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1. Submitted By:
Md. Hasan Masrur (545)
Submission date: 3rd March, 2014
An Assignment on
Biotechnological methods
In
Pollution abatement
Course no: Env. 420
Submitted by:
GROUP NO: 2
SHARMINE I (547)
Submitted to:
ABDUL KADIR IBNE KAMAL
Assistant Professor,
Department of Environmental
Sciences, Jahangirnagar
University, Savar, Dhaka-1342
Department of Environmental Sciences,
Jahangirnagar University
Savar, Dhaka- 1342

2. Table of content
Content Page no.
Metal pollution and its bio
abatement
Introduction
Materials and methods
Biological markers (biomarkers or
bioindicators)
Fish as Metal Biomarker for Water
Pollution in Worldwide
1-5
1
1-2
3-5
5
Eutrophication in biological
phosphorus removal
Enhanced Biological Phosphorus
Removal (EBPR)
Cultivation of microorganisms in
wastewater
6-10
6-8
9-10
Cell immobilization as a tool
in waste treatment
Cell immobilization
Methods for immobilization of
microbial cells
Factors affecting microbial cell
adsorption
11-19
11-18
16-18
19
References 20-23

3. 1 | P a g e
Metal pollution and its bio abatement
Introduction
Heavy metals are non-biodegradable pollutants whose release in the
environment is mainly related to industrial wastewaters discharged from
industrial and mining activities. The use of bacterially mediated sulphate
reduction in Permeable Reactive Barrier is an alternative technique for the
remediation of heavy metals polluted streams. SRB are known to grow using
small organic molecules, essentially small molecular weight compounds, like
acetate, lactate, propionate, butyrate, valerate, methanol, ethanol, glycerol,
glucose (Postgate, 1979). However, pure substrates as carbon source may not
be cost effective for this kind of treatment. Usually, organic mixtures are used
in biological PRB construction as electron donor in the sulphate reduction:
biodegradable materials are generally mixed with more recalcitrant ones to
ensure long term SRB growth (Cocos et al., 2002; Gibert et al., 2004). Full
scale applications of organic-carbon based sulphate reducing PRB are also
characterized by the addition of gravel to improve barrier permeability and
limestone to increase pH and stimulate SRB growth (Ludwig et al., 2002).
Batch experiments were preliminarily performed to determine the optimal
mixture for treating heavy metals in biological PRB. Selected mixture was then
tested in continuous fixed column experiments to simulate permeable reactive
barriers running. Ethanol was also tested as electron donor for the
sulphatereduction for several reasons, including a well-defined and “clean”
composition, ease of availability, low cost and the possibility to use
bioethanol. Finally, preliminary batch tests starch were conducted to test the
ability of SRB to grow on this substrate.
Materials and methods
Batch tests with solid reactive mixtures
Eight reactive mixtures were prepared consisting of three main functional
components: a mix of organic materials, a neutralizing agent (limestone), and
a non-reactive porous medium (silica sand or perlite). A sample (20 g) of each
mixture was added in flasks and filled with 80 mL of liquid C Medium
(Postgate, 1979). Flasks were sealed and 20mL inoculum of bacteria

4. 2 | P a g e
cultivated in C Medium were added. Best performing reactive mixture (RM:
6% leaves, 9% compost, 3% Fe(0), 30% silica sand, 30% perlite, 22%
limestone) was further tested in presence and in absence of bacteria and
single organic components (compost and leaves) were also investigated for
sulphate removal without inoculum. All experiments were conducted at 37°C
under shacking condition. pH, Eh, SO42- and S2- production were monitored
for 22 days. Each test was performed twice and average values were
considered.
Column tests with solid reactive mixture M8
Column tests were performed in a fixed bed column (height 1 m; diameter 0.2
m; column volume, V=6.65*10-3 m3) made of Plexiglas with 10 equally distant
outputs along the axial length, numbered from the bottom to the top of the
column. It was packed with perlite (an expanded clay) and silica sand on the
bottom (10 cm length) followed by reactive mixture (RM) (80 cm) and topped
with perlite and silica sand (10cm) (pore volume V0 = 1.5 L) .SRB were
inoculated in the core of the column. Column was regularly fed with a solution
containing heavy metals (Cd 0.1 mM, Cr(VI) 0.1 mM, Cu 0.1 mM, Zn 0.1 mM
and As(V) 27μM) and sulphate (31 mM). Samples from three different outputs
(1, 5 and 9) were analyzed for pH, Eh and the residual amounts of sulphates
and metals.
Batch tests with ethanol
Glass reaction flasks (120 mL), containing a sampling port, were used for all
the experiments. 80 mL of modified C Medium (ethanol 6 g/L instead of
lactate) was added in flasks. Therefore the flasks were sealed and 20 mL
inoculum of bacteria cultivated in C Medium were added by a sterile syringe
through the sampling port. All experiments were conducted at 37°C under
shacking condition. pH, Eh, SO42- and S2- production were monitored for 30
Column tests with ethanol
Column tests were performed in two fixed bed column like that used in
column tests with mixture RM. Columns were filled with perlite (pore volume
V0 = 3.5 L), one inoculated by SRB and the other used as blank. Both columns

