Chapter-1

INTRODUCTION

1.1 INTRODUCTION

Xenobiotics are those compounds that are alien to a living

individual and have a propensity to accumulate in the environment. Both

natural and anthopogenic activities result in accumulation of wide range

of xenobiotic compounds in the environment, and thus cause a global

concern (Gienfrada & Rao, 2008). Surfactants, one of the major

xenobiotic compounds used today, are widely used in household cleaning

detergents, personal care products, textiles, paints, polymers, pesticide

formulations, pharmaceuticals, mining, oil recovery and the pulp and

paper industries.

Anionic surfactants are the earliest and the most common surfactants

that are not only used as detergents, but also widely applied in many fields of

technology and research. They are usually considered to be the “workhorse”

in the world of detergent. They have been successfully employed to enhance

the efficiency of the active ingredients in pharmaceutical and agricultural

formulations, cosmetics, biotechnological compounds, and in several

industrial processes. One of the major xenobiotic anionic surfactant that has

large scale industrial application and thus broad environmental release is

linear alkylbenzene sulphonates (LAS).

LAS was introduced in 1965 as a biodegradable alternative to non

biodegradable branched chain alkylbenzene sulphonate (ABS) and since

has become the most widely used anionic surfactant in commercial

detergent formulations. LAS frequently used as the sodium salts, finds its

application as the sole surfactant or in combination with other anionic,

nonionic or cationic surfactants in a detergent formulation.

2 Chapter 1

LAS are nonvolatile compounds produced by sulphonation of

linear alkylbenzene. The surfactant molecule is composed of a

hydrophobic moiety, an alkyl chain, and a hydrophilic part composed

of a benzene ring and a sulphonate group. The linear alkyl chain has

typically 10 to 13 carbon units, approximately in the following mole ratio

C10

:C11

:C12

:C13

=13:30:33:24, an average carbon number near 11.6 and a

content of the most hydrophobic 2-phenyl isomers in the 18-29% range

(Valtorta et al., 2000). Commercial products are always mixtures of

homologues of different alkyl chain lengths (C10-C13 or C14) and isomers

differing in the phenyl ring positions (2 to 5 phenyl). The ratio of the

various homologues and isomers, representing different alkyl chain

lengths and aromatic ring positions along the linear alkyl chains, is

relatively constant across the various household applications. This

constant ratio is unique and does not apply to the other major surfactants.

Therefore, the present assessment adopted a category approach, i.e., it

considered the fate and effects of the LAS mixture as described above

rather than of each isomer and homologue separately. However,

fingerprints in the different environmental compartments are reported.

LAS have the following structure:

Figure 1: Structure of LAS

H3C (CH3)x CH CH2 (CH2)y CH3

SO3 Na(x+y = 6-9)

+-

Introduction 3

Irrespective of its initial usage, the vast majority of these surfactants

eventually reach water ways, either directly or via sewage treatment works.

The routes by which LAS enter the environment vary among countries, but

the main route is via discharge from sewage treatment works. When

wastewater treatment facilities are absent or inadequate, sewage may be

discharged directly into rivers, lakes, and the sea. Another route of entry of

LAS to the environment is by the spreading of sewage sludge on agricultural

land. LAS thus entering the environment are gradually removed by a

combination of adsorption and biodegradation.

LAS are the most important anionic surfactants that reach the

waste water treatment plants (WWTP) unchanged. Studies conducted on

the fate of LAS during waste water treatment have indicated that they are

efficiently removed by physical, chemical and biological processes. Apart

from precipitation and adsorption onto suspended solids, which can range

from 30 to 70% (Berna et al., 1989) of the initial contents, microbial

degradation generally accounts for the major elimination route resulting in

an overall reduction of 95-99.5% of the LAS load in activated sludge

systems (Painter & Zabel, 1989). But due to its molecular characteristics,

LAS tends to adsorbed on to sediment particles and hence may escape

WWTP without degradation. The presence of LAS in sewage sludge

leaving the WWTP is dependent upon the type of treatment the sludge

undergoes. Sewage sludge that is aerobically digested may contain LAS

concentrations of about 100-500 mg/kg dry weight, considerably lower

than those found in anaerobically treated sludge (5,000-15,000 mg/kg dry

weight). Therefore, the extent of LAS contamination of sewage is

4 Chapter 1

generally dependent upon the individual WWTP and the method of sludge

digestion employed (Cirelli et al., 2008).

In contrast to this, if domestic wastewater is discharged directly in

to natural water streams because of deficient treatment facilities, the

surfactant levels in water can be considerably higher. This causes

particular concern since under these circumstances aquatic organisms are

exposed to considerable levels of surfactants, which exhibit relatively

high toxicities (Schoberl, 1997). In untreated wastewater also the LAS

concentration may be reduced due to adsorption on to sediments as well

as by biodegradation through endogenous bacterial communities present

in the stream, with slower kinetics compared to WWTP (Eichhorn et al.,

2002). LAS may be transported to long distances from the source of its

contamination due to its high water solubility. Discharging of polluted

rivers on to estuaries and subsequently into the sea contributes to the

contamination of LAS in coastal waters.

Surfactants enter the hydrosphere not only in relation with the use

of detergents but also due to the use of these substances in industry, in

mining, refining, and transporting of various raw materials. Synthetic

surfactants easily form complexes with other compounds and are rapidly

adsorbed at interfaces, which hampers their determination by analytical

methods (Gonzalez- Mazo & Gomez- Parra, 1996) and can lead to

underestimating the determined values compared to the real pollution of

the aquatic ecosystem. The presence of surfactants may be important for

the fate of pesticides at effluent-irrigated sites because they may increase

the apparent solubility of hydrophobic pesticides (Vigon & Rubin, 1999).

Waste streams from the rinsing of mixing equipment at shampoo

Introduction 5

formulation factories enter sewage systems and adds to the LAS

concentration there. Under these conditions, high concentrations of

surfactants may persist long enough to generate undesirable foam in

sewage treatment plants. Excessive foaming in sewage treatment plants

causes operational difficulties and may also lead to health hazards in the

form of air born pathogens carried on wind blown foams.

A possible solution to eliminate the high concentration of LAS

from the waste water stream is to treat it with the surfactant degrading

bacteria at the inlet to the WWTP. LAS catabolism confronts the

microorganisms - generally bacteria - involved in it with the task of being

able to convert a very wide variety of structures, namely the aliphatic

chain with a non-uniform number of carbon atoms, the aromatic ring,

which is, in addition, distributed randomly over the alkyl chain, and

cleavage of the carbon-sulphur bond on the benzene ring. Since all the

organisms involved in LAS degradation do not have full enzymatic

potential for conversion of all the structures mentioned, different species

or genera of bacteria are frequently involved in catabolism of the LAS

molecule (Schoberl & Marl, 1989).

Cain et al., (1972) detected five different types of reaction

a) ω-oxidation with subsequent β-oxidation of the aliphatic chain, but

no desulphonation and no degradation of the benzene ring.

b) ω-oxidation and subsequent β-oxidation with simultaneous

desulphonation and ring splitting.

c) As in b), but accompanied by reductive desulphonation. In this way,

phenylalkanoate is produced instead of p-hydroxyphenylalkanoate.

6 Chapter 1

d) α-oxidation with subsequent β-oxidation and ring desulphonation

without attacking on the ring itself.

e) If the alkyl chain has a low number of carbons (< 4), biodegradation

begins on the benzene ring, either by the hydrolytic route or in some

cases by reductive ring desulphonation.

The method of cell immobilisation seems to be promising in the

development of the biotechnology for the removal of various xenobiotic

bearing effluents (Murugesan, 2003) Recently, immobilised microbial cells

have frequently been applied for bioremediation and biosynthetic processes

(Sasaki et al., 2007). Physical entrapment of cells inside polymer matrix is

one of the most widely used and straight forward techniques for cellular

immobilisation, since it does not depend significantly on cellular properties.

Immobilisation of microbial cultures has proved to be advantageous in

municipal and industrial sewage treatment because of high degradation

efficiency and good operational stability.

A possible means to enhance the availability of contaminants is

application of surfactants. Now a days chemical surfactants are used for

this purpose. However a prerequisite for surfactant–enhanced

biodegradation is that the degradative microorganism should not be

adversely affected by the surfactant. Bacteria using detergents for growth

face an additional challenge. They have to invest much of their energy

into protection, while taking an increased risk of damage because they

have to take up the toxic detergents to metabolise them. So it is very

important to find bacteria with both desired biodegradability and ability to

thrive in the presence of surfactants.

Introduction 7

Biosurfactant(s) spontaneous release and function are often related

to hydrocarbon uptake. Therefore, they are predominantly synthesized by

hydrocarbon degrading microorganisms. Some biosurfactants, however,

have been reported to be produced on water-soluble compounds, such as

glucose, sucrose, glycerol or ethanol (Heyd et al., 2008). It was reported

that most of the surfactant resistant bacteria are capable of producing

biosurfactants (Plante et al., 2008).

During the past few years, biosurfactant production by various

microorganisms has been studied extensively. Also various aspects of

biosurfactants, such as their biomedical and therapeutic properties (Singh &

Cameotra, 2004; Rodrigues, 2006), natural roles (Ron & Rosenberg, 2001),

production on cheap alternative substrates (Maneerat, 2005; Gautam &

Tyagi, 2005) and commercial potential (Desai & Banat, 1997) have been

recently reviewed. Most of the work on biosurfactant applications has been

focusing on bioremediation of pollutants (Mulligan, 2005) and microbial

enhanced oil recovery (Banat, 1995).

