INTRODUCTION
Chapter 1
1.1 INTRODUCTION
Society’s ever-expanding utilization of materials, energy and
space is accompanied by an increasing flux of anthropogenic organic
chemicals into the environment. The presence of man-made
hazardous compounds in the environment is a subject of intense
concern to many countries in the world .The different chemicals
discharged by the industries to the environment can upset the delicate
balance of the ecosystem. Minute quantities of these compounds
entering into the living organisms may accumulate in the various levels
of food chain. Studies about the degradation and transformation of
such compounds and the proper processing of the effluents containing
them are essential for the safe and clean environment.
Surfactants are the largest class of compounds present in raw
domestic wastewater. They are used in household and industrial
laundry and cleaning operations. The ever-increasing demand of
surfactant since the middle of this century is causing great concern
about its role in environmental pollution. Surfactants cause foam at
sewage treatment plants (1) and pollute underground water (2). Due to
their toxic nature, their presence endangers the aquatic flora and
fauna (3). Also, they create many health hazards like dermatitis (4).
They exert a solubilizing effect on many organic compounds and
increase the carcinogenic impact.(5) Having penetrated into water, the
surfactant changes its quality by causing it to have an unpleasant
smell and taste.
Surfactants basically are of three types- anionic, cationic and
non-ionic. Anionic and cationic detergents have a permanent negative
and positive charge respectively attached to nonpolar (hydrophobic)
2 Chapter 1
carbon- carbon chains. Non-ionic detergents do not have any
permanent charge. Surfactant can also be classified on the presence
of either sulphate or phosphate. Surfactant generally contains strong
lipophilic, hydrophobic group and strong, hydrophilic group.Therfore it
simultaneously attracted and repelled by water. Consequently, it tends
to its cleaning and foaming action. The hydrophobes in general use for
detergent formulation are aliphatic or alkyl aromatic hydrocarbon
radicals containing 12 to 20 carbon atoms. The hydrophobic groups
are generally anionic, such as sulfonates sulfates, ester carboxylates
or nonionic polyethers or polyalcohols (Amund et al, 1997).
Sodium dodecyl sulphate (SDS) is a widely applied anionic
surfactant that accounts for approximately 25% to 30% of the
world’s total synthetic surfactants. SDS is of high demand due to its less
cost and excellent foaming characteristics. SDS otherwise known as
sodium lauryl sulfate (SLS) is used in household products such as
toothpastes, shampoos, shaving foams and bubble baths, in laboratories
and in various other industries. The high prevalence and usage of this
detergent thus demands knowledge on its degradation aspects.
The molecular structure of SDS (C12H25NaO4 ) is composed of
two units namely, (1) A hydrocarbon chain (C1- C12), (2) A sulphate
group attached to the carbon (Schleheck et al, 2003). The molecule
has a tail of 12 carbon atoms, attached to a sulphate group giving the
molecule the amphiphilic properties required of a detergent.
Introduction 3
The pollution of soil and water with SDS is widespread in
the environment and is creating major health problems. High
concentrations of SDS in the environment cause may cause dermatitis
and inflammation. SDS is found to be toxic even in microgram
concentrations to aquatic flora and fauna. Moreover, reports
supporting and opposing the carcinogenicity of this component in high
concentrations do exist.
Degradation of SDS is a major challenge for bacteria because
this detergent solubilizes biological membrane and denatures proteins.
(Helenius et al., 1975). Several resistant mechanisms against anionic
detergents like diffusion barriers (Nikaido et al., 1985), multidrug efflux
pumps (Poole, 2004), or CIP proteases (Rajagopal et al, 2002) have
been described. All these resistance mechanisms require energy and
have been shown to protect cells which grow in the presence of
detergents Bacteria using detergents for growth face an additional
challenge. They have to invest part of their energy into protection
while taking an increased risk of damage because they have to take
up the toxic detergents to metabolize them. (Klebensberger et al.,
2006)
In relation to surfactant biodegradability, it is important to
distinguish between primary and ultimate biodegradation. The breakdown
of a substance as measured by a substance specific analytical method,
for example, the loss of the sulphate groups from surfactants, such as
Sodium dodecyl Sulphate would be a primary biodegradation step that
would lead to an immediate loss of surfactant properties. (Measured by
the methylene blue anionic surfactants (MBAS) reduction test. (Hayashi,
1975). However, ultimate biodegradation implies the complete
conversion of these surfactants into products such as CO2, H2O,
4 Chapter 1
inorganic salts and cellular products, and many more metabolic steps
would therefore be involved. nevertheless, confirmation of ultimate
biodegradation is important because primary biodegradation, although
leading to losses in surfactant properties. may still yield recalcitrant
and possible toxic metabolites. (Roig et al., 1998).
Several enzymes are involved in SDS degradation.
(Alkylsulphatases, Dehydrogenases etc.). Degradation of SDS is
started by alkylsulphatases which hydrolyses SDS to sulphate and 1-
dodecanol (Thomas and White, 1989). The liberated alcohols are
subsequently oxidized by the enzyme alcohol dehydrogenases
(Dodgson & White, 1983). Of these enzymes alkylsulphatases is of
great importance as high prevalence of this enzyme can serve as an
indicator of detergent contamination. Further utility of immobilized
forms of these enzymes in bioreactors may increase their usage
potential.
There is an urgent need to search for new possibilities of
enhanced degradation of surfactants. Bacterial degradation of
pollutions has proved to be very efficient especially in combination
with immobilization methods. Immobilization of bacterial cells offers
several advantages such as prevention of cell losses in continual
processes, and allows working with high cell densities. Furthermore,
immobilized organisms are more resistant to adverse effects occurring
during the degradation processes like changes of physico-chemical
parameters such as pH, temperature, fluctuations of substrate
concentration, presence of toxic substances etc.
To improve the biodegradation of surfactants at higher
concentration biotechnological approaches may be applied to bring
Introduction 5
out efficient solutions for biological cleanup of industrial wastewater. In
order to increase bacterial concentration in the bulk solution and to
enhance biodegradation rates, membrane bioreactors have been
successfully used as one of the alternatives. (Mortazavi et al.2008).
This work has been largely motivated by concerns over the
biodegradation of SDS like surfactant at high concentration using a
suitable bioprocess. The present work was addressed towards
biodegradation of high concentration of SDS using a novel bioreactor
for the effective bioremediation strategy. Attempts were also made to
study the metabolic products of SDS biodegradation to ensure that all
the degradative products are non toxic to the ecosystem.
1.1.1 Objectives
1. Isolation, screening, and identification of efficient Sodium
dodecyl sulphate degrading bacteria
2. Optimization of factors for efficient biodegradation
3. Analysis of metabolic byproducts of sodium dodecyl sulphate.
4. Extraction and purification of enzyme responsible for SDS
degradation.
5. Designing of a multistage reactor for detoxifying SDS polluted
wastewater.
6 Chapter 1
1.2 REVIEW OF LITERATURE
Surfactants are organic compounds that reduce surface tension
in water and other liquids (Kowalska et al; 2004). In the domestic
wastewater produced by the households, surfactants invariably exists
in significant amount due to detergents used for all kinds of washing.
Surfactants have also been widely used in textiles, fibers, paints,
polymers, cosmetics, pharmaceuticals, mining, oil recovery pulp and
paper (Sheng et al., 1999). These applications of the surfactant,
increasing its discharge in the wastewater, produce foam and enter
into the underground water resources and constituting an ecological
risk for aquatic organisms (Nasiruddin and Uzva, 2005).
