Download Granulation - a unique example of biofilm formation

The following slides are provided by
Vincent O’Flaherty.
Dr.
Use the left mouse button to move forward
through the show
Use the right mouse button to view the
slides in normal view, edit or print the
slides
2. The role of kinetic parameters
in granule formation
• Growth rates, substrate affinities etc.
• The granule represents a balanced ecosystem,
this is not a straightforward outcome kinetically
• The growth rates of fermentative organisms are
5-10
times
faster
than
syntrophs
and
methanogens - so environmental factors will
greatly affect the likelihood of a stable community
developing - delicate balance
• The cell yields of methanogens and
syntrophs are thus rate limiting for the
growth of granules - loading rates and
other parameters are based on this
• However, probably the most important
kinetic determinant is the outcome of
competition for acetate between two
methanogens - Methanosaeta (ex.
Methanothrix) sp. and Methanosarcina
sp.
Acetate Competition and
Granulation
• Methane can be produced via acetate
or via H2/CO2 - 70-90% of the biogas
produced by anaerobic digesters is via
acetate - the key CH4 source
• Surprisingly
however
only
two
methanogens
have
ever
been
discovered that can utilise acetate Methanosaeta and Methanosarcina
• Methanosaeta
concilli and M.
Soehgennii are
rod-shaped
organisms which
tend to grow as
filaments
• Methanosarcina barkeri is coccoid and grows
3-6 times faster than Methanosaeta on acetate,
but Methanosaeta is the dominant methanogen
in granular sludge (up to 90% of methanogenic
cells)
• Methanosaeta has a much higher substrate
affinity and in a balanced anaerobic digester
the concentration of acetate will be very low
• The presence of Methanosaeta is believed to
be crucial as the filaments provide a nucleus
for granule initiation
The role of cell surface characteristics in granule
formation
• Hydrophobicity, electrophoretic mobility, and
isoelectric point are used to predict the
adhesion of bacteria to surfaces
• Hydrophobicity measured by water contact
angle is the most commonly used - allows
estimation of the surface energy of bacteria
• Work to date suggests that fermentative
organisms are hydrophilic ( water contact
angle of < 45˚)
• Most syntrophs and methanogens are
hydrophobic - > 45˚
• Hydrophobic cells, like oil droplet, tend to
stick to each other
• BUT, by association with hydrophilic cells
aggregates which behave hydrophilically can
be formed
• Role of trace elements and nutrients in granule
formation
• Granule formation is optimal when nutrient
conditions are ideal for the microorganisms
involved
• Ca2+ specifically appears to promote
granulation up to a concentration of 150 mg/l
• Role of seed sludge in granule formation
• Ideally granular sludge should be used as an
inoculum, but its expensive (c. € 6000 per
load) and transport costs can also be very
high
• Can use local sources like animal manures,
sewage sludge which contain high numbers
of anaerobic microbes
• Very important to get the balance of the
developed community right - its possible to
measure the methanogenic activity of the seed,
needs to be high initially
• Relates to the much slower growth rates of
methanogens vs fermentatives
• If methanogens are not present in high numbers,
acidification or souring of the reactor can occur
• The loading rate to the reactor is normally
increased stepwise and each step is only
carried out when >80% removal is
obtained - keep acetate low
(Methanosaeta)
• Irrespective of the source of seed - the
content of Methanosaeta is of crucial
importance in achieving granulation
Environmental Factors Affecting
Granule Formation
• Can be broken down as follows:
• Liquid and biogas upflow velocities
• Substrates
• Temperature and pH
• Role of liquid and superficial biogas
upflow velocities in granule formation
• There is a requirement for “selection
pressure” to encourage the formation of
granules
• In AD designs like the UASB arises as a
result of the liquid and biogas loading
rates (m3/m3/h or m3/m3/day)
• Results in washout of non-settling cells or
flocs and the retention of granules
• For UASB start-up loading rates are 0.250.4 m3/ m3/h liquid and 4-7 m3/m3/day
biogas
• Once stable granular bed is formed, much
higher loading rates can be safely applied
• Role of substrates in granule formation
• Specific substrates are directly related to
the proliferation of certain types of
bacteria
• Energy-rich substrates (e.g.
