of nutrients.

(Morita,1988). There are many papers reported the effect of

starvation on morphology (Morita 1985), alternation of the cell surface

structures (Tuomanen et al.,1988) photosynthesis and genetic

regulation (Nystrom et al., 1989), formation of cross protection against

heavy metals such as Cd2- (Feriane et al., 1995).

Truex et al., 1992 studied the effect of starvation on quinoiline

degradation by Pseudomonas cepacia finding that long time starvation

(60-80 days) leads to more efficient conversion of quinoline to

degradation products with quinoline concentrations of 39 and 155

Mm.However, long time starved cell required 3-5 × longer time for

induction of quinoline degradation in comparison with short time

starved cells.It was evident that starvation positively affected the

biotransformation potential of Commomonas terrigena cells towards

DHSS. (Roig et al., 1998)

1.2.8. Treatment of SDS wastewater

The problem of considerable contamination of the aqueous

environment with organic pollutants requires the development of quick

and simple methods for the removal, separation and determination of

these compounds. The main classes of organic compounds that most

36 Chapter 1

of the industries used and discharge into the effluents are surfactants,

phenols and dye. All these compounds are troublesome contaminants

which pose not only to toxicity and health hazards but also hamper the

environmental treatment processes. Surfactants are generally

removed by chemical precipitation foam separation ultrafiltration,

physical adsorption and biological method.

a) Physical adsorption

Removal of organic pollutants by adsorption on active carbon is

used successively for a long time (Wheeler et al., 1959). Literature

study indicate several types of adsorbent for removal of surfactants

from waste water. (Schewnger et al., 1970; Waymen, 1984;

Krishnamurthy, 1993) The adsorption of alkyl sulphate on various

adsorbents such as alumina (Dick et al., 1971) and activated carbon

(Sing et al., 1974) were studied and they showed 80 and 89%

removal. The sodium form of type A Zeolite (Savitsky, et al., 1981)

was also tested for alkyl sulphate removal, but in this case, the

efficiency was not good. The main disadvantage of these adsorbing

materials is that they are not cost-effective. A very low-cost scrap

rubber in the form of granules had been selected to remove AS from

the water environment, and the efficiency of the rubber granules was

evaluated. (Sarkar et al., 2000). Adsorption of nonionic surfactant from

its aqueous solution into commercial rubber was reported by Shalaby

and El-Feky (1999). Purakayastha et al., (2005) studied the surfactant

removal efficiency of four adsorbents–granular activated charcoal,

waste tire rubber granules, wood charcoal and silica gel. Waste tire

rubber granules exhibited maximum removal efficiency.

Review of Literature 37

b) Biological treatment method

Microbial system is the potent tool to deal with environmental

pollutants. Biotechnology for hazardous waste management involves

the development of systems that use biological catalysts to detoxify

environmental pollutants. Biotechnology offered a number of strategies

for waste treatment. (Nakamura and Sawada, 2000).

i) Improvement of existing processes by application of

adapted or engineered microbial strain.

ii) Construction of bioreactors containing immobilized

biocatalysts or biofilim of suitable organism in the

detoxification of environmental chemicals.

iii) Development of biosensors to detect trace amounts of

toxic organics or heavy metals.

Alginate is most widely used as a polysaccharide matrix for the

immobilization of viable cells. (Kierstan et al., 1985). Polyvinyl alcohol

(PVA) is no-toxic to organisms and can be cheaply produced in an

industrial scale. (Shindo & Kamimura 1990). Wu & Wisecarver 1992

used Calcium alginate modified PVA beads for immobilization of cells

so as to prevent the agglomeration of the beads and improve their gas

permeability. Thomas and White, 1991. described the development of

an immobilized biocatalyst for the surfactants treatments which

involved the entrapment of a versatile surfactant-degrading bacterium

Pseudomonas C12 B in a polyacrylamide gels.

Roig et al., 1997 studied the sorption isotherms and kinetics in

the primary biodegradation of anionic surfactants by immobilized

38 Chapter 1

bacteria, Comamonas terrigena N3H. Comamonas terrigena N3H was

immobilized by covalent linking on silanized inorganic supports and by

physical entrapment of cells within calcium alginate beads and

reticulate polyurethane foam.The surfactant degrading biocatalyst

Pseudomonas C12 B was immobilized by covalent linking on silanized

inorganic supports and by physical entrapment of cells within

reticulate polyurethane foam. Both immobilized biocatalysts have been

shown to be appropriate for the primary biodegradation of SDS. (Roig

et al., 1998).

Jerabkova et al.,1999 investigated the biodegradation of SDS,

by bioreactor with immobilized cells of Pseudomonas C12 B. Cells

were immobilized by adsorption on porous glass beads with either

unmodified or silanized surface. Both types showed equivalent

efficiency to remove SDS (85%). The surfactant degrading bacterium

Pseudomonas C12 B in immobilized state has been used in several

investigations for removing anionic surfactants (Ellis et al., 2002).

Farzaneh et al., 2009 studied the efficiency of immobilized cells

(stenotrophomonas maltophilia) on glass beads for the removal of

SDS.

Roig et al., 1997 studied the treatment of surfactant with the

plug flow recirculation bioreactor packed with polyurethane reticulated

foam particles with or without Pseudomonas C12 B.Four cycles (one

day) was required to complete the biodegradation.Surfactant removal

capacity was maintained in 5-6 days of continuous operation. Roig et

al., 1998 also studied the treatment of surfactant with the recirculation

bioreactor packed with polyurethane reticulated foam particles with or

without the Comamonas terrigena N3H .The above same result

observed.

Review of Literature 39

Feikenhauer et al., 2002 Investigated that high strength

wastewater of some industries contains high concentrations of

surfactants and readily biodegradable compounds like starch and

other carbohydrates. Wastewater of this type found in the textile wet

processing industry. High concentration of surfactants has been

shown to inhibit the digestion process.

Dhouib et al., 2002, developed a high performance process for

the treatment of anionic surfactant containing waste water. A

Citrobacter braakii was used for the continuous degradation of SLES

synthetic medium and cosmetic industry waste water. By testing

several dilution rates it was concluded that Citrobacter braakii was

able to degrade up to 0.065 g l -1h-1.

Mortazavi et al., 2008 investigated the removal of SDS from

synthetic wastewater in an Intermittent Cycle Extended Aeration

System (ICEAS). The reactor was made up of polyethoxyglass tank

which is equipped with diffuser air.The removal of SDS was more than

98%.Luis (2004) also reported that the removal efficiency of SDS in

ICEAS is more than 95%. Ebrahimi et al., 2006 studied the removal

efficiency of COD in conventional active sludge system was 87 %. In

addition in a study of SDS biodegradation by the Acinetobacter

isolated from active sludge, the removal efficiency was 96.4 %.

(Hosseini et al., 2007)