5. 3 | P a g e
were regularly fed with a solution containing sulphate (31 mM) and ethanol
(65 mM). Samples from three different outputs (1, 5 and 9) were analyzed for
pH, Eh and the residual amounts of sulphates.
Batch tests with starch
Glass reaction flasks (120 mL), as those previously described, were used for
the experiments with starch. 80 mL of C Medium (without carbon sources)
and starch (100 g/L) were adder in flask. Therefore the flasks were sealed and
20 mL inoculum of bacteria cultivated in C Medium were added by a sterile
syringe through the sampling port. All experiments were conducted at 37°C
under shacking condition. pH, Eh, SO42- and S2- production were monitored
for 100 days. Each test was performed twice and average values were
considered.
Biological markers (biomarkers or bioindicators)
In the attempt to define and measure the effects and presence of pollutants
on aquatic system, biomarkers have attracted a great deal of interest. The
principle behind the biomarker approach is the analysis of an organism to
their metal contents in order to monitor the metal excess in their tissues.
Various aquatic organisms occur in rivers, lakes, seas and marines potentially
useful as biomarkers of metal pollutants, including fish, shellfish, oyster,
mussels, clams, aquatic animals and aquatic plants and algae.
Fish as biomarkers
Fish from Lakes: Nasser Lake
Tilapia nilotica is one of the aquatic organisms affected by heavy metals, so in
many cases, Tilapia nilotica was used as metal biological marker in
toxicological studies in which it was substantiated with the highest sensitivity
to toxic effect (Patin, 1984). Rashed (2001a, b) studied Co, Cr, Cu, Fe, Mn, Ni,
Sr, Pb, Cd and Zn in different tissues of fish (Tilapia nilotica) from Nasser lake
to assess both the water pollution with these metals and the lethal level of
these metals in fish. Fish samples were collected from two Kohrs in Nasser
Lake ( Kohr Kalabsha and Kohr El-Ramel) .The fish tissues includes muscle,
gill, stomach, intestine , liver, veritable column and scales .The fish ages were

6. 4 | P a g e
1,1.5 , 2, 2.5 and 3 years. This study resulted in that fish scales exhibited the
highest concentrations of Cd, Pb, Co,Cr,Ni and Sr (0.088,0.95,0.29,0.30,0.25
and 3.21 μg/g DW respectively). Whole fish contains the higher
concentrations of the studied metals compared to the previous study by
Awadallah et al.(1985) in the same fish from Nasser Lake, and this mean the
increase in metal pollution in Lake water as the results of man activities (Table
1). This increasing in metal concentration was as the result of increasing
pollution loads to the Lake from agricultural wastes, which include chemical
pesticide and fertilizers. These agricultural wastes reached the Lake body
from the agricultural farms on the beach of the Lake. The source of Pb in the
Lake water and fish was resulted from gasoline contains Pb from the fishery
boats and tour ships travels from Aswan to Sudan (Mohamed et al.1990).
Fish from River Nile
River Nile is the main source for potable water and as the result of man
activities in and on the river body it become loaded by metal pollution. Fish
in the River Nile was used as biological marker for the River pollution by
metals. Mohamed et al. (1990) used Tilapia nilotica fish as a biomarker for
the Nile water pollution with metals at the discharge.Point of fertilizer factory
with the Nile. Ag, Au, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr and Zn were
determined in tilapia nilotica fish collected from the Nile area at the point of
fertilizer discharge to the Nile and south and north this point. The results
revealed that fish near the point of the factory discharge possess the highest
levels of metals as the result of pollution with metals. Other study for using
fish as biomarker for water pollution with metals was conducted (Khallaf et
al., 1994). Two species of fresh water fish (Tilapia nilotica, named Bolti, and
Karmout ) caught from River Nile at Hawamdia and Kafer El-Zayat , at North
Egypt and also from governmental fish farms (Abbassa and Barseik) were
used to detectthe presence of industrial wastes especially heavy metals as
environmental pollution in the river track and its accumulation in edible fish
tissues. The result reveals that heavy metals in different water samples except
Cu and Zn were more than the recommended permissible levels (Table2). Iron
level in Hawamdia and Kafer-El-Zayat tilapia nilotica samples (63.4 and 54.7

