ffi|l reTtb_o-9 ! 9f- _! !

oteg !n-o-to 11
-s

Benefiis from relecrse d'

lhe environmenl.
Protection of envirrnment
- Bioremediation of polluted environment
Control of global environmental process
- Reversal of land desertification
- Reversal of green house effect
Agriculture
- Increasing efficiency of plant nutrition
- Pest control (safe biopesticides)
- Protection of plants from climatic stress
- Protection of plants from tumour formation and disease
Food Industry
- Microorganisms producing enzymes for food industry
- Miiroorganisms with improved efficiency of fermentation
- Improved microorganisms for milk industry.
Health care
-Microoganisms as live attenuated vaccines
Source: Velkov (1996).

A. BIOREMET}IATION
Bioremediation is the use of living microorganisms to degrade environmental pollutants or
prevent pollution. It
is a technology for removing pollutants from the environment, restoring
contaminated sites and preventing future pollution. However, it has global, regional, and local
application.frh" basis oi bior"-e-diation is the enorrnous natural capacity of microorganisms to
OelraOe orgffi. compounds. This capacity could be. improved by applying the GMM:) -1$
In Japan, academic, industrial and governmental research is tightly coordinated for global
application of environmental biotechnology. Researchers are exploiting large scale application of
bioremediation that can affect desert formation, global climate change and the life cycle of
materials. Attempts are being made to develop microorganisms that can help reverse desert
formation.Fhir work is based on developing biopolymers that retain water and reverse desert
formation !'Al.caligens luteus is being used to produce 'superbioabsorbent', a polysaccharide which
is composed of glucose and
glucuronic acid. These can
4psorb and hold more than
thousand times of its own weight
of water}
Qti( rrtt informations from
fundamental research biore-
mediation technology has been
used to remove environmentally
'j'hazardous chemicals.
accumulated in their cells or
detoxify them into non-toxic
forms.fleveral members of algae,
funei'and bacteria are known to
solJbilize, transport and deposit
the metals, and detoxify dyes and
complex chemicals)
7 ./'-

r-\
,,,
.i
'
n';".i' ffi
.1

'P^-
Environmental ei$S-q!t1o!oQv-!fl
Stt" toxic waste materials remain in vapour, liquid or solid phases, therefore, bioremediation
technology varies accordingly whether the waste material involved is in its natural setting or is
removed and transported into a fermenter (bioreactor). On the basis of removal and transportation
of wastes for treatment, basically there are two methods: in situ bioremediation and ex situ
bioremediatiori )
t, ln s$tr Bicremediation
In situ bioremediation is the clean up approach which directly involves the contact between
microorganisms and the dissolved and sorbed contaminants for biotransfomafian (Bouwer and
Zehnder,1993). Biotransformation in the surface environment is a very complex process. Potential
advantages of in situ bioremediation methods include minimal site disruption, simultaneous
lreatment of contaminated soil and ground water, minimal exposure of public and site personnel,
and low costs. But the disadvantages are (i) time consuming method as compared to other
remedial methods, (ii) seasonal vatiation of micrpbial activity resulting from direct exposure to
prevailing environmental factors, and lack of control of these factors, and (iii) problematic
application of treatment additives (nutrients, surfactants and oxygen) (Christodoultos and
Kontsospyros, 1998). The microorganisms act well only when the waste materials help them to
generate energy and nutrients to build up more cells. When the native microorganisms lack
biodegradation capacity, genetically engineered microorganisms (GEMs) may be added to the
surface during in situ bioremediation. But stimulation of indigenous microorganisms is preferred
over addition'of GEMs. There are two types of in situ bioremediation: intrinsic and engineered
in situ bioremediation.
{cl Intrinsic Bioreurediation: Conversion of environmental pollutants into the harmless
fgrms through the innate capabilities of naturally opcurring microbial population is called intrinsic
bioremediation. However, there is increasing interest on intrinsic bioremediation for control of all
or some of the cofitamination at waste sites. The intrinsic i.e inherent capacity of microorganisms
to metabolize the contaminants should be tested at laboratory and field levels before use for
intrinsic bioremediation. Through site monitoring programmes progress of intrinsic bioremediation
should be recorded time to time. The conditions of site that favour intrinsic bioremediation are
ground water flow throughout the year, carbonate minerals to buffer acidity produced dunlg

Secretion of enzymes by microbiol cell in bioremediotion,

 biodegradation, supply of electrq4,.aceg.ptors and nutrients for microbial growth and absence of
toxic compounds.'The other environmental factors such as pH, concentation, temperature and
nutrient availability determine whether or not biotransformation takes place. Bioremediation of
waste mixtures containing metals such as Hg, Pb, As and cyanide at toxic concentration can create
problem (Madsen, 1991).
The ability of surface bacteria to degrade a given mixture of pollutants in ground water is
dependent on the type and concentration of compounds, electron acceptor and duration of bacteria
exposed to contamination. Therefore, ability olindigenous bacteria degrading contaminants can
be determined in laboratory by ptate count and microcosm studies.
{$) nngi{t€€ild in situ Bioremdiatim: Intrinsic bioremediation is satisfactory at some places,
but it is slow process
due to poorly adapted microorganisms, limited ability of electron accepror
and nutrients, cold temperature and high concentration of contaminants. When site conditions are
not suitable, bioremediation requires construction of engineered systems to supply materials that
stimulate microorganisms. Engineered in situ bioremediation accelerates the desired biodegradation
reactions by encouraging growth of more microorganisms via optimising physico-chemical conditions
(Bouwer et al' 1998). Oxygen and electron acceptors (e.g.NOrt- and SOo2-)and nutrients (e.g.
nitrogen and phosphorus) promote microbial growth in surface.