carbohydrates and sugars) give rapid
growth of granular sludge
• Poor granulation and fluffy floc formation
are often seen with protein rich
wastewaters
• Temperature has an important bearing on
which substrates support granulation e.g. acetate alone can allow granular
growth at 37˚C but not at 55˚C
• Role of Temperature and pH in granule
formation
• AD operates currently at two main
temperature levels 35-40˚C (mesophilic)
and 55-60˚C (thermophilic)
• Reaction proceed faster at high
temperatures - so higher loading rates can
be applied thermophilically
• But, a less diverse microbial population
develops and the system is much more
vulnerable to pH changes and other
shocks
• Also much more expensive to operate
(heating)
• Current research is focused on trying to
to operate psychrophilically (<20˚C)
Problems with Granulation
• Operational conditions - not suitable for
granulation - pH alterations, toxic shocks
• Inhibitors H2S, NH3 and toxic chemicals
• Foaming/floatation - lack of sugars or lots
of slowly degraded lipids in the
wastewater
Current Model of Granule
Structure
• Confocal microscopy, fluorescent DNA
probes and microsensors have been the
main experimental approaches used to
reveal structure/function relationships
• Granules grown on different substrates
have different properties - but a general
model can be used to explain common
features
Anaerobic digestion of sulphatecontaining wastewaters
• Industrial wastewaters can, in general, be
effectively treated using anaerobic digestion produces large quantities of methane which can
be burned to generate electricity or for heating use of combined heat and power plants allows for
generation of electricity and heat recovery
• Normally less than 10% of the biogas produced is
required to operate the plant - also produces far
less waste biomass than aerobic system = less
disposal costs
• Modern digester design makes the process more
attractive - can operate at high rates and
therefore smaller, cheaper digester can be used
• Usual procedure is to have first stage anaerobic
and then small activated sludge plant to “polish”
the effluent = achieve discharge standards. May
need some nutrient removal or other tertiary
steps depending on the fate of the effluent
Problematic industrial
wastewaters
• Application to industrial wastewaters
can occasionally be complicated by
microbiological problems - typical
example is treatment of sulphate
containing wastewaters - examples of
the
type
of
negative
microbial
interactions which can occur in an
engineered ecosystem
• Industrial wastewaters can contain high
levels of recalcitrant organic chemicals
(e.g. chloroform, carbon tetrachloride
etc.), xenobiotic products and side
products
(insecticides,
herbicides,
detergents etc.)
• Can also contain significant quantities of
inorganics some of which may be highly toxic
e.g. cadmium in tannery wastewater
• In anaerobic systems the presence of alternative
external oxidising agents ( e.g. sulphate SO42-)
can promote the development of a sulphatereducing rather than a methanogenic population
• This will result in the channelling of electrons
towards the formation of H2S not methane
SRB and anaerobic digestion
• Very complex systems - absolute need to fully
understand the microbiology in order to control
treatment plant operation - i.e on one hand a
useable fuel is generated and on the other hand a
malodourous atmospheric pollutant is produced
under sulphidogenic conditions
• Presents a challenge to microbiologists because
of the complexity of the systems and the
technical difficulty in studying them
•
Examples of wastewaters that contain highlevels of sulphate include:
1. Molasses-based fermentation industries - e.g.
citric acid production, rum distillery
2. Paper and board production
3. Edible oil refinery
•
Many other industries use sulphuric acid in their
processes - leads to sulphate in the ww
So what?
• In the absence of external oxidising agents
(sulphate, nitrate, etc.) anaerobic ecosystems are
methanogenic - flow of reducing equivalents is
directed towards the reduction of CO2 to CH4
• In the presence of sulphate - the flow may be
redirected towards the reduction of sulphate to
sulphide by sulphate reducing bacteria (SRB)
• In other words there is a competition between
different microorganisms for substrate
• What will determine the outcome of
competition?
• Very important to know as on one hand a
useable fuel is produced, while on the
other hand a toxic, corrosive malodourus
compound is produced
• Bacteria that reduce sulphate to H2S are either
assimilatory or dissimilatory:
• 1. Assimilatory Sulphate Reduction: Carried out
by many different bacteria - purpose is to reduce
sulphate to sulphide prior to uptake of S for
assimilation into S-containing proteins etc.
• No major environmental effect only amount of
sulphate needed for bacterial growth is reduced
• e.g Klebsiella sp. - only reduce 1 mg sulphate for
every 200 mg (d.wt.) of cells produced
• 2. Dissimilatory Sulphate Reduction: Totally
different process only carried out by a unique
group of bacteria carrying out anaerobic
respiration using sulphate as electron acceptor
• Consequently transform large amounts of
sulphate to H2S during growth
• e.g. Desulphovibrio sp. - for every 1 mg of
sulphate reduced, only 0.5- 1.0 mg (d.wt) of cells
are produced
• SRB exhibit considerable morphological
and nutritional diversity - grouped
together only on the basis of carrying out
dissimilatory sulphate reduction
• Widely
distributed
in
the
natural
environment - include both sporeformers
(Desulfomaculum
sp.)
and
nonsporeformers (Desulfovibrio sp.)
• Can be divided into two broad categories
based on their metabolism:
• 1. Incomplete Oxidisers: carry out
incomplete
oxidation
of
organic
compounds to acetate, CO2 and H2S - can
use a very wide range of starting organics
e.g. aliphatic mono- and dicarboxylic
acids, alcohols, amino acids, sugars,
aromatic compounds etc.
• 2. Complete oxidisers: Complete oxidation
of starting organic substrates to CO2 and
H2S - same wide range of substrates, but
can also grow on acetate, breaking it
down completely to CO2
• Chemolithotrophic species also common grow on H2/CO2 or on CO very common
ability to grow on H2 very important in
certain ecosystems
• 4H2 + SO42- + H+ -----> 4 H2O +HSG˚´ = -150KJ/mole
• Very favourable reaction energetically
• These species must be able to fix CO2 autotrophic
• SRB very versatile metabolically - in the
absence of sulphate in their environment,
they
can
switch
from
anaerobic
respiration
to
chemoorganotrophic
fermentation - energy gain by substrate
level phosphorylation only
• V. important as allows maintainence of
SRB in the absence of sulphate
What happens during anaerobic
treatment of sulphate containing
wastewaters?
• Competition between SRB and other anaerobes
for common organic and inorganic substrates for energy and reducing equivalents
• Outcome is determined by a number of factors
- COD/BOD conc.