7. 5 | P a g e
μg/g respectively) was more than its permissible levels, these may be due to
the discharge of the adjacent chemical factories that used Fe in their
processing.
Fish as Metal Biomarker for Water Pollution in Worldwide
Arsenic as biomarker for water pollution was assessed by Takatsu and
Uchiumi (1998) in which the contents of the metal in the tissues of the fish,
Tribolodon hakonsis, from Lake Usoriko, located in Aomori Prefecture, Japan,
were examined. It was discovered that large amounts of As were accumulated
in the eye tissues. This might be partly related to the fact that the lake water
contains a relatively large amount of As. Mercury levels in muscle of some fish
species from Dique channel, Colombia was measured to assess the water
pollution with Hg (Olivero et al.1997). The highest values of Hg (105 μg/kg)
found in fish from the Dique channel were lower than those found in fish
species from the Lower Gallego and Cince Rivers in Spain (Raldua and
Pedrocchi, 1996). In the Tapajos River, an Amazon water body highly exploited
by gold mining activities, the average value for Hg in muscle of Carnivorous
fish was 690 μg/kg, almost ten times higher than those found in the Dique
channel (Malm et al., 1995). They also concluded that, however the highest
Hg concentration did not reach the limits level internationally accepted for
considering a fish not acceptable for human consumption (WHO, 1990).

8. 6 | P a g e
Eutrophication in biological phosphorus removal
Enhanced Biological Phosphorus Removal (EBPR)
EBPR relies on the selection and proliferation of a microbial population
capable of uptaking orthophosphate in greater amounts than their normal
biological growth requirements.
Enhanced biological phosphorus removal is a process that uses alternating
anaerobic and aerobic zones to provide an environment that encourages the
growth of Phosphorus Accumulating Organisms (PAO). PAOs store excess
polyphosphate in their cell mass and phosphorus is removed with the waste
sludge.
Fig: A typical Enhanced Biological Phosphorus Removal configuration

9. 7 | P a g e
Mechanism of biological phosphorus removal
The unique feature of EBPR is the anaerobic selector used in the treatment
process.
Fig: mechanism of biological phosphorus removal
The reactions that occur in the anaerobic zone
Phosphorus Accumulating Organisms (PAOs) use poly-phosphate and
glycogen stored in their cells as energy sources to enable them to uptake VFA.
VFA are converted to Polyhydroxyalkanoates (PHA) and stored in the cells of
PAOs. As they take up VFA, the PAOs release orthophosphate into the mixed
liquor. PAOs do not grow in the anaerobic zone but their ability to uptake
food in the form of VFAs gives them a competitive advantage over other
bacteria.
The reactions that occur in the aerobic zone
In the aerobic zone PAOs use PHA as a source of carbon and energy for
metabolism and cell growth. PAOs will also restore their supplies of glycogen
and poly-phosphate in the aerobic zone. To replenish their stored poly-
phosphate, PAOs will take up excess phosphate from the mixed liquor which
is the mechanism of biological phosphorus removal.

10. 8 | P a g e
In the anaerobic zone PAOs will rapidly take up BOD (as VFAs) and release
orthophosphate into the mixed liquor. As the wastewater passes through the
anaerobic zone VFA will rapidly decrease and orthophosphate will increase.
In the aerobic zone BOD will continue to decrease. As PAOs restore their poly-
phosphate supplies in the aerobic zone the concentration of orthophosphate
in the mixed liquor will rapidly decrease.
Fig: the phosphorus and BOD profile through a treatment plant
Under anaerobic conditions PAOs take up VFA from the mixed liquor and
store it as PHA within their cells. To do this PAOs use the glycogen and poly-
phosphate as energy sources depleting their stores of these compounds.
Under aerobic conditions PAOs use up their stored PHA for metabolism and
growth and to restock their supplies of glycogen and poly-phosphate. To build
up their supply of polyphosphate PAOs will take up excess orthophosphate
from the mixed liquor in the aerobic zone.
The importance of sludge age in the treatment system on biological
phosphorus removal
As with all activated sludge systems, sludge age determines how long the
biomass stays in the biological part of the treatment system. Because
biological phosphorus removal is compartmentalized into anaerobic, anoxic,
and aerated zones, all biological zones must be taken into consideration when
calculating sludge age. Selectors are usually much smaller than that of

