2

Contents


Title Page
Acknowledgement 3
Abstract 4
Introduction 5
Part one: DeIinition oI bioremediation

6
Part two: Heavy metals and its hazards eIIect
in aquatic environment
8
Part three: Methods & mechanisms oI heavy metal
removal

12
Part Iour : Mechanisms oI microbial removal oI heavy metals 24
Arabic abstract 27
ReIerences 28
3


Acknowledgement


First and above oI all . thanks to Allah . without his
grace and help this study would have been unable to
be completed .



I would like to express my sincere
and my deep gratitude and appreciation to my
supervisor Dr. Sahar El-Shatoury . associate
proIessor oI Microbiology . Botany Department .
Faculty oI science . Suez Canal University . Ior her
keen supervision . guidance . encouragement during
the course oI study .



I would like to thank Dr. Samira Resk
Mansour . proIessor oI Microbiology . Head oI
Botany Department and all the staII members oI
Botany Department . Faculty oI science . Suez
Canal University Ior their encouragement .








4

Abstract


The aquatic environment with its water quality is
considered the main Iactor controlling the state oI health and
disease. Pollution oI the aquatic environment by inorganic
and organic chemicals is a maior Iactors posing serious threat
to health. this report Iocuses on the pollution caused by
heavy metals as mercury (Hg). cadmium (Cd). arsenic (As).
chromium (Cr). thallium (Tl). and lead (Pb) which are very
dangerous because they tend to bioaccumulate in the body
result in many diseases.
Also. this report Iocuses on the diIIerent methods oI heavy
metals removal and bioremediation. Actinomycetes .in
particular. show many mechanisms oI heavy metal removal
as biosorption. metal reduction and produce siderophores .


5

Introduction



Water is essential to humans and other liIeIorms. Thus any pollution in the
water or in the aquatic environment has great eIIects on human health. animals and
Iishes. One oI the dangerous pollutants is the heavy metals. Heavy metals can enter a
water supply by industrial and consumer waste. or even Irom acidic rain breaking
down soils and releasing heavy metals into streams. lakes. rivers. and groundwater.
Metal ions can be incorporated into Iood chains and concentrated in aquatic organisms
to a level that aIIects their physiological state. OI the eIIective pollutants are the heavy
metals which have drastic environmental impact on all organisms. Trace metals such
as Zn. Cu and Fe play a biochemical role in the liIe processes oI all aquatic plants and
animals; thereIore. they are essential in the aquatic environment in trace amounts. In
the Egyptian irrigation system. Cu. Pb and Cd are serious polluting metals. The main
source oI Cu and Pb are industrial wastes as well as algaecides (Ior Cu). while that oI
Cd is the phosphatic Iertilizers used in crop Iarms (Mason, 2002).

There are various methods oI heavy metals removal; those are categorized as
chemical. physical and biological. This report Iocus on the biological removal oI
heavy metals by actinomycetes . Actinomycetes are a group oI Gram-positive
bacteria with high guanine and cytosine content and they include some oI the most
common soil liIe. Ireshwater liIe. and marine liIe. playing an important role in
decomposition oI organic materials. such as cellulose and chitin. Thereby. they are
playing a vital part in organic matter turnover and carbon cycle. This replenishes the
supply oI nutrients in the soil and is an important part oI humus Iormation.

















6

1.Definition of bioremediation :


Bioremediation can be defined as any process that uses microorganisms, fungi, green plants or
their enzymes to return the natural environment altered by contaminants to its original
condition.

Generally, bioremediation technologies can be classified as in situ or ex situ. In situ
bioremediation involves treating the contaminated material at the site while ex situ involves
the removal of the contaminated material to be treated elsewhere. Some examples of
bioremediation technologies are bioventing, land farming, bioreactor, composting,
bioaugmentation and biostimulation.
Bioremediation can occur on its own (natural attenuation or intrinsic bioremediation)
or can be spurred on via the addition of fertilizers to increase the bioavailability within the
medium (biostimulation). Recent advancements have also proven successful via the
addition of matched microbe strains to the medium to enhance the resident microbe
population's ability to break down contaminants. Microorganism whose perform the function
of bioremediation is known as Bioremediators (bioaugmentation).


1.2 Examples of bioremediation cases :

1.2.1 Pesticides, San Francisco Bay Estuary

Pesticide contamination of rivers and streams is a matter of concern throughout the United
States. Field and laboratory studies in the Sacramento River and San Francisco Bay have
shown the effects of biological and non-biological processes in degrading commonly used
pesticides, such as molinate, thiobencarb, carbofuran, and methyl parathion.

1.2.2 Agricultural chemicals in the Midcontinent
Agricultural chemicals affect the chemical quality of ground water in many Midwestern States.
Studies in the Midcontinent have traced the fate of nitrogen fertilizers and pesticides in ground
and surface waters. These studies have shown that many common contaminants, such as the
herbicide atrazine, are degraded by biological (microbial degradation) and non-biological
(photolytic degradation) processes.
1.2.3 Sewage effluent, Cape Cod, Massachusetts
Disposal of sewage effluent in septic drain fields is a common practice throughout the United
States. Systematic studies of a sewage effluent plume at Massachusetts Military Reservation
(formerly known as Otis Air Force Base) led to the first accurate field and laboratory
measurements of how rapidly natural microbial populations degrade nitrate contamination
(denitrification) in a shallow aquifer.

7

1.2.4 Crude oil spill, Bemidji, Minnesota

In 1979, a pipeline carrying crude oil burst and contaminated the underlying aquifer. U.S.
Geological Survey (USGS) scientists studying the site found that toxic chemicals leaching
from the crude oil were rapidly degraded by natural microbial populations. Significantly, it was
shown that the plume of contaminated ground water stopped enlarging after a few years as
rates of microbial degradation came into balance with rates of contaminant leaching. This
was the first and best-documented example of intrinsic bioremediation in which naturally
occurring microbial processes remediates contaminated ground water without human
intervention.






