-----\---

FjiB" 2 -1" Surface treatment using above-ground reactor, injection of oxygen, acid nutrient and
extraction walls.
When contamination is deeper, amended water is injected through wells. But in some in situ
bioremedfittousyqb:U!.lqtheiiraction and injection wells are used in combipation ro control the
 Environmental Biotechnology
flow of contaminated ground water combined with above-ground bioreactor treatment and
subsequent reinjection of nutrients spiked effluent are done (Fig. 25.1). -
2. Ex situ Bioremediation
Ex situ bioremediation involves removal of waste materials and their collectioqgg*a place to
facilitate microbial degradation. Ex siru'bioremediation technology includes most of disadvantages
and limitations. It also suffers from costs associated with solid handling proceqg e.g. excavation,
screening and fractionation, mixing, homogenising and final disposal. On the basis of phases of
contaminated materials under treatment ex situ bioremediation is classified into'two: (i) solid-
phase system (including land treatment and soil piles) i.e. composting, and (li) slurry-phase
systems (involving treatment of solid-liquid suspensions in bioreactors).
{s) Solid-phase Treafment: Sotid-phase system includes organic wastes (e.g. leaves, animal
Jnanures and agricultural wastes), and problematic wastes (e.g. domestic and industrial wastes,
sewage sludge and municipal solid wastes)..The traditional clean-up practice involves the informal
processing of the organic materials and production of composts which may be used as soil I

amendment.
(i) Composting. Composting is a self-heating, substrate-dense, managed microbial system,
and one solid-phase biological treatment technology which is suitable to the treatment of large
amount of contaminated solid materials. tlowever, many hazardous compounds are resistant to
microbial degradation due to complex
chemical structure, toxicity and compound
concentration that hardly support growthl
Microbial growth is also affected by mois-
ture, pH, inorganic nutrients and particle ?:

size. Because composting of hazardous

wastes typically involves the
bioremediation of contaminated substrate- Ditfused bed
sparse soils, support of microbial self-heat- Fig. 25.2, Physical configuration of an open composting
ing needs incorporation of proper amount (Forced aerated treatment system i.e. cutway
of supplements. The hazardous compounds side view).
reported to disappear through composting
includes aliphatic and aromatic hydrocarbons and certain halogenated compounds. The possible
routes leading to disappearance of hazardous compounds include volatilization, assimilation,
adsorption, polymerization and leaching (Hogan, 1998).
Composting can
Automated
be done in open
system i.e. land
treatment, and in
Exhaust
closed system. The outlet
open land system
can be inexpensive
treatment method, Recircu-
but the temperature Perforated _c o_MP_o-sr_ _ lation, duct
fluctuates from floor
summer to winter.
Therefore, rate of
biodegradation of
Fig. 25.3. Physical configuration of a closed composting system with forced
waste materials ventilation (cutway side view) (based on Hogan 1998).
dEclines. Secondly,

.4.
c
 EE e ri"r,tmor or giot."t n otogy _-
land featment system may become oxygen limited, depending on nmount of substrate, depth of
waste, application, etc. However, efficiency of open treafinent system can be increased by
fassing
air (Fig. 25.2). This approach is referred to as engineered soil piles andforced aeration treatment.
The closed treatment system is preferred over the open land treatment system because controlled
air is supplied to maintain the mr'crobial activity. As a result of microbial growth andt'VoftitiliZation
of hazndous compgunds, internal
.i"sffi;;B$jgr--ffitemperytqE.gradualffiG.-Tii#i";f;G'df
_up-is' r"{gspffi r"sg{e -tsg
blowers for air

25.3). ventilator,s dupply.orJge3-,?sd remo-rgJrg"gt-[.rgysttgl'ffi"tion of

- il* -
(ri) Compsting Process. As composting is a solid-phur" biological treatment, target
c.omg9u1d1 must-bp+HruE$.gl"a ti x. The hazardous .o-pffid,
shouldbebiologicallytrarm-nne0.romaterialshouldbesuitably
prepared so that biological treatment potential should maximise. This is done by adjustment
of
several physical, chemical and biological factors (Fig. 25:4). The hazardous wastes must
be well
solubilized so that they may be bioavailable..The hazardous compounds and soil organic matters
serve the source of carbon and energy for microorganisms. Microbial enzymes ,"Jr"i"O during
growth phase degrade toxic compounds. Howev"., prop", maintenance of water, Or, inorganic
nutrients and pH increase the rate of decomposition.
f
q i-----t
j Contaminated materiat
L__.___ _ I

,]

lnorganic Organic Bulking pH

t Water lnoculum
.4.
nutrients amendmen: agent adjustment

Initialmixing

"-ttrfl

*
<- JDecontaminated material P
I
I

Y
Final use / disposal

fig' 25'4. Outline of composting treatment sequence (based on Hogan l99S).