11. 9 | P a g e
aeration zones and should be taken into account when determining sludge
age.
The proper sludge age ensures the optimization of the biochemical reactions
that need to take place and for the biomass to uptake excess phosphates in
the aerobic basins (see key knowledge 1.3.5). Too short a sludge age and
insufficient treatment can occur with resultant poor effluent quality. As
sludge age increases in an activated sludge system, nitification becomes a
factor and the need for anoxic zones becomes critical for denitrification and
the removal of nitrates. Long sludge ages such as in extended aeration
systems, can lead to secondary release of phosphorus through biomass decay.
Long sludge ages can also result in biochemical reaction problems for PAOs.
Sludge ages of 5-30 days are likely to be observed in successful EBPR plants.
BOD5 (or COD) /Total P ratio: As discussed in key knowledge 1.2.1, the
influent BOD5 or COD to total phosphorus ratio is critical for PAOs to grow,
function, and take up phosphorus from solution. The influent BOD5 or COD
must be in a form that is readily available to PAOs, such as volatile fatty acids.
A minimum BOD5/total P ratio of 20:1 or a COD/total P ratio of 45:1 is
needed for successful bio-P removal.
Sludge Age (SA) and Hydraulic Retention Time (HRT): The contact time
between the influent and the bacteria (HRT) as well as the proper sludge age
(SA) for the microbial biochemical reactions to take place in the biomass are
critical for PAO growth, metabolism and selection. These parameters are
discussed further in key knowledges 2.2.3 and 2.2.4.
Temperature: Research shows that effective phosphorus removal has been
shown to occur between 5- 30°C as long as proper sludge ages are provided
for cold and warm weather temperatures. In fact, PAO’s may be at a selective
advantage in colder temperatures (5-10°C).
Internal Recycle Flows: Internal recycles are used in EBPR systems to
create favorable conditions for PAO selection and growth. Common internal
recycle flows include return activated sludge (RAS), anoxic zone to anaerobic
zone recycles, and aerobic to anoxic zone recycles.

12. 10 | P a g e
Cultivation of microorganisms in wastewater
Biological removal of nutrients in bioreactors is, by definition, performed
bymicroorganisms. However, the species commonly used are non-specific and
environmentally enriched from the sludge by the incubation conditions, as
explained later for EBPR. Several attempts to intentionally use specific
microorganisms as cleaning agents were reported, as has been done in earlier
decades (for review of earlier cases: De la Nou¨e and De Pauw, 1988). These
include bacteria and microalgae.
Bacteria
The tropical cyanobacterium Phormidium bohneri in domestic wastewater
removed nitrogen and phosphate after growing 50 and 75h, respectively.
Adding monopotassium phosphate enhanced production of biomass by 56%,
but did not significantly affect the time for completely removing these
nutrients. This could not be repeated at the lower temperature.This failure
indicates that tertiary biological wastewater treatment at low temperatures
(51C) cannot be developed with the polar strains tested because they grow too
slowly under extreme cold. However, they may have potential at moderately
cool temperatures (about 151C and above), common from spring through fall
in northern climates (Chevalier et al., 2000).
Bacteria cultivated for relatively short periods in wastewater present a feasible
alternative to the longer EBPR process (described later). Staphylococcus
auricularis,growninsequencingbatchreactorsunderrepeated anaerobic and
aerobic conditions, was able to remove
between5and50mgPL1,correspondingtomorethan 90% removal of
phosphorus. These results, achieved after a short incubation period,
demonstrated that a long adaptation time, one of the major problems in
starting an EBPR process, could be addressed by a shorter approach (Choi
and Yoo, 2000).
The purple photosynthetic non-sulfur bacterium Rhodobacter
capsulatus,immobilizedoncellulosebeads, removed organic carbon,
ammonium ions, and phosphateionsfromadilutedgrowthmediumoveraperiod

