Microbial Encapsulation-An approach for enhancing Anaerobic Digestion efficiency
1. An approach for enhancing
Anaerobic Digestion efficiency
Meenakshi Mehra (CSIR-JRF)
Biotechnology Division
DRDE, DRDO, Gwalior
Imprisonment of ‘cell or enzyme’ in a distinct support/ matrix
2. CONTENTS
₪ GLOBAL CHALLENGES
₪ WHY ANAEROBIC DIGESTION?
₪ DETAILED STAGES OF ANAEROBIC DIGESTION
₪ ADVANTAGES OF ANAEROBIC DIGESTION
₪ LIMITING FACTORS FOR ANAEROBIC DIGESTION
₪ IMPROVEMENT FOR ANAEROBIC DIGESTION
₪ MICROBIAL ENCAPSULATION
₪ TECHNIQUES FOR MICROBIAL ENCAPSULATION
₪ COMMON MATRICES USED FOR MICROBIAL ENCAPSULATION
₪ APPLICATIONS OF MICROBIAL ENCAPSULATION
₪ ADVANTAGES OF MICROBIAL ENCAPSULATION
₪ CHALLENGES OF MICROBIAL ENCAPSULATION
₪ CONCLUSION
3. Global Challenges
₪ As world’s population is increasing rapidly, it is imprinting
adverse impact on the environment.
₪ Waste generation on a large scale, and the increased
energy consumption due to modernization is the second
most emerging issue now days.
₪ One of the main problem is to cope with an increasing
amount of primary waste in an environmentally
acceptable way
₪ Society is slowly moving towards seeking more
sustainable production methods to “Kill both the birds
with a single stone”, like waste minimization, distributed
energy generation, etc
₪ Anaerobic Digestion is the best way to decompose the
organic waste as well as to produce energy in the form of
biogas
4. Why Anaerobic Digestion?
Anaerobic Digestion (AD) is a microbiological process whereby organic matter is
decomposed in the absence of oxygen.
Using an engineering approach and controlled design, the AD process is applied to process
organic biodegradable matter in air proof reactor tanks, commonly named digesters, to
produce biogas.
The interest in the process is mainly due to the following two reasons:
₪ A high degree of reduction of organic matter in comparison to the Aerobic Digestion
₪ The production of biogas, which can be utilized to generate different forms of energy
5. Countries That Use Incentive To Encourage Anaerobic Digestion
Source: A Global Perspective of Anaerobic Digestion Policies and Incentives - Global Methane Initiative
6. Policies And Regulations Globally
Source: A Global Perspective of Anaerobic Digestion Policies and Incentives - Global Methane Initiative
Countries with agriculture policies
directly related to anaerobic
digestion,
70% (21 of 30 countries
researched)
Countries without agriculture
policies directly related to
anaerobic digestion,
30% (9 of 30 countries
researched)
Countries not
researched
for this report
8. 1 2 3 4
Hydrolysis
Acidogenesis Acetogenesis Methanogenesis
₪ Complex organic matter, Carbohydrates,
Fats & Proteins are broken down into
glucose molecules, fatty acids and amino
acids
₪ This stage is carried out by hydrolytic
enzymes exerted by fermentative
microorganisms
₪ Products of hydrolysis (hydrogen and
acetate) may be used by methanogens.