The reason for their popularity as high value microbial products is

primarily because of their specific action, low toxicity, higher

biodegradability, effectiveness at extremes of temperature, pH, salinity,

widespread applicability, and their unique structures which provide

properties that classical surfactants may lack (Desai & Banat, 1997,

Kosaric, 1992). Unlike chemical surfactants, which are mostly derived

from petroleum feedstock, these molecules can be produced by microbial

fermentation processes using cheaper agro-based substrates and waste

materials. Another reason for the search of biological surfactant is the

depletion of oil resources required for the production of synthetic

8 Chapter 1

surfactants. This makes microbial surfactants even more promising

(Turkovskaya et al., 2001).

Keeping this in mind the present study was focused on the

biodegradation of LAS. Various factors determining the efficiency of

degradation and the byproducts formed during degradation were also

considered. The degradation of LAS by free and immobilised cells of

Pseudomonas sp. was investigated to check the suitability of the isolated

organisms for onsite LAS removal. Immobilised cells are important when

it deals with high concentrations of LAS. Characterisation of the

biosurfactant produced by one of the isolate was also done. Presence of

LAS was determined in commercially available detergents. Toxicity

studies were done in soil microcosm, paddy seeds and fish juveniles.

1.1.1 Objectives

The main objectives of the study were:-

• Isolation, screening and identification of Linear alkylbenzene

sulphonate (LAS) degrading bacteria from detergent contaminated soil.

• Optimisation of different parameters for efficient Linear

alkylbenzene sulphonate (LAS) degradation.

• Analysis of metabolic by-products.

• Characterisation of biosurfactant produced by the selected LAS

degrading organism.

• Toxicity studies of Linear alkylbenzene sulphonate (LAS).

1.2 REVIEW OF LITERATURE

1.2.1 Surfactants

Surfactants are any organic substance used in detergents,

intentionally added to achieve cleaning, rinsing and/or fabric softening

due to its surface active properties. They consist of hydrophilic and

hydrophobic groups to such an extent that they are capable of forming

micelles (ISO 862). Industries worldwide discharge a wide range of

surfactant, or surface-active agents, to their wastewater treatment

facilities. Water pollution caused by synthetic surfactants has been

increasing during the past few years due to their extensive use in

household, agriculture and other cleaning operations. Synthetic

surfactants released into the aquatic system have adversely affected

ecosystems (Baleux & Caumette, 1977). Today the detergent wastes

constitute a major component of organic pollutants that are carried by

various means into lakes, rivers, and seas and cause serious environmental

problem (Takada et al., 1992; Abd-Allah, 1995).

Alkylbenzene sulphonates are the most commonly used surfactants in

domestic detergent formulations (Greek, 1991). In the United States and

Europe, linear alkylbenzene sulphonates (LAS) have been used since the early

1960s, when the low rate of biodegradation of branched – chain alkylbenzene

suphonates (BAS) was recognized (Alexander, 1973; Cain, 1987; 1994; Greek,

1991). LAS currently represent the most abundant constituents in either

domestic or industrial detergents, which account for close to 30% of the world

wide usage of synthetic surfactants (Thoumelin, 1991).

10 Chapter 1

Synthetic detergents are a mixture of linear alkylbenzene

sulphonate (LAS) and its isomers together with other additives. LAS is a

surface-active material found in relatively high amounts in domestic and

industrial wastewaters, discharged mainly from the textile, cosmetic and

tanning industries (Lin & Peng, 1994; Brillas et al., 1995). The worldwide

production of LAS is about 2.5x106 tonnes per annum (Knepper & Berna,

2003). Commercial LAS nominally comprises 20 compounds (Dong

et al., 2004; Schleheck et al., 2000; Eichhorn & Knepper, 2002).

Surfactants are especially noted for their wetting qualities and

their effectiveness as emulsifiers. More over some surfactants readily

adsorb in to surfaces which leads to surface modification. These

properties account for the exploitation of the surfactants in many product

areas as detergents and household cleaners and to a lesser extend as textile

softner, other textile aids, antistatic agents, additives in paints, metal

processing, shampoos, cosmetics, and oil drilling operations. Some

surfactants have antimicrobial properties which provide the basis for their

utility as biocides (van Ginkel, 1996).

A surfactant combines in a single molecule—a strongly

hydrophobic group with a strongly hydrophilic one. Such molecules tend

to congregate at the interfaces between the aqueous medium and the other

phases of the system such as air, oily liquids, and particles, thus imparting

properties such as foaming, emulsification, and particle suspension

(APHA, 2005).

Review of Literature 11

1.2.2 Occurrence of LAS in the environment

The concentrations of LAS have been monitored in several

environmental compartments. LAS is reported not to be biodegraded by

anaerobic biological processes usually employed in sludge stabilization

(McEvoy & Giger, 1985; Swisher, 1987) and it may be found in the gram

per kilogram range in anaerobic sludge.

Painter (1992) reported that concentrations of LAS in sewage

sludges have been measured in the range of 2 to 12 g/kg for primary and

anaerobically digested sludge (mostly in the range 4-10 g/kg), whereas

aerobically digested sludge and activated sludge contained 2.1-4.3 g/kg

and 0.09-0.86 g/kg, respectively. LAS are found in soils that are treated

with sewage sludge as a fertilizer.

The concentrations of LAS in raw wastewater have been reported

to range from 3 mg/L to 21 mg/L (De Henau et al., 1989; Holt et al.,

1995). According to Mackay et al., (1996) contamination of soil

environments with LAS is possible due to sludge application on

agricultural soil and land filling. The presence of surfactants in sludge

may have undesirable environmental effects since the surfactant

molecules may lead to groundwater contributing to groundwater

contamination. A monitoring of contaminants in sludge samples from

municipal sewage treatment plants in Denmark showed that the

concentrations of LAS varied between 0.01 and 16 g/kg. The median

concentration of all examined sludge samples (20) was 0.53 g/kg, whereas

the medians were 0.02 g/kg for 11 activated sludge samples and 0.94 g/kg

for 9 samples consisting of a mixture of activated and anaerobically

digested sludge (Madsen et al., 1998).

12 Chapter 1

Although LAS and other common surfactants have been reported

to be readily biodegradable by aerobic processes, much of the surfactant

load into a sewage treatment facility (reportedly 20–50%) is associated

with suspended solids (Greiner & Six 1997; McAvoy et al., 1998) and

thus escapes aerobic treatment processes. The monitoring conducted in

the Netherlands showed that the concentrations of LAS in the effluent of

seven representative municipal sewage treatment plants varied between

0.019 and 0.071 mg/l with an average value of 0.039 mg/l (Matthijs et al.

1999).

The presence of LAS in sewage works is varies depending on their

use in industrial processing in addition to domestic activities. An average

LAS concentration of 1~10 mg/L can be found in municipal wastewater

treatment dealing only domestic wastewater (Field et al., 1995) but this

range is noticeably increased when industrial wastes from washing

processes are also treated (Beltrán et al., 2000).

The presence of LAS in the environment close to sewage

treatment plant outfalls was reviewed (Bjerregaard et al., 2001). The

concentration of LAS in sewage treatment plant effluent was in the range

of 0.02–1.0 mg/L that was in the range reported to have a physiological

impact on marine life.

1.2.3 Bioremediation of LAS

The increasing release of organic pollutants by industries cause many

health–related problems. However, increased awareness of the harmful

effects of environmental pollution has led to a dramatic increase in research

on various strategies that may be employed to clean up the environment. It is

Review of Literature 13

now realised that microbial metabolism provides a safer, more efficient, and

less expensive alternative to physico-chemical methods for pollution

abatement (Hebes & Schwall, 1987).

Bacteria capable of degrading aromatic sulphonates have been

isolated from industrial sewage treatment plants (Zimmermann et al., 1982;

Thurnheer et al., 1990) or obtained through continuous adaptation (Kuhm

et al., 1991). Biodegradation of sulphonated aromatic compounds has been

studied for many years (Locher et al., 1991; Goszczynski et al., 1994).

Various authors have reviewed the subject of biodegradation of

organo pollutants over the past decade (Kumar et al., 1996; Johri et al.,

1996). Biotransformation of organic contaminants in the natural environment

has been extensively studied to understand microbial ecology, physiology

and evolution for their potential in bioremediation (Johan et al., 2001; Mishra

et al., 2001; Watanabe, 2001).

Biodegradation of LAS begins at the terminus of the alkyl chain with

an omega-oxidation and is followed by successive cleavage of C2 fragments

(ß-oxidation) (Huddleston & Allred, 1963; Swisher, 1963). These

intermediates are further biodegraded by oxidative scission of the aromatic

ring and cleavage of the sulphonate group (Setzkorn & Huddleston, 1965;

Swisher, 1967).

Goodnow & Harrison, (1972) conducted a survey for surfactant

degradation among aerobic bacteria. Forty-five strains of 34 species in 19

genera degrade one or more of detergent compounds, tallow – alkyl – sulfate,

alkyl – ethoxylate – sulfate and linear – alkyl – benzene – sulphonate.

Microorganisms implicated in LAS degradation include Nocardia sp.,

14 Chapter 1

Fusarium sp., Aspergillus sp., Pseudomonas sp., Micrococcus sp. and

Acinetobacter sp. (Kobayashi & Rittmann, 1982).