Surfactant contains both strong hydrophobic and hydrophilic
moieties. According to the charge of their hydrophilic moiety,
surfactants can be classified into four categories: anionic, non-ionic,
cationic and amphoteric (Mozia et al., 2005). Anionic surfactants are
one of the most frequently employed surfactants and constitute
approximately two-third of these surfactants. Cationic surfactants
constitute less than 10% of the ionic surfactants and rest is anionic
surfactant. The predominant class of anionic surfactant is linear
alkylbenzene sulfonate and linear alkyl sulfate (Liwarska and Bizukojc
2006). As a result, their fate in the environment has been widely
studied. An example of linear alkyl sulfate is Sodium Dodecyl Sulfate
(SDS), which is a representative of anionic surfactant (Adac et al.,
2005).
The increasing releases of organic pollutions by industries
cause many health related problems. However increased awareness
of the harmful effects of environmental pollution has led to the dramatic
Review of Literature 7
increase in research on various strategies that may be employed to
clean up the environment. It is now realized that microbial metabolism
provides a safer, more efficient, and less expensive alternative to
physico- chemical methods for pollution abetment (Hebes et al.,
1987).
1.2.1 Ecological impact of SDS
Surfactants are widely used in everyday personal care and
household products as well as in a variety of industrial applications (Li,
2008). In fact, many surfactants and their degradation products have
been found worldwide in waste water discharges, sewage treatment,
plant effluents, natural water and sediments (Ying, 2006). Because
many surfactants are ubiquitous (Ying et al., 2002; Venhuis and
Mehrvar, 2004), the potential toxic effects of these chemicals have
attracted much research attention in the past several decades (Abel,
1974; Lewis and Suprenant, 1983; Lewis, 1991). Many different
mechanisms of toxicities exist for different types of surfactants and
one single surfactant can produce its toxicity through more than one
mechanism (Li, 2008).
SDS have ambiguous effect on the environment .Intensive
research has done to investigate the effects of surfactants on natural
communities (Abel, 1974; Malagrino et al., 1987; Augier, 1991; Lewis,
1992; Huang and Wang, 1994; Hansen et al., 1997; Rocha et al.,
2007; Rosety et al., 2007). The interaction of surfactants with cell
membrane lipids appears to disrupt membrane integrity, thus causing
toxic effects (Abel, 1974).The toxicity of surfactants is primarily
determined by their ability to be adsorbed and penetrating the cell
membrane (Rosen et al., 2001).
8 Chapter 1
Anionic surfactants such as SDS exert toxic and harmful effects
on higher organisms .Some of the reports are described below. SDS
may be damaging to the immune system, especially within the skin.
Skin layers may separate and inflame due to its protein denaturing
properties (CIR publication, 1983). Carcinogenic nitrosamines can form
in the manufacturing of sodium dodecyl sulfate or by its interaction
with other nitrogen bearing ingredients within a formulation utilizing
this ingredient (CIR publication, 1983). SDS can induce damage of the
skin barrier function (Léveque et al., 1993). SDS act against cell
membranes and can solubilize proteins causing their denaturation
(Cserhati et al., 2002).They can also modify the activity of an enzyme
by binding to it (Cserhati et al., 2002). The normal levels of blood
chemical parameters are altered due to topical exposure to SDS,
evidently indicate that SDS is capable of deep adsorption and
penetration through the skin barrier and can reach deeper internal
organs such as liver and kidneys to cause systemic effect.(Wadaan et
al., 2009). This poses the question whether it could be a serious
potential health threat from its use in shampoos, cleansers and
toothpastes (CIR publication, 1983).
Ducks have been observed to be at risk for hypothermia when
exposed to detergent-polluted waters in low temperatures (e.g., 0.07
Mm SDS at 0 degrees C) (Singer and Tjeerdema, 1993). Surfactants
such as SDS have the potential to enhance the penetration of water
into the birds’ feathers. This may decreases the feathers’ insulating
capacity, which decreases the bird’s ability to maintain body
temperature.
The SDS is nowadays widely spread in many different aquatic
environments, where it has an important pollution potential.
Review of Literature 9
(Ainsworth, 1992). According to literature data, anionic surfactants
give toxic effects to various aquatic organisms at concentrations as
low as 0.0025 mg l-1 (Petterson et al., 2000). Since the 1970’s decade,
investigations on the toxic effects of the SDS have been conducted
with aquatic organisms,as fish (Barbieri et al., 2002, Rocha et al.,
2007), crustaceans (Lewis & Suprenant 1983, Singh and Kumar,
2002) and molluscs (Marin et al., 1994, Da Ros et al. ,1995, Hansen et
al., 1997) ans Daphanides (PAN 2005).The main adverse effects
already reported are cellular, histological and physiological damages,
which comprise alterations in the fish gill tissues (Abel 1974)
lysosomal disturbances and enzymatic inhibition or stimulation (Drewa
1988, Da Ros et al. ,1995), growth reduction (Hansen et al. ,1997) and
alteration of the cardiac activity. Bromnage and Fuchs (1976) reported
the acute toxicity of SDS to gold fish. The LC50 values of SDS were
estimated to be 5.25 mg/l for 24 hour & 48 hour and 5.17 mg/l for 72
hr & 92 hr exposures. Madhyastha et al., (1979) reported the LC50
values of SDS for Rasbora daniencnius is 6.30 mg/l regarding
molluscs. The detrimental effects of this detergent on stimulation of
Chlorella growth and photosynthesis without affecting respiration
(Petrea,1979) decrease in salinity tolerance of water hyacinth exposed
to SDS (Muramato, 1988), alteration in growth pattern of Chlorella and
Microcystis (Lipnitskaya et al., 1989), inhibition of filter feeding habits
of bivalves (Ostroumov, 2003), marine invertebrate embryos and
larvae (Bellas et al., 2005), morphological and physiological damage
of surfactant polluted sea spray on Pinus pinea & Pinus halepensis
(Nicolotti et al., 2005) inhibition of mussel suspension feeding
(Ostromouv & Widdows, 2006), are some of the studies pointing out
the toxicity of SDS.
10 Chapter 1
The toxic effects of detergents were reported by Abdel-Hamid
(1986) on phytoplankton, on bacteria and soil fungi.The effects of SDS
to algae have been studied by Singer and Tjeerdema,1993.At low
levels, growth of some algae species was stimulated, but at higher
levels, growth was inhibited. SDS also inhibited nitrogen fixation of a
cyanobacterium Gloeocapsa at 50 ppm, corresponding to 1.73 ×10-4
mol L-1. (Tozum-Calgan et al., 1994). These results emphasized the
need to control concentrations of surfactants entering the soil system.
Improper and/or excessive use of SDS could adversely affect
the survival and function of soil organisms, including earthworms,
bacteria, algae, and protozoa. SDS misuse or spills could also result
in the damage and even death of areas of organic crops given its non-
selective herbicidal properties.Nandlal et al., (2003) reported the effect
of synthetic detergent (surf excel) on germination parameters,
seedling growth and photosynthetic pigments in Mungbean.
Mungbean seeds failed to germinate at detergent levels beyond
0.17%; it showed marked decrease in rate and vigour index
SDS showed sensitivity to the microorganisms with symptoms
including acute toxicity (Mariani et al., 2006; Tozum Calgan et al.,
1994). The sewage sludge isolates A.johnsonii and Oligotropha
carboxidovorans showed nearer 59% and 20 % viability during the
treatment with 0.2 and 2 mg mgl-1 of SDS.Toxicity of anionic and
cationic surfactant to Acinetobacter Junii, (Phosphate accumulating
bacterium) in pure culture was studied by Mariani et al., 2006;
Hrenvoic et al., 2007. Results showed a 100 % inhibition of both CFU
and P-utake rate at a concentration of 10-3 molL-1 and higher .The
decreased P-accumulation as a result of surfactants represents one
important mechanism of surfactant toxicity against P-accumulating
Review of Literature 11
bacteria. In another study consisting of various taxa ,the EC 50 value
obtained for the bacterium Vibrio fischeri was 2.6 mgL-1 of SDS
,corresponding to 9.02 ×10-6 mol L -1 . (Tozum Calgan et al.,1994).
