Environmental-Friendly Bio-technologies for Sustainable Agrowaste Management

Agricultural wastes – global problem
Environmental impact
Recycling of wastes
Disadvantages
(cost intensive, unsafe, requires treatment, high energy
requirement, environmental unfriendly)
Physical, Chemical, Biological methods
Value added for Economic development
3R concept
is a must
Concept of sustainable
development
Why biological methods?
Cheap, safe and effective
Utilization of agrowastes for the production of added value and commercial products
Viable large scale operation of enzymatic hydrolysis with low production cost
Environmental friendly system/process
Reproducible, efficient, low operational cost, non-detrimental approach & good quality products
Strategic management of agricultural solid wastes

Dried palm kernel cake
Empty fruit bunches
Sugarcane
bagasse
Oil palm shells
Oil palm fresh
fruit fibers
Paddy straws
Some of the lignocellulosic materials from Malaysian agrowastes
Malaysian golden
crop

Sustainable development of natural resources –
generation of agrowastes with a volume of >100
million tons a year
Environmental impact
Commercializable and high value products/processes in recycling of waste materials via biotechnological approach
Solid State Fermentation (SSF)
Potential fungal
isolates
Enzymes, organic acid,
biopesticides, compost, mushroom,
animal feed, fertilizers
Why SSF?
Simple
Cost effective
Environmental friendly
High product concentration
Ease in product purification
Less contamination risk
Applicable for production
of various microbial
products
SCB
PKC
RH
RW

Potential microorganisms with good growth on agrowastes

The utilization of agrowastes as substrates for SSF
Medium formulation and fermentation conditions Enzyme preparations
Cellulase
USM I
Xylanase
USM II
Solid state fermentation (SSF) for the production of microbial metabolites

AGRICULTURAL
WASTES
BIOMANAGEMENT
Animal feed formulation
Fermentable sugars
Yeast biomass
Poultry
Aquaculture industries
Composting, biofertilizers, antibiosis
Enzymes, Flavouring compounds
Plants/Agriculture
Yeast extract
Nitrogenous compounds/SSF
SSF/semi solid fermentation
Chemicals
SSF/enzymatic/physical
Submerged fermentation
SSF
Applications in food and chemical
industries
Bioplastics
Submerged fermentation
Submerged
fermentation
UTILIZATION OF MALAYSIAN
AGRICULTURAL WASTES
Bioremediation
SSF

Enzyme production via SSF
Downstream
processes
Analysis
Packaging
SSF fermenter
Fungal isolates substrate/medium Inoculum development
Enzyme extraction
Biomass separation
Enzyme concentration
Freeze drying
Stability
Activity
Safety
Enzyme powder

Lipase production by Mucor miehei by solid state fermentation
After optimization : 27 U/g/day
Before optimization : 10 U/g/day
Growth of M. meihei
in flask system

PRODUCTION OF ENZYMES AND
ITS INDUSTRIAL APPLICATIONS
Lipase (Fine chemical synthesis and detergency)
Protease (Allergenic protein degradation in latex and feeds and detergency)
Xylanase/hemicellulase (Enzymatic deinking in paper recyling, production of
fermentable sugars)
Cellulase (Similar to xylanase)
Lignin peroxidase (Lignin degradation, dye decolorisation)
Manganese peroxidase (Similar to LP)
Laccase (Similar to LP dan MnP)
Manannase (Degradation of mannan in palm kernel cake)
Phytase (Feed formulation)
β-glucosidase (Feed formulation and fermentable sugar production)

SCALING UP OF SOLID STATE FERMENTATION - FERMSOSTAT®

Substrate port (A) and Sampling port (B) provided in FERMSOSTAT®.

Mixing system provided in FERMSOSTAT®. (X) Speed control motor, (Y) Various digital readouts and (Z) Impeller.