LAS are completely degraded in wastewater treatment plants, and

different organisms participate in their mineralisation, each degrading a part

of the molecule. A four – member consortium was identified as responsible

for LAS mineralisation (Jimenez et al., 1991), and a larger consortium was

found to be involved in mineralisation in a marine environment (Sigoillot

et al., 1992). In these consortia, some members attacked the side chain, while

others degraded the aromatic moiety. Soberon – Chaves et al., (1996)

isolated a Pseudomonas aeruginosa strain W51D which is able to mineralise

at least 70% of a BAS commercial mixture and completely degrade LAS.

Amund et al., (1997) studied the biodegradability potentials of three

detergent products with the trade names Omo, Teepol and sodium dodecyl

sulfate (SDS) by the native bacteria of the Lagos lagoon using the lagoon

die- away method. The detergent – utilising bacteria identified were mainly

gram – negative and of the following genera: Vibrio, Klebsiella,

Flavobacterium, Pseudomonas, Escherichia, Enterobacter, Proteus, Shigella

and Citrobacter.

LAS is removed to about 99.9 % by a functional sewage treatment

plant, largely through biodegradation (Schöberl, 1997). The degradation of

LAS is more complex than previously realised. LAS is not a single

compound, but, ideally, a mixture of 20 compounds, all subterminally

substituted, linear, alkyl chains (C10-C13) carrying a 4-sulfophenyl moiety.

Of these 20 compounds, 18 are optically active, so there are 38 structures in

the ideal mixture. Thus, many SPCs, and similar compounds, are formed

from commercial LAS, and subsequently mineralised in the second

Review of Literature 15

degradative step by specialised organisms (Sigoillot & Nguyen, 1992; Hrsák

& Begonja, 1998).

Laboratory degradation studies of linear alkyl benzenes by Nocardia

amarae MB-11 isolated from soil showed an overall degradation of linear

alkyl benzenes isomers to the extent of 57-70%. Degradation of 2-phenyl

isomers of linear alkyl benzenes was complete and faster than that of other

phenyl position (C3–C7) isomers which were degraded to the extent of 40-

72% only (Bhatia & Singh, 1996).

Linear alkylbenzene sulphonates (LAS) is easily biodegraded than

non-linear alkylbenzene sulphonate (ABS) eventhough, total biodegradation

still requires several days (Gledhill, 1975; Nomura et al., 1998). Branner

et al., (1999) investigated the ability of a microbial community to degrade

linear alkylbenzene sulphonate (LAS) in soil columns under water saturated

conditions. Pseudomonas aeruginosa W51D is able to grow by using

branched-chain doecylbenzene sulphonates (B-DBS) or the terpenic alcohol

citronellol as a sole source of carbon. A mutant derived from this strain

(W51M1) is unable to degrade citronellol but still grows on B-DBS, showing

that the citronellol degradation route is not the main pathway involved in the

degradation of the surfactant alkyl moiety (Campos-Garcia et al., 1999). The

degradation of of linear alkylbenzene sulphonate (LAS) was studied in a two-

stage anaerobic system where the acidogenic reactor was bioaugmented with

a strain of Pseudomonas aeruginosa (M113). This is a strain, which under

aerobic and denitrifying conditions uses LAS as carbon source (Almendariz,

et al., 2001).

Perez et al., (2002) investigated the role of benthic microorganisms

in the biodegradation of detergents in the marine environment. According to

16 Chapter 1

Schleheck et al., (2004) Parvibaculum lavamentivorans Strain DS-1 is the

first pure culture of a heterotrophic organism (strain DS-1T) proven to utilise

commercial LAS. It catalyses the ω-oxygenation of the LAS side chain and

about three rounds of β –oxidation; a wide range of products,

sulfophenylcarboxylates, sulfophenyldicarboxylates and a,b-unsaturated

sulfophenylcarboxylates. Dong et al., (2004) reported that Parvibaculum

lavamentivoransT degrades commercial LAS via ω-oxygenation, oxidation

and chain shortening through β- oxidation to yield a wide range of SPCs.

Mineralisation of LAS by defined pair of herotrophic bacteria was also

reported (Schleheck et al., 2004). According to them Parvibaculum

lavamentivorans DS-1T and Comamonas Testosteroni sp B-2 and KF-1

community can mineralise about 8 congeners of LAS.

An experimental study conducted in aquarium with seawater

enriched in a pure linear alkylbenzene sulphonate (LAS), namely 1–(p–

Sulfophenyl) nonane, has shown that the primary degradation was 10 times

more rapid in the presence of the sponge Spongia officinalis than in the

presence of only marine bacteria (Perez et al., 2000). The very rapid

degradation kinetics observed in this study may be due to the symbiotic

microbial community present in S officinalis. Lijun et al., (2005) studied the

LAS degradation of immobilised Pseudomonas aeruginosa with low –

intensity ultrasonic and the influence of original LAS concentration, pH,

rotary velocity and different conditions of low-intensity ultrasonic irradiation

on the degradation of LAS.

Abboud et al., (2007) reported degradation of the anionic surfactants

linear alkylbenzosulphonate (LAS) and sodium dodecyl sulfate (SDS) by a

Review of Literature 17

consortium of the mixed facultative anaerobes Acinetobacter calcoaceticus

and Pantoea agglomerans which were isolated from waste water.

1.2.4 LAS degrading organisms

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

done extensive work with the genus Pseudomonas. Two unknown bacterial

isolates, C12 & C12B were obtained from enriched soils and cultured on

media containing detergent compounds as sole sources of carbon (Payne &

Feisal, 1963b). Both the isolates destroyed the foaming capacity of dodecyl

sulfate in the media. C12B, grow on dodecyl benzene sulphonate (DBS)

whereas C12 could not utilise this surfactant. Species of Hansenula and

Candida are resistant to high concentrations of anionic alkylbenzene

sulphonates and degrade sub inhibitory concentrations of these detergents

(Standard & Ahearn, 1970). A Vibrio sp. is capable of metabolising the alkyl

chain of dodecylbenzene sulphonate as the sole source of carbon and energy

(Bird & Cain, 1972).

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

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

metabolise it. The process was energy dependent, and cell lysis occurred

during stationary phase. Studies using pure cultures have shown that many

bacterial species partially degrade LAS but do not completely mineralise the

surfactant (Swisher, 1987; Sigoillot & Nguyen, 1990). Experiments with

seawater and marine sediment have shown that bacterial communities

degrade LAS with greater efficiency than isolated strains (Sigoillot &

Nguyen, 1990). These bacterial isolates degrade only the alkyl chain and do

not cleave the sulphonated aromatic ring.

18 Chapter 1

A bacterial consortium capable of linear alkylbenzene sulphonate

(LAS) mineralisation under aerobic conditions was isolated from a chemostat

inoculated with activated sludge. The consortium, designated KJB, consisted

of four members. Three isolates had biochemical properties characteristic of

Pseudomonas spp.; the fourth showed characteristics of the Aeromonas spp.

Cell suspensions were grown together in minimal medium with [14C] LAS as

the only carbon source. After 13 days of incubation, more than 25% of the

[14C] LAS were mineralised to 14CO2 by the consortium.

Schleheck et al., (2004) reported that Parvibaculum lavamentivorans

strain DS-1T, a small heterotrophic bacterium, able to ω-oxygenate the

commercial surfactant linear alkylbenzenesulphonate (LAS) and shorten the

side chain by β-oxidation to yield sulfophenylcarboxylates.

The biofilm formed by the Parvibaculum lavamentivoransT on glass

particles catabolise LAS through undefined ‘ω-oxygenation’ and β-oxidation

and excrets sulphophenyl carboxylates (SPC) quantitatively (Schleheck &

Cook, 2005). The degradation of centrally substituted congeners of LAS by

strain Parvibaculum lavamentivorans DS-1T yields sulphophenyl carboxylate

and sulphophenyldicarboxylate (Schleheck et al., 2007).

Hosseini et al., (2007) reported that the Pseudomonas beteli and

Acinetobacter johnsoni isolates from Tehran municipal active sludge were

able to degrade 96.4% and 97.2% of the original Linear alkylbenzene

sulphonate (LABS) levels after 10 days of growth, respectively. Mixed

culture of the two isolates did not significantly increase LABS utilisation

(97.6%).

Review of Literature 19

Table 1: Microorganisms in the biodegradation of LAS

Sl. No. Organism Reference

1 Pseudomonas C12B Payne & Feisal, 1963b

2 Hansenula and Candida Standard & Ahearn, 1970

3 Vibrio sp. Bird and Cain, 1972

4 Pseudomonas aeruginosa strain W51D

Soberon – Chaves et al., 1996

5 Nocardia amarae MB-11 Manju Bhatia and Devendra Singh, 1996

6 Spongia officinalis Perez et al., 2000

7 Pseudomonas aeruginosa M113 Almendariz, et al., 2001

8 Phanerochaete chrysosporium Yadav et al., 2001

9 Parvibaculum lavamentivoransT Dong et al., 2004

10 Parvibaculum lavamentivorans DS-1Tand Comamonas Testosteroni sp B-2 and KF-1

Schleheck et al., 2004

11 Pseudomonas aeruginosa Lijun et al., 2005

12 Parvibaculum lavamentivoransT Schleheck & Cook ,2005

13 Pseudomona sp. Prats et al., 2006

14 Parvibaculum lavamentivorans Strain DS-1

Schleheck et al., 2004, Schleheck et al., 2007

15 Acinetobacter calcoaceticus and Pantoea agglomerans

Abboud et al., 2007

16 Pseudomonas beteli and Acinetobacter johnsoni

Hosseini et al., 2007

17 Parvibaculum lavamentivorans DS-1T and Comamonas testosteroni sp. KF-I

Schleheck et al., 2010

20 Chapter 1

Comamonas testosteroni KF-I can mineralise the SPCs by forming

two transient intermediates 4-Sulphoacetophenon (SAP) and 4- Sulphophenol.