₪ Relatively slow step - can limit the rate of
the overall process
Carbs,
Proteins
& Fats
Sugar,
Amino
Acids &
Fatty Acids
Volatile
Fatty
Acids
Acetic
Acid
H2
and
CO2
Methane
&
CO2
Detailed Stages In Anaerobic Digestion
9. 1 3 4
Hydrolysis
Acidogenesis
Acetogenesis Methanogenesis
2
₪ Bacteria break down glucose molecules,
fatty acids & amino acids into volatile
fatty acids & alcohols resulting in
byproducts like hydrogen sulphide,
carbon dioxide and ammonia
₪ By acid-forming bacteria, hydrolyzed
organic compounds are converted to
higher organic acids such as propionic
acid, butyric acid and to acetic acid,
hydrogen and carbon dioxide
Carbs, Proteins
& Fats
Sugar,
Amino
Acids &
Fatty Acids
Volatile
Fatty
Acids
Acetic Acid
H2
and
CO2
Methane
&
CO2
Detailed Stages In Anaerobic Digestion
10. 1 4
Hydrolysis Acidogenesis
Acetogenesis
Methanogenesis
2 3
₪ Higher organic acids produced during
acidogenesis are subsequently converted
into trans-acids and alcohols are
converted into hydrogen, carbon dioxide
and transformed to acetic acid and
hydrogen by acetogenic bacteria
₪ Acetate and hydrogen produced during
acidification and acetogenic reactions are
substrates for methanogenic bacteria
Carbs, Proteins
& Fats
Sugar,
Amino
Acids &
Fatty Acids
Volatile
Fatty
Acids
Acetic Acid
H2
and
CO2
Methane
&
CO2
Detailed Stages In Anaerobic Digestion
11. 1
Hydrolysis Acidogenesis Acetogenesis
Methanogenesis
2 3 4
• Archaea convert hydrogen and acetic acid
into methane and carbon dioxide
• Methanogenesis is a critical step in the
entire anaerobic digestion process, and its
biochemical reactions are the slowest in
comparison to those in other steps.
Methane-producing bacteria are strict
anaerobes and are vulnerable to even
small amounts of oxygen
Carbs, Proteins
& Fats
Sugar,
Amino
Acids &
Fatty Acids
Volatile
Fatty
Acids
Acetic Acid
H2
and
CO2
Methane
&
CO2
Detailed Stages In Anaerobic Digestion
12. Environmental
Benefits
Energy
Benefits
Economic
Benefits
• Elimination of malodorous
compounds
• Reduction of pathogens
• Production of sanitized compost
• Promotion of carbon
sequestration
• Protection of ground/surface
water
• Deactivation of weed seeds
• Decrease in GHGs emission
• Beneficial reuse of recycled water
• Net energy producing process
• Biogas facility generates high
quality surplus energy as
electricity and heat is produced
• Reduces reliance on energy
imports
• Such a facility contributes to
decentralized, distributed
power systems
• Biogas is a rich source of
electricity, heat, and
transportation fuel
• Transforms waste liabilities into
new profit
• Time devoted to moving,
handling and processing
organic waste is minimized
• Adds value to negative value
feedstock
• Biomass-to-biogas, and
reduces cost of water treatment
• Reduces expenditures on
energy imports
Advantages Of Anaerobic Digestion
13. Bacteria:
Must have enough living organisms and the two different types bacteria types
required in balance
pH:
The optimum pH for the digestion of organic waste is in the range 6.8 to 7.2 with
the limit of the range for operation without significant inhibition being 6.5 to 7.6
Toxins:
Certain materials implies inhibitory effects on digestion, if their concentration
become too high
Temperature:
The rate of food stabilization increases and decreases with temperature within
certain limits
Time:
The SRT/HRT (Solid Retention Time/Hydraulic Retention Time) ratio, directly implies
the efficiency of a treatment system. Higher the ratio, more efficient and
economic the system
Waste type:
Solid concentration and frequency of feeding can impact the process
Limiting Factors For Anaerobic Digestion
14. During Anaerobic Digestion, the doubling time of the hydrolytic and acedogenic
bacteria is about 1-1.5 days, while it is extended till 1-4 and 5-15 days for acetogens
and methanogens respectively.
For complete digestion bacteria require a long time inside digester and may easily be
washed out.
In addition to this, the methanogens are very sensitive to the process conditions;
their low growth rate results in a relatively long start-up period of upto 3 months to
have a stable operation.
To retain the bacteria inside the digester, it is required to trap the bacteria inside the
digester.