If there is low substrate-density or site-specific conditions, analogue or non-analogue, noh-

^-*.,F .--,-
'idr-n-
-=*-:j-!*
 \
Environmental Biotechto,ogyj$
hazardous carbon sources that can stimulate microbial growth and enzyme production can be
added to compost. Organic amendment also stabilize microbial pqpulation in inhibitory environment.
Secondly, presence of sufficient amount of water enhances microbial growth. Addition of inorganic
nutrients influences microbial growth and rate of decomposition of hazardous wastes. Under
nitrogen limiting conditions Phanerochaete chrysosporium produces extracellular lignin peroxidase
that degrades benzopyrene and 2,4, 6-trinitrotoluene. It has also been noted that a pH range of
5.0-7.g promoted the highest rates of degradation of hazardous wastes. But lignin degradation has
been found the most rapid at pH of 3.0-6.5. This shows that optimal pH levels can be species,
site and waste specific.
Gradual colonization of organic materials is done by indigenous microflora, but hazardous
chemicals may inhibit microbial growth. Therefore, bioaugmentation (1.e. use of commercial or
GEMs) of wastes is also recommended.
To provide experimental proof of biodegradation during.composting, a cornmon hazardous
contaminant pesticide, l4c-labelled Carbaryl was added in sewage sludge-wood chip mixture at
I.3-2.2 ppm concentration. After 18-20 days in laboratory composting apparatus, 1.6-4.9 per
cent of Carbaryl was recovered as
toCO, and remaining bound to soil organic matter.
(b) Slurry-phase TFeatment: The contaminated solid materials (soil, degraded sediments,
etc), microorganisms and water formulated into slurry are brought within a bioreactot i.e. fermenter.
Thus slurry-phase treatment is a triphasic system involving three major components: water, suspended
particulate matter and air. Here water serves as suspending medium where nutrients, trace elements,
pH uO3uttrnent chemicals and desorbed contaminants are dissolved. Suspended particulate matter
includes a biologically inert substratum consisting of contaminants (soil particles) and biomass
\
attached to soil matrix or free in suspending medium. Air prov*ides oxygen for bacterial growth.
Slurry-phase reactors are new design in bioremediation. The objectives of bioreactor designing are
to (l) alleviate microbial growth limiting faetors in soil environment such as substrate, nutrients and
oxygen availability, (tt) promote suitable environmental conditions for bacterial growth such as
moisture, pH, temperaturl, and (ill) minimise mass transfer limitations and facilitate desorption of
organic material from the soil matrix (Christodoultos and Kontsospyros, 1998).
Biologically there are three types of slurry-phase bioreactors : aerated lagoons, low-shear
airlift reactor, and fluidized-bed soil reactor. The first two types.are in use of full scale
bioremediation, while the third one is in developmental stage.
(f) Aerated l,agoons, Fig. 25.5 shows the slurry-phase lagoon system which is very similar
to aerated lagoon used for inoculum
treatment of small common Water susPension Surface aerator ;Microbial
municipal waste-water. Nu-
f- Nutrient
I
trients and aeration are sup- I

plied to the reactor. Mixers

o o oio o op c
are fitted to mix different o o olo o^o^L.O
vvp'
o
O

components and form slurry, \ L'tv

o o olo _"-----+--
ooop -:-: oo
whereas surface aerators
provide air required for mi-
9
--r= Solid/sludge/
sediment
crobial growth. The process
may be used as single-stage Fig. 25,5. Slurry-phase lagoon
or multistage operatation. If
the waste contains volatiles, this reactor is not appropriate.
(di) Low-shear Airlift Reactqrs (t,[fARs), The LSARs are useful wheg wate__c,ggtains volatile
components; tight process ,ontrol and increased efficiency of bioreactors are iequired. Fig. 25-6
shows a low-shear airlift slurry-phase bioreactor. T SARs are cylindrical tanks which is madp up
 a"
"' EE[A*rrbook of Biorechnology
of stainless steel. In this bioreactor, pH, Motor driver
'temperature,
nutrient addition, mixing and
oxygen can be controlled as desired. Shaft is
equipped with impellers. It is driven by motor
set up at the top.The rake arms are connected
with blades which is used for resuspension of
coarse materials that tend to settle on the
bottom of the bioreactor. Air diffusers are
placed radially along the rake arm. Airlift
provides to bottom circulation of contents in
reactor. Baffles make the hydrodynamic
behaviour of slurry-phase bioreactors. pre:
treatment process includes size fractionation
of solids, soil washing, milling to reduce
particle size and slurry preparation. Certain
surfactants such as anthracene, pyrene,
perylene, etc. are added to enhance the rate of
Fig. 25.6. A low shear airlift slurry phase
biodegradation. These act as co-substrate and
utllize as carbon source. Co-substrates also induce the production
of beneficial enzymes (Christodoultos
and Kontsospyros, 1998). I

(c) Factors Affecting Slurry-phase Biodegradation:

Factors that affect slurry-phase biodegra-
dation are (i) pH (optimum 55-8.5), (t, moisture content, (iii)
remperature (20-30.c), oxygen (aer-
obic metabolism preferred), (iv) aging, (v) mixing (mechanical
and air rnixing), (vj) nutrients (N, p
and micronutrients), (vii) microbial population (naturally
occurring microorganisms are satisfactory,
genetically engineered microorgunirm, for layer'co-pound
may be added), and (viii)reactor opera-
tion (batch and continuous cultures).