Based on these results Schleheck et al., (2010) postulated a path way of

mineralisation of LAS involving Parvibaculum lavamentivorans DS-1T and

Comamonas testosteroni sp. KF-I . Farzaneh et al., (2010) reported that a

Stenotrophomonas maltophilia strain isolated from activated sludge that

utilised branched anionic surfactants (BAS) as a sole carbon source.

1.2.5 Factors affecting biodegradation

Biodegradation may not occur at optimal rates, if the

environmental factors are not adequate (Providenti et al., 1993).

Proper aeration is essential if aerobic, catabolic reactions are to

occur. Deksissa and Vanrolleghem (2003) reported that aeration can limit

LAS degradation in rivers. Khleifat (2006) reported that a consortium of

facultative anaerobic bacteria completely degraded LAS with in 72 h at

high aeration however at normal aeration only 70% degradation was

achieved with in a 96 h incubation time. When calls are under limited

aeration less than 40% of LAS degradation was obtained.

Abboud et al., (2007) investigated three different shaking rates

(aeration) on surfactant biodegradation and found that all shaking rate

were effective in enhancing LAS biodegradation and the highest selected

shaking rate produced maximum degradation.

Temperature affects the rate of degradation of the xenobiotics by

influencing the physical and chemical properties of the LAS, microbial

metabolism, the specific growth rate of the microorganism, the rate of

enzymatic activity, involving the oxidation process and the composition

Review of Literature 21

of microbial community (Gibbs et al., 1975; Takamatsu et al., 1996).

Prats et al., (2006) reported that there was significant influence of

temperature on the final removal of surfactants. At lower incubation

temperatures the organisms need a longer acclimatisation period and will

affect the initial degradation rate. According to Abboud et al., (2007)

higher temperature brings down the rate of LAS biodegradation.

Citrobacter braakii, Delftia acidovorans strain SPB1, Pseudomonas strain

C12B, Acinetobacter calcoaceticus and Pantoea agglomerans required

30°C for optimal SDS degradation (Payne & Feisal, 1963; Abboud et al.,

2007) while Comamonas terrigena strain N3H showed optimum growth

at 28°C (Roig et al., 1998). Pseudomonas sp. that can degrade SDS at

25°C was also reported (Marchesi et al., 1997)

Wong et al., (2002) found that introduction of isolated PAH-

degradative bacteria for bioremediation requires a specific set of abiotic

factors, including availability of source energy, suitable pH, temperature,

water content, and soil oxygen concentrations, and the presence of

essential elements. Atagana et al., (2003) studied physical and chemical

conditions in order to optimize the bioremediation of creosote-

contaminated soil and reported that management of aeration, moisture

content and pH are important considerations. They observed that these

factors play very significant roles in the utilisation of creosote in soil.

1.2.6 Molecular biology of LAS degradation

The metabolic diversity of Pseudomonas bacteria has been well

documented. Chakrabarty, (1972) identified a plasmid encoded pathway

for salicylate degradation in P. putida RI. Since then, other degradative

plasmids have been shown to be involved in the metabolism of camphor

22 Chapter 1

(Rheinwald et al., 1973), naphthalene (Dunn & Gunsalus, 1973), toluene,

m- and p-xylenes (Worsey & Williams, 1975; Friello et al., 1976),

octane and other substrates. Many of these plasmids are transmissible

among Pseudomonas species and compatible with one another

(Chakrabarty, 1976). Biodegradative plasmids some times confer other

characteristics, such as mercury resistance (Chakrabarty, 1976), or UV-

response enhancement (McBeth, 1989).

Elevated growth temperature has been successfully used to cure

tetracycline resistant, pencillinase positive strains of S.aureus (May et al.,

1964). A method for the curing of R (resistance) and F (Sex factor)

plasmids in E.coli K12 by SDS treatment has been reported by Tomoeda

et al., (1968). Hohn & Korn, (1969) have used acridine orange to cure the

F plasmid from cells of Escherichia coli. In 1969, Bouanchand et al.,

(1969) described the use of ethidium bromide to eliminate plasmids in

antibiotic-resistant Entero bacteria and staphylococci. Loss of function at

a rate higher than the expected rate of mutation and/or the ability to

transfer a function to recipient cells are generally accepted properties that

provide genetic evidence for the presence of a plasmid involved in

conferring a particular function to a cell (Williams, 1978).

Work on a plasmid bearing strain of Pseudomonas testosteronii

and the existence of the OCT plasmid, which codes for the oxidation of

straight chain alkanes, lead Cain (1981) to propose that LAS degradation

may be plasmid-encoded. He also proposed that desulphonation and meta-

cleavage of the aromatic ring are mediated by plasmid-encoded enzymes

in a fashion analogous to pWWO, TOL plasmid-mediated toluene

metabolism. The presence of a plasmid in Pseudomonas C12B, able to

Review of Literature 23

degrade alkyl sulfates, alkylbenzene sulphonates, and linear alkanes and

alkenes was physically demonstrated by gentle cell lysis followed by

agarose gel electrophoresis (Kado & Liu, 1981).

A protocol for using elevated incubation temperature (5-7oc above

the normal or optimal growth temperature) as a curing method was

described by Carlton and Brown, (1981). A general procedure for plasmid

curing has been outlined by Caro et al., (1984). Trevors (1986)

summarised and reviewed plasmid curing agents and procedures.

Intercalating dyes such as a acriflavine, acridine orange, ethidium

bromide and quinacrine have been successfully used in curing bacteria of

plasmids.

Studies by Wittich et al., (1988) demonstrated that the ability of

naphthalene-degrading populations to oxidise sulphonated naphthalenes.

It was reported that naphthalene catabolism is commonly a plasmid-

encoded phenotype (Sayler et al., 1990)

Sharma & Laxminarayanan, (1989) studied effect of high

temperature on plasmid curing of Rhizobium spp. in relation to nodulation of

pigeon pea (Cajanus cajan (L.) Millsp). They observed that the high

temperature in the semi arid zones of Haryana could be responsible for the

low nodulation of pigeon pea because the plasmid carrying the nodulation

genes is cured at 40o- 45oC giving rise to non-nodulating mutants. Nath and

Deb, (1997) conducted curing of plasmids of Corynebacterium renale

using mutagens ethidium bromide, acridine orange and rifampicin.

The nucleotide sequences of two genes involved in sodium

dodecyl sulfate (SDS) degradation, by Pseudomonas, have been

24 Chapter 1

determined. One of these, sdsA, codes for an alkylsulfatase (58957 Da)

and has similarity over a 201-aminoacid stretch to the N terminus of a

predicted protein of unknown function from Mycobacterium tuberculosis.

The other gene, sdsB, codes for a positive activator protein (33600 Da)

that has extensive similarity with the lysR family of helix-turn-helix

DNA-binding activator protein (Davison et al., 1992).

A higher incidence of plasmid was reported in bacteria growing in

waste water treatment plants and among bacteria that grew on LAS

containing medium. However the presence of plasmid did not necessarily

confer the ability to degrade LAS nor was the ability to degrade LAS

dependent on the presence of a plasmid. LAS mineralisation is mediated

by a consortium and the evidence that initial attack of LAS is plasmid

mediated is inconclusive (Breen et al., 1992).

Several n-alkane degradation pathways have been investigated in

bacteria, but only two plasmid conferring n-alkane dissimilation have

been reported. In early studies of alkane degradation, the OCT plasmid

was found in P. putida (van Beilen et al., 1994). Chakrabarthy (1973)

constructed a cointegrate of OCT with plasmid CAM. The cointegrate

retained properties of both plasmids. Furthermore, CAM-OCT was much

more readily transmissible to a wide range of recipients than the poorly

transmitted OCT. Recently anotherplasmid, which is very similar to

plasmid OCT, has been isolated from P. maltrophila (Lee et al., 1996).

Mitomycin C curing experiments and conjugation experiments in

Pseudomonas C12B demonstrated that the ability to utilise n-alkanes (C9-

C12) and n-alkenes (C10 and C12) of medium chain length was plasmid

encoded (Kostal et al., 1998).

Review of Literature 25

On the basis of curing and transformation experiments,

monocrotophos (MCP) degradation by Pseudomonas mendocina was

plasmid-borne and transferable to other bacteria (Bhadbhade et al., 2002).

Zhen et al., (2006) reported plasmid-mediated degradation of 4-

chloroniotrobenzene by newly isolated Pseudomonas putida strain

ZWEL73.

Transferable degradative plasmids play an important role in the

adaptation of microbial communities to the presence of xenobiotics in their

environments as other mobile genetic elements, including conjugative

transposons, integrons, genomic islands and phages (Top & Springal,

2003). Yeldho et al., (2010) reported that the degradation of the anionic

surfactant sodium dedecyl shlphate by Pseudomonas aeruginosa S7 is a

plasmid coded character.