8
2.1-Definition of heavy metals:

The term heavy metal refers to any metallic chemical element that has a relatively high
density and is toxic or poisonous at low concentrations. Examples of heavy metals
include mercury (Hg), cadmium (Cd), arsenic (As), chromium (Cr), thallium (Tl),
and lead (Pb).
Heavy metals are natural components of the Earth's crust. They cannot be
degraded or destroyed. To a small extent they enter our bodies via food, drinking water
and air. As trace elements, some heavy metals (e.g. copper, selenium, zinc) are essential
to maintain the metabolism of the human body. However, at higher concentrations they can
lead to poisoning. Heavy metal poisoning could result, for instance, from drinking-water
contamination (e.g. lead pipes), high ambient air concentrations near emission sources, or
intake via the food chain.
Heavy metals are dangerous because they tend to bioaccumulate.
Bioaccumulation means an increase in the concentration of a chemical in a biological
organism over time, compared to the chemical's concentration in the environment.
Compounds accumulate in living things any time they are taken up and stored faster than
they are broken down (metabolized) or excreted.
2.2-How metals get into freshwater :

Metals are introduced into aquatic systems as a result oI a variety oI human activities involving the
mining. processing. or use oI metals and/or substances that contain metal pollutants. The most
common heavy metal pollutants are arsenic. cadmium. chromium. copper. nickel. lead and mercury.
There are diIIerent types oI sources oI pollutants: point sources (localized pollution). where
pollutants come Irom single. identiIiable sources. The second type oI pollutant sources are nonpoint
sources. where pollutants come Irom dispersed (and oIten diIIicult to identiIy) sources. There are
only a Iew examples oI localized metal pollution. like the natural weathering oI ore bodies and the
little metal particles coming Irom coal-burning power plants via smokestacks in air. water and soils
around the Iactory.
The most common metal pollution in freshwater comes from mining companies. They
usually use an acid mine drainage system to release heavy metals from ores, because
metals are very soluble in an acid solution. After the drainage process, they disperse the
acid solution in the groundwater, containing high levels of metals.

2.3- What happens when an excess of metals enters freshwater ecosystems.
When the pH in water falls, metal solubility increases and the metal particles become more
mobile. That is why metals are more toxic in soft waters. Metals can become locked up
in bottom sediments, where they remain for many years. Streams coming from draining
mining areas are often very acidic and contain high concentrations of dissolved metals
with little aquatic life. Both localized and dispersed metal pollution cause environmental
damage because metals are non-biodegradable. Unlike some organic pesticides, metals
cannot be broken down into less harmful components in the environment.

The ionic form of a metal is more toxic, because it can form toxic compounds with other
ions. Electron transfer reactions that are connected with oxygen can lead to the production
of toxic oxyradicals, a toxicity mechanism now known to be of considerable importance in
both animals and plants. Some oxyradicals, such as superoxide anion (O2-) and the
hydroxyl radical (OH-), can cause serious cellular damage.
9
2.4-Metal tolerance
Some metals, such as manganese, iron, copper, and zinc are essential micronutrients. They are
essential to life in the right concentrations, but in excess, these chemicals can be poisonous.
At the same time, chronic low exposures to heavy metals can have serious health effects in
the long run.
Tolerance to metals has also been recorded in invertebrates and in fish. After exposure
for 24 hours to a copper concentration of 0.55 mg/l, rainbow trout showed a 55 per cent
inhibition of sodium uptake and a 4 per cent reduction in affinity for sodium, which resulted in
an overall decrease in total sodium concentration of sulphydryl-rich protein (Lauren and
McDonald 1987a,b). The protein was considered to be a metallothionein. These low
molecular weight proteins contain many sulphur-rich amino acids which bind and detoxify
some metals. The pretreatment of an organism with low doses of a metal may stimulate
metallothionein synthesis and provide tolerance during a subsequent exposure (Pascoe and
Beattie, 1979).


2.5-Toxicity of metals .

For the protection of human health, the maximum permissible concentrations for metals in natural
waters that are recommended by the Environmental Protection Agency (EPA), are listed below:
Table 1 : Maximum Permissible Concentrations (MPC) of Various Metals in Natural Waters For the
Protection of Human Health
Metal Chemical Symbol mg m
-3

Mercury Hg 0.144
Lead Pb 5
Cadmium Cd 10
Selenium Se 10
Thallium Tl 13
Nickel Ni 13.4
Silver Ag 50
Manganese Mn 50
Chromium Cr 50
Iron Fe 300
Barium Ba 1000

This table gives an idea of the relative toxicity of various metals. Mercury, lead and cadmium are
not required even in small amounts by any organism.

Because metals are rather insoluble in neutral or basic , pHs of 7 or above give a highly
misleading picture of the degree of metal pollution. So in some cases it may underestimate
significantly the total of metal concentrations in natural waters.

The three most pollutans heavy metals are Lead, Cadmium, and Mercury.


10

Effects of heavy metals are summarized as following :

2.5.1 Effects of Chromium on the environment

Chromium is used in metal alloys and pigments for paints, cement, paper, rubber, and other
materials. Low-level exposure can irritate the skin and cause ulceration. Long-term exposure can
cause kidney and liver damage, and damage too circulatory and nerve tissue. Chromium often
accumulates in aquatic life, adding to the danger of eating fish that may have been exposed to
high levels of chromium.

2.5.2 Effects of Copper on the environment
Copper is an essential substance to human life, but in high doses it can cause anemia, liver and
kidney damage, and stomach and intestinal irritation. People with Wilson's disease are at greater
risk for health effects from overexposure to copper. Copper normally occurs in drinking water from
copper pipes, as well as from additives designed to control algal growth.

2.5.3 Effects of Lead on the environment
In humans exposure to lead can result in a wide range of biological effects depending on the level
and duration of exposure. Various effects occur over a broad range of doses, with the developing
foetus and infant being more sensitive than the adult. High levels of exposure may result in toxic
biochemical effects in humans which in turn cause problems in the synthesis of haemoglobin,
effects on the kidneys, gastrointestinal tract, joints and reproductive system, and acute or chronic
damage to the nervous system.

2.5.4 Effects of Mercury on the environment
Mercury is a toxic substance which has no known function in human biochemistry or physiology
and does not occur naturally in living organisms. Inorganic mercury poisoning is associated with
tremors, gingivitis and/or minor psychological changes, together with spontaneous abortion and
congenital malformation.
Monomethylmercury causes damage to the brain and the central nervous system, while
foetal and postnatal exposure have given rise to abortion, congenital malformation and
development changes in young children.