3. BIOREMEI}IATIOIT OF HYDROCARBOI\S
I Petroleum and its products are hydrocarbons. These two have
much economic importance.
oils ibnstitute a variety of hydrocarbons viz., xylanes, naphthalenes,
octanes, camphor, etc. If
piesent in the environment these caus€ pollution. For example,
during cold war between Iraq and
America, millions of gallons of petroleum was leaked into sea
*tri.t resulted in fish mortality.
In addition, oil and petrol leakage in marine environment is of usual phenomenon.
For
11 million gallon oil spill from the supertanker Exxon Valdez
ran around near prince"*u-p1.,
William
Sound, Alaska in March 1989. It is interesting to note that in
Pennasylvania about27,111litres
(6'000 gallon) of petrol leakage occurred. It contaminated
the underground water supplies. The
input of oil to the environment can be ecologically devastating
and cost of cleaning can go ro
several million dollars.
' ' In toxic environment, microorganisms act only if the conditions e.g. temperature, pH and
inorganic nutrients are adequate. Oil is insoluble in water
and is less dense. It floats on warer
surface and forms slicks. It should be noted that in bulk storage
tank microbial growth is not
possible iirii\'ioea water and air are supplied. The microorganisms
which are capable of degrading
petroleum include pseudomonads, various corynebacteria,
mycobacteria and some yeasts. However,
there are jyo methods for bioremediation of hydrocarbons/oil
spills, by using mixture of bacteria,
and using genetically engineered microbial stains.
 Environmental Biotechnology
inip r-'-i'f
G lrungal perllets ; Mycelial pellets of C. versicolor can be used to decolourise lignin-con-
tgtnil&yaste in an air-tight fermentor in a minimum cost. Addition of Mg* increases the
raj9. of decolourisation because it acts as act..iJator of many enzymes. A colour reductioqly*
927o with 6JVo reduction in COD may be obtained 7 days of using C. versicolor:
. [lum,.rt--,i!is*rl i:illtnre I The fungus immobilised in beads
Toxic metals.
of calcium alginate gel can give the best result treated
with water. In a case study, immobilised C. versicolor
tecolouri sed 80Vo after 3 days in the presence of su- Soil Water Air

crose. A fluidised bioreactor containing polyurethrane-

ffimobilised C. versicolor reduced colour of a bleach.
{r-:.} Bioreurediad,f qur r"rf }{eav.v- l\'tetals : Due to dicharge of Exposed to living organism
industrial waste, metal pollution is generally increasing in the
environment. Metals absorbed by organisms at the lower torphic
Bioaccumutation
level of food chain becomes gradually codcentrated in those
organisms which are at the higher levels where man is the
- Biomagnification
ultimate to be victimized. Hence, the concentration of heavy
metals gradually increases at each trophic level and enter in
Toxicity posed
hfrman uooy in higtrer amount (Fig.25.7). Therefore, maximum
ildmage is caused in the consumers of the last trophic level (Cellular, metabolic and structural)
such as human. Several microorganisms have shown Fig" r5"7. Fate of toxic metals in
''deto*ification
potential to deal with such environmental biosystems.
problem.
Various approaches have been made to.detoxify and clean up these meals such as :*use of
certain chemical which in turn cause seg-o..p{ary po}luti9n, and plrysical methods that requires large
iU:S gts"ergyGxpansioii riiaf6iials. Another option ii the use-oFdi'fferent type of microorganisms
t*.-.q-*_,.,-
s uc h-as al gae junei b ac teri a that romoye_ solution.
3na -m_e. lals "frosr*he.

NIefal-micrcbe Interactions and iVlechnnisrn of i\{etal Removal: On the basis of localiza-

tion site of the metal, microbial interactions with heavy metals can be classified into different cat-
egories such as extracellular,-dioc-elluluiluna-inir";;ii;-r;;;rrere microorganisms mobilize, immo-

--hilifp, transffi'F'reclfiitate; ?tilifr,iiate, coordinatgjlg_trange, adsorb itreg"taG and 6unloftn

.o*pt.*ti :,.

rc;oorganisms remove metals by the following mechanismslSdsorption (negatively charged