1.2.7 Immobilisation

Immobilised cells have been defined as cells that are entrapped

within or associated with an insoluble matrix. Mattiasson (1983)

discussed various methods of immobilisation: covalent coupling,

adsorption, entrapment in a three dimensional polymer network,

confinement in a liquid-liquid emulsion, and entrapment within a

semipermeable membrane. Catalytic stability can be greater for

immobilised cells than for free cells, and some immobilised

microorganisms tolerate higher concentrations of toxic compounds than

do their non immobilised counterparts (Dwyer et al., 1986; Westmeier &

Rehm, 1985). Under many conditions, immobilised cells have advantages

over both free cells and immobilised enzymes. Use of immobilised cells

26 Chapter 1

permits the operation of bioreactors at flow rates that are independent of

the growth rate of the microorganisms employed (Nunez & Lema, 1987).

A number of immobilisation supports and matrices have been

developed and their characteristics tested under diverse conditions.

Currently, the most popular cell entrapment matrix is Ca- alginate, a

biopolymer having a structure similar to bacterial cell wall (Akin, 1987;

Philips & Poon, 1988). Polyurethane was shown to be an effective

immobilisation matrix for a pentachlorophenol (PCP) -degrading

Flavobacterium strain. Advantages of polyurethane immobilisation

included the maintenance of PCP degradation activity for up to 150 days

and the reversible adsorption of PCP to the polyurethane, which protected

the cells from the toxic effects of the PCP (O'Reilly & Ronald, 1989).

Immobilised Pseudomonas C12B offer considerable potential for

treatment of waste water streams containing a range of chemical length

homologues of primary alkyl sulphate, alkyl ethoxysulphate and some

alkylbenzene sulphonate surfactants (Thomas & Graham, 1991).

Immobilisation can provide an advantageous environment for the

biocatalyst, increasing both its bioconversion activity and resistance to

various types of damage (Anselmo & Novais, 1992; Hallas et al., 1992).

Bacteria Comamonas terrigena N3H immobilised in polyurethane

foam have been successfully used for the biodegradation of the anionic

surfactants dihexyl sulfosuccinate and dioctyl sulphosuccinate (Roig

et al., 1998). Not only polyurethane foam but also alginate gel was

successfully employed for the immobilisation of the strain C. terrigena

N3H (Huska et al., 1996b, 1997a). The surfactant-degrading biocatalyst

Pseudomonas C12B was immobilised by covalent linking on silanised

Review of Literature 27

inorganic supports and by physical entrapment of cells within reticulated

polyurethane foam. Both immobilised biocatalysts have been shown to be

appropriate for the effective primary biodegradation of the anionic

surfactants sodium dodecyl sulphate (SDS), dodecylbenzene sulphonic

acid (DBS), dioctyl sulphosuccinate (DOSS) and dihexyl sulphosuccinate

(DHSS) (Roig et al., 1998).

Perei et al., (2001) immobilised cells of Pseudomonas

paucimobilis on the surface of Mavicell- Si beads and by entrapment in a

calcium alginate phyta gel composite gel matrix for the degradation of

sulphanilic acid. They reported that calcium alginate phytagel proved a

good matrix for immobilisation of Pseudomonas paucimobilis, with

essentially unaltered biodegradation activity.

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was

immobilised in various matrices namely, polyurethane foam (PUF),

polyacrylamide, alginate and agar. The degradation rates of 20 and 40

mM p-cresol by the freely suspended cells and immobilised cells in

batches and semicontinuous with shaken cultures were compared. The

PUF-immobilised cells achieved higher degradation of 20 and 40 mM p-

cresol than freely suspended cells and the cells immobilised in

polyacrylamide, alginate and agar (Tallur et al., 2009).

1.2.8 Adsorption

One of the methods employed for removing contaminants from

waste water is adsorption. Adsorption capacity for specific single organic

solutes of a homologous series is thought to be a direct function of: 1) The

adsorbate properties, such as functionality, branching or geometry,

28 Chapter 1

polarity, hydrophobicity, dipole moment, molecular weight and size, and

aqueous solubility; 2) The solution conditions, such as pH, temperature,

pressure, adsorbate concentration, ionic strength, and presence of

background and competitive solutes; and 3) The nature of the adsorbent,

such as surface area, pore size and distribution, surface distribution, and

surface characteristics (Belfort, 1980).

The surfactants adsorb to the river sediment, stimulating the

simultaneous attachment of bacteria. The adsorption process accelerates

the biodegradation of alkyl sulfate surfactants (Marchesi et al., 1991a,b;

White, 1995). Anionic surfactants are amphiphatic compounds consisting

of a hydrophobic (alkyl chains of various length, alkylphenyl ethers,

alkylbenzenes, etc.) and a hydrophilic part (carboxyl, sulfate, sulphonates,

phosphates, etc). The hydrophobic and hydrophilic parts readily interact

with the polar and apolar substructures in marcomolecules such as

proteins (Yamaguchi et al., 1999; Xiao et al., 2000), and cellulose or with

the polar or apolar molecules in a mixture of compounds (Chirila

et al., 2000; Von Berleps et al., 2000). Because of these interactions,

anionic surfactants can decrease the energy of interaction and the energy

of solvation between a high variety of heterogeneous phases in many

technological processes and biological systems by adsorbing on oil–water

(Staples et al.,2000), polystyrene water (Turner et al.,1999), and air–water

(Hawerd & Warr, 2000) interfaces.

In order to find materials for effective surfactant removal, the

adsorption of anionic surfactants on various solid surfaces have been

extensively studied. Thus, it has been established that sodium lauryl

sulfate is readily adsorbed onto arsenic-bearing ferric hydrite (Quan et al.,

Review of Literature 29

2001), other surfactants have been adsorbed by layered double hydroxides

(Pavan et al., 2000), by hydrolytically stable metal oxides (Vovk, 2000).

The adsorption of anionic surfactant on solid surfaces (Somasundaran &

Huang, 2000; Rodriguez & Scamehorn, 2001) can modify surface

characteristic and electron transfer (Wang et al., 2000a), can increase the

film thickness of other adsorbed molecules (Churaev, 2000; Esumi et al.,

2000; Miyazaki et al., 2000) and can result in the formation of surface

aggregates similarly to micelles (Luciani et al.,2001). Schleheck & Cook

(2005) reported the need of a biofilm formation by Parvibaculum

lavamentivoransT on a soild support (e.g. glass particles) when utilising

commercial LAS for growth.

1.2.9 Analysis of metabolic byproducts.

The concentrations of surfactants in environmental samples are

usually below the limit of the analytical method. Therefore, pre concentration

is necessary before analysis. Solid liquid extraction is identified as the best

method for extraction of surfactants from sludge. In most cases further

purification is also required before analysis. LAS are desorbed from sewage

sludge either in a continuous procedure by extraction into chloroform as ion

pairs with methylene blue (McEvoy & Giger, 1986) or in a continuous

procedure by the application of soxhlet apparatus and addition of soild NaOH

to the dried sludge in order to increase extraction efficiency (Marcomini &

Giger, 1987). Heating of sludge or sediment samples in methnol under reflux

for 2hr is also sufficient to extract LAS with recoveries of 85% (Matthijs &

De Henau, 1987).

The anionic surfactants differ in their biodegradability so there is

accumulation of different anionic surfactants together in waste water.

30 Chapter 1

Therefore, the identification of persistent anionic surfactants in sewage

effluent is of considerable practical and theoretical importance (Carre &

Dufils, 1991). Various combined chromatographic techniques identified the

persistent anionic surfactants as linear alkylbenzene sulphonates, sulfophenyl-

carboxylated linear alkylbenzene sulphonates, tetralin and indane

sulphonates, and alkylphenol polyethoxylate carboxylates (Field et al., 1992).

High-performance liquid chromatography and capillary zone

electrophoresis have been used for the separation of homologues and

structural isomers of linear alkylbenzene sulphonates with subsequent UV

detection. The analytical advantages of these techniques are applied to the

analysis of technical products containing high amounts of LAS as well as

river water samples with very low LAS concentrations using pre

concentration steps (Vogt et al., 1995). High performance liquid

chromatography (HPLC) has the advantages of rapid analysis and high

sensitivity. A suitable analytical condition has been established for HPLC

and the LAS in modem sediments from core Zhu-9 at the Pearl River

mouth has been determined by HPLC (Hu et al., 2000). LAS were

determined by HPLC method and the results were compared with those

obtained by MBAS method. HPLC was found to be the precise and

reliable method for determining LAS (Huseyin et al., 2001).

The use of Solid Phase Microextraction (SPME) for the qualitative

and quantitative determination of Linear Alkylbenzene sulphonates (LAS)

in waste water samples was investigated. A Carbowax / Templated Resin

(CW/TPR) coated fiber was directly immersed into influent and effluent

samples of a sewage treatment plant (STP). The extracted LAS were

desorbed with a solvent in a specially designed SPME-LC interface for

Review of Literature 31

analysis with HPLC-FLD and Electrospray Ionization Mass Spectrometry

(ESI-MS). The combination of SPME with ESI-MS proved to be an

alternative technique for the LAS determination in waste water (Ceglarek

et al., 1999).

Reverse phase HPLC using UV (Mathijs & Henau 1987; Cavalli

& Lazzarin 1987; Heinig et al., 1996) or fluorescence (Castles et al.,

1989; Leon et al., 2000) detection can be considered as routine methods

for LAS analysis. The application of gas chromatography mass

spectrometry (McEvoy & Giger 1985; 1986; Reiser et al., 1997; Ding &

Fann, 2000; Pratesi et al., 2000) has provided qualitative and quantitative

analysis of previously derivatized LAS samples.