2.5.5 Effects of Nickel on the environment
Small amounts of Nickel are needed by the human body to produce red blood cells, however, in
excessive amounts, can become mildly toxic. Short-term overexposure to nickel is not known to
cause any health problems, but long-term exposure can cause decreased body weight, heart
and liver damage, and skin irritation. The EPA does not currently regulate nickel levels in
drinking water. Nickel can accumulate in aquatic life, but its presence is not magnified along
food chains.
11

2.5.6 Effects of Selenium on the environment
Selenium is needed by humans and other animals in small amounts, but in larger amounts can
cause damage to the nervous system, fatigue, and irritability. Selenium accumulates in living
tissue, causing high selenium content in fish and other organisms, and causing greater health
problems in human over a lifetime of overexposure. These health problems include hair and
fingernail loss, damage to kidney and liver tissue, damage to circulatory tissue, and more severe
damage to the nervous system.

2.5.7 Effects of Antimony on the environment
Antimony is a metal used in the compound antimony trioxide, a flame retardant. It can also be
found in batteries, pigments, and ceramics and glass. Exposure to high levels of antimony for
short periods of time causes nausea, vomiting, and diarrhea. There is little information on the
effects of long-term antimony exposure, but it is a suspected human carcinogen. Most antimony
compounds do not bioaccumulate in aquatic life.

2.5.8 Effects of Cadmium on the environment
Cadmium derives its toxicological properties from its chemical similarity to zinc an essential
micronutrient for plants, animals and humans. Cadmium is biopersistent and, once absorbed
by an organism, remains resident for many years (over decades for humans) although it is
eventually excreted.
In humans, long-term exposure is associated with renal disfunction. High exposure can lead
to obstructive lung disease and has been linked to lung cancer, although data concerning the
latter are difficult to interpret due to compounding factors. Cadmium may also produce bone
defects (osteomalacia, osteoporosis) in humans and animals. In addition, the metal can be linked
to increased blood pressure and effects on the myocardium in animals, although most human
data do not support these findings.





12

Methods of heavy metals removal are divided into three classes according to the
nature of the process :

3.1Chemical methods:


3.1.1 Carbamates

Carbamates are chemical reducing agents that can be obtained as either sodium
dimethyldithiocarbamate or sodium diethyldithiocarbamate. Carbamate precipitation is again
an equilibrium reaction that does not go to completion. Metal residuals of 1.0 - 1.5 mg/l can
usually be obtained. Carbamates are not effective at acidic pH levels and are not always
effective at treating chelated wastes.


3.1.2 Selective heavy metals removal from waters by amorphous zirconium phosphate

Uptake of heavy metals including lead, cadmium, and zinc onto ZrP was studied by using a
polystyrene sulfonic-acid exchanger D-001 as a reference sorbent and Ca2+ as a
competing cation due to its ubiquity in natural or industrial waters. The results indicated that
the uptake of heavy metals onto ZrP is essentially an ion-exchange process and dependent
upon solution pH. In comparison with D-001, ZrP exhibited more favorable sorption of
heavy metals particularly in terms of high selectivity, as indicated by the distribution
coefficients of ZrP even several orders higher than D-001 towards heavy metals when
calcium ion coexisted at a high level in solution.( Bingcai Pan, Qingrui Zhang, Wei Du,
Weiming Zhang, Bingjun Pan, Qingjian Zhang, Zhengwen Xu, Quanxing Zhang ) .

3.1.3 Organometallic Precipitation
In 1991 Steve Holtzman, pioneered a new more environmentally responsible method of
removing heavy metals. This process revolves around the formation of insoluble
organometallic compounds formed by reacting metal bearing wastes with a proprietary
organic agent.
3.1.4 Chemical precipitation
Precipitation of metals is achieved by the addition of coagulants such as alum, lime, iron
salts and other organic polymers. The large amount of sludge containing toxic
compounds produced during the process is the main disadvantage.
Hydroxide precipitation : Chemical precipitation of heavy metals as their hydroxides
using lime or sodium hydroxide is widely used. Lime is generally favoured for precipitation
purposes due to the low cost of precipitant, ease of pH control in the range of 8.0 10.0
and the excess of lime also serves as an adsorbent for the removal of metal ions. The
efficiency of the process depends on a number of factors, which include the ease of
hydrolysis of the metal ion, nature of the oxidation state, pH, presence of complex
forming ions, standing time, degree of agitation and settling and filtering and
characteristics of the precipitate. The limitations of this method include difference
between metals in the optimum pH for hydroxide formation may lead to the problems in
the treatment of effluents containing combined metal ions. Variability in metal hydroxide
solubility at a fixed pH is another drawback.
13

Carbonate precipitation : Carbonate precipitation of metals using calcium or sodium
carbonate is very limited. Patterson et al., 1997 reported improved results using
carbonate precipitate for Cd (II) and Pb (II) from electroplating effluents. When the pH
was brought to 7.5, residual concentration of Pb (II) and Cd (II) were 0.60 and 0.25 mg/L
respectively.

Sulphide precipitation : Since most of the heavy metals form stable sulphides, excellent
metal removal can be obtained by sulphide precipitation. Treatment with sulphides is
most advantageous when used as a polishing step after conventional hydroxide
precipitation or when very high metal removals are required.

3.1.5 Chemical reduction
Reduction of hexavalent chromium can also be accomplished with electro-chemical units. The
electrochemical chromium reduction process uses consumable iron electrodes and an
electric current to generate ferrous ions that react with hexavalent chromium to give
trivalent chromium as follows United States Environmental Protection Agency (USEPA,
1979).

3Fe
2+
+ CrO
4
2-
+ 4H
2
O 3Fe
3+
+ Cr
3+
+ 8OH
-

Another application of reduction process is the use of sodium borohydride, which has been
considered effective for the removal of mercury, cadmium, lead, silver and gold (Kiff, 1987).

3.1.6 Xanthate process
Insoluble starch xanthate (ISX) is made from commercial cross linked starch by reacting it with
sodium hydroxide and carbon disulphide. To give the product stability and to improve the
sludge settling rate, magnesium sulphate is also added. ISX works like an ion exchanger,
removing the heavy metals from the wastewater and replacing them with sodium and
magnesium. Average capacity is 1.1-1.5 meq of metal ion per gram of ISX (Anon, 1978).
ISX is most commonly used by adding to it the wastewater as slurry for continuous flow
operations or in the solid form for batch treatments. It should be added to the effluent at pH 3.
Then the pH should be allowed to rise above 7 for optimum metal removal (Wing, 1978).
Residual metal ion level below 50 g/L has been reported (Hanway et al., 1978, Wing et al.,
1978). The effectiveness of soluble starch xanthate (SSX) for removal of Cd (II), Cr (VI) and Cu
(II) and insoluble starch xanthate (ISX) for Cr (VI) and Cu (II) have been evaluated under
different aqueous phase conditions. Insoluble starch xanthate had better binding capacity for
metals. The binding capacity of SSX and ISX respectively for different metal ions follows the
sequence of Cr (VI)> Cu (II)> Cd(II) and Cr (VI)> Cu (II) (Tare et al., 1988).