A reduction in counts of bacteria, fungi, actinomycetes,
phosphate solubilizers and nitrogen fixers were observed on exposure
to anionic surfactants. The dehydrogenase activity of the detergent
exposed soil was significantly decreased with increased
concentrations of detergents. On exposure to detergent germination
percentage of paddy was found to be decreasing. When the treatment
concentration increased from 1mg/l to 10 mg/l the germination
percentage drops below 50% (Asok et al., 2009).
Most cleaning products are used in water solutions that are
typically disposed down-the-drain, where they combine with other
wastes. These compounds can act on biological wastewater treatment
processes and cause problems in sewage aeration and treatment
facilities due to their high foaming, lower oxygenation potentials and
making death of waterborne organisms (Eichnorn et al., 2002).
Generally the presence of surfactants helps in the degradation
of polycyclic hydrocarbons. But the degradation of PAH was inhibited
by sodium dodecyl sulfate because this surfactant was preferred as a
growth substrate (A Tiehm, 1994). This point out the presence of this
detergent in the water bodies will indirectly lead to bioaccumulation of
other hydrocarbons. Similar inhibitory effects were seen when effect of
sodium dodecyl sulfate (SDS) and biosurfactants from Pseudomonas
aeruginosa UG2 was assessed during biodegradation of 13 of the 16
USEPA priority polycyclic aromatic hydrocarbons (PAH) in a wood-
preserving soil contaminated with creosote and pentacholorophenol
12 Chapter 1
for a period of at least 20 years (Deschênes et al., 1996). Assimilation
of sulfur from alkyl- and arylsulfonates by Clostridium sp. was
analyzed by Denger and Cook (1997).
1.2.2 SDS Degradation
Microbial degradation is the most efficient way of SDS
degradation (White et al., 1990). One of the several sets of
transformations that remove organic compounds from the environment
is that group of reactions mediated by organisms.Many
microorganisms were responsible for the degradation of organic
compounds.
Payne and co workers (1963a, 1963b, 1965) and Hsu (1965)
have done extensive work with the genus Pseudomonas aeruginosa
(C12 and C12B). These isolates were obtained from enriched soils
and cultured on media containing detergent compounds as sole
sources of carbon (Payne and Feisal, 1963b). Both isolate destroyed
the foaming capacity of dodecyl sulfate; but C12B could only grow on
dodecyl benzene sulfonate (DBS) and destroy the foaming capacity of
this surfactant. Sigoillot et al., 1988 isolated and characterized 26
surfactant degrading bacteria from marine environment.A laboratory
system was developed for modeling the colonization of slide-discs by
reverine epilithic biofilm capable of the bio-degradation of anionic
surfactants. Initial experiments using batch or fed-batch systems
produced biofilms yielding at least 10 times fewer viable bacteria and
slower surfactant bio degradation rates than occur in indigenous bio
film formed on slate-discs.
Review of Literature 13
Many investigators pointed out that continued growth and
biomass accumulation of the bacteria were coincidental. This indicates
that the bacteria are actually utilizing SDS as their sole carbon source.
(Di Cocia et al., 1994; Jimenez et al., 1991 and Sigillot et al., 1990).
Mechesi et al., 1994, identified the SDS-degrading bacterial
attachment to reverine sediment in response to the surfactant primary
degradation and identified as Pseudomonas sp
Past experience have demonstrated anionic surfactants
biodegradation are exclusively conducted by bacteria (Cain et al.,1981
and Juker et al., 1994). In another work, a surfactant-degrading
Klebsiella sp. is isolated from a lagoon contaminated with surfactant
(Amund et al., 1997). Other surfactant-degrading bacteria that have
been reported are Pseudomonas sp. strain C12B (Payne and Feisal,
1963; Thomas and White, 1989) Pseudomonas sp., strain DES1 (Hales
et al., 1982) Nocardia amarae (Bhatia and Singh, 1996), Comamonas
terrigena (Roig et al., 1998), Bacillus cereus (Singh et al., 1998),
Citrobacter braakii (Dhouib et al., 2003), and other bacterial genus such
as Acinetobacter, Pantoea, Vibrio, Flavobacterium, Pseudomonas,
Enterobacter, Actinomyces, Eschericia, Shigella, Proteus, Anaebena,
Corynebacterium and Staphylococcus (Gledhill, 1974; Abboud et al.,
2007; Ogbulie et al., 2008). Table1 gives a list of various micro
organisms capable of degrading SDS.
Singh et al., 1998 isolated Bacillus cereus, capable of degrading
SDS. B.cereus was isolated from a detergent polluted pond. This strain
showed growth with exceedingly high concentration of both anionic and
nonionic detergents. Increased aeration, and the continuous-feeding of
the microcosm with nutrient amended discharge from the sewage
14 Chapter 1
treatment plant separately gave rise to increased eplithic bacterial cell
numbers and SDS biodegradation rates (Lee et al., 1996).
Zhang et al., 1998 reported the aerobic biodegradation kinetics
of four anionic and nonionic surfactants of sub and supra-critical
micelle concentration. (CMCs). Primary degradation at supra- CMCs
level ( 2500 mg/l for SDS) and ultimate biodegradation at both sub and
supra CMCs were best described by first order kinetics. Increasing
surfactant concentration from sub to supra-CMC significantly
decreased primary biodegradation, ultimate biodegradation and foam
degradation.
Many researchers used activated sludge in order to isolate
bacteria that are able to degrade anionic surfactants (Schleheck et al.,
2000). Activated sludge provides elevated nutritional carbon and other
factors necessary for growth of a wide variety of microorganisms;
therefore it is an ideal source for isolation of degrading strains. (Cain,
1981).
Schleheck et al., (2000) isolated Proteobacterium strain DS-1
that could utilize commercial SDS in aerobic culture.The influence of
natural organic matter (NOM) on the absorption of SDS and bacteria
to riverine sediment surfaces and the effects of these interactions on
the rates of SDS biodegradation in riverine environments were
investigated by Marshall et al., 2000.
Pure culture degradation studies revealed that 66% of SDS
was degraded (Payne, 2004). Enrichment cultures from river
sediments yielded several nonfermentative dentrifying bacteria,
capable of anaerobic respiratory growth on the surfactant SDS as a
Review of Literature 15
sole source of carbon and energy. Four selected isolates grew
aerobically or anaerobically (with added nitrate) on primary alkyl
sulfates of chain length C6-C12 but not on the shorter homologues
(C2-C5), or DL- octan-2-yl sulfate, or sodium triethoxy sulphate.
Hosseini et al., 2007 studied the biodegradation of anionic
surfactants by isolated bacteria from activated sludge. In this survey, 2
different bacteria, Pseudomonas beteli and Acinetobacter Johnsoni
were isolated from Tehran municipal activated sludge. Cell aggregation
of Pseudomonas aeruginosa strain PAO1 was found as an energy
dependent stress response during growth with sodium dodecyl sulphate
(Klebensberger et al., 2006). The growth started with the formation of
macroscopic cell aggregates which consists of respiring cells
embedded in an exracellular matrix composed of acidic polysaccharides
and DNA. Damaged cells accumulated in these aggregates compared
to those cells that remained suspended.These isolates were able to
degrade 97.27 & 96.4% of the SDS after 10 days of growth
respectively. The highest peak of SDS degradation occurred during the
log phase of bacterial growth.
Ojo et al., 2009 studied the biodegradation of synthetic
detergents in waste water. The heterotrophic bacterial count from the
76 randomly collected effluent samples was 42.9x106 cfu/ml, while the
mean bacterial detergent-degrader population was 20.94x106 cfu/ml.
