Abstract
Recent trends in the area of bioremediation
mainly focus on pure culture techniques; in
other words, people try to develop processes
based on good degrading isolates. But when we
discuss about in-field applications, results
are not quite satisfactory. Most of the operational
processes, starting from trickling filter to
highly advanced pneumatic reactor involve mixed
culture or microbial consortia. Considering
the facts, process involving multi species consortia
would be a better choice over mono culture concept.
On this conceptual background, multi species
microbial granules are emerging as most promising
agents for state-of-theart wastewater treatment
and bioremediation technologies. High satiability,
high stability in variable organic load, effective
mineralization and amenability for bioaugmentation
are some of the superior parameters which make
them one of the best choices for bioremediation
and wastewater treatment. Here, an attempt is
made to evaluate various merits and demerits
of microbial granules based bioprocesses with
special emphasis on aerobic granules.
Introduction
Most of the real wastewaters are complex in
nature and none of the individual bacterial
species is capable of completely degrading complex
wastes. Complete degradation of complex industrial
wastes involves a complex suite of metabolic
reactions
mediated by several closely related species.
To address this difficult task we have a system
where different microorganisms with different
metabolic capability lie in close proximity
of others. Biofilm is a classical example seen
in the natural environment, where different
species of microorganisms arrange themselves
in specific spatial organization.
To make use of this natural phenomenon people
have developed various bioreactors having biofilm
on various inert substrata. Biofilm reactors
are extensively used in environmental biotechnology
because high biomass can be retained to treat
large volumes of diluted aqueous solutions that
are typical of industrial and municipal wastewaters.
Here, specific surface area is a very important
parameter and becomes crucial in case of high
strength organic waste. The specific surface
area will increase dramatically by growing biofilm
in suspended forms. These can be achieved by
two ways 1) Biofilm is allowed to grow on light
weight spherical carrier particles (sand, plastic
beads. etc.) and 2) Spherical biofilm without
any substratum (aerobic and anaerobic granules).
Therefore, these microbial aggregates satisfy
two basic conditions of optimal treatment plant
viz., broad metabolic capability of microbial
community present in the system and efficiency
of solid-liquid separation at the last stage
of treatment procedure. Comparing suspended
biofilm based reactors with conventional bioreactors,
it has many distinguished characteristics such
as a) excellent satiability (easy clarification)
b) minimal effect of variable organic load c)
simple design with minimum parameter control
d) involves biodegradation, biotransformation
and biosorption (complete biological process).
Carrier bound biofilms have some drawbacks compared
to carrier-less microbial granules as selection
of proper carrier material is not an easy task.
Similarly, it has less specific surface area
compared to granular biomass. Anaerobic granulation
in Up flow Anaerobic Sludge Blanket (UASB) reactors
has been investigated and applied both at laboratory
scale and as well as at large-scale. The formation
of microbial granules under aerobic conditions
has been invented very recently.
Present status
Formation of anaerobic microbial granules has
been observed in case of upflow anaerobic sludge
blanket (UASB) reactor in 1980’s. Recent
review reports that nearly 6000 industrial wastewater
treatment plants based on this technology are
in operation world wide. However, anaerobic
granulation technology has the following disadvantages:
1) requires long startup period (2 to 4 months)
2) relatively high operation temperature (30
to 35°C for mesophilic UASB reactors),3)
unsuitable for low-strength organic wastewater
4) not suitable for nutrient (nitrogen and phosphorous)
removal. In order to overcome these drawbacks,
Mishima and Nakamura in 1991 developed microbial
granules in aerobic upflow sludge blanket reactor.
Later, Morgenroth et al., (1997) cultivated
aerobic microbial granules in a sequencing batch
reactor (SBR). Ever since, microbial granules
have been successfully cultivated in sequencing
batch reactors using synthetic wastewater or
real wastewater as influent. The engineering
parameters such as settling time, organic loading,
dissolved oxygen etc., have been studied and
optimum range determined for developing microbial
granules. But the mechanism of microbial granulation
is still not fully understood. Particularly,
the biological factors such as aggregating abilities
of individual bacterial strains which gives
structural framework, the catabolic diversity
of granules which gives the functional capability
and the driving force for microbial cell aggregation
require better elucidation. With the help of
various advance technology like confocal laser
scanning microscopy (CSLM) and 16Sr RNA based
microbial identification along with fluorescence
insitu hybridization (FISH), researchers have
tried to explain the phenomenon associated with
biogranulation.
Microbial granulation:
A sequential developmental process
Aerobic granulation is a process involving the
development from seed sludge (used as inoculum)
to compact aggregates, further to granular sludge
and then finally to mature nearly spherical
granules. This self-immobilization process involves
clearly distinguishable (based on size and morphology)
multiple steps. Figure 1 displays the microstructure
of granular biomass. Granulation may be initiated
by self-adhesion of bacterial strains. This
can be through autoaggregation or co-aggregation
among individual bacterial strains. Auto-aggregation
refers to the physical cell-to-cell interaction
between genetically identical cells, while co-aggregation
refers to the interaction between genetically
distinct bacterial cells (Rickard et al.,
2003). High shear and short settling time employed
in reactor may select bacterial strains having
aggregating ability and strains with poor aggregating
ability may get washed out. Aerobic starvation
in cyclic manneroperated in SBR may influence
bacterial physiological activity and hence aggregating
ability. At cellular level, these (autoaggregation
and coaggregation) interactions are mediated
by lectinsugar kind of interactions. Development
of granules may share the similar events involved
in multispecies biofilm development. Intercellular
communication plays a role in organizing the
three dimensional spatial distribution of bacteria
in the granules. Cell surface hydrophobicity
and extracellular polymeric substances (EPS)
facilitate the aggregation of bacteria and maintenance
of granular structure. Figure 2 indicates schematic
representation of aerobic granules formation.