14
3.1.7 Solvent extraction
Liquid-liquid extraction (also frequently referred as solvent extraction) of metals from solutions
on a large scale has experienced a phenomenal growth in recent years due to the
introduction of selective complexing agents (Beszedits, 1988). In addition to
hydrometallurgical applications, solvent extraction has gained widespread usage for waste
reprocessing and effluent treatment.
Solvent extraction involves an organic and an aqueous phase. The aqueous solution
containing the metal or metals of interest is mixed with the appropriate organic solvent and the
metal passes into the organic phase. In order to recover the extracted metal, the organic
solvent is contacted with an aqueous solution whose composition is such that the metal is
stripped from the organic phase and is reextracted into the stripping solution. The
concentration of the metal in the strip liquor may be increased, often 110 to 100 times over
that of the original feed solution. Once the metal of interest has been removed, the organic
solvent is recycled either directly or after a fraction of it has been treated to remove the
impurities.

3.2- Physical methods :
3.2.1 Membrane process
Important examples of membrane process applicable to inorganic wastewater treatment
include reverse osmosis and eletrodialysis (Environmental Protection Agency EPA,
1980). These processes involve ionic concentration by the use of selective membrane with
a specific driving force. For reverse osmosis, pressure difference is employed to initiate the
transport of solvent across a semipermeable membrane and electro dialysis relies on ion
migration through selective permeable membranes in response to a current applied to
electrodes. The application of the membrane process described is limited due to
pretreatment requirements, primarily, for the removal of suspended solids. The methods
are expensive and sophisticated, requiring a higher level of technical expertise to operate.
A liquid membrane is a thin film that selectively permits the passage of a specific
constituent from a mixture (Beszedits, 1988). Unlike solid membranes, however liquid
membranes separate by chemistry rather than size, and thus in many ways liquid membrane
technology is similar to solvent extraction. Since liquid membrane technology is a fairly recent
development, a number of problems remain to be solved. A major issue with the use of
supported membranes is the long term stability of the membranes, whereas the efficient
breakup of microspheres for product recovery is one of the difficulties encountered frequently
with emulsion membranes.
3.2.2 Evaporators
In the electroplating industry, evaporators are used chiefly to concentrate and recover valuable
plating chemicals. Recovery is accomplished by boiling sufficient water from the collected
rinse stream to allow the concentrate to be returned to the plating bath. Many of the
evaporators in use also permit the recovery of the condensed steam for recycle as rinse
water. Four types of evaporators are used throughout the elctroplating industry (USEPA,
1979a) (I) Rising film evaporators; (ii) Flash evaporators using waste heat; (iii)
submerged tube evaporators; (iv) Atmospheric evaporators. Both capital and operational
costs for evaporative recovery systems are high. Chemical and water reuse values must
offset these costs for evaporative recovery to become economically feasible.
15
3.2.3 Cementation
Cementation is the displacement of a metal from solution by a metal higher in the electromotive series.
It offers an attractive possibility for treating any wastewater containing reducible metallic ions. In
practice, a considerable spread in the electromotive force between metals is necessary to ensure
adequate cementation capability. Due to its low cost and ready availability, scrap iron is the metal
used often. Cementation is especially suitable for small wastewater flow because a long contact
time is required. Some common examples of cementation in wastewater treatment include the
precipitation of copper from printed etching solutions and the reduction of Cr (VI) in chromium
plating and chromate-inhibited cooling water discharges (Case, 1974). Removal and recovery of
lead ion by cementation on iron sphere packed bed has been reported (Angelidis et al., 1988,
1989). Lead was replaced by a less toxic metal in a harmless and reusable form.
3.2.4 Ion exchange
Ion exchange resins are available selectively for certain metal ions. The cations are exchanged for H+
or Na+. The cation exchange resins are mostly synthetic polymers containing an active ion
group such as SO3H. The natural materials such as zeolites can be used as ion exchange
media (Van der Heen, 1977). The modified zeolites like zeocarb and chalcarb have greater
affinity for metals like Ni and Pb (Groffman et al., 1992). The limitations on the use of ion
exchange for inorganic effluent treatment are primarily high cost and the requirements for
appropriate pretreatment systems. Ion exchange is capable of providing metal ion
concentrations to parts per million levels. However, in the presence of large quantities of
competing mono-and divalent ions such as Na and Ca, ion exchange is almost totally ineffective.
3.2.5 Electrodeposition
Some metals found in waste solution can be recovered by electrodeposition using insoluble anodes.
For example, spent solutions resulting from sulphuric acid cleaning of Cu may be saturated
with copper sulphate in the presence of residual acid. These are ideal for electro-winning
where the high quality cathode copper can be electrolytically deposited while free sulphuric
acid is regenerated.
3.2.6 Adsorption
Since activated carbon also possesses an affinity for heavy metals, considerable attention has been
focussed on the use of carbon for the adsorption of hexavalent chromium, complexed cyanides
and metals present in various other forms from wastewaters. Watonabe and Ogawa (1929) first
presented the use of activated carbon for the adsorption of heavy metals. The mechanism of
removal of hexavalent and trivalent chromium from synthetic solutions and electroplating effluents
has been extensively studied by a number of researchers. According to some investigators, the
removal of Cr (VI) occurs through several steps of interfacial reactions (Huang and Bowers,
1979).
The direct adsorption of Cr
6+
onto carbon surface.
The reduction of Cr
6+
species to Cr
3+
by carbon on the surface.
The adsorption of the Cr
3+
species produced, which occurs to a much lesser extent than
the adsorption of the Cr
6+
species.
Adsorption of Cr (III) and Cr (VI) on activated carbon from aqueous solutions has been studied
(Toledo, 1994). Granular activated carbon columns have been used to treat wastewaters containing
lead and cadmium (Reed and Arunachalam, 1994, Reed et al., 1994). Granular activated carbon
was used for the removal of Pb (II) from aqueous solutions (Cheng et al., 1993). The adsorption
process was inhibited by the presence of humic acid, iron (III), aluminum (III) and calcium (II).
16