cell surfaces of microorganisms bind to the metal ions)/.6ern-p1u*f6on (migporfanisms proOuce
gpulillgjds (e.g.gjrig "1-cid, oxalic acid, gluconic acid, formic acid, lactic acid, malic acid) which
chelate metal ions. Bio,sgqption of metalq.$s9 takes placg due to carboxylic groups found in micro-
bial polysacch?pdes and other polymerP, precipitatign (some bacteria produce ammonia, organic
bases or HrS which precepitate metals as hyd,roxi.{gs or sulfates. For example, Desulfuvibrio and,
Ddsilfolomacululm' tiansform SOo to HrS which promotes extracellular precipitation of insoluble
1ne.d,fUlffi. Klebsietli ae:rogenes detoxifies cadmium sulphate which precipitates on cell_ sur-
face,nVolatilization
lFrm
(some bacteria causes methylation of -Hg2* Q and converts to dimethyl ffi"rtury
which is a volatile compound), etc.
The species of C,!,tJorel,la, Anabaena inaequalis, Westiellopsis prolffica, Stigeoclonium tenue,
Sygglgcoccaj.sp: tolerate heavy metals. However, sevgmlEp-qqies-pI _Chlorel,l_q, Ansbae.na, marine
algae have been used for the remo,val of heavy metals. But the operational conditions limit the
 A Textbook of Biotechnology
practical application of
these organisms. Rai et al. (1998) studied biosorption of Cd+* by a
capsulated nuisance cyanobacterium, Microcystis both from field and laboratory. The naturally
occurring cells showed higher efficiency for Cd++ and Ni++ as compared to laboratory cells.
Fungi also are capable of accumulating heavy metals in their cells. However, several
mechanisms operate in them for removal of heavy metals from the solution, a few of these have
been discussed below :
. hletabolism-independent accumulatfon. The positively charged ions in the solution are
attracted to negatively charged ligands in cell materials. Biosorption of metal ion occurs
on microbial cell surface. But composition of biomass and other factors affect biosorption.
For example, in Rhizopus arrhizus adsorption depends on ionic radius of Li3+, Mn2*,
Cu2*, Zn2*, Cd2*, Ba2*, Hgt* and pb2+. However, binding of Hg2+,, Agr*, Cd2+, Al3+,
Ni2+, Cu2* and Pb2+ strongly depends on concentration of yeast cells.
) Metabolism-dependent aecumulation.In fungi and yeast, heavy metal ions are trahsported
into the cells through cell membrane. However, as a result of metabolic processes ions are
[recipitated around the cells, and synthesised intracellulEif as metal-binding'prdtbins.
ffi6]Gffii'.orb.u2*,Cd2+,Co2*,Ni2+,Zn2+byfungihasbeendemopstratEff
. Moreover, intracellular uptake is influenced by certain external factors such as pH, anionq,
cations and organic materials, ggys plgry, etc. Metal uptake by growing batch culture
ffir"iound maximum durigg,l"g-pft"* log phaqe, in Aspergillus niger; Penicillium
sp iffii6 ^:id"i,i, nffiffi f,m GffiHjTff.,
"ftEatl:t
I e 8s ).
t trlxtracell,tlut pt**i!it"il" and cornplexation. Fungi produce several extracellular
nj!$-u9t9 which can complex or precipitate heavy metals. p#example, many fungi and
y?'ast relea-se high affinily
kujggilg co,ppoundq thatg.hplpt" iron. It is called slderophores.
The Fe3+.Tam}fri"it aG 6tm"O outside the cefl wall are taken-up into*ihe cell. In
Sacchiromy:f9s cerevisia.e r:emoval of -"iul, i, Oon. by*their precipitation as stilphides
ii'precipitared as CuS iBeveridge, 1989).
*+ ".;ci?.
Bacteria secrete extracellular poi;;; iifr .uprule that have anionic c-haracts.r. External biofilm
associated with cell, complexes with metals and accirmulate substantially. Wholg,SeJ!"9!-p.gc"it!us
y.,ubtilis have shown to reduce ,gold from through extracellutSr ggfn*lig$g$ensforma-
$g,]*,"t "+u0
tion. Under anoxiccltvitqn-mentals, sulphite-reducing bactena(Dt/su v:ibrii andng;ijfgtomaculum)

%1{iZg organ!.g, matter using sulphate isffiiectron aceptor. Citrobacie,' sp: gelerates HPO2-. after
*:n:n7{4gqic cleavage of glycerol-l -phosphate..rMgllt_igttr:Xi$ly form Complexes with HPO42+
to form insoluble p"*g.r*lj"atgs"w_bjqh {gm+iq bpund-rs the ceils.
-itJ -gi"tttption:
It is a passive Tetaboligm-in-depgndent physico-chemical interacrions be-
heavy metal ions and -i:X..9j4-lu1face. It is defined as 'a no'n-directed physicg-chemical
1Y""n
interaction that may occur benuZien metal/radionuclide. species aid microbial cell' . Mostbiosorption
fihenomena are combination of process"fru.tr ur ion _exchange complex-
ation, ionic band formation, precipitation, nucleation, etc. (Fig. 25.8). ' '
"t"rt-rtatilinteractions,
These interactions occurs due to complexity of microbial surfaces and chemicaVphysical prop-
erties of mgtal ions. For biosorption, utTiil""tiute of cells is not pi"i"quirite. The pfiCess can oc-bur
even with inactivated/dead cells. Biosorption offers several advantages, for examples,
t Process is not governed by physiological constraints of microbial cells. There is no need of
coltly nutrients for growth and aseptic operation of cells. It operates at a wider range of
conditions such as pH , temperature and metal concentrations.
 En ritott-"nt"l Biot""hrrology fEil
r Inactivated biomass works as an ion exchanger. Therefore, the prcess
is rapid, for example
Streptomyces nouresei carries out biosorption at a high speed
lisO lrmoUg).
---- o!' Metals can be
desorbed readily from the.biosorbent and recovered
{#} organisms ruvolv'ed in Sirmlptinn: There are several microorganisms such as.lgglqeria
(Arthrobacter viscosus, Pseudomonas syryngae, P.
aeruginosa, p putida, Bacillus subtilis, E. coli,
Streptomyces/nouresei, S. pintprina, etc.), fungi und y.art (Sacc'harotmyces
spp., Aureobasidium
pullulans, R' nigricans, etc.) and qlgSe (Chlorella valgaris,
Ascophyltum nod,osum, Cladophora
crispata, etc.). Fungal mycelia (e.g.-Kperyillus and, Peniciilium.)utro
r.-ove metals from wasrewa
ter and offer a good alternative fo*r detoxification of effluents.
Biosorption have shown that As-
pergillus oryzae can remove cadmium efficiently from solution (Thbl
e 25.2).
in metor removcilftorn indusfricd w&rnobr (b@d
_9l Bitbn, t99p) :