Xylanase production in the
FERMSOSTAT

Enzymes production in
the FERMSOSTAT®

Summary of optimum conditions for production of cellulases and xylanase enzymes by A. niger USM AI 1.

Profiles of batch production of enzymes using the FERMSOSTAT®

SSF INTELLIGENT FERMENTER
Water/inoculum
storage
Control panel
Tray system

Optimization of enzymes production via solid state
fermentation using PKC

Application of lipase for esterification reaction of acetone glycerol acyl esters
1,2-O-isopropylidene glycerol + fatty acids 1,2-O-isopropylidene acyl glycerol
+ water
Mild hydrolysis
monoacylglycerol + acetone
Lipase
of Pseudomonas sp.
a. Celite 545, b. Florisil, c. Kieselguhr,
d. Amberlite XAD-4, e. Amberlite XAD-7,
f. Eupergite C, and g. Eupergite C250L.
Hexane
Log P: 3.5
Fatty acid specificity
CONTINUOUS PACKED BED
BIOREACTOR SYSTEM
(Each cycle was for the reaction time
of 24 hr )
Lauryl esters

The use of SSF materials as enzyme source in organic
synthesis
0.5 ml vinyl acetate
1.0 ml hexane
0.5 ml tetrahydrofuran
20 mg SSF materials
Room temperature, 1000 rpm
Phenylethanol Phenylacetate
2-Cl-cyclohexanol 2-Cl-cycloacetate
2-I-cyclohexanol 2-I-cycloacetate
Kinetic resolutions by SSF 292 (Mucor hiemalis with highest Ec.
Alcohols
Acetates
SSF materials in tray
system

Comparison on the use of SSF materials from different sources of lipase source in organic synthesis

Depletion of forest resources – global problem
(Each ton of paper making – 17 –20 trees, 31,500 L of water, 41,000
Kw/h energy, 50-70 kg chemicals)
27 kg air pollution and creating 2.5 m3 landfill materials
Environmental impact
Recycling of waste paper – conventional chemical methods
(sodium hydroxide, sodium silicate, hydrogen peroxide, hypochlorite,
chelating agents and surfactants)
Environmental impact
(cost intensive, unsafe, requires treatment for finished papers, high energy
requirement, environmental unfriendly)
ENZYME APPLICATION IN PAPER INDUSTRY

PULP AND PAPER INDUSTRIES IN
MALAYSIA
Demand for paper continues to be strong although in paperless global society (a state of self sufficiency)
The industry is heavily dependent on imported fibre, particularly virgin pulp.
> 1.0 million tons per annum (19 paper manufacturing companies)
Paper import: 1,189,120 metric tonnes per year (RM2.7 billion)
Thus, a new source of fibre is needed to strengthen the industry
Non-wood materials Recycling of waste papers (< 5%)
Kenaf fibres (Conventional chemical method)
(Hibiscus cannabinus) (No biological/enzymatic method)

ISSUES ON ENVIRONMENTAL IMPACTS
Pollutions from conventional chemical methods
Environmental friendly, biological methods via
biotechnology
Alternative biological methods
for paper recycling using
biocatalysts/enzymes
____________________________________________________________
Why biological method for paper recycling?
Cheap, safe and effective
Utilization of agrowastes for the production of added value and
commercial products
Viable large scale operation of enzymatic hydrolysis of pulp and ink removal
with low production cost
Environmental friendly system/process for deinked waste papers
Reproducible, efficient, low operational cost, non-detrimental approach
good quality deinked papers

SPECIFIC OBJECTIVES
ON ENZYMATIC DEINKING SYSTEM
_______________________________________________
1. To design, construct and fabricate the enzymatic bioreactor for paper pulp hydrolysis , flotation system and pulp separation unit for continuous enzymatic deinking, ink removal, ink separation and reuse
of enzymes and flotation solution.
To evaluate the performance of the enzymatic deinking system under continuous operation based on the quality and properties of the deinked papers.