Disadvantages of conventional chemical & physical methods for treatment of
wastewater containing heavy metals
Precipitation, by adjusting the pH value is not selective and any iron (ferric ion) present in the liquid
effluent will be precipitated initially followed by other metals. Consequently precipitation
produces large quantities of solid sludge for disposal, for example precipitation as hydroxides of
100 mg/l of copper (II), cadmium (II) or mercury (II) produces as much as 10-, 9- and 5 fold mg/l
of sludges respectively. The metal hydroxide sludge resulting from treatment of electroplating
wastewater has been classified as a hazardous waste. The performance characteristics of
heavy metal waste water treatment technologies are identified in the table below. The versatility,
simplicity and other technology characteristics will contribute to the overall process costs, both
capital and operational. At present many of these technologies such as ion exchange represent
significant capital investments by industry.
Table 2: Performance characteristics of various heavy metal removal
/recovery technologies
Technology pH
change
Metal
selectivity
Influence
of
Suspended
solids
Tolerance
of
organic
molecules
Working
level for
appropriate
metal
(mg/I)
Adsorption, e.g.
Granulated
Activated
carbon
Limited
tolerance
Moderate Fouled Can be
poisoned
<10
Electro
chemical
Tolerant Moderate Can be
engineered
to tolerate
Can be
accommodated
>10
Ion exchange Limited
tolerance
Chelate -
resins can
be selective
Fouled Can be
poisoned
<100
Membrane Limited
tolerance
Moderate Fouled Intolerant >10
Precipitation
(a) Hydroxide Tolerant Non-selective Tolerant Tolerant >10
(b) Sulphide Limited
tolerance
Limited
selective
pH dependent
Tolerant Tolerant >10
Solvent
extraction
Some
systems
Metal
selective
Fouled Intolerant >100
pH
tolerant
extractants
available

As seen from the table above, conventional methods are ineffective in the removal of
low concentrations of heavy metals and they are non-selective. Moreover, it is not
possible to recover the heavy metals by the above mentioned methods.




17
3.3- Biological methods :

3.3.1 Biosorption
During the 1970s increasing environmental awareness and concern led to a search for new
techniques capable of inexpensive treatment of polluted wastewaters with metals. The search
for new technologies involving the removal of toxic metals from wastewaters has directed
attention to biosorption, based on binding capacities of various biological materials.
Till date, research in the area of biosorption suggests it to be an ideal alternative for
decontamination of metal containing effluents. Biosorbents are attractive since naturally occurring
biomass/adsorbents or spent biomass can be effectively used. Biosorption is a rapid phenomenon
of passive metal sequestration by the non-growing biomass/adsorbents. Results are convincing and
binding capacities of certain biomass/adsorbents are comparable with the commercial synthetic
cation exchange resins.
The biosorption process involves a solid phase (sorbent or biosorbent; adsorbent; biological
material) and a liquid phase (solvent, normally water) containing a dissolved species to be sorbed
(adsorbate, metal). Due to the higher affinity of the adsorbent for the adsorbate species, the latter is
attracted and bound there by different mechanisms. The process continues till equilibrium is
established between the amount of solid-bound adsorbate species and its portion remaining in the
solution. The degree of adsorbent affinity for the adsorbate determines its distribution between the
solid and liquid phases.
There are many types of adsorbents; Earths forests and plants, ocean and freshwater
plankton, algae and fish, all living creatures, that including animals are all biomass/ adsorbents.
The renewable character of biomass that grows, fuelled directly or indirectly by sunshine, makes it
an inexhaustible pool of chemicals of all kinds.
Biosorption has advantages compared with conventional techniques (Volesky, 1999). Some
of these are listed below:
Cheap: the cost of the biosorbent is low since they often are made from
abundant or waste material.

Metal selective: the metalsorbing performance of different types of biomass can
be more or less selective on different metals. This depends on various factors
such as type of biomass, mixture in the solution, type of biomass preparation and
physico-chemical treatment.

Regenerative: biosorbents can be reused, after the metal is recycled.

No sludge generation: no secondary problems with sludge occur with
biosorption, as is the case with many other techniques, for example,
precipitation.

Metal recovery possible: In case of metals, it can be recovered after being
sorbed from the solution.

Competitive performance: biosorption is capable of a performance comparable to
the most similar technique, ion exchange treatment. Ion exchange is, as
mentioned above, rather costly, making the low cost of biosorption a major
factor.
18

Biosorbents intended for bioremediation environmental applications are waste biomass of
crops, algae, fungi, bacteria, etc., which are the naturally abundant. Numerous chemical groups
have been suggested to contribute to biosorption. A review of biosorption of heavy metals by
microorganisms is presented below. Biosorption by microorganisms have various disadvantages,
and hence many low cost adsorbents (industrial/agricultural waste products/byproducts) are
increasingly used as biosorbents. This chapter also provides review of the low cost adsorbents
used for removal of heavy metals (Ahalya et al., 2004).
- Biosorption of heavy metals by microorganisms
A large number of microorganisms belonging to various groups, viz. bacteria, fungi, yeasts,
cyanobacteria and algae have been reported to bind a variety of heavy metals to different extents
(table 3 ). The role of various microorganisms by biosorption in the removal and recovery of heavy
metal(s) has been well reviewed and documented (Stratton, 1987; Gadd and Griffiths, 1978;
Volesky, 1990; Wase and Foster, 1997; Greene and Darnall, 1990; Gadd 1988). Most of the
biosorption studies reported in literatures have been carried out with living microorganisms.
However due to certain inherent disadvantages, use of living microorganisms for metal removal
and recovery is not generally feasible in all situations. For example, industrial effluents contain
high concentrations of toxic metals under widely varying pH conditions. These conditions are not
always conducive to the growth and maintenance of an active microbial population. There are
several advantages of biosorption of using non living biomass and they are as follows:
1. Growth independent nonliving biomass is not subject to toxicity limitation by cells.
2. The biomass from an existing fermentation industry, which essentially is a waste after
fermentation, can be a cheap source of biomass.
3. The process is not governed by physiological constraints of microbial cells.
4. Because nonliving biomass behaves as an ion exchanger, the process is very rapid,
requiring anywhere between few minutes to few hours. Metal loading is very high on
the surface of the biomass leading to very efficient metal uptake.
5. Because cells are non-living processing conditions are not restricted to those
conducive for the growth of the cells. Hence, a wider range of operating conditions
such as pH, temperature and metal concentrations are possible. Also aseptic operating
conditions are not essential.
6. Metals can be desorbed readily and then recovered. If the value and the amount of
metal recovered are insignificant and if the biomass is plentiful, the metal loaded
biomass can be incinerated, eliminating further treatment.
Biosorption essentially involves adsorption processes such as ionic, chemical and physical
adsorption. A variety of ligands located on the fungal cell walls are known to be involved in metal
chelation. These include carboxyl, amine, hydroxyl, phosphate and sulphydryl groups. Metal ions
could be adsorbed by complexing with negatively charged reactions sites on the cell surface. In the
table below presents an exhaustive list of microrganisms used for the uptake of heavy metals.