Microorganisms Metals removed

Aspergillus niger Copper, cadmium, zinc
A. oryzae Cadmium
P enicillium spinulosum
Copper, cadmium zinc
Rhizopus arrhizus Uranium
S ac charomy ce s ce revi s iae Uranium
Trichoderma viride Copper
ATM-BioclaimrM Biotechnology-based use of granulated product de-
rived form biomass

COORDINATION

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hvdrox I|1\amlno \ aililrrL,
ihino a-{'
imino
|I
.
r' ----- ..
hvdrox ^,,-, ,
CHELATION

METAL
METAL
COMPLEXATION

ffig' 25-s' Microbial biomass showing various ligands involved in

metal biosorption
(Panikar et al., 2003).
f!f,fr:f.fg"r "r
ai"t""h""t"sy
Biosorption of some organisms sorbing various metals at different conditions (such as pH,
temperature (T), initial metal concentration (Co), biomass concentration (B) and value of metal
uptake (Q) are given in Table 25.3.
,ii0*osorpftoh d rnefiefs by,eoro micfoo{gEnAsrns under dltfrrcnt
,i' condlfiolts.
Organism and metals Operating conditions Q @s/s)
pH T('C) Co B (s/I)
(m:g/ml) .
Arthrobacter sp.
Cd 7 20 I 0.6 1.4
Co 3.5-6 30 180 0.4 1.48
Pb 5-5.5 .30 254 1.4 130
Streptomyces nouresei Cd 6 30 1-1 t0 3.5 3.4
Cu ).) 30, 0.6-65 3.5 3.4
Pb 6.1 30 2-207 3.5 36.5
Ag 6 30 l-100 3.5 38.6
Zn s3 30 0.6-65 3.5 1.6

Rhizopus arrhizus cd 3.5 26 10-400 n.a. 25

Cr l-2 25 25-400 n.a. 4.5
Cu 5.5 25 0.6-25 n.a. 9.5
Pb5 '25 150 1 6.5
Zn 6-7 n.a. 10-600 3 13.5
Penicillium digitatum
cd 5.5 25 10-50 6.5 3.5
Cu 5.5 25 10-50 6.5 3
Pb 5.5 25 10-50 6.5 J

Source: Paknikar et al. (2003); n.a., not available.

(iii) Factor aff'ecting bioserrption of metals: Biosorption depends on various factors like type
of biomass, metal chemistry, temperature, pH, biomass concentration, initial metal concentration,
composition of biomass, etc.
iiv) Metal bisorption technologies; Several technoligies for metal removal have been com-
mercialized and employed as briefly given below:
c AT]MBIOCL,A.IMTII process: The Advance Mineral Technology (ATM) Inc. (U.S.A.)
developed a wastewater treatment process with Bacillus sp. (immoblised in beads) pre-
treated with caustic solution. It is specific for metal cations in the order: Cu2* > Zn2* >
Cd2*=Ni2+>pb2*.
t AlgaSORBru process: Biorecovery systems, Inc. (U.S.A.) developed this propri etffiy based
material which consists of several types of living and nonliving algae. Atgal cultures are
immobilised in silica gel in the form of beads. The desorption of metal is carried out.
. BIO-FIX'ret process: The Bureau of Mines (U.S.A.) developed this process that consists
of biomass immobilised in polysulfone. It consists of thermally killed biomass of Sphag-
num peat moss, algae, yeast, bacteria and lor aquatic flora. The beads are suitable for
practical application in stirred tank reactor, fixed and fluidized-bed columns.
 Environmental Biotechnology fi[!|
DDT (l,l,l-trichloro-2,2-bis (p-chlorophenyl) ethane) is an insecticide that persists in soil
for four years. Degradation pathway of DDT involves an initial dechlorination of the trichioromethyl
group to form l,l-dichloro 2-ethane which then undergoes further dechlorination, oxidation and
decarboxylation to form bis methane. Subsequent cleavage of one of the normal aromatic
rings
yields p-chlorophenyl acetic acid, which may also undergo ring cleavage. Microorganisms a-ssociated