Klebsiella oxytoca was able to degrade approximately 80% of 2.0 gl-1
SDS after 4 days of incubation concomitant with increase in cellular
growth. (Shukor et al., 2009).
Kramer et al., 1980 have described the growth of Enterobacter
cloacae in 25% SDS. The bacteria appeared to tolerate SDS rather
16 Chapter 1
than metabolize it. The process was energy dependent, and cell lysis
occurred during stationary phase. Extreme detergent resistance may
be characteristic of the Genus Enterobacter.
Another survey of riverine bacteria by White et al., (1985)
showed that the ability to grow in the presence of the surfactant
sodium dodecyl sulfate (SDS) was widespread but the proportion of
SDS-resistant isolates was significantly greater at a polluted site, as
compared with clean source water
The capacities of epilithic and planktonic river bacterial
populations to degrade sodium dodecyl sulfate (SDS) in samples
taken at two times during 1987 from one clean and four polluted sites
in a South Wales river were estimated in die-away tests under
simulated environmental conditions (Anderson et al., 1990). There
was a relatively slow disappearance of SDS in die-away tests for both
planktonic and epilithic populations taken from the clean source site,
as compared with those taken from the downstream polluted sites, for
which the rate of biodegradation was accelerated, sometimes after an
apparent initial lag period.
Anderson et al., 1989 investigated the die-away kinetic analysis
of the capacity of epilithic and planktonic bacteria from clean and
polluted river water to biodegraded SDS.
Review of Literature 17
Table 1. Microorganisms in the biodegradation of SDS
Sl.No Microorganism Reference
1 Comamonas terrigena Matcham et al ., 1977
2 Pseudomonas spp.DESI and C12B Hales et al., 1985
3 Coryneform Bia White et al., 1987
4 Pseudomonas strain Stavskia et al .,1989
5 Pseudomonas BPS – D Marchesi et al .,1991
6 Pseudomonas strain Marchesi et al .,1994,
7 Vibrio, klebsiella, flavobacterium, Pseudomonas, E-coli, shigella, citrobacter
Amund et al., 1997
8 Bacillus cereus Sing et al., 1998
9 Pseudomonas C12B Jerabkova et al., 1999
10 Proteobacterium strains DS-1 Schleheck et al., 2000
11 Pseudomonas AE-A, AE-B, AE-D Ellis et al., 2001
12 Citrobacter braakii Dhouib et al., 2002
13 Rhodococcus ruber DSM 44541 Pogoreve et al., 2003
14 Pseudomonas strain Ellis et al.,2004
15 Pseudomonas aeruginosa strain PA01
Klebensberger et at., 2006
16 Klebsiella oxytoca,Acinetobacter calcoacetiacus,Serratia odorifera
Khleifat et al., 2006
17 Pseudomonas beteli, Acinetobacter Johnsoni
Hosseini et al., 2007
18 Acinetobacter calcoaceticus and Pantoea agglomerans
Abound et al., 2007
19 Enterobacter cloacae Khleifat et al., 2008
20 Stenotrophomonas maltophIlia Farzaneh et al., 2009
21 Enterobacter liquifasciens, klebsiella liquifasciens, klebsiella aerogenes,
Escherichia coli
Ojo et al., 2009
18 Chapter 1
1.2.3 Molecular studies based on SDS degradation
Loss of function at a rate higher than expected rate of mutation
are generally accepted properties that provide genetic evidence for
the presence of plasmid involved in conferring a particular function to
a cell. (Williams, 1978). Pseudomonas C12B, an SDS degrading
strain, harbors a plasmid coding for degradation of medium chain
length n-alkanes. (Kostal et al., 1998). The plasmid was designated as
pDEC.Its size was estimated at several hundred kb according to the
mobility in agarose gels. (Payne and Feisal, 1963; White et al., 1985).
Genes coding for degradation of halogenated aromatic compounds
are often plasmid-borne (Chakrabarty, 1972). Degradation of salicylic
acid or n-alkanes (C6, C18) which are so far reported to be carried on
one or more well characterized plasmids such as the SAL and OCT
plasmid respectively (Chakrabarty 1976).
Similarly antibiotic resistance is a common plasmid encoded
character. Plasmid encoded character often plays significant role in
bacterial adaptation to xenobiotics in the environment. (Kado and Liu,
1981). There have been reports on the involvement of bulky plasmids
in the biodegradation of e-caprolactam and related compounds
(Boronin et al., 1984; Esikova et al., 1990, 1993; Grishchenkov et al.,
1993). Ojo et al., 2009 isolated plasmid DNA from synthetic detergent
degraders in waste water from a tropical environment. The size of the
plasmid was 14-15 kbp.It was evident that the genetic information for
detergent-hydrocarbon utilization was not plasmid mediated since the
cured isolates grew on detergent supplemented medium after plasmid
was removed. Two alkane utilizing plasmid encoding genes have been
described, CAM-OCT and a plasmid in a strain of P.maltophilia (Lee et
Review of Literature 19
al., 1996). Pseudomonas putida MCM B-408 capable of utilizing e-
caprolactam (monomer of nylon-6) as the sole source of carbon and
nitrogen was found to harbor a single 32-kb plasmid (Kulkarni et al.,
1998). Analysis of alkyl sulphatase in parent and cured strain
confirmed that both enzymes are encoded by the chromosome. The
ability of Pseudomonas C12 B to utilize alkyl benzene sulphonate also
appears to be coded by the chromosome (Kostal et al.,
1998).Deshpande et al., 2001 reported the Plasmid-mediated
dimethoate degradation in Pseudomonas aeruginosa MCMB-427 and
located in 6.6 kbp plasmid.
Curing agents used by Deshpande et al., (2001) were
plumbagin (50 µg /ml), acridine orange (50 µg /ml)and ethidium
bromide (500 µg /ml).Curing of the plasmid by plumbagin or ethidium
bromide resulted in the loss of ability of MCMB-427 to degrade
dimethoate. Mitomycin C is the only mutagen that has been reported
to cure caprolactam degradative plasmids from Pseudomonas species
(Boronin et al., 1984). SDS at 0.1 g ml was not cured by Mitomycin C
(Kulkarni et al., 1998). Pseudomonas C12B was also cured by
mitomycin C resulting in the loss of pDEC plasmid. (Kostal et
al.,1998). Other Pseudomonas plasmids such as CAM, OT, SAL, NAH
and TOL were removed by treatment with Mitomycin C. (Williams,
1978).
1.2.4 Factors affecting the SDS biodegrdation
There were many reports to examine the impact of
environmental factors on the process of surfactant biodegradation in
seawater, namely: temperature, pH, aeration, concentration and the
presence of other sources. (Sales et al., 1998).
20 Chapter 1
a) Temperature
The dependence on temperature on the rate of the degradation
process is related to the metabolism of the micro-organisms
responsible for degradation. Many other mesophilic SDS degrading
bacteria reported in the literature required 300C for optimum SDS
degradation.Citrobacter braakii,Delftia acidovorans strain SPB1,
Pseudomonas strain C12B, Acinetobacter calcoaceticus and Pantoea
agglomerans required 30oC for optimal SDS degradation (Payne and
Feisal, 1963; Schulz et al., 2000; Dhouib et al., 2003; Abboud et al.,
2007) . Comamonas terrigena strain N3H showed optimum growth at
28O C for optimum SDS degradation (Roig et al., 1998). Marchesi et al.
(1997) also reported a lower temperature (25oC.) for the degradation
of SDS by Pseudomonas sp.
Shukor et al., 2009 have isolated an SDS-degrading Klebsiella
oxytoca from an SDS-polluted water sample from Malaysia. The effect
of temperature on the cellular growth of strain DRY14 on 2.0 g l -1 SDS
was studied at temperatures ranging from 10 to 50oC. Cellular growth
was increased as the temperature was increased from 10oC reaching
an optimum at 37oC before a dramatic decline in growth was seen at
higher temperatures.