19
Table 3 : Biosorbent uptake of metals by Microbial Biomass

Metal Biomass Type Biomass class Metal uptake
(mg/g)
Freshwater alga Biosorbent 86-94
Fungal biomass Biosorbent 65
Rhizopus arrhizus Fungus 54
Streptomyces noursei Filamentous bacter 38.4
Ag




Sacchromyces cerevisiae Yeast 4.7
Sargassum natans Brown alga 400
176 Aspergillus niger Fungus
15
Rhizopus arrhizus Fungus 164
Palmaria tevera Marine alga 164
Palmaria palmata Marine alga 124
Chlorella pyrenoidosa Freshwater alga 98
Cyanidium caldarium Alga 84
Chlorella vulgaris Freshwater alga 80
Bacillus subtilis Bacteria Cell wall 79
Chondrus crispus Marine alga 76
Bacillus subtilis Bacterium 70
71 Spirulina platensis Freshwater alga
58
Rhodymenia palmata Marine alga 40
Au













Ascophyllum nodosum Brown marine alga 24
Ascophyllum nodosum Brown markertman
ine alga
215
Sargassum natans Brown marine alga 135
Fucus vesiculosus Brown marine alga 73
Candida tropicalis Yeast 60
56 Pencillium chrysogenum Fungus
11
Rhizopus arrhizus Fungus 30
Sacchromyces cervisiae Yeast 20-40
Rhizopus arrhizus Fungus 27
Rhizopus nigricans Fungus 19
Pencillium spinulosum Fungus 0.4
Pantoea sp. TEM 18 Bacteria 204.1
Chlamydomonas reinhardtii Alga 42.6
Spirulina sp. Blue green algae 1.77 meq/g
Enterobacter cloaceae
(Exopolysaccharide)
Marine bacterium 16
Padina sp. Brown seaweed 0.75
Sargassum sp. Brown seaweed 0.76
Ulva sp. Green seaweed 0.58
Gracillaria sp. Red seaweed 0.30
Cd


















Gloeothece magna Cyanobacteria 115425 g mg1
Ascophyllum nodosum Brown marine algae 100
Sacchromyces cerevisiae Yeast 4.7
Co


Ulva reticulata Marine green algae 46.1
20
Enterobacter cloaceae Marine bacterium 4.38
Bacillus biomass Bacterium 118 Cr3+
60 Cr 6+
Rhizopus arrhizus Fungus 31
Candida tropicalis Yeast 4.6
Streptomyces nouresei Bacteria 1.8
Pantoea sp. TEM 18 Bacteria 204.1
Spirulina sp. Cyanobacteria 10.7 meq/g
Cr






Spirogyra sp. Filamentous algae 4.7
Bacillus subtilis Biosorbent 152
Candida tropicalis Yeast 80
Manganese oxidising bacteria MK-2 50
Cladosporium resinae Fungus 18
Rhizopus arrhizus Fungus 16
Saccharomyces crevisae Yeast 17-40; 10; 6.3
Pichia guilliermondii Yeast 11
Scenedesmus obliquus Freshwater algae 10
Rhizopus arrhizus Fungus 10
Pencillium chrysogenum Fungus 9
Streptomyces noursei sp. Filamentous bacteria 5
Bacillus sp Bacterium 5
Pencillium spinulosum Fungus 0.4-2
Aspergillus niger Fungus 1.7
Trichoderma viride Fungus 1.2
Pencillium chrysogenum Fungus 0.75
Pantoea sp. TEM 18 Bacteria 31.3
Ulva reticulata Marine green alga 56.3
Spirulina sp. Blue green algae 6.17 meq/g
Enterobacter cloaceae
(Exopolysaccharide)
Marine bacterium 6.60
Padina sp. Brown seaweed 1.14
Sargassum sp. Brown seaweed 0.99
Ulva sp. Green seaweed 0.75
Gracillaria sp. Red seaweed 0.59
Thiobacillus thiooxidans Bacteria 38.54
Cu

























Ulothrix zonata Algae 176.20
Bacillus subtillis Bacterial cell wall
preparation
201
Bacillus biomass Bacterium 107
Fe


Sargassum fluitans Brown alga 60
Rhizopus arrhizus Fungus 54
Pencillium chrysogenum
(biomass not necessarily in its
natural state)
Fungus 20
Cystoseira baccata Marine alga 178
Hg



Chlamydomonas reinhardtii Algae 72.2
Fucus vesiculosus Brown marine algae 40
Ascophylum nodosum Brown marine algae 30
Sargassum natans Brown marine algae 24-44
Ni




Bacillus licheniformis Bacterial cell wall
preparation
29
21
Candida tropicalis Yeast 20
Rhizopus arrhizus Fungus 18
Bacillus subtillis Bacterial cell wall
preparation
6
Rhizopus nigricans Fungus 5
Absidia orchidis Fungus 5
Ulva reticulata Marine green algae 46.5
Padina sp. Brown seaweed 0.63
Sargassum sp. Brown seaweed 0.61
Ulva sp. Green seaweed 0.29
Gracillaria sp. Red seaweed 0.28










Polyporous versicolor White rot fungus 57
Bacillus subtilis(biomass not
necessarily in its natural
state)
Biosorbent 601
Absidia orchidis Fungus 351
Fucus vesiculosus Brown marine algae 220-370
Ascophyllum nodosum Brown marine algae 270-360
Sargassum natans Brown marine algae 220-270
Bacillis subtilis(biomass not
necessarily in its natural
state)
Biosorbent 189
Pencillium chrysogenum Fungus 122; 93
Rhizopus nigricans Fungus 166
Streptomyces longwoodensis Filamentous bacteria 100
Rhizopus arrhizus Fungus 91; 55
Streptomyces noursei Filamentous bacteria 55
Chlamydomonas reinhardtii Algae 96.3
Padina sp. Brown seaweed 1.25
Sargassum sp. Brown seaweed 1.26
Ulva sp. Green seaweed 1.46
Gracillaria sp. Red seaweed 0.45
Pb
