with DDT degradation are Aspergillus flavus, Fusarium oxlsporum, Mucor aternans,
p. chrysosporium, Trichoderma viride, etc. Environmental factors including pH, temperature,
bioavailability, nutrient supply and oxygen availability affect biodegradation of pesticides.
tb) {irne }Iagipulatign of l}esticide-degrading }licroorganisms: Day-by-day the number
of xenobiotic-degrading miroorganisms is increasing. However, pesticide-degrading genes
of only
afew microorganisms have been characterized. Most of genes'responsible for catabolic degradation
are located on the chromosomes, but in a few cases these genes are found on plasmids
or
genes of
transposons. Chakrabarty and Gunsalus (lg7I) found that camphor degrading
pseudomonas putida are located on plasmid. Soon after, naphthalene (NAH) degrading plasmid
was isolated. Discovery of these genes made it possible to construct a new genetically en-eineered

strain of p putida that alone was potent to degrade camphor, naphthalene, xylene. toluene'
octanes and hexanes. For detailed discission, see Chaper 17.
For the first time pesticide degradation through plasmid mediated genetically engineered
cloned and
microorganism was reported by Chakrabarty et a/. (1981). Nagata et al. (1993) have also
sequenced two genes involved in early steps of Y-HCH degradation in UT26. The /rzA gene encodes
y-gCU dehydrochlorinase which converts Y-HCH to !,2,4TCB via Y-PCCH and I,4-TCDN' The
linB gene encodes l,4-TCDN chlorohydrolase which converts l,4-TCDN to Z,4-DDOL via 2,4,5-
DNOL. This gene is a member of the haloalkanedehalogenase family with a broad range specificity
for substrate. The genetically engineered P putida comprises of both linA and linB genes
(Nagata

et aL, 1993). ': '

The opd gene, initially isolated from Flavqbacterium sp. ATCC2755I and Pseudomonas
diminuta, has been well char acterized. It is associated for degradation of pesticides such
as

parathion, methylparathion, etc. Sims et al. (1990) transferred a recombinant DNA plasmid
containing opd gene into a fungus, Gliocladium virens which expressed at low level. G-
virens is
a useful soil saprophyte which has shown a strong mycoparasitic activity against many fungal
pathogens. Strains of G. virens are potential for use in the bioremediation of contaminated.soil.
bpti-irution of opd expression in bioremedially useful organism such as P. chrysosporium, G.
virens, etc. holds a great promise in lessenilg the pesticide pollution. l

B. BIOALiGiVIEI\TATIOI\ (LTSE OF BLEI\DS OF

MICROORGAl\ISM)
Acceleration of biodegradation of specific
compounds by inoculating bacterial cells is
called @ Bacterial cells
contian specific plasmid which encodes en-
zymes for degradation of those compounds. A
variety of plasmids have been reported from
Alcaligenes. Acinetobacte r, Arthrobacter
B eij ei rinkia, Klbb s iella, F lav ob act e rium and
P s e udomorzas. Several genetically
strains have been developed exploiting
en gineered
I
Pseudomonas. Production of microbiol products .f or
bioougmentotion,
 EEEI n re'tuook or Biotechnology
1. Priniepfes of Bioaugmentation
Mict'borganisms capable of degrading herbicides/other chemicals
in industrial water are iso-
Iut:f from wastewater' compost, sludege, etc. Some of the strains. may be irradiated
to enhance
their ability and mutants are selected (Fig. 25.10). Before
their use in the environment they are
tested in laboratory for their biodegradation ability.
Bioassays are also used to assess the toxicity
of the wastewater for commercial preparation of microbial
seeds. Selected strains are used in
large fermentor to get mass culture. Then they are preserved
freezing. - through
e lyophilization, drying and

lsolation and
I
\r selection

Scaleup

il
lsolated and adapted mutants
I

pur&ufture
I

Long t# storage
undervaccum
.
Fig' 25'10' lsolation and purification of microbial
blends used for pollution contrql. -
commercial bioaugmentation products are single cultures
of consortia of microorganisms with
certain degradative properties or their desirable characters.
At present most important users are the
industrial wastewater treatment plants. The selected
microorganism is added to a bioreactor so that
potential for biodegradation of wastes must be
maintained or enhanced. Due to tradq secrets infor-
mation on bioformulation of mixture of microbial cultures
are not scanty.
Application of biaugmentation includes: (a) the increased
BoD removal in wastewater treat-
ment plants' (D) reduction of sludge volume by
about 30vo afteraddition of selected miroorganisms,
(c) use of mixed cultures in sludge digestion, (d;
biotreatment of hydrocarbon wastes, and (e)
biotreatment of hazardous wastes.
The use of added microorganisms for freating hazardous
wastes usch as phenol, ethylene gly-
col' formaldehyde has been attempted. Bioaugmentation
with parachlorophenol-degrading bacteria
decomposed 96vo parachlorophenol in t hours. cells of candida tropicalis have been used for
 Environmental Biotechnology
removal of high concentration of phenol present in freshwater. Ability of a bioreactor to dechlori-
nate 3-chlorobenzoate was increased after addition of Desulfumonile tiedjei to a methanogenic upflow
anaerobic granular sludge banket. Anoxygenic phototrophic bacteria have also been considered for
the degradation of toxic compounds in wastes (Bitton, 1999).
Some demerits of bioaugmentation are : (a) need of an acclimation period prior to onset of
biodegradation, (b) a short survival or lack of growth of microbial inocula in the seeded bioreactors,
and (c) some times negative or non-conclusion of some of commercial products.
3. [$se *rf Enzyrfle$
Several enzymes have been detected in wastewaters such as catalase, phosphate esterases and
amino-peptidases. These enzymes can be added to fresh waters to improve biodegradation of
xenobiotic compounds. For example, parathion hydrolases (isolated from Pseudomonas and Fla-
vobacterium) have been used to cleanup the containers of parathion and detoxification of wastes
containing high concenfration of organophosphates. A little information is available on use of enzymes
in wastewater treatment plants. This technology is applied to reduce the production of excessive
amount of extracellular polysaccharides during wastewater treatment because overproduction of
polysaccharides results in increased water retention with reduced rate of dewatering. Addition of
enzyme can degrade these expolymers.
Some specific enzymes (e.g. horseradish peroxidase) can catalyse the polymerisation and pre-
cipitation of aromatic compounds (e.g. substituted aniline and phenols). Horseradish peroxidase
catalyses the oxidation of phenol and chlorophenols by hydrogen peroxide. The extracellular fungal
laccases (obtained from Trametes versicolor or Botrytis cinerea) can be used for the treatment of
effluents gbnerated by the pulp and paper industry because this enzyme can be useful for dechlorina-
tion of chlorinated phenolic compounds or oxidation or aromatic compounds even at adverse envi-
ronmental conditions such as low pH, high temperature, presence of organic solvents, etc. There-
fore, attention has now been paid to use extremozyme of microorganisms that can work at extreme
environments also.
{l, [3fr{itri $ ,'#.$?,$"p. ${,-hi,;