Abboud et al., 2007 observed the 300C or 370C incubation
temperature for optimum SDS and LAS biodegradation by mixed cultures
of Acinetobacter calcoaceticus and Pantoea agglomerans. Temperature at
42 0C did not produce more than 10% of the SDS degradation where as in
the case of LAS 50% degradation observed at 420C. The optimum SDS
degradation temperature for Pseudomonas betelli and Acinetobacter
sp.was also 30oC (Hosseini et al., 2007).
Review of Literature 21
b) pH
The effect of pH on SDS degradation was studied by many
investigators. The preference to neutrality or near neutrality in terms of
optimal growth on SDS is shared by many other SDS-degrading
bacteria such as Pseudomonas strain C12B at pH 7.5 to 8.0 (Payne
and Feisal, 1963), Citrobacter braakii at pH 7.0 (Dhouib et al., 2003),
Comamonas terrigena strain at pH 7.4 (Roig et al., 1998) and,
Klebsiella oxytoca at pH 7.25 (Shukor et al., 2009). Other pHs like 7.5
and 6.5 gave intermediate effect on the degradation ability of LAS and
SDS while pH 5.5 produced the lowest extent of degradation (Shukor
et al., 2009). In another report, growth on SDS by a novel consortium
of Acinetobacter calcoaceticus and Pantoea agglomerans required pH
8.5 for efficient degradation. Growth was dramatically reduced at lower
or higher pHs than the optimum (Abboud et al., 2007).
In bioremediation works, a cheap source of pH controlling
chemical such as calcium carbonate can be added to soil during
bioremediation to achieve near neutrality in order to optimize
remediation .pH was studied using an overlapping buffer system
consisting of phosphate and carbonate (50 Mm) spanning the pH
range from 6.5 to 8.5. (Abboud et al., 2007).
c) SDS concentration
Many studies show that there is an inverse relationship
between SDS concentration and degradation. The growth of
Enterobacter cloalace in 25% SDS is described by Kramer et al.,
(1980).The bacteria appeared to tolerate SDS rather than metabolize
it. The process was energy dependent, and lysis occurred during
stationary phase. Klebsiella oxytoca degraded almost 80% of 2.0 g l-1
22 Chapter 1
SDS was after 4 days of incubation concomitant with increase in
cellular growth. Ten days of incubation were needed to degrade SDS
completely. Growth then enters plateau on 8 th days onwards; possibly
due to substrate depletion. Disappearance of bubbles during
incubation period also indicates the absence of SDS due to
degradation of this surfactant from the culture. (Shukor et al., 2009)
Khleifal et al., 2006 studied the biodegradation of sodium lauryl
ether sulfate by 2 bacterial isolates (Acinetobacter and klebsiella).
Effect of SLES concentration on the rate of biodegradation was
studied and observed that maximum SLES degradation occurs within
a concentration range of 1000-3000 ppm in 96 hours, whereas higher
concentration of SLES (5000, 7000 ppm) were degraded over a longer
incubation time (120-144 hours). This could be interpreted as the
higher concentration of SLES possibly leading to a decrease in
substrate bioavailability, which slows down its removal
Effect of substance mass on surfactants biodegradation was
observed by Abboud et al., 2007. A much higher degradation of SDS
at 4.0 g l-1 has been reported using a consortium of Acinetobacter
calcoaceticus and Pantoea agglomerans with complete degradation
occurring after approximately 5 days. The higher ranges of SDS
concentration (3000–8000 ppm) were degraded by the same mixed
culture but still an inverse relationship existed between the increase in
mass and the extent of degradation. Many scientists investigate the
toxicity effects of high surfactant amounts on bacterial growth. The
mixed culture showed better resistance to high surfactant mass toxicity
than either bacterial strain alone. Khleifal et al., 2006 studied the toxic
effect of SDS at mass range of 4000–8000 ppm to mixed bacterial
consortium (Acinetobacter calcoacetiacus, klebsiella oxytocan (A-K)
Review of Literature 23
and Serratia odorifera and Acinetobacter calcoacetiacus (A-S)) growing
in broth medium. The growth rate achieved for A-K and A-S were 0.26
h-1 and 0.21h-1 respectively. A mixed culture of activated sludge from a
municipal waste water treatment plant grown on higher SDS
concentrations (500-2500 mg/l) increased the microbial specific growth
rate in comparison to cultures, incubated with lower SDS
concentrations( in the range of 0.379 to 0.0567h-1) (zhang et al ., 1999).
The growth expressed as colony forming unit (CFU), was found
to be proportionally suppressed with increasing the amounts of
surfactant present in culture. Such mass intolerance was more
pronounced on the Acinetobacter than on Pantoea strain particularly,
when elevated amounts of SDS were encountered. (Abboud et al.,
2007).
The effect of three different rates of shaking (aeration) on
surfactants biodegradation was investigated. High and medium
shaking rates were also substantially effective on SDS breakdown
giving 100% degradation while the low shaking rates gave less than
10% degradation of this surfactant. (Abboud et al., 2007)
The effect of various carbon sources including glucose,
sucrose, maltose, mannitol and succinic acid were added separately
at a fixed concentration of 0.2% to broth medium before the
biodegradation. The addition of same carbon sources caused a slow
down in the degradation process of SDS (140 h). (Abboud et al.,
2007).
24 Chapter 1
1.2.5 Analysis of metabolic byproducts
Gas chromatography (GC) provided the convincing evidence
of synthetic degradation in sewage treatment plant.The detection of
unusual peaks in the GC profiles provided the scientific evidence of
inclusion of certain hydrocarbons in detergent formulation outside that
of industry specifications.The unusual peaks are attributable to
inclusion of certain chemical optical brightners.(Ojo et al., 2009 ).
Sawyer et al., 1956 have shown that gas chromatography provides a
very effective analytical method for determining intermediate in and
time required for, degradation in such system.
The biodegradation of SDS is initiated by sulfate release
catalyzed by alkyl sulfatase enzymes, to produce an alcohol which is
readily assimilated by the central metabolic pathways found in bacteria
(Anderson et al., 1999). Sulfate ester detergents like sodium dodecyl
sulphate are considered as readily biodegradable .Several SDS
degradation is started by an alkyl sulfatase which hydrolyses SDS to
sulfate and 1-dodecenol. This primary alcohol is oxidized to lauric acid
and further degraded by oxidation to acetyl COA residues. (Thomas
et al., 1989).
Studies with four different naturally occurring organisms have
shown that the three possible routes for primary biodegradation of
alkyl ethoxy sulphate surfactant (etherase, sulphatase, ω and β
oxidation). Thomas et al., 1989 studied the metabolic pathway for the
biodegradation of sodium dodecyl sulphate. Analysis of the extractable
lipids established the sequential production from SDS to 1- dodecanol,
dodecanal and dodecanoic acid.
Review of Literature 25
Metabolic pathway of alkyl sulphate is studied by Lijmbach and
Brinkuis, 1973. They observed that alkyl sulphatase hydrolyse
inorganic sulphate from its ester linkage with its alcohol, the later
being readily assimilated through metabolic pathway. Biodegradation
of alkyl polyethoxy sulphate, by microorganism is intiated by the
breakdown of related nonionic alkyl ethoxy alcohol surfactants by
cleavage of the alkyl ether bond. (Patterson et al., 1970; Tobin et al.,
1976; Williams et al., 1966). The liberated polyethylene glycol moieties
are subsequently degrade more slowly.