Ecklonia radiata Marine alga 282
Freshwater alga(biomass not
necessarily in its natural
state)
Biosorbent 436 Pd

Fungal biomass Biosorbent 65
Pt Freshwater alga (biomass not
necessarily in its natural
state)
Biosorbent 53
Sargassum fluitans Brown algae 520
Streptomyces longwoodensis Filamentous bacteria 440
Rhizopus arrhizus Fungus 220; 195
Sacchromyces crevisae Yeast 55-140
U








Bacillus sp. Bacterium 38
22
Chaetomium distortum Fungus 27
Trichoderma harzianum Fungus 26
Pencillium chrysogenum
(biomass not necessarily in its
natural state)
Fungus 25
Alternaria tenulis
Rhizopus arrhizus Fungus 160; 93 Th

Sacchromyces cerevisae Yeast 70
Bacillus subtilis(biomass not
necessarily in its natural
state)
Biosorbent 137
Sargassa sp.



Brown algae 70
Manganese oxidising bacteria (MK-2) 39
Sacchromyces cerevisae Yeast 14-40
Candida tropicalis Yeast 30
Rhizopus arrhizus Fungus 20; 14
Pencillium chrysogenum Fungus 6.5
Bacillus sp. Bacterium 3.4
Pencillium spinulosum Fungus 0.2
Padina sp. Brown seaweed 0.81
Sargassum sp. Brown seaweed 0.50
Ulva sp. Green seaweed 0.54
Gracillaria sp. Red seaweed 0.40
Zn













Thiobacillus thiooxidans Bacteria 43.29
Among micro-organisms, fungal biomass offers the advantages of having high
percentage of cell wall material, which shows excellent metal binding properties (Gadd,
1990; Rosenberger, 1975; Paknikar, Palnitkar and Puranik, 1993). Many fungi and
yeast have shown an excellent potential of metal biosorption, particularly the
genera Rhizopus,Aspergillus, Streptoverticullum and Sacchromyces (Volesky and
Tsezos, 1981; Galun et al., 1984; de Rome and Gadd, 1987; Siegel et al., 1986; Luef et
al., 1991, Brady and Duncan, 1993 Puranik and Paknikar, 1997).
23


3.3.2 Phytoremediation

Phytoremediation is the use of certain plants to clean up soil, sediment, and water contaminated
with metals. The disadvantages include that it takes a long time for removal of metals and the
regeneration of the plant for further biosorption is difficult.



3.3.3 Complexation
The metal removal from solution may also take place by complex formation on the cell surface after
the interaction between the metal and the active groups. Aksu et al. 1992 hypothesized that
biosorption of copper by C. vulgaris and Z. ramigera takes place through both adsorption and
formation of coordination bonds between metals and amino and carboxyl groups of cell wall
polysaccharides. Complexation was found to be the only mechanism responsible for calcium,
magnesium, cadmium, zinc, copper and mercury accumulation by Pseudomonas syringae. Micro-
organisms may also produce organic acids (e.g., citric, oxalic, gluonic, fumaric, lactic and malic
acids), which may chelate toxic metals resulting in the formation of metallo-organic molecules.
These organic acids help in the solubilisation of metal compounds and their leaching from their
surfaces. Metals may be biosorbed or complexed by carboxyl groups found in microbial
polysaccharides and other polymers.




3.3.4 Removal of heavy metals using waste eggshell

When calcined eggshell was applied in the treatment of synthetic wastewater containing heavy
metals, a complete removal of Cd as well as above 99% removal of Cr was observed after 10
min.
Although the natural eggshell had some removal capacity of Cd and Cr, a complete removal
was not accomplished even after 60 min due to quite slower removal rate. However, in contrast
to Cd and Cr, an efficient removal of Pb was observed with the natural eggshell rather than the
calcined eggshell. From the application of the calcined eggshell in the treatment of real
electroplating wastewater, the calcined eggshell showed a promising removal capacity of heavy
metal ions as well as had a good neutralization capacity in the treatment of strong acidic
wastewater. (PARK Heung Jai, JEONG Seong Wook, YANG Jae Kyu, KIM Boo Gil, LEE Seung Mok) .


24

4- Mechanisms of microbial removal of heavy metals:


4.1 Metal reduction

Bacterial reduction of metallic ions has been shown to occur for U(VI), Se(VI), Cr(VI), Mo(VI), Se(IV),
Hg(II), Ag(I) and others (Bradley and Obraztsova, 1998). Three Cr(VI) reduction mechanisms have
been described (Cervantes and Campos, 2007): (i) In aerobic conditions, chromate reduction has
been commonly associated with soluble chromate reductases that use NADH or NADPH as
cofactors. (ii) Under anaerobiosis, some bacteria, can use Cr(VI) as an electron acceptor in the
electron transport chain (Bopp and Ehrlich, 1988). (iii) Reduction of Cr(VI) may also be carried out
by chemical reactions associated with compounds such as amino acids, nucleotides, sugars,
vitamins, organic acids or glutathione. For instance, ascorbate is capable of reducing Cr(VI), and
riboflavin derivatives FAD and FMN are essential coenzymes for chromate reducing flavoenzymes
(Masayasu, 1991).

Example

Biological reduction of chromate by Streptomyces griseus (Ashwini C. Poopal, R. Seeta
Laxman)

The most toxic form of chromium Cr(VI) can be converted to less toxic Cr(III) by reduction. Among the
actinomycetes tested for chromate reduction, thirteen strains reduced Cr(VI) to Cr(III), of which one
strain of Streptomyces griseus (NCIM 2020) was most efcient showing complete reduction within
24 h. Chromate reduction was associated with the bacterial cells and sonication was the best method
of cell breakage to release the enzyme. The enzyme was constitutive and did not require presence of
chromate during growth for expression of activity. Chromate reduction with cell free extract (CFE)
was observed without added NADH. However, addition of NAD(P)H resulted in 23-fold increase in
activity. Chromate reductase showed optimum activity at 28 C and pH 7.