Long-chain sulfophenyl carboxylate compounds (SPC) with more

than five carbon atoms, which are degradation products of linear

alkylbenzene sulphonates (LAS), have been isolated by solid-phase

extraction (SPE) followed by identification with liquid chromatography/

ionspray-mass spectrometry (LC/ISP-MS). The isolation procedure

involved extraction in a C18 minicolumn and subsequently in a strong

anionic exchange (SAX) column, the final determination was

accomplished by Negative Ion LC/ISP/MS using a two step approach

(Riu & Barcelo, 2001).

To enable LAS determination in biota samples, LAS and its

coproducts (methylbranched LAS, dialkyltetralin sulphonates) are

extracted from tissues using matrix solid-phase dispersion, isolated by

strong anion exchange chromatography and determined by HPLC-

electrospray-tandem mass spectrometry. Tolls et al., (2003) adapted this

32 Chapter 1

method, to study the extent of bioaccumulation of linear alkylbenzene

sulphonate (LAS) in feral organisms the sediment dwelling Tubifex sp.

LAS and sulfophenyl carboxylate compounds (SPC) were isolated

by solid-phase extraction (SPE) and then determined by liquid

chromatography-electrospray mass spectrometry (LC-ESI-MS). The

method enabled unequivocal identification of C10-C13 LAS by

monitoring the ion at m/z 183 and the base peak corresponding to the [M-

H]- ion. Unequivocal identification and determination of some

metabolites of the LAS, the sulfophenyl carboxylate compounds (SPC),

was achieved by monitoring [M-H]- ions (Riu et al., 2001).

The ultimate goal in detergent’s environmental analysis is the

quantification of individual compounds separated from all their isomers

and/or homologues. Chromatographic methods like HPLC, GC or SFC

are amongst the most powerful analytical instruments with regard to

separation efficiency and sensitivity. Because of the low volatility of

surfactants HPLC is used far more often than GC. Since the commence of

atmospheric pressure ionization (API) interfaces, LC-MS coupling is

increasingly used for determination of surfactants (Cirelli et al., 2008).

1.2.10 Biosurfactants

Biosurfactants are microbially produced surface-active compounds.

They are amphiphilic molecules with both hydrophilic and hydrophobic

regions causing them to aggregate at interfaces between fluids with

different polarities such as water and hydrocarbons (Georgiou, 1992;

Kosaric, 1993). These biomolecules may also decrease interfacial surface

tension (Rouse et al., 1994; Shafi & Khanna, 1995). Although the

Review of Literature 33

function of biosurfactants in microorganisms is not fully understood, it is

known that these secondary metabolites can enhance nutrient transport

across membranes, act in various host-microbe interactions, and provide

biocidal and fungicidal protection to the producing organism (Banat,

1995a, 1995b: Lin, 1996). Many of the known biosurfactant producers are

also hydrocarbon-degrading organisms (Willumsen & Karlson, 1997;

Volkering et al., 1998).

Bacteria make low molecular weight molecules that efficiently

lower surface and interfacial tensions and high molecular weight

polymers that bind tightly to surfaces (Desai & Banat, 1997; Roesenberg

et al., 1999). The low molecular weight biosurfactants are generally

glycolipids in which carbohydrates are attached to a long-chain aliphatic

acid or lipopeptides. Glycolipid bioemulsifiers, such as rhamnolipids,

trehalose lipids and sophorolipids, are disaccharides that are acylated with

long-chain fatty acids or hydroxy fatty acids. One of the best-studied

glycolipids is rhamnolipid, produced by several species of

Pseudomonads, which consists of two moles of rhamnose and two moles

of β-hydroxydecanoic acid (Lang & Wullbrandt, 1999).

1.2.11 Rhamnolipids

Production of rhamnose containing glycolipids was first described

in Pseudomonas aeruginosa by Jarvis & Johnson (1949). L-Rhamnosyl-

L-rhamnosyl-β- hydroxydecanoyl-β-hydroxydecanoate and L-rhamnosyl-

β-hydroxydecanoyl-β-hydrocydecanoate, referred to as rhamnolipids 1

and 2 respectively, are the principal glycolipids produced by P.

aeruginosa (Edward & Hayashi, 1965). These glycolipids, in which one

or two molecules of rhamnose are linked to one or two molecules of β-

34 Chapter 1

hydroxydecanoic acid, are the best studied (Figure 2). While the OH

group of one of the acids is involved in glycosidic linkage with the

reducing end of the rhamnose disaccharide, the OH group of the second

acid is involved in ester formation (Karanth et al., 1999).

Figure 2: Structures of mono and dirhamnolipids (Price et al., 2009).

(A) Mono rhamnolipid; (B) Dirhamnolipid

1.2.12 Properties of biosurfactants

Biosurfactants are of increasing interest for commercial use because

of the continually growing spectrum of available substances. There are many

advantages of biosurfactants compared to their chemically synthesised

counterpart.

Rhamnolipids from P. aeruginosa decrease the surface tension of

water to 26 mN/m and the interfacial tension of water/hexadecane to <1

mN/m (Hisatsuka, et al., 1971). In general, biosurfactants are more effective

and efficient and their CMC is about 10–40 times lower than that of chemical

surfactants, i.e. less surfactant is necessary to get a maximum decrease in

surface tension (Desai & Banat, 1997).

Review of Literature 35

Many biosurfactants and their surface activities are not affected by

environmental conditions such as temperature and pH. McInerney et al.,

(1990) reported that lichenysin from B. licheniformis JF-2 was not affected

by temperature (up to 50°C), pH (4.5–9.0) and by NaCl and Ca

concentrations up to 50 and 25 g/L respectively. A lipopeptide from B.

subtilis LB5a was stable after autoclaving (121°C/20 min) and after 6 months

at –18°C, the surface activity did not change and tolerate from pH 5 to 11 and

NaCl concentrations up to 20% (Nitschke & Pastore, 1990).

Unlike synthetic surfactants, microbial-produced compounds are

easily degraded and particularly suited for environmental applications such

as bioremediation (Mulligan, 2005) and dispersion of oil spills.

Very little data are available in the literature regarding the toxicity of

microbial surfactants. They are generally considered as low or non-toxic

products and therefore, appropriate for pharmaceutical, cosmetic and food

uses. A biosurfactant from P. aeruginosa was compared with a synthetic

surfactant (Marlon A-350) widely used in the industry, in terms of toxicity and

mutagenic properties. Both assays indicated higher toxicity and mutagenic

effect of the chemical-derived surfactant, whereas the biosurfactant was

considered slightly non-toxic and nonmutagenic(Flasz et al.,1998).

1.2.13 Analysis of rhamnolipids

Amphiphilic molecules accumulate at the interface of different media

and form micelles or vesicles above a certain concentration, called the critical

micelle concentration. Below this value, surface or interfacial tension

depends on the concentration of the active compound and can be used for an

indirect quantification of the total rhamnolipid content using a calibration

36 Chapter 1

curve with pure rhamnolipid for comparison. Tensiometric measurements

were applied widely in the early days of rhamnolipid research (Guerra-

Santos et al., 1984; Reiling et al., 1986), owing to their simplicity. They are

still used, especially for new rhamnolipid species (Tuleva et al., 2002;

Ochoa-Loza et al., 2007).

Quantitative haemolytic activity tests are carried out using

erythrocyte suspensions and by measuring the released haemoglobin

absorbance at 540 nm (Johnson & Boese-Marrazzo, 1980). Siegmund and

Wagner (1991) developed a semiquantitative agar plate cultivation test is

based on the formation of an insoluble ion pair of anionic surfactants with the

cationic surfactant CTAB and the basic dye methylene blue. Rhamnolipids

are detected as dark-blue halos around the colonies, with the spot diameter

being dependent on rhamnolipid concentration.

One of the main disadvantages of the indirect and colorimetric

methods described above is the ignorance of sample composition and, hence,

the occurrence of various rhamnolipid species. Chromatographic separation

of a rhamnolipid mixture, coupled with appropriate detection methods like

MS, revealed that the hydroxy fatty acid moiety of the rhamnolipids may

comprise various fatty acid chain lengths (Deziel et al., 1999; Haba et al.,

2003; Monteiro et al., 2007).

Thin-layer chromatography (TLC) has been used extensively for

determining the composition of culture broth extracts of rhamnolipids and for

their preliminary purification on a thicker chromatographic layer. An

alternative for rhamnolipid identification is to couple TLC analysis with a

detection system based on, e.g., fast atom bombardment (Rendell et al.,

1990; de Koster et al., 1994). This combination even allows one to

Review of Literature 37

distinguish different fatty acid chains and their sequences inside the

rhamnolipid that cannot be dissociated by chromatography.

Separation of rhamnose or fatty acids by GC, coupled with flame

ionisation detectors or mass spectrometers, has been reported (Arino et al.,

1996; Mata-Sandoval et al., 1999) Rhamnose is analysed quantitatively after

conversion into trimethylsilyl esters, not giving any structural information

about rhamnolipid composition (Arino et al., 1996).

High performance liquid chromatography (HPLC) is not only

appropriate for the complete separation of different rhamnolipid species

(Deziel et al., 1999; Lépine et al., 2002), but can also be coupled with various

detection devices (UV, MS, evaporative light scattering detection, ELSD) for

identification and quantification of rhamnolipids. While normal-phase

chromatography of rhamnolipids is quite popular in TLC, analytical column

chromatography uses mostly reversed-phase silica columns with a gradient of

acetonitrile and water (30–70% acetonitrile to 70–100% acetonitrile) (Deziel

et al., 2000; Trummler et al., 2003; Benincasa et al., 2004).