13. 11 | P a g e
of 19–22 days with a residence time of 10h at 351C (Sawayama et al., 1998b).
The photosynthetic bacteria, Rh. sphaeroides S, Rb. sphaeroides NR-3, and
Rhodopseudomonas palustris, immobilized in porous ceramic under aerobic
conditions, simultaneously removed COD, phosphates, nitrates, and H2S
from a synthetic sewage wastewater. In the batch treatment, 77% of the
phosphates were removed effectively within 48h. In semi-continuous
treatments, this removal took about one month (Nagadomi et al., 2000).
Microalgae
Unicellular microalgae Chlorella vulgaris and Scenedesmus dimorphus were
capable of removing upto 55% of the phosphates from dairy industry and pig
farming wastewaters(Gonzalezetal.,1997,forearlierstudiessee De la Nou¨e and
De Pauw, 1988). Another strain of Scenedesmus, grown in artificial
wastewater, also removed more than 50% of the phosphates (Voltolina et
al.,1999).Productionofstarchyieldedwastewaterwith a unique C:N:P ratio of
24:0.14:1. This effluent supported good growth of Spirulina platensis.
Reductionsinphosphatelevelsofthedigestedeffluentreached over 99% (Phang
et al., 2000). S. platensis could efficiently remove nitrates, ammonia, and
phosphates from synthetic wastewater (Ogbonna et al., 2000).
These studies employed microalgae in a suspension.
Theirapplicationisseverelylimitedbythedifficultiesof harvesting the enormous
microalgal population developedinthewateraftertreatment.Therefore,theideaof
entrapping microalgae for easy removal by sedimentation with spherical gels
gained some momentum. For example, C. vulgaris, immobilized in two natural
polysaccharide gels (carrageenan and alginate), was used to treat primary
domestic wastewater.
The most radical combination of microalgae and bacteria suggested so far is
the use of plant growthpromoting bacteria, used in agriculture, to enhance
the growth and nutrient removal capacity of microalgae from wastewater. The
bacteria of the genus Azospirillum are used as inoculants to promote the
growth and yield of numerous crop plants, mainly by affecting the hormonal
metabolism and mineral absorption of the plants (Bashan and Holguin, 1997).
The underlying hypothesis assumed that the bacteria will enhance the

14. 12 | P a g e
performance of unicellular plants, like microalgae, and that the single-cell
plant will respond similarly to bacterial inoculation like a higher plant. Co-
immobilization of C. vulgaris and C. sorokiniana with A. brasilense, in small
alginate beads, significantly enhanced all the growth parameters of the
microalgae (Gonzalez and Bashan, 2000). Furthermore, these artificial
combinations (thus far not found in nature) profoundly changed many
cytological, physiological, and biochemical pathways and products within the
microalgal cells (Gonzalez- Bashan et al., 2000; Lebsky et al., 2001; de-
Bashan et al., 2002a). This co-immobilization, under semi-continuous
synthetic wastewater culture conditions, significantly increased the removal
of ammonium and soluble phosphate ions, compared to immobilization of the
microalgae alone (de-Bashan et al., 2002b). Recently, these combinations
were successful in significantly reducing ammonium and phosphate levels of
municipal wastewater (de-Bashan et al., 2004).
In summary, no new technology has emerged from decades of research on
intentionally using specific microorganisms for nutrient removal. Several
proposals, especially the entrapment of microorganisms in polysaccharide
gels and combinations of several organisms for simultaneous treatment of the
wastewater, have the best potential for commercial use. However, as yet, it is
a minor research avenue in the phosphate removal field in wastewater
treatment.

15. 13 | P a g e
Cell immobilization as a tool in waste treatment
Cell immobilization
Immobilization is a general term describing a wide variety of the cell or the
particle attachment or entrapment (Lopez et al., 1997). It can be applied to
basically all types of biocatalysts including enzymes, cellular organelles,
animal and plant cells. Currently, different kinds of immobilization have
found wide applications not only in the field of biotechnology, but also in
pharmaceutical, environmental, food and biosensor industries (Peinado et al.,
2005).
The cell immobilization emerged as an alternative for enzyme immobilization
(Cheetham et al., 1979; Parascandola and Scardi, 1980; Woodward, 1988).
Immobilization of cells containing specific enzymes has further advantages
such as elimination of long and expensive procedures for enzymes separation
and purification and it is vital to expand their application by enabling easy
separation and purification of products from reaction mixtures and efficient
recovery of catalyst (Junter and Jouene, 2004; Stolarzewicz et al., 2011). In
comparison with immobilized enzymes, immobilized cells provide new
possibilities since they can be used as natural, water-insoluble carriers of
required enzyme activities (Vojtisek and Jirku, 1983).
In the case of the immobilization of microbial cells, their field of application
spreads from industrial to environmental process. Microorganisms retained
on a carrier can be used in continuous and semi-continuous production
processes allowing for significant cost decrease, as the biocatalyst does not
need to be refilled (Wada et al., 1979; Park and Chang, 2000; Mrudula and
Shyam, 2012).
Cell immobilization has been defined as the physical confinement or
localization of viable microbial cells to a certain defined region of space in
such a way as to limit their free migration and exhibit hydrodynamic
characteristic which differ from those of the surrounding environment while
retaining their catalytic activities for repeated and continuous use (Dervakos
and Webb, 1991; Freeman and Lilly, 1998; Covizzi et al., 2007; Amim et al.,