CHROMIUM REMOVAL FROM TANNERY WASTEWATER USING CHEMICAL AND BIOLOGICAL
TECHNIQUES AIMING ZERO DISCHARGE OF POLLUTION (Hesham M. Abdulla , Engy M. Kamal
, Amr H. Mohamed , Ahmed D. El-Bassuony , Suez Canal University, Ismailia )

Tannery effluent containing chromium is one of the most obvious problems in leather industry. In
Egypt, the tannery wastewater is discharged directly to the main domestic sewage pipeline
which adds difficulties to the sewer system and to the wastewater treatment plants , Therefore,
the removal and reuse of the chromium content of these wastewaters is necessary for
environmental protection and economic reasons. In this study, the recovery of chromium (III) was
carried out by using precipitation process , To this purpose, two common precipitating agents
lime and cement dust were used.

For biological removal of Cr(VI), thirty four actinomycete isolates were found to tolerate up to
100 mg/l Cr(VI), five of these isolates could tolerate Cr(VI) at concentration up to 2500 mg/l and have
abilities to produce chromate reductas .







25



4.2 Biosorption

Biosorption is a rapid phenomenon of passive metal sequestration by the non-growing biomass
(Gourdon, et al., 1990). Biosorption mainly involves cell surface complexation, ion exchange and
microprecipitation (Gadd, 1990). Different microbes have been found to vary in their affinity for
different heavy metal(s) and hence differ in their metal binding capacities. Some biomass(es) exhibit
preference for certain heavy metal(s) whereas others do not show any specific binding and have a
broad range (Greene and Darnall, 1990 ).

The bacterial cell wall is the first component that comes into contact with metal ions, where the
solutes can be deposited on the surface or within the cell wall structure (Doyle et al., 1980). Since
the mode of solute uptake by dead/inactive cells is extracellular, the chemical functional groups of the
cell wall play vital roles in biosorption. Due to the nature of the cellular components, several
functional groups are present on the bacterial cell wall, including carboxyl, phosphonate, amine and
hydroxyl groups (Van der wal et al., 1997) .


Example

Biosorption of cadmium ions by Actinomycetes and separation by flotation (M.I.
Kefala, A.I. Zouboulis, K.A. Matis)

The removal of cadmium, a toxic metal of high environmental priority due to its toxicity, from dilute
aqueous solutions has been studied , applying microorganisms and using living, as well as non-
living bacterial biomass of two specially isolated Actinomycetes strains, AK61 and JL322. The
main parameters influencing this treatment process, namely contact time, pH of the solution,
temperature and toxic metal and biomass concentrations have been examined and Langmuir
isotherms have been depicted. Dispersed-air flotation was applied as the subsequent separation
method for harvesting the suspended metal-laden microorganisms, following the biosorption of
cadmium. The investigated parameters (in batch mode, laboratory scale) were in this case the
dispersion pH, the flotation time, the air flow rate and the surfactant concentration. Electrokinetic
measurements have been also performed for the biomass dispersions, under similar conditions with
biosorption, providing useful information for the process mechanisms. Applying the optimum
defined conditions, over 95% removal of cadmium has been achieved in one stage and
simultaneously, quantitative separation of the used (metal-loaded) biomass has been obtained.

Bioremediation of Zinc by Streptomyces aureofacienes (Osama H. EL Sayed, Hala
M. Refaat, Mahmoud A. Swellam, Mahmoud M. Amer, Aziza I. Attwa and Mohamed E.
El Awady Benha university , benha, Egypt)

In this study. twelve Streptomyces species isolated Irom Egyptian soil and identiIied by
using physiological tests as Bergeys manual was assessed quantitatively to the eIIects
oI zinc using plate diIIusion method. Biosorption oI zinc by biomass microorganisms
is an innovative and alternative technology Ior removal oI these pollutants due to their
good perIormance. low cost and large available quantities. Experiments in liquid
culture were used to determine concentration ranges oI the metals at which the most
tolerant species could grow showed that Streptomyces aureofacienes had maximum
uptake oI zinc (734.8 g g-1 biomass).


26



4.3 Siderophores

Siderophores are small organic molecules with strong affinity for ferric iron and are primarily
produced to mediate iron acquisition, but they are also able to bind other metals (Hu and Boyer,
1996) or in rare cases protect against metal toxicity (Cortese et al., 2002). Despite many
different structures of the siderophores, they are usually classified according to the type of the
binding ligand (Boukhalfa and Crumbliss, 2002). Ferrioxamines and ferrichromes were the first
siderophores recognized as iron transport agents produced by bacteria and fungi
(Budzikiewicz, 1997). Only few siderophores using carboxylate ligands are known, for example
staphyloferrin. Pyridine-2,6- bis(thiocarboxylic acid) (Pdtc) is an example of a siderophore using
sulfer as ligand donor atoms (Cornelis and Matthijs, 2002). Pdtc was identified as a
siderophore having such multiple properties, beside carbon tetrachloride degradation (Lee et al.,
1999). Pdtc forms soluble stable complexes with many transition metals such as iron, cobalt,
chromium and copper (Stolworthy et al., 2001). Many heavy metals and metalloids have been
found to form insoluble precipitates with pdtc, and pdtc was effective in protecting some bacteria
against metal toxicity (Cortese et al., 2002). Pdtc was found to reduce Cr(VI) to Cr(III) in
bacterial cultures and in abiotic reactions with chemically synthesized pdtc; Cr(III) subsequently
formed complexes with pdtc were found to slowly release Cr(III) as chromium sulfide and
possibly Cr(III) oxides and hydroxides (Chirwa and Wang, 2000). The properties of pdtc make it
a good candidate for heavy metal remediation by metal reduction and complexation, selective
precipitation of heavy metals from solution, and also solubilization of metal and mixed metal-
carbon tetrachloride contaminated wastes (Gadd,2004). .


27
-' .='-'

'-' `' ---''- '-=' -'-= -'-' '--
'--'' '-- ==' .- -' -' - '- --
--' --= --' - '`'-' - --- '+'
--=-= '--`' '-' '`'-' -- == .- .' .

-`' '-' '+-'`- .- --= -= -'-= '-
' --'' ---' --' .- '+---- -- -'
'-'-' .=-- ''-''- -'--' '-- ' ---' -'--'
-=' '-=` .= -- '+- '+-== .-- ---'
-` - -=' '-- - ''-''- .`- -`
-` .- '-= --'' '' .--' .

'--' .- '--`' '-' ' = .- -`' '-
-='--' --'---' --'---' =' '+-- --'-' -
='- -'= '--`' '-'' -='--' =''-' '= '--' -
- -- -' ---' '--'' =- .


28
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