Application
of aerobic granule technology
Compact biomass combined with diverse metabolic
capability makes aerobic microbial granules
primary choice for treatment of high organic
load. Limited space availability to set up reactors
can be overcome as aerobic granules provide
high conversion rate along with efficient biomass
separation. Treatment capacity can be easily
altered by varying loading rate, wastewater
composition and bioaugmentation. Following are
some of the applications demonstrated at laboratory
scale as well as at pilot scale using SBR and
aerobic granules.
1.High
strength organic waste water treatment
High biomass retention (up to 6-12 g/l) due
to dense and compact structure enhances COD
removal by aerobic granules. Tay (2004) have
achieved 15 Kg COD/M3. A problem
observed in case of very high organic loading
is the development of loose and fluffy granules
due to excessive growth of filamentous organisms.
However, this can be controlled by intermittent
loading and cycle variation.
2.Simultaneous
removal of nitrogen and organics
Complete nitrogen removal by nitrification
and denitrification. Nitrite and nitrate produced
from nitrification are reduced to gaseous nitrogen
by dinitrifiers. Yang et al. (2004)
investigated the simultaneous removal of organics
and nitrogen by aerobic granules. Heterotrophic,
nitrifying and denitrifying populations were
shown to successfully co-exist in the microbial
granules. Increased substrate N/COD ratio led
to significant shifts among these populations
within the granules.
3.Phosphorus
removal
Environmental regulations in many countries
require reduction of phosphorus concentration
in wastewater to levels of 0.5 to 2.0 mg/l before
discharge. The well-known enhanced biological
phosphorus removal (EBPR) process removes P
without the use of chemical precipitation and
is an economical and reliable option for P removal
from wastewater. The EBPR process operates on
the basis of alternating anaerobic and aerobic
conditions with substrates feeding limited to
the anaerobic stage. Most EBPR processes are
based on suspended biomass cultures and require
large reactor volumes. Although full scale experience
shows a strong potential of the EBPR, difficulties
in assuring stable and reliable operation have
also been recognized. Often, the reasons for
failure of biological phosphorus removal are
not clear (Barnard et al., 1985; Bitton,
1999).
4.Phenolic waste
water treatment
Phenol is a toxic and inhibitory substrate,
but also a carbon source for the bacteria. The
consequence of the presence of phenol in biological
wastewater treatment is process instability,
which can lead to the washout of the microorganisms
(Allsop et al., 1993). In low concentrations,
phenol is biodegradable, but high concentrations
can kill phenol degrading bacteria. Industrial
wastewaters from fossil fuel refining, pharmaceutical
and pesticide processing are the major sources
of phenolic pollution. Jiang et al.,
(2002, 2004) investigated the feasibility of
treating phenol-containing wastewater with aerobic
granular sludge. Granular sludge is less susceptible
to toxicity of phenol because much of the biomass
in the granules is not exposed to the same high
concentration as present in the wastewater.
The phenol-degrading aerobic granules displayed
an excellent ability to degrade phenol (Jiang
et al., 2002, 2004). For an in fluent
phenol concentration of 500 mg/l, a stable effluent
phenol concentration of less than 0.2 mg/l was
achieved in the aerobic granular sludge reactor
(Jiang et al., 2002, 2004). The high
tolerance of aerobic granules to phenol can
be exploited in developing compact high-rate
treatment systems for wastewaters loaded with
a high concentration of phenol. Aerobic granules
may prove powerful bioagents for removing other
inhibitory and toxic organic compounds from
high strength industrial wastewaters.
Aerobic granules appear to be highly tolerant
of toxic heavy metals (Xie, 2003).
5.Biosorption
of heavy metals by aerobic granules
Heavy metals are often found in a wide variety
of industrial wastewaters. More stringent metal
concentration limits are being established in
view of their relatively high toxicity. Many
biomaterials have been tested as biosorbents
for heavy metal removal. These include marine
algae, fungi, waste activated sludge and digested
sludge (Lodi et al., 1998; Taniguchi
et al., 2000; Valdman and Leite, 2000).
In view of the physical characteristics of aerobic
granules as discussed earlier, these granules
are ideal biosorbent for heavy metals. The granules
are physically strong and have large surface
area and high porosity for adsorption. In addition,
the granules can be easily separated from the
liquid phase after biosorption capacity is exhausted.
Future trends
in aerobic granules
Aerobic granulation has been observed only
in SBRs. The feasibility of attaining aerobic
granulation in continuous culture systems needs
to be investigated. Selection pressure is the
main driving force for aerobic granulation;
however details of the mechanism need to be
worked out elaborately. As aerobic granules
possess optimum characteristics required for
waste water treatments, the technology has a
great future in the treatment of wastewater.
Figure 1: Microstructure
of granular biomass.
Figure 2: Schematic
representation of aerobic granules formation.
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