A number of different thin layer chromatographic systems on
silica gel and cellulose plates were employed to separate and
identify35 S labeled metabolites of SDTES. Metabolites have been
identified as mono-di and tri ethylene glycol monosulphate (major
metabolites) and acetic acid -2-ethoxy sulphate and acetic acid
diethoxy sulphate. Major metabolites were produced by rupture of one
or other ether linkage present in the surfactant molecule by a single
etherase enzyme. Acetic acid -2-ethoxy sulphate and acetic acid
dietoxy sulphate were formed by the oxidation of the free alcohol
groups of di and triethylene glycol monosulphates respectively and
increased in amount during the stationary phase of growth. Inorganic35
S-sulphate also appeared from the parent surfactant by the action of
sulphatase enzyme. (Hales et al., 1982). Primary degradation of
SDTES by Pseudomonas sp.DES1 involved in the action of one or
more sulfatase and one or more ether cleaving enzymes. By analogy
with alkyl sulphatase of Pseudomonas sp.C12B (Dodgson, 1981), the
sulphatase may expected to liberate the free alcohol and inorganic
sulphate. In the case of SDTES the products of etherase are alcohol,
aldehyde or carboxylic acid.
26 Chapter 1
Pathway of SDTES was identified and the intermediate could
have produced by the subterminal oxidation and cleavage between
C-11, C-10 or C-9 and C-8 of the dodecyl chain followed by beta
oxidation (Hales et al., 1986). Normally beta oxidation route appears
to be used only when other mechanisms are unavailable. Example for
beta oxidation were the bacterial degradation of alkyl benzene
sulphonate (Cain 1981) and mammalanian degradation of alkyl
sulphate (Denner et al, 1969; Maggs et al., 1982) alkyl ethoxy
sulphates (Taylor et al., 1978). Steber and Wierich (1985) observed
the combination of omega /beta oxidation and ether cleavage during
the degradation of nonionic alkyl ethoxylate by mixed sewage culture.
In order to obtain a detailed insight into the environmental fate
of complex surfactant mixtures a sensitive and specific analytical
technique is required to enable the detection and identification of
individual components and metabolites. Eichhorn and Knepper., 2000
used LC-MS for separating and identifying the components.
Extensive investigation of anionic surfactant,LAS with respect
to its degradation behaviour and metabolic pathway under aerobic
conditions that occur in sewage treatment plants was carried out by
Schoberl.,1989.Sulfophenyl carboxylates (SPCs) have been identified
in the environment as primary degradation intermediates of LAS.
(Dicorcia and Samperi., 1994).
The intial step in the aerobic metabolism of a Cn–LAS is the
omega oxidation of the terminal methyl group of the alkyl chain,
releasing a Cn SPC.This metabolite was further breakdown by beta
oxidation leading to the short chain of SPC. (Eichhorn and Knepper.,
2000). Analytical detection of SPC with non specific detectors was
Review of Literature 27
difficult due to the lack of commercial available standard. Using mass
selective detector allowing identifying the compounds via its molecular
ion, and additionally by one or more characteristic fragments.
1.2.6 Role of enzymes on SDS degradation
Microorganisms use a range of sulfatases of widely varying
substrate specificities to use sulfate esters as carbon and/or sulfur
source. At least three mechanically distinct groups of sulfatases exist:
arylsulfatases, the best studied group (hereby assigned to group I),
are predominantly eukaryotic. They are characterized by an active-site
serine or cysteine post translational modified to formylglycine that
mediates the cleavage of the CO-S bond of sulfate esters, producing
inorganic sulfate and the corresponding alcohol The Fe(II)
α-ketoglutarate-dependent dioxygenase super family of enzymes
constitutes a second group (group II) of sulfatases. These enzymes
oxidatively cleave sulfate esters into inorganic sulfate and the
corresponding aldehyde and require α-ketoglutarate as a cosubstrate
.Third group of alkyl sulphatase is microbial origin.
Alkyl sulphates are among most environmentaly acceptable
synthetic surfactants because they are readilly biodegraded by
environmental and sewage baceria.Biodegradation is intiated by alkyl
sulphatase enzymes. Sulphate esters make up a large propotition of
sulfur that is found in aerobic soils, and so it is not surprising that many
soil microorganisms have evolved enzymes that catalyse the hydrolysis
of theses compounds ,either to release sulfate moitey as a sulfur source
for growth. or as the first step in their mineralization. (Dodgson et al.,
1982; Dodgson and White,1983; White et al .,1985 ).
28 Chapter 1
Sulphatase catalyse hydrolytic cleavage of the sulphate ester
bond by liberating inorganic sulphate and corresponding alcohol.They
are present in wide variety of species ,ranging from bacteria to
humans. (Kertesz., 1999; Roy., 1971). In contrast to the role of human
sulphatase, which are involved in the sulphation of sulfated
glycolipids, the primary role of bacterial alkyl sulfatase is the
assimilation of sulfur(Denger et al., 1997; Dodgson et al.,1982) or in
the provision of carbon and energy sources for cell growth. (Fitzgerald
et al., 1977).
Strains that are able to grow with alkyl sulphatases such as
SDS as the source of carbon are widespread in the environment,even
in samples isolated from uncontaminated sites. (White et al., 1983)
Alkyl sulphatases have mostly been found in gram negative
bacteria; the only exceptions to date are strains of Bacillus cereus
(singh et al., 1998) and coryneform strain BI a (white et al., 1987).
Alkyl sulphatase activity has also been found in Psedomonas
aeruginosa, Aerobacter aerogenes, Comamonas terrigena and
Pseudomonas putida .Most of the work on alkyl sulfatases was carried
out with gram negative bacteria, Pseudomonas sp strain C12B
(NC1MB 11753 = ATCC 43648) and Comamonas terrigena (NCIMB
8193) (cloves et al., 1980.
White et al., 1992 studied the biodegradation of short-chain
alkyl sulphates by coryneform species .Growth of the organism was
accompanied by disappearance of butyl-1-sulphate. Non-denaturing
polyacrylamide gel electrophoresis of extracts of cells grown on butyl-
1-sulphate, followed by incubation of gels in butyl-1-sulphate and
Review of Literature 29
precipitation of liberated SO4-2 as BaSO4 revealed a single white band
of alkylsulphatase activity.
A strain of Bacillus cereus isolated from a detergent polluted
pond rapidly degraded SDS to dodecan-1-ol by the enzyme
alkylsulfatase (Singh et al., 1998). Marches et al., 1994 studied the
SDS-degrading bacteria attached to reverine sediment in response to
the surfactant or its primary biodegradation. Three of the Pseudomonas
strains were chosen for their known ability to express alkylsulphatse
enzymes capable of hydrolyzing SDS. The alkylsulphatase phenotypes
were confirmed by gel zymography of all extracts.
Kahnert and Kertesz (2000) characterized a sulfur-regulated
oxygenative alkylsulfatase from Pseudomonas putida S-313. Ellis et al.,
2002 isolated novel alkylsulphatase capable of degrading branched
primary alkylsulphate surfactant 2-butyloctyl sulphate.
Pogorevc and Faber (2003) isolated and purified
alkylsulphatases from the whole cells of Rhodococcus ruber DSM
44541 capable of hydrolyzing ()-2-octyl sulphate in a stereo- and
enantiospecific fashion .When grown on complex medium, the cells
produced two sec- alkylsulphatases and (at least) one primary
alkylsulphatase in the absence of an inducer, such as sec- alkyl
sulphate or a sec- alcohol.
The alkylsuphatases of Pseudomonas C12 B appears to have
a periplasmic localization in the cell (Fitzgerald et al., 1975). Bacteria
containing the alkylsulfatases that initiate SDS degradation were also
widespread. The polluted site yielded significantly more alkylsulfatase-
containing strains than the clean site, and this difference was due to a
30 Chapter 1
greater number of strains at the polluted site bearing constitutive
rather than inducible enzymes. The bacteria involved in surfactant
biodegradation are often capable of producing a multiplicity of
alkylsulphatases.