16. 14 | P a g e
2010). Since the early 70s, when Chibata’s group announced successful
operation of continuous fermentation of L-aspartic acid, numerous
research groups have attempted various microbial applications with
immobilized cells (Ramakrishna and Prakasham, 1999). Environmental
applications of immobilized microbial cells are reported by Bettmann and
Rehm (1984), Anselmo et al. (1985), Sahasrabudhe et al. (1988), Oreilly and
Crawford (1989), Beunink and Rehm (1990), Balfanz and Rehm (1991),
Stormo and Crawford (1992), Cassidy et al. (1996), Wang et al. (1997), Wang
et al. (2002), Wang et al. (2007), Zhang et al. (2007), Zhou et al. (2008), Bazot
and Lebeau (2009), Wang et al. (2010), Ahmad et al. (2012) and Nickzad et al.
(2012).
Support materials
The support selection is one of the crucial decisions to be made in the course
of preparation of the immobilization process (Zacheus et al., 2000). For
treatment of wastewater, support materials need to meet the following criteria:
insoluble, non-biodegradable, non-toxic, nonpolluting, light weight; flexibility
in overall shape, high mechanical and chemical stability, high diffusivity,
simple immobilization procedure, high biomass retention, minimal
attachment of other organisms and preferably a low cost price (Leenen et al.,
1996; Zacheus et al., 2000). Other criteria, such as physical characteristics
(porosity, swelling, compression, material and mean particle behavior), as well
as, possibility for microbial growth and solubility, are more application
specific (Górecka and Jastrzębska, 2011).
The carriers are classified as inorganic material (zeolite, clay, anthracite,
porous glass, activated charcoal, and ceramics) and organic polymers.
Inorganic carriers were selected to immobilize microorganisms because they
can resist microbial degradation and are thermostable (Cassidy et al., 1996;
Verma et al., 2006). The organic polymeric carriers are more abundant than
inorganic carriers and can be natural and synthetic polymeric carriers
(Cassidy et al., 1996). Several syn-thetic (acrylamide, polyurethane, polyvinyl,
resins) and natural polymer derivatives of algal polysaccharides (alginate,
carrageenan, agar, agarose), and chitosan, an amino polysaccharide derived

17. 15 | P a g e
from chitin, has been experimentally used. The most commonly used
polymers are the natural polymers alginate and carrageenan but these
natural polymers are less stable in wastewater than synthetic polymers
(Bashan, 1998; Arica et al., 2004; Moreno-Garrido, 2008; Stolarzewicz et al.,
2011).
Alginates (polymers made of different proportions and sequences of
mannuronic and guluronic acids extracted from brown algae) are easy to
handle, nontoxic to humans, the environment, and the entrapped
microorganisms, legally safe for human use, available in large quantities, and
inexpensive.
Chitosan is inexpensive, non-toxic property and possesses potentially reactive
amino functional groups which can enhance the affinity of the carrier with
the microorganisms. However, the mechanical stability of the carrier would
decrease because of the biodegradability in the course of usage.
Other natural gels, such as agar, collagen andagarose, also can be used as
microbial encapsulation carriers (Zhou et al., 2008). Some natural polymers
are more vulnerable to environmental degra-dation by microbes. However,
diffusivity is higher in natural polymers and they are less hazardous to
produce (Leenen et al., 1996; Cassidy et al., 1996).
Synthetic polymeric supports are not easily biodegradable and have much
better mechanical performance compared with nature carrier. Materials, such
as polyacrylamide (PAM), polyvinyl alcohol (PVA), polyethyleneglycol (PEG)
and polycarbamoyl sulphonate (PCS) were synthesized as encapsulation
carriers (Leenen et al., 1996).
In order to improve the stability of gel carrier, various synthetic plastics, for
example polypropylene (PP), polyethylene (PE), polyvinylchloride (PVC), poly-
urethane (PU) and polyacrylonitrile (PAN) have been explored extensively as
immobilized microorganism carriers more recently (Zacheus et al., 2000).
Among the various extensively used plastics carriers, polyurethane (PU) is one
kind of outstanding carrier for entrapping microorganisms in piloted
applications in practical wastewater treatment (Guimarães et al., 2002).