Encapsulation may be an excellent approach to retain the bacteria inside the digester
for long time
Improvement For Anaerobic Digestion
15. What is Bioencapsulation
Entrapped in a matrix
Entrapped in droplets
o Encapsulation method involves covering and protecting the
microorganisms
o Encapsulation method has been used since 1993 as an
alternative technology to the entrapment over which it enjoys
advantage of higher cell loading and no cell leakage
o The bio-encapsulation of microorganisms is performed with the
incorporation of an active ingredient suspension into a matrix
followed by a mechanical operation, and finally stabilization by
a chemical or physical-chemical process
o The suspensions are non-Newtoniun fluids
o Substrates and products diffuse in and out easily through gel
matrix
17. ⃝ Encapsulation refers to physicochemical or mechanical process to entrap a
substance in a material in order to produce particles/capsules/beads of desired
size
⃝ The selection of the best encapsulation technology needs to consider numerous
aspects in order to guarantee the survival of bacteria
⃝ Encapsulation is achieved by employing several techniques such as, coacervation,
emulsion/interfacial polymerization etc
Bioencapsulation Techniques
Bioencapsulation
Spray-Drying Spray-Chilling
Liquid Droplet
Forming
Emulsion-based
Techniques
Coacervation
Fluid-bed
Agglomeration
18. Spray-Drying
Commonly used technique for food
ingredients production
The first spray dryer was
constructed in 1878, used for the
first time to encapsulate a flavour
Active ingredient is dissolved in the
encapsulating agent
Obtained solution is dried,
providing a barrier to oxygen and
aggressive agents
Schematic diagram of a spray-dry encapsulation processMini Spray Dryer
19. Spray-Cooling/Spray-Chilling
This process is similar to spray-drying
In the case, a molten matrix with low melting point is used to encapsulate the bacteria
The mixture is injected in a cold air current
Capsules produced in this way are generally not soluble in water
Schematic diagram of a spray-chill encapsulation process
20. Fluid-Bed Agglomeration And Coating
Schematic Diagrams of two types of the most commonly used fluid-bed coaters
Evolved from a series of inventions patented by Dr.
Wurster and colleagues
These patents are based on the use of fluidizing air
to provide a uniform circulation of particles past an
atomizing nozzle
The most used techniques are
referred to as the bottom-spray
(Wurster) fluid-bed process and
the top-spray fluid-bed process
However, variations such as
tangential-spray are also
practiced.
Air distribution plate
Bacteria being
Coated
Coated
Bacteria
21. Emulsion-based Techniques
An emulsion is the dispersion of two
immiscible liquids in the presence of
a stabilizing compound
Emulsions are produced by the
addition of the core phase to a
vigorously stirred excess of the
second phase that contains the
emulsifier
While a hydrophobic core phase is termed an
oil-in-water emulsion (o/w) (fig. A)
When the core phase is aqueous this is
termed a water-in-oil emulsion (w/o) (fig. B)
There are also double emulsions, such as
water-in-oil-in-water (w/o/w) (fig. C)
A
B
C
Schematic diagram of emulsion based encapsulation process
22. Coacervation
This process involves the precipitation of a polymer or several polymers by phase
separation simple or complex coacervation, respectively
During simple coacervation polymer is “salted out” by addition of agents, that have
higher affinity to water than the polymer.
With regard to complex
coacervation, two
colloids are mixed at a pH
at which both polymers
are oppositely charged
(i.e. gelatin (+) and arabic
gum (-)), leading to phase
separation and formation
of enclosed solid particles
or liquid droplets.
Schematic diagram of coacervation encapsulation process
23. Liquid Droplet Forming
It includes two principal steps:
(1) the internal phase, containing the inoculants is dispersed in small drops, and then
(2) these drops will solidify by gelation or formation of a membrane on their surface
Different technologies are available for dripping, and the selection of the best one is
related with desired size, acceptable dispersion size, production scale and the maximum
shear that cells can tolerate.