Nowadays, mixture composition and structure analysis can be

performed by tandem quadrupole mass spectrometers (developed in the late

1970s). Good rhamnolipid ionisation is achieved by electrospray ionisation

for direct infusion or HPLC-MS, as it represents a “soft” method with little

fragmentation of primary molecules. Ionised molecules are selected by a

mass analyser according to their mass-to-charge ratio (m/z) and are

subsequently detected (Schenk et al., 1995; Lépine et al., 2002).

One of the most commonly used techniques for rhamnolipid analysis

by IR spectroscopy is FTIR attenuated total reflectance (ATR) spectroscopy

38 Chapter 1

(Tuleva et al., 2002; Borgund et al., 2007). NMR spectroscopy allows for an

even more accurate structure and purity analysis than IR spectroscopy and

consists of solid-state and high-resolution techniques. Different techniques,

such as correlation spectroscopy and heteronuclear multiple quantum

coherence, can be applied for NMR spectroscopy (Monteiro et al., 2007). A

novel approach for rhamnolipid screening using MALDI-TOF mass

spectrometry was developed by Price et al., (2009).

1.2.14 Biosurfactants in bioremediation

Considerable work has been done on rhamnolipid biosurfactant

produced by various Pseudomonas aeruginosa strains capable of selectively

complexing cationic metal species such as Cd, Pb, and Zn. A 5 mM solution

of rhamnolipid produced by P. aeruginosa ATCC9027 was found to complex

92% of cadmium, a complexation of 22 lg/mg rhamnolipid (Tan et al., 1994).

Although the function of biosurfactants in microorganisms is not

fully understood, it is known that these secondary metabolites can enhance

nutrient transport across membranes, act in various host-microbe interactions,

and provide biocidal and fungicidal protection to the producing organism

(Banat, 1995a; 1995b; Lin, 1996).

There is an extensive body of literature documenting the effects of

biosurfactants, including rhamnolipids, on biodegradation of hydrocarbons,

both aliphatic and aromatic (Miller 1995a). Addition of rhamnilipid to pure

culture has been shown to enhance the biodegradation of hexadecane (Shreve

et al., 1995), octadecane (Churchill et al., 1995), n-paraffin and phenanthrene

(Zhang et al., 1997). In addition rhamnolipids have been shown to enhance

degradation in soil systems containing tetradecane, pristane (Jain et al.,

Review of Literature 39

1992), creosote (Providenti et al., 1995) and hexadecane (Herman et al.,

1997 a,b).

However, it is the ability of the biosurfactant producers to reduce

interfacial surface tension, which has important tertiary oil recovery and

bioremediation consequences (Lin, 1996; Volkering et al., 1998). Many of the

known biosurfactant producers are also hydrocarbon-degrading organisms

(Rouse et al., 1994; Willumsen & Karlson, 1997; Volkering et al., 1998).

Studies have shown that this surfactant complexes preferentially with

toxic metals such as cadmium and lead than with normal soil metal cations

such as calcium and magnesium, for which it has a much lower affinity

(Herman et al., 1995; Torrens et al., 1998). The feasibility of using a

biodegradable surfactant, surfactin from Bacillus subtilis, for the removal of

heavy metals from contaminated soil and sediments was evaluated (Mulligan

et al., 1999). After one and five batch washings of the soil, 25% and 70% of

the copper, 6% and 25% of the zinc, and 5% and 15% of the cadmium could

be removed by 0.1% surfactin along with 1% NaOH, respectively. Mulligan

et al., (2001) evaluated the feasibility of using surfactin, rhamnolipid, and

sophorolipid for the removal of heavy metals, Cu and Zn, from sediments.

Several biosurfactants have shown antimicrobial action against

bacteria, fungi, algae and viruses. Rhamnolipids inhibited the growth of

harmful bloom algae species, Heterosigma akashivo and Protocentrum

dentatum at concentrations ranging from 0.4 to 10.0 mg/L. A rhamnolipid

mixture obtained from P. aeruginosa AT10 showed inhibitory activity

against the bacteria Escherichia coli, Micrococcus luteus, Alcaligenes

faecalis (32 mg/ml), Serratia arcescens, Mycobacterium phlei (16 mg/ml)

and Staphylococcus epidermidis (8 mg/mL) and excellent antifungal

40 Chapter 1

properties against Aspergillus niger (16 mg/mL), Chaetonium globosum,

Enicillium crysogenum, Aureobasidium pullulans (32 mg/mL) and the

phytopathogenic Botrytis cinerea and Rhizoctonia solani (18 mg/mL)

(Abalos et al., 2001).

The increased incidence of human immunodeficiency virus

(HIV)/AIDS in women aged 15–49 years has identified the urgent need for a

female-controlled, efficacious and safe vaginal topical microbicide. To meet

this challenge, sophorolipid produced by C. bombicola and its structural

analogues have been studied for their spermicidal, anti-HIV and cytotoxic

activities (Shah et al., 2005).

1.2.15 Toxicity studies

Surfactants entering the environment through the discharge of

sewage effluents into surface waters and application of sewage sludge on

land have the potential to impact the ecosystem owing to their toxicity on

organisms in the environment. The toxicity data from laboratory and field

studies are essential for us to assess the possible environmental risks from the

surfactants.

1.2.16 Toxicity of LAS in terrestrial environment

LAS may reach the soil environment when anionic surfactants are

used as emulsifying, dispersing and spreading agents in the processing of

fertilizers and distribution of pesticides in agriculture. Concentrations of LAS

in raw sewage sludge are very high due to its wide spread use and strong

sorption on sludge during treatment. Mc Evoy and Giger (1985) measured

LAS concentration before and after anaerobic digestion and found no

degradation of LAS occurs during anaerobic treatment of sludges. The load

Review of Literature 41

of LAS in sewage sludge may be considerable with concentrations of more

than 10g/kg dry weight (Jensen, 1999).

Forsyth (1964) showed that the fungicidal effect of several anionic

surfactants could be due to leakage of aminoacids. The effects of

surfactants on microorganisms can be due to simple reduction of surface

tension or to specific effects of the surfactant like germicidal effects (Parr

& Norman, 1965). Toxic action of surfactants may also be due to

reactions at the cell surface like depolarisation of cell membrane. This

result in decreased absorption of essential nutrients and oxygen

consumption. Another effect may be delayed release of toxic metabolic

products from the cell leading to a build up. Both may ultimately result in

the death of the organism. It also reported that morphology, pigmentation,

exudates production, and rigidity of sporangiophores in microfungi to be

affected by anionic surfactants (Lee, 1970).

Litz et al., (1987) observed considerable short-term acute

physiological damage on ryegrass in a field experiment using an application

rate of 500 kg/ha, but no reduction in yield was found after harvest. Figge &

Schoberl (1989) conducted an extensive study of LAS effects on plants (and

potato) using a plant metabolism box. They estimated the field NOEC values

to be 16 mg/kg for bush beans, grass and radish and 27 mg/kg for potatoes.

From the terrestrial toxicity data available, LAS can be considered as not

being highly toxic to terrestrial organisms. The toxicity of anionic surfactants

towards algae has been experimented. It has been found that the toxic effect

show high differences according to type of the surfactants and the species

under investigation (Lewis, 1990).

42 Chapter 1

Unilever (1987), as cited from Mieure et al., (1990), studied the

effect of LAS on sorghum (Sorghm bicolour), sunflower (Helianthuus

annuus) and munga bean (Phaseolus aureus) by the OECD Terrestrial Plant

Growth Test (OECD 208). Using test concentrations of 1, 10, 100, 1000

mg/kg LAS in a potting soil, they determined the 21-day growth EC50 of

167, 289, and 316 mg/kg for sorghum, sunflower and munga bean,

respectively. The highest reported NOEC was 100 mg/kg for the three

species.

Due to the usage of sludge for fertilization of arable land and the use

of surfactants in pesticide formulations, relatively large efforts have been

taken to the investigation of effects of LAS to crop plants. The effects on

wild species are less investigated. Gunther and Pestemer (1990) performed a

series of toxicity tests with LAS on oat (Avena sativa), turnip (Brassica rapa)

and mustard (Sinapis alba) in a sandy loam at different concentrations and

measured the fresh weight of shoots after 14-day exposure. The lowest 14-

day EC5 value was determined for oats (50 mg/kg soil). But its EC50 value

was similar to that of turnip or mustard. Marschner (1992) mentions the

following direct effects to plants after LAS exposure: destruction of root-cell-

membrane, changes in the fine structure, and effects on physiological

processes such as photosynthesis. A comparative study to assess the toxicity

of anionic surfactant SDS and Tween 80, a non ionic surfactant, on the

nitrogen fixing ability of the cyanobacterium Gleocaspa was done. It was

found that toxicity of SDS considerably higher than that of non ionic

surfactant (Tozum- algan & Atay- Guneyman, 1994).

The terrestrial toxicity data are quite scattered and they are mainly

measured for LAS on plants, but limited toxicity also available on soil fauna

Review of Literature 43

(Kloepper-Sams et al., 1996). It was reported that by using test

concentrations of 1, 10, 100 and 1000 mg/kg LAS in a sandy loam, the 14th

day EC50 (growth) of different species were calculated and was found to be

ranged in 90.1 to 204 mg/kg (Jensen, 1999).