18. 16 | P a g e
Martins et al. (2012) reported potential of the Gramnegative bacterium
Serratia marcescens and the yeast Candida rugosa to immobilization on
polyurethane foam.
Methods for immobilization of microbial cells
Immobilization of microbial cells in biological processes can occur either as a
natural phenomenon or through artificial process (Ramakrishna and
Prakasham, 1999). Different immobilization types have been defined: covalent
coupling/cross linking, capture behind semipermeable membrane or
encapsulation, entrapment and adsorption (Mallick, 2002). The types of
immobilization can be grouped as ‘‘passive” (using the natural tendency of
microorganisms to attach to surfaces-natural or synthetic, and grow on them)
and ‘‘active” (flocculant agents, chemical attachment and gel encapsulation)
(Cassidy et al., 1996; Cohen, 2001; Moreno-Garrido, 2008).
Covalent bonding/Cross linking: The mechanism involved in this method is
based on covalent bond formation between activated inorganic support and
cell in the presence of a binding (crosslinking) agent. For covalent linking,
chemical modification of the surface is necessary. Covalent attachment and
cross-linking are effective and durable to enzymes, but it is rarely applied for
immobilization of cells. It is caused mainly by the fact that agents used for
covalent bonds formation are usually cytotoxic and it is difficult to find
conditions.
Entrapment: Entrapment is an irreversible method, where immobilized cells
are entrapped in a support matrix or inside fibers. This technique creates a
protective barrier around the immobilized microbes, ensuring their prolonged
viability during not only processing but also storage.in polymers (Górecka and
Jastrzębska, 2011). Entrapment is the most method extensively studied in
cell immobilization. The matrices used are agar, alginate, carrageenan,
cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross-
linkable resins, polyacrylamide, polyester, polystyrene and polyurethane
(Lopez et al., 1997; Ramakrishna and Prakasham, 1999).
Entrapment of the microorganisms in porous polymer carrier was often used
to capture the microorganisms from suspended solution and then obtain the
immobilized microorganisms. The polymer matrix used in this method
confining microorganisms has porous structure, and thus the pollutant and
various metabolic products could easily diffuse through into the matrix. In

19. 17 | P a g e
this method, a lot of porous polymers can entrap microorganisms under
ambient conditions (Verma et al., 2006).
As a rule, the entrapment methods are based on the inclusion of cells within
a rigid network to prevent the cells from diffusing into surrounding medium
while still allowing penetration of substrate. Entrapment of cells in alginate
gel is popular because of the requirement for mild conditions and the
simplicity of the used procedure. Several reports are available employing
alginate gel (Kierstan and Bucke, 1977).
Entrapment allows high mechanical strength, but contains some
disadvantages, such as, cell leakage, costs of immobilization, diffusion
limitations, and deactivation during immobilization and abrasion of support
material during usage. Another disadvantage is low loading capacity as
biocatalysts have to be incorporated into the support matrix (Krekeler et al.,
1991; Song et al., 2005; Gao et al., 2010; Stolarzewicz et al., 2011).
Encapsulation: Encapsulation is another irreversible immobilization method,
similar to entrapment. In this process, biocatalysts are restricted by the
membrane walls (usually in a form of a capsule), but free-floating within the
core space (Górecka and Jastrzębska, 2011). The membrane itself is semi-
permeable, allowing for free flow of substrates and nutrients (when cells are
used as a biocatalyst), yet keeping the biocatalyst inside. The factor
determining this phenomenon is the proper pore size of the membrane,
attuned to the size of core material. This limited access to the microcapsule
interior is one of the main advantages of microencapsulation, for it protects
the biocatalyst from the harsh environmental conditions. As most
immobilization method, it prevents biocatalyst leakage, increasing the process
efficiency as a result (Park and Chang, 2000).
The encapsulation method was used to enclose the microorganisms in a
polymer-gel by Jen et al. (1996) and is one of the most frequently used in
laboratory experiment up to now and there is far away engineering application
for wastewater treatment (Lozinsky and Plieva, 1998).
However, even though in encapsulation, high cell loading can be achieved,
but the capsules are still very weak (Song et al., 2005). The diffusion limitation
is one of the inevitable drawbacks associated with encapsulation method
(Lozinsky and Plieva, 1998).