Thomas et al. (1989) studied the location of long chain alkyl
sulphatase in the bacterial cell. A location for long-chain
alkylsulphatases in the outer cell wall is rational because it eliminates
the threat to the cell while still allowing assimilation of the carbon as
the long-chain (non-surfactant) alcohol. There was no activity inside
the cells. In contrast, the short-chain (C1-C4) alkylsulphatase in a
coryneform isolated for its ability to grow on butyl-1-sulphate was
located entirely in the cytoplasm. The butyl sulphatase was apparently
associated with granules of poly-P-hydroxybutyric acid. The different
locations for the long- and short-chain alkylsulphatases may be related
to the relative potential toxicities of their ester substrates.
Bacillus cereus metabolized SDS into 1-dodecanol alkyl
sulphatase within 8 hours of incuation.The appearance of enzyme
activity in the SDS supplemented medium started after 1 hour of
incubation and last up to 8 hours. Therefore the enzyme sulphatase is
inducible in nature because the appearance of enzyme activity was
not instant. Rather it appeared after 1 h of incubation in SDS-
supplemented medium and thereafter there was abrupt and linear
increase lasting up to 8h. Addition of chloramphenicol (25 mg/ml) to
SDS-supplemented cells did not show alkylsulphatase activity
suggesting that synthesis of enzyme upon SDS addition is required to
synthesis of enzyme upon SDS addition is required to manifest
activity. (Singh et al., 1998).
Review of Literature 31
Pseudomonas ATCC 19151 and Pseudomonas BPS-D contain
inducible alkylsulphatase enzyme, it converts SDS into1-dodecanol
which is responsible for the attachment of bacteria to the riverine
sediment.The period of maximum attachment coincided with that of
maximum production of dodecanol .The alcohol was further
metabolized and its concentration declined, the bacteria detached
from the sediment surface. (Marchesi et al., 1994).
The maximum rate of SDS hydrolysis and dodecanol
accumulation correspond to the point at which bacterial surface has
become most hydrophobic and which the residual SDS concentration
had reached zero, the surface hydrophobic index returned to
zero.Increase in hydrophobicity is due to the conversion of SDS to
dodecanol close to the bacterial cell surface by primary alkyl
sulphatase, which is located in the periplasm. (Thomas et al., 1986)
In addition, the incidence of strains containing multiple
alkylsulfatases was much higher at the polluted than at the clean site.
(White et al., 1985). The bacteria involved in the biodegradation of
SDS are often capable of producing a multiplicity of alkylsulphatases.
Thus Pseudomonas C12B can produce five such enzymes (Dodgson
et al., 1974 and Pseudomonas DESI four (Hales, 1981). Of the five
alkylsulphatases produced by Pseudomonas C12B, two (designated
P1 and P2) are active towards primary alkyl sulphates, whereas the
other three (SI, S2 and S3) act on secondary alkyl sulphates
(Matcham et al., 1977a; Dodgson & White, 1983). P1 is constitutive
and P2 is Inducible in nature. The latter enzymes exhibit positional and
stereo-specificity, SI being active towards D-alk-2-yl sulphates, S2
towards L-alk-2-yl sulphates and S3 towards symmetrical and near
symmetrical secondary alkyl sulphates.All three enzymes surprisingly
32 Chapter 1
operate by rupture of the C-O bond of the ester sulphate linkage, and
this is accompanied by inversion of configuration (Bartholomew et al.,
1978; Shaw et al., 1980).
On some occasions, gels incubated with SDTES and Bacl2
contained a faint, transient band of precipitated BaSO4 in a position
corresponding to the P2 enzyme. On other occasions, the band did not
appear at all even though P2 was present, suggesting that the later
enzyme may not responsible for induction of the sulphatase activity.
Cell extracts from aerobic or anaerobic cultures of the most sulfatase
active towards octylsulphate but inactive towards propyl, butyl and
pentenyl sulfates and seconday alkyl sulphates (Dodgson et al., 1984)
The most striking difference between the two Pseudomonas
C12B enzymes is that relating to their modes of action. The P1
enzyme ruptures the O-S bond of the C-0-S linkage and hence would
be regarded as exhibiting the normal behaviour for a hydrolytic
enzyme active towards esters of the type alcohol--acid. (Bateman et
al., 1986). Dodgson et al. (1982) have previously drawn attention to
the fact that of six other known classes of hydrolytic enzymes active
towards this type of ester, all operate by scission of the 0-acid bond. In
sharp contrast with the P1 enzyme, the P2 enzyme attacks the C
(alcohol)-O bond, in common with the mechanisms of action of the
secondary alkylsulphatases of Pseudomonas C12B that have so far
been studied in detail (the SI and S3 enzymes). These C-0-bond
splitters are thus emerging as atypical of sulphatases in particular and
hydrolytic enzymes in general.
Alkyl sulphates phenotypes were confirmed by gel zymography.
Marchesi, 1994 studied the location of alkyl sulphatase by gel
Review of Literature 33
zymography by the generation of white bands of insoluble dodecanol in
the gel. The same method was used by Hales et al (1985) to study the
alkyl sulphatase activity .Sulphatase activity towards SDTES was
detected by incubating the gels at 300C in a solution containing 10 m M
–SDTES and 20Mm BaCl2 in 20 Mm tris /HCl, pH 7.8. Although the
liberated alcohol was not sufficiently insoluble to form a visible
precipitate, white bands were formed because the presence of Ba 2+
ions led to the precipitation of liberated SO4 2-
Extracts of Pseudomonas sp. strain C12 B cells grown on SDS
supplemented basal salt medium in batch culture produced P1 and P2
bands when gels were incubated with SDS.When the same cell
extracts were separated on gels and gels were with 2- butyl octyl
sulphate instead of SDS, no alkyl sulphatase bands were
observed.(Ellis et al ., 2001)
Stoichiometric liberation of butanol and SO42- from butyl
1-sulphate confirmed the true hydrolytic nature of reaction catalysed.
(White and Russel., 1992). Stoichiometric incorporation of 18 O from
H218O into SO4
2- during the degradation of butyl -1- sulphate by pure
enzyme showed that fission of the C-O-S ester linkage occurred at the
O-S bond.The O-S mechanism is common in all aryl sulphatase
(Dodgson et al., 1982)c,the P1 alkylsulphatase of Pseudomonas C12
B (Cloves et al., 1988) the 2,4 dichloro phenoxy ethyl sulphate in
P.putida (Lillis et al.,1983) and the D-lactate -2-sulphate sulphatase in
P. syringae (Crescenzi et al., 1984).
Primary alkylsulphohydrolase of the soil bacterium Pseudomonas
C12 B was purified to homogeneity by column chromatography on
DEAE- cellulose Sephadex G-100 and butyl agarose (Cloves et al.,
34 Chapter 1
1980a). The results showed the presence of a hydrophobic site on the
enzyme capable of accommodating an alkyl chain of considerable
length.
Matts et al., 1994 described the purification and characteristics
of the novel short chain alkyl sulphatase from strain Corenybacterium
B1a and compared with its properties with other alkyl sulphatase.
Butyl –sulphate was routinely employed as a substrate for the assay of
alkyl sulphatase activity.Alkyl sulphatase for short chain alkyl suphate
was measured by BaCl2 /gelatin method. (Dodgson, 1961). Purification
was done with streptomycin sulphate and DEAE cellulose column
chromatography and hydrophobic column chromatography.Purification
factor achieved from crude extract to final preparation was in the
range 65 to 75 fold. SDS/E of denatured protein yielded a single band
corresponding to 77.6 kDa.
Pogorev (2003) was also tried to purify secondary alkyl
sulphatase from Rhodococcus rubber by using DEAE –cellulose
batch, phenyl sepharose Q6, Blue sepharose and superdex 200.
