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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.