Dripping
Methods
Drop
Generation
By gravity
Coaxial
Flow
Jet
Breakage
Vibrational
Mechanism
Cutting
Method
24. Drop Generation
Dripping by gravity
The simplest method to make individual drops,
droplet size is determined by its weight and
surface tension, as well as the nozzle perimeter
Diameter of the drop is higher than 2 mm
not interesting for an industrial application
Drop generation by gravity using a
160 μm nozzle
25. Drop Generation
Coaxial Flow
A coaxial air flow is applied around the extrusion nozzle
microsphere diameter ranges a few micrometers to 1 mm
The air flow might be replaced by a liquid : with a suitable selection of the liquid flow
Schematic diagram of a submerged two-fluid static nozzle
Equipment for Flow-Focusing
technology to make droplets
26. Jet Breakage
Vibration Technology For Jet Break-up
A vibration is applied on a laminar jet for controlled break-up
A laminar liquid jet is broken up into equally sized droplets by a superimposed
vibration
Image of Encapsulator and Droplet formation based on the nozzle vibration technology
27. Jet Breakage
Jet-cutter Technology
The bead production is achieved by cutting a jet into cylindrical segments by a
rotating micrometric cutting tool
Jet-Cutter technique is especially capable of processing medium and highly
viscous fluids up to viscosities of several thousand mPas
the fluid is pressed with a high velocity out of a nozzle as a solid jet
Jet
Cutter Beads formed by
Jet-Cutter
Schematic diagram of jet cutting technique
28. Matrices Used For Encapsulation
Also, certain synthetic polymers have been used for bio-encapsulation
of living cells such as, polyacrylamide, polystyrene and polyurethane.
Polyacrylamide gel was the first matrix material used to immobilize
cells
Hydro-gels extracted from seaweeds, such as alginates, agar-agar,
carrageenan, and agarose are used for polymerization or cross-linking
Gums and proteins are frequently used as protective materials to
cells, although they usually turnout to be more expensive
Sodium alginate is one of the most used products for the bio-
encapsulation of microorganisms
29. Alginate is natural polysaccharide produced by brown algae.
First isolated and named by Scottish scientist, Dr.C.C.Stanford in
1883 and since it has been utilised as a hydrocolloid in variety of
applications
Alginate production is not only exclusive to seaweeds, indeed,
some bacteria are able to produce extracellular alginate
(Azotobacter vinelandii, Pseudomonas spp)
Alginates are linear macromolecules comprising two monomers
linked by alpha 1-4: β-acid and D-mannuronic acid to α-L-
guluronic acid
Alginic Acid (Alginate)
Azotobacter vinelandii
30. Chitosan
It is a deacetylated derivative of chitin
A positively charged polymer, forms ionic hydrogels by addition of anions
Chitosan is biodegradable and biocompatible but, it is necessary to consider the
antibacterial activity of this polymer
Carrageenans
Carrageenans are sulphated polysaccharides obtained from different species of marine
red algae
Largely used as thickening, gelling agent, texture enhancer or stabilizer on food,
pharmaceutical and cosmetic formulations
Starch
Bacteria can be encapsulated by adhesion to starch granules
Usually chemically modified starch (maltodextrin or cyclodextrin) is used for
encapsulation in combination with the spray-drying technology , fluid bed granulation
Gellan gum
It is produced as an aerobic fermentation product by a pure culture of Pseudomonas
elodea
A disadvantage is that, it is having a high gel- setting temperature (80-90°C for about 1h)
which results in heat injuries to the cells
Common Matrices Used For Encapsulation
34. ₪ Protection of pheromones from oxidation and light during storage
and release
₪ Entrapment of nutrients and hormones responsible for growth
₪ Adding ingredients to food products to improve nutritional value
₪ Adding antimicrobial agents for food packaging
₪ Replacement of therapeutical agents (not taken orally)
₪ Preparation of enteric coated dosage forms selectively absorbed
in the intestine
₪ Addition of oily medicines to tablet dosage forms
₪ Separation of incompatible substances e.g., pharmaceutical
eutectics
Bioencapsulation: Applications
Food& Dairy Industries
Pharmaceutical Industry
Agriculture
35. ₪ To decrease the volatility of the drug
₪ To prevent hygroscopic properties
₪ Safe handling, easy recovery, disposal at an
acceptable economic cost
₪ To protect drugs from environmental hazards
such as humidity, light, oxygen or heat
₪ To decrease the potential danger of handling
of toxic or noxious compounds
Bioencapsulation: Applications
Chemical Industry
36. (FC)-Free cells, (GEC)-Galacto- oligosaccharides co- encapsulated cells, (EC)-Encapsulated cells with alginate alone,
(FEC)-Fructo-oligosaccharides co- encapsulated cells, (XEC)-Xylo-oligosaccharides co- encapsulated cells,
(IEC)-Isomalto-oligosaccharides co-encapsulated cells
Survival ability of microencapsulated L. fermentum with
oligosaccharides in simulated gastric juice
Survival ability of microencapsulated L. fermentum with
oligosaccharides in simulated intestinal juice
37. Number of publication related to nanotechnology in
food packaging in the last 15 years.