These purification sequence allowed to purify alkyl sulphatase RS2
119 fold from crude cell extract in reproducible way.SDS/PAGE
revealed the presence of a single band at about 43 KDa for RS2.
Highly enantioselective stereo-inverting sec- alkylsulphatase
activity of hyperthermophilic Archae was noted by Wallner et al., 2005.
Wallner et al., 2005 also noted similar highly enantioselective sec-alkyl
sulfatase activity of the marine planctomycete Rhodospirulla baltica.
Gadler and Faber (2007) have reviewed the various scopes
and dimensions of alkylsulphatases, which can be employed for the
Review of Literature 35
enantio-convergent transformation of racemic sulfate esters into a
single stereoisomeric secondary alcohol, with a theoretical yield of
100%.
1.2.7 Effect of starvation on SDS biodegradation
One of the major environmental factors influencing microbial
physiology and cell growth is nutrient availability. Most natural
environments are characterized by low bioavailability of nutrients.
(Morita,1988). There are many papers reported the effect of
starvation on morphology (Morita 1985), alternation of the cell surface
structures (Tuomanen et al.,1988) photosynthesis and genetic
regulation (Nystrom et al., 1989), formation of cross protection against
heavy metals such as Cd2- (Feriane et al., 1995).
Truex et al., 1992 studied the effect of starvation on quinoiline
degradation by Pseudomonas cepacia finding that long time starvation
(60-80 days) leads to more efficient conversion of quinoline to
degradation products with quinoline concentrations of 39 and 155
Mm.However, long time starved cell required 3-5 × longer time for
induction of quinoline degradation in comparison with short time
starved cells.It was evident that starvation positively affected the
biotransformation potential of Commomonas terrigena cells towards
DHSS. (Roig et al., 1998)
1.2.8. Treatment of SDS wastewater
The problem of considerable contamination of the aqueous
environment with organic pollutants requires the development of quick
and simple methods for the removal, separation and determination of
these compounds. The main classes of organic compounds that most
36 Chapter 1
of the industries used and discharge into the effluents are surfactants,
phenols and dye. All these compounds are troublesome contaminants
which pose not only to toxicity and health hazards but also hamper the
environmental treatment processes. Surfactants are generally
removed by chemical precipitation foam separation ultrafiltration,
physical adsorption and biological method.
a) Physical adsorption
Removal of organic pollutants by adsorption on active carbon is
used successively for a long time (Wheeler et al., 1959). Literature
study indicate several types of adsorbent for removal of surfactants
from waste water. (Schewnger et al., 1970; Waymen, 1984;
Krishnamurthy, 1993) The adsorption of alkyl sulphate on various
adsorbents such as alumina (Dick et al., 1971) and activated carbon
(Sing et al., 1974) were studied and they showed 80 and 89%
removal. The sodium form of type A Zeolite (Savitsky, et al., 1981)
was also tested for alkyl sulphate removal, but in this case, the
efficiency was not good. The main disadvantage of these adsorbing
materials is that they are not cost-effective. A very low-cost scrap
rubber in the form of granules had been selected to remove AS from
the water environment, and the efficiency of the rubber granules was
evaluated. (Sarkar et al., 2000). Adsorption of nonionic surfactant from
its aqueous solution into commercial rubber was reported by Shalaby
and El-Feky (1999). Purakayastha et al., (2005) studied the surfactant
removal efficiency of four adsorbents–granular activated charcoal,
waste tire rubber granules, wood charcoal and silica gel. Waste tire
rubber granules exhibited maximum removal efficiency.
Review of Literature 37
b) Biological treatment method
Microbial system is the potent tool to deal with environmental
pollutants. Biotechnology for hazardous waste management involves
the development of systems that use biological catalysts to detoxify
environmental pollutants. Biotechnology offered a number of strategies
for waste treatment. (Nakamura and Sawada, 2000).
i) Improvement of existing processes by application of
adapted or engineered microbial strain.
ii) Construction of bioreactors containing immobilized
biocatalysts or biofilim of suitable organism in the
detoxification of environmental chemicals.
iii) Development of biosensors to detect trace amounts of
toxic organics or heavy metals.
Alginate is most widely used as a polysaccharide matrix for the
immobilization of viable cells. (Kierstan et al., 1985). Polyvinyl alcohol
(PVA) is no-toxic to organisms and can be cheaply produced in an
industrial scale. (Shindo & Kamimura 1990). Wu & Wisecarver 1992
used Calcium alginate modified PVA beads for immobilization of cells
so as to prevent the agglomeration of the beads and improve their gas
permeability. Thomas and White, 1991. described the development of
an immobilized biocatalyst for the surfactants treatments which
involved the entrapment of a versatile surfactant-degrading bacterium
Pseudomonas C12 B in a polyacrylamide gels.
Roig et al., 1997 studied the sorption isotherms and kinetics in
the primary biodegradation of anionic surfactants by immobilized
38 Chapter 1
bacteria, Comamonas terrigena N3H. Comamonas terrigena N3H was
immobilized by covalent linking on silanized inorganic supports and by
physical entrapment of cells within calcium alginate beads and
reticulate polyurethane foam.The surfactant degrading biocatalyst
Pseudomonas C12 B was immobilized by covalent linking on silanized
inorganic supports and by physical entrapment of cells within
reticulate polyurethane foam. Both immobilized biocatalysts have been
shown to be appropriate for the primary biodegradation of SDS. (Roig
et al., 1998).
Jerabkova et al.,1999 investigated the biodegradation of SDS,
by bioreactor with immobilized cells of Pseudomonas C12 B. Cells
were immobilized by adsorption on porous glass beads with either
unmodified or silanized surface. Both types showed equivalent
efficiency to remove SDS (85%). The surfactant degrading bacterium
Pseudomonas C12 B in immobilized state has been used in several
investigations for removing anionic surfactants (Ellis et al., 2002).
Farzaneh et al., 2009 studied the efficiency of immobilized cells
(stenotrophomonas maltophilia) on glass beads for the removal of
SDS.
Roig et al., 1997 studied the treatment of surfactant with the
plug flow recirculation bioreactor packed with polyurethane reticulated
foam particles with or without Pseudomonas C12 B.Four cycles (one
day) was required to complete the biodegradation.Surfactant removal
capacity was maintained in 5-6 days of continuous operation. Roig et
al., 1998 also studied the treatment of surfactant with the recirculation
bioreactor packed with polyurethane reticulated foam particles with or
without the Comamonas terrigena N3H .The above same result
observed.
Review of Literature 39
Feikenhauer et al., 2002 Investigated that high strength
wastewater of some industries contains high concentrations of
surfactants and readily biodegradable compounds like starch and
other carbohydrates. Wastewater of this type found in the textile wet
processing industry. High concentration of surfactants has been
shown to inhibit the digestion process.
Dhouib et al., 2002, developed a high performance process for
the treatment of anionic surfactant containing waste water. A
Citrobacter braakii was used for the continuous degradation of SLES
synthetic medium and cosmetic industry waste water. By testing
several dilution rates it was concluded that Citrobacter braakii was
able to degrade up to 0.065 g l -1h-1.
Mortazavi et al., 2008 investigated the removal of SDS from
synthetic wastewater in an Intermittent Cycle Extended Aeration
System (ICEAS). The reactor was made up of polyethoxyglass tank
which is equipped with diffuser air.The removal of SDS was more than
98%.Luis (2004) also reported that the removal efficiency of SDS in
ICEAS is more than 95%. Ebrahimi et al., 2006 studied the removal
efficiency of COD in conventional active sludge system was 87 %. In
addition in a study of SDS biodegradation by the Acinetobacter
isolated from active sludge, the removal efficiency was 96.4 %.
(Hosseini et al., 2007)