20. 18 | P a g e
Adsorption: The immobilization passive or adsorption natural of
microorganisms onto porous and inert support materials is similar to the
adsorption of colloid particles (Araujo et al., 2010). Apparently, it is the first
example of cell immobilization and probably is the simplest method of
reversible immobilization (Monsan et al., 1987; Klein and Ziehr, 1990).
This technique is based on the physical interaction between the
microorganism and the carrier surfaces, while frequently reversible is simple,
cheap and effective. The immobilization of microorganisms on properly chosen
adsorbents stimulates microbial metabolism, protects cells from unfavorable
agents, and preserves their physiological activity (Nikovskaya, 1989; Kozlyak
et al., 1991, 1993).
Different from the inherent problems associated with cell entrapment, cell
immobilization through adsorption provides a direct contact between
nutrients and the immobilized cells thus, eliminating such concerns
(Braschler et al., 2005). This cell immobilization technique involves the
transport of the cells from the bulk phase to the surface of support (porous
and inert support materials), followed by the adhesion of cells, and
subsequent colonization of the support surface (Kilonzo and Bergougnou,
2012).
Adsorption is based on weak forces, however, still enabling an efficient
binding process. Usually in bonds formation, several forces are involved: van
der Waals forces, ionic and hydrophobic interactions and hydrogen bonds.
Both electrostatic and hydrophobic interaction controlling the cell
immobilization on the support (Hsu et al., 2004, Górecka and Jastrzębska,
2011).
In contrast to ceramics, wood chips and straw, fibrous matrices provide
adequate supporting surfaces for cell adsorption (Talabardon et al., 2000;
Chu et al., 2009) due to their high specific surface area, void volume,
mechanical and permeability, low pressure drop, diffusion problems and
toxicity, maximum loading, biodegradability and durability and low cost and
high availability (Huang and Yang, 1998). Their natural configuration also
allows them to trap more cells than other materials (Yang and Shu, 1996;
Yang and Lo, 1998).

21. 19 | P a g e
Polyurethanes foams for immobilization by adsorption:
Polyurethanes (PU) are one of the most versatile materials in the world today.
They are known for being a perfect material for footwear, machinery industry,
coatings and paints, rigid insulation, elastic fiber, soft flexible foam, medical
devices (Romaškevič et al., 2006). Some time ago PU was found to be
applicable in the biochemical and biotechnological fields and flexible
polyurethane foams have gained relevance as microbial carriers for their good
mechanical properties, high porosity, large adsorption surface, resistance to
organic solvents and microbial attack, easy handling, regenerability and cost
effectiveness (Patil et al., 2006). In general, the high rates of sorption of
polyurethane foam in removing lubricants boats, while Silva et al. (2006) have
described that the immobilization of bacteria in polyurethane foam increased
resistance to high concentrations of sulphate.
Factors affecting microbial cell adsorption
There are many factors (such as the age and the physiological state of cells)
that influence the sorption of microbial cells. The surface structures of
bacterial cells (flagella and other appendages), superficial charges and
hydrophobicity also play an important part in the cell adherence to solid
surfaces (Donlan, 2002; Chae et al., 2006; Oulahal, et al., 2008). The
composition of the medium, its pH, and environmental conditions
considerably influence the adsorption of cells by changing their electrokinetic
potential (Stanley, 1983; Fletcher and Pringle, 1986; Kilonzo and Bergougnou,
2012).
The surface properties of adsorbents also affect the process of cell
immobilization (Busalmen and Sanchez, 2001, Ubbink and Schar-
Zammaretti, 2007). The degree of cell immobilization depends on the
structure and the size of adsorbent pores (Arinbasarova et al., 1982). The
nature of adsorbents is also important. Organic adsorbents are chemically
stable and show a great variety of surface properties and pore structures,
whereas inorganic adsorbents are resistant to biological degradation are
affordable, and can be easily regenerated. The positive charge and
hydrophobic character of the polyurethane foam, allow interaction with most
microbial cell surfaces (Afghan et al., 1984; Wang et al., 2009). They are
inexpensive and easily regenerated by extraction or washing with solvents
(Belyakova and Schevchenko, 1986).

22. 20 | P a g e
The microbial immobilization in polyurethane, combined with the use of
bioreactors improved significantly the biodegradation process of phenols and
derivatives (Pai et al., 1995). The highest efficiency in the degradation of
ophthalate by cells Bacillus-spp. immobilized in polyurethane foam, in
relation to alginate.
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