Biofiltration is a new technology used to purify contaminated air evolved from volatile organic
compounds by involving microorganisms. It is a low cost technology gradually becoming popular
due to simple operational and waste-removal efficiencies. Biofiltration is the oldest biotechnologi-
cal method for removal of undesired foul gas components from air. Since 1920, biofilters were used
to remove odorous compounds from wastewater treatment plants or animal farming. It could be
achived by digging trenches, laying an air distribution system and refillihg the trenches with perme-
able soil, wood chips and compost.
In the 1960s, the first biofilters were built in the U.S.A.Between 1980s and 1990s, about 30
large and full-scale systems of about 1,000 m3 capacity have been constructed. Biofiltration has
more industrial success in Europe and Japan where over 500 biofilters are in operation (School er
a1.,2003).
Moreover, biofiltration is not suitable for highly haloginated compounds (e.g. trichloroethyl-
ene, trichloroethane and carbon tetrachloride) due to its low aerobic degradation. Also the size of a
biofiltration in inversely proprotional to the degradation rate.
if'l *iillftlterr; Biofiltration is done by using biofilters. Biofilters are the, packed-bed units in
which gas is blown through bed of compost or soil covered by an active biofilm made by the natural
microorganisms. Diagram of a biofilter is given in Fig. 25.n.
!!$l A Textbook of Biotechnotogy
The microorganisms consume the gaseous organic
Treated gas
pollutants and use as source of carbon and energy. stream
Instead, it may contain an inner support where a spe-
cial or pool of microorganisms are cultivated. The
harmful compounds are degraded by an active biofilm
covering the bed. The unwanted odorous organic com-
pounds from gaseous phase are removed. They are Packaging
absorbed or adsorbed on porous solid base of the section
biofilter, or dissolved into liquid phase and then
oxidised by the microorganisms. Biofiltration is ben-
eficial because it does not require large amount of en- Moisture
supply
ergy during operation.
(fi:) Micraroganisrns Esd in hiofrItem: Differ-
ent aspects have been studied regarding the microbial
potential of biofilters: (a) isolation and characterisation,
(b) use of pure cultures of bacteria or fungi, (c) mixed Drainage
microbial population, (d) effect of enrichment culture
Humification
including application of special strains, types of mi-
chamber
croorganisms and their metabolic activities, (e) effect
of external conditions on microbial activity, and A re-
lease of microorganisms from biofilters. Brower
Microorganisms present' iri biofilter are aerobic @
Gas stream
ones. Most of them are bacteria (mostly coryneforms
and endospore formers), occasionally pseudomonads, Fig' 25'11'' Diagram of a biofilter'
protozoa, invertebrates and a few actinomycetes (mainly Streptomyces spp.) and.some
fungi (mostly Alternaria, Aspergillus, Botrytis, . Cladosporium, Fusarium, Mortierells,
Rhizopus, Penicillium, Trichoderma). Fungi form a large
specific area which remain in direct contact of air flowing
through filter.
Microorganisms are the most critical components of
biofilters. Because they transform or degrade the contami-
nants. Naturally occurring microorganisms are available for
'the process because they get
adapted to the contaminants.
In some cases, a specific microbe or genetically engineered
microorganisms may be used (Schroeder,2002).
(ffr) B,iafilter rnedia: The filter media must have
some characteristics for performance of the biofilter. Be-
cause all the filter media allow the polluted air to interact
closely with the degradative microorganisms, oxygen and
water. Constitution of the physical media provides fine po-
rous, large surface area and distribution of uniform pore
size. Unifofln pore size strognly defines the efficiency of
biofilm. Inorganic bed material has a good flow properties
Biofilter for obsorbption of contomi- and consists of a variety of metal oxides, glass or ceramics
nonts from oir,
beads. PVC is commonly used as packing material.