Elsgaard et al., (2001) reported that that short-term effects observed

for aqueous LAS on soil microbiology were modified by the dosage of LAS

with sewage sludge and by a prolonged incubation time, which may allow

for microbial recovery. Vinther et al., (2003) reported that the functional

diversity of the aerobic, heterotrophic bacterial community was rather

insensitive to LAS.

It was reported that application of high concentrations of LAS exerts

a selective pressure on the hetrotrophic platable bacterial diversity and result

in reducing bacterial diversity (Maria et al., 2008). Sanchez-Pienado et al.,

(2009) reported that the continuous application of the anionic surfactant LAS

to the soil increased the acid and alkaline phosphatase activity and

arylsulphatase activity. But the soil dehydrogenase activity was decreased on

continuous LAS exposure.

1.2.17 Aquatic toxicity of LAS

Aquatic toxicity data are widely available for anionic, cationic and

nonionic surfactants. Lewis (1991) has summarised the chronic and sub

lethal toxicities of surfactants to aquatic animals and found that chronic

toxicity of anionic and nonionic surfactants occurs at concentrations usually

greater than 0.1 mg/L. Considerable quantity of anionic surfactants released

in to ground and waste waters hence the fate of this class of pollutants has

been extensively studied (White & Russell, 1992). The efficiency of the

44 Chapter 1

wastewater purification processes concerning the concentration of alkyl

sulfate detergents in the effluent has to be controlled (Fendinger et al., 1992)

because the incomplete purification of waste waters may result in the

contamination of the groundwater by anionic detergents (Zoller, 1993). The

concentration of anionic surfactants in rivers and lakes showed marked

variation and it depended heavily on the environmental conditions such as

the intensity of offshore oil and gas exploration (Tkalin, 1993), density of sea

traffic (Decembrini et al., 1995), the distance of residential districts

(Muramoto et al., 1996; Souza & Wasserman, 1996), and the diurnal

discharge of sewage.

A reversible inhibitory effect of alkyl benzene sulphonate detergents

on photo assimilation of carbon by algae has been reported (Hicks &

Neuhold, 1966). Azov et al., (1982) reported that the regular concentrations

of hard detergents in domestic raw wastewater do not affect algal production

in High Rate Oxidation Pond (HROP). Only a considerable increase in the

use of hard nonionic detergents or feeding the pond with industrial

wastewaters which contain high concentrations of hard detergents would

cause a severe inhibition of algal production.

An indirect action of surfactants on coastal vegetation has also been

observed: uptake of marine chloride and sodium is enhanced by the erosion

of the epicuticular wax, which reduces the water surface tension (Badot &

Badot, 1995; Badot et al., 1995)

The structure-activity relationship for both acute and chronic toxicity

of a variety of alcohol ether sulfates on Ceriodaphnia dubia has been

investigated (Dyer et al., 2000). Acute toxicity was found to increase with

alkyl chain length and decrease with an increasing number of ethoxylate

Review of Literature 45

units. Chronic toxicity tests were done using Brachionus calyciflorus.

Chronic toxicity was found to be related to the percentage of the molecular

surface associated with atoms possessing partial negative charges and with

increasing the length of the ethoxylate chain. A study of the influence of Ca2+

to the toxicity of LAS on algae (D. magna) showed that the toxicity of LAS

increased with alkyl chain length and an increase in water hardness.

Concentrations of LAS ranged from 33–335 mg/L and water hardness (as

CaCO3) was varied from 200–2000 mg/L. Water hardness was found to

stress D. magna, thereby, increasing LAS toxicity (Verge et al., 2001).

The relationship between interfacial properties and toxicity of several

surfactants (including octyl-, dodecyl-, tetradecyl-, hexadecyl-

trimethylammonium chloride, octyl- and decyl-dimethyl-2-hyrdroxy ethyl

ammonium chloride, and LAS) on an immobilised artificial membrane was

reviewed. The surfactant toxicity was primarily a function of the ability of

the surfactant to adsorb and penetrate the cell membrane of aquatic

organisms (Rosen et al., 2001). Inhibitory effects of water-soluble detergents

on algae are occasionally reported in the literature.

Strong inhibitory effects of the anionic surfactant linear alkylbenzene

sulphonate (LAS) on four strains of autotrophic ammonia-oxidising bacteria

(AOB) are reported. Nitrosospira strains were considerably more sensitive to

LAS than two Nitrosomonas strains. In each strain, the metabolic activity

(50% effective concentration [EC50], 6 to 38 mg/L) was affected much less

by LAS than the growth rate and viability (EC50, 3 to 14 mg/L). However, at

LAS levels that inhibited growth, metabolic activity took place only for 1 to

5 days, after which metabolic activity also ceased (Brandt et al., 2001).

46 Chapter 1

The effect of LAS on the enzyme activity of α-amylase from Bacillus

licheniformis was studied. It was found that LAS apart from its now known

capacity for destabilising proteases (Russell & Britton, 2002), is also capable

of significantly decreasing the activity of the α-amylase studied, even at

concentrations lower than its CMC. This noteworthy loss in enzymatic

activity is most likely due to the electrostatic interactions inherent to the

anionic character of LAS (Bravo Rodríguez et al., 2006).

It is also found that a very high water hardness (> 2000 mg/L as

CaCO3) may be a stress factor giving a much lower LC50–48 h than at lower

water hardness and the same LAS concentrations. Although 0.2 mg/L is

considered as the no observed effect concentration (NOEC), lamellar gill

epithelia of rainbow trout fry hypertrophied and its swimming capacity was

reduced after 54 days of exposure (Hofer et al., 1995). Because of its extensive

application, a considerable amount of anionic surfactants are released in the

environment and can accumulate sludge sewage treatment flow (Holt et al.,

1995) causing serious pollution of sea (Romano & Garabetian, 1996) and

rivers (Odokuma & Okpokwasili, 1997). Utsunomiya et al., (1997) studied the

toxic effects of C12LAS and three quaternary alkylammonium chlorides on

unicellar green alga Dunaliella sp. by measuring 13C glycerol. The 24-h

median effective concentrations (EC50–24 h) were 3.5 mg/L for LAS, 0.79

mg/L for alkyl trimethyl ammonium chloride (TMAC), 18 mg/L for dialkyl

dimethyl ammonium chloride (DADMAC) and 1.3 mg/L for alkyl benzyl

dimethyl ammonium chloride (BDMAC): the toxic potencies were in the order

of TMAC>BDMAC>LAS>DADMAC.

LAS acute toxicity to D. magna increases with the alkyl chain or

homologue molecular weight probably due to higher interaction of heavier

Review of Literature 47

homologues with cell membranes (Verge et al., 2000). Temart et al., (2001)

conducted risk assessment of LAS in the North Sea. The LAS concentration

range in the estuaries around the North Sea ranged from 1 to 9 Ag/L, while in the

offshore sites, it is below the detection limit (0.5 Ag/L). The predicted no-effect

concentrations (PNEC) were 360 and 31 Ag/L for freshwater and marine pelagic

communities, respectively. Given that the maximum expected estuarine and

marine concentrations are 3 to > 30 times lower than the PNEC, the risk of LAS

to pelagic organisms in these environments is judged to be low.

1.2.18 Human toxicity

The amphoteric character of anionic surfactants facilitate their

accumulation in living organisms. The negatively charged head group can

bind to the positively charged molecular substructures by electrostatic forces

while the hydrophobic moiety may interact with the apolar parts of the target

organs or organisms by hydrophobic forces. Modifying of protein structure

and misfunctioning of enzymes and phopholipid membranes by anionic

surfactants causes toxic symptoms in organs and animal and human

organisms. Thus, the damaging effect of surfactants on human lymphocytes

was reported the effect of cationic surfactants being the highest (Antoni &

Szabo, 1982). Anionic surfactants mainly show eye and skin irritation

potentials. Anionic surfactants also damage human skin as determined by

differential scanning calorimetry and permeation studies. Interestingly,

nonionic surfactants were able to reduce the damaging effect of anionic

surfactants; however, the molecular basis of the phenomenon has not been

elucidated (Eagle et al., 1992).

A quantitative structure– activity relationship (QSAR) study revealed

that the hydration capacity of n-alkyl sulfates was closely correlated with the

48 Chapter 1

irritational potential, the maximum was found at C12 analogue (Wilhelm

et al., 1993). The earlier results on the bioconcentration of surfactants were

previously collected and critically evaluated (Kloepper- Sams & Sijm, 1994).

The dependence of the skin irritancy potential of anionic surfactants on the

molecular structure was well established. The results indicated that the length

of the alkyl chain of sodium alkyl sulfates has a considerable impact on their

skin irritating potential. C18 compounds caused cell injury whereas C10 and

C16 compounds caused more severe membrane destruction and protein

denaturation (Kotani et al., 1994). Sodium lauryl sulfate causes more severe

skin dehydration than dodecyl trimethyl ammonium bromide; complete

repair of the irritant reaction was achieved 17 days after surfactant exposure

(Wilhelm et al., 1994). The test of the cutaneous toxicity of surfactants in

normal human keratinocytes assessed by cytotoxicity, arachidonic acid

release and regulation of interleukin-1a mRNA revealed that the effect of

SDS was higher than that of the nonionic surfactants Triton-X-100 and

Tween 20 (Shivji et al., 1994).

The physicochemical properties and low production rate of anionic

surfactant result in their large scale production and consumption world wide.

Besides the beneficial effects they have discernible toxicity and cause

marked environmental pollution problem. So a rapid removal from the

environment after use will increase the environmental acceptability and more

safe use of these compounds.