38. The ZnO-NPs encapsulated alginate
nanocomposites demonstrated its bactericidal
activity with water containing Staphylococcus
aureus
For surface water, the nano-composites
showed excellent antimicrobial activity within
1 min (inactivated all the bacteria)
However, with synthetic water, the nano-
composites showed good results after 120
min with a low bacteria concntration of 200
cfu/ml
Furthermore, the leached Zn2+ was well
within the recommended limits for use in
water treatment
39. Lab-scale Bio-filter Real-lifeBio-filter
Development of a Rapid, Effective Method for Seeding Biofiltration
Systems using Alginate Bead-immobilized cells
Microbial colonisation inside the fissure of biodegradable hydrogel beads
40. Accumulated methane production by digesting bacteria
encapsulated in, (A) different natural membranes and
(B) different sachet sizes of PVDF filter membrane
Digesting bacteria was encapsulated
inside the natural membrane (alginate)
and synthetic membrane (polyvinylidine
fluoride) having pore size 0.1 µm
Methane production from encapsulated
digesting bacteria was successful
Synthetic membrane exhibited higher
stability in the digester
Synthetic membrane sachets Natural membrane capsules
A
B
41. The methanogenic bacteria
and methanotrophic bacteria
were immobilized in the
ratio, 10:1 inside the barium
alginate beads
Results showed that, the
biological PCE dechlorination
rate was 92% and there was
highly effective degradation
of PCE
Concentration of PCE in effluent during degradation
42. Effect of a transient low pH during continuous operation of the
process fed with acetate (pH 5.0)
Relief of inhibitory effect of phenol in batch experiments fed with
various concentrations of phenol and 2100 mgl-1 of acetate
43. Encapsulation of Anaerobic Microbial Inoculum (AMI)
for microbial enrichment and volume reduction of
seeding volume of inoculum in Biodigester
44. Bioencapsulation: Advantages
₪ Gain temporary protection against any potentially degenerative changes
₪ Promote a higher localised cell loading
₪ Prevents high dilution rates
₪ Prevents inoculum washout, due to the continuous loading of substrate and removal
of effluent
₪ Degradable nature of hydrogels provides continuous slow release mechanism
₪ Easier to separate products and reactants
₪ No further purification is required as there is no cell contamination in the product
46. Bioencapsulation: Challenges
₪ May alter the activity of some cells
₪ Some of enzymes secreted by cells become unstable
₪ Sometimes, compromises the viability of cell due to heat generated during preparation
₪ Forms a depot in tissues or muscles for longer period and hence may produce pain after
medical use
₪ Polymer may produce toxic effects
₪ Expensive
47. CONCLUSION
₪ Since industrial revolution, there have been major progressions in agriculture,
manufacture, transport and most importantly global industrialization.
₪ In the last few decades, the onset of this rapid urbanization has led to an increase in the
demand of a huge amount of energy.
₪ An increase in the waste generation is also one of the consequences of the
modernisation of the world.
₪ For a couple of years, disposal of waste and increased consumption of energy has been a
considerable issue.
₪ Anaerobic digestion is playing an important role to overcome both the problems.
₪ As anaerobes are very sensitive to the environmental changes, the anaerobic digestion
technology needs to be modified.
₪ By encapsulating anaerobes, we can prevent sudden environmental shocks to the
bacteria.
₪ Various researches have been done to encapsulate the anaerobic bacteria and they have
found successful too.
₪ But, there should be some more modifications towards the betterment of anaerobic
digestion to get “a clean and green Earth” .