Application system for enzyme hydrolysis of waste papers and flotation for ink removal under optimized conditions
Enzymatic deinking
Flotation
High quality Deinked paper :
Comparable properties of commercial papers or papers by
conventional
chemical method
An effective and fast prototype
of the flotation
system
containing
enzymatic hydrolysed
paper pulp
Ink removed
Before After

DEINKING OF PULP – PAPER RECYCLING
BEFORE
AFTER
Enzymatic deinking
and flotation
process

Optimization of the laboratory enzymatic hydrolysis of pulp
Optimization of the flotation process
*Enzymes from Indigenous sources

Physical characteristics of deinked paper
* Bleaching involved in treatment process

Schematic diagram of pilot
System of continuous
enzymatic deinking process
Stirred tank enzyme
Bioreactor
Flotation system

Details of the continuous enzymatic deinking process
I : Enzyme bioreactor
Features
i. The bioreactor is a stirred tank reactor, which will be equipped with impeller for agitation
ii. The motor will be used for agitation with controlled agitation rate
iii. The impeller will be designed as blades to prevent clumpings of pulp.
iv. The temperature of the bioreactor will be controlled by temperature controlled
jacket
v. Upon completion of reaction, the pulp will be pump into the flotation system while the enzyme solution will be recycled into the bioreactor the next batch of pulp.
vi. Equipped with probes for temperature and pH
b. Capacity
Volume : 150 L (estimated to be 10 kg of pulp at 1% pulp consistency per cycle*)
75 cm (ID) and 120 cm height
Pulp consistency can be varied, more pulp at higher consistency and higher agitation rate and enzyme concentration. At 1%, per cycle 30 – 40 min or 36 - 40 cycles per day or 360 – 400 kg pulp deinked per day

II. Flotation system
Features
Equipped with a motor for agitation with controlled agitation rate
Equipped with multiblade impeller to disperse the hydrolysed pulp
Temperature control via heating coil
Equipped with sparger connected to flowmeter for air flow rate from compressor
Ink removed via ink trap and collected in the ink reservoir
Upon completion deinked pulp drain in pulp collection container via a siever.
The flotation solution will be recycled into the flotation system for next batch
of pulp.
vii. Place vertically supported with pillars
Power supply : 240 V
b. Capacity
Volume : 750 L (estimated to be 10 kg of pulp at 1% pulp consistency*)
Flotation column: 200 cm height, total height 300 cm, ID 90 cm
Pulp collecting container: 100 cm height
Ink reservoir : ID 20 cm and 30 cm height

Hydrolysis
tank
Flotation
system

Developed pilot scale of enzymatic deinking system; (A) Front view, (B) Left view and (C) Right view.
Notes: K: Vacuum pipe; L: Peristaltic pump; M: Mixer controller box; N: Heater; O: Enzyme storage vessel; P: Enzyme/water recycling pipe; Q: Enzyme/water recycling vessel; R: Drain pipe; S: Water pump; T: Deinked pulp outlet; U: Sieve; V: Ink reservoir; W: Water inlet; X: Flotation vessel; Y: Solenoid valves; Z: Flowmeter.

Enzyme storage vessel; Front view (i) and Top view ii).
Bioreactor equipped with mixer; Front view (i) and Top view (ii).
Note: A: Enzymatic hydrolysis vessel; B: Mixer controller box; C: Mixer, D: Impeller shaft, E: pH probe; F: Thermocouple; G: Sieve; H: Impeller tips; I: Pulp slurry outlet.

Flotation vessel; Front view (i) and
Top view (ii).
Note: A: Flotation vessel; B: Viewing glass; C: Nozzles; D: Ink reservoir; E: Foam outlet; F: Scraper; G: pH Probe; H: Thermocouple; I: acid/base reservoir; J: surfactant reservoir; K: Blower; L: water level sensor.
Pulp collecting vessel; Front view (i) and Top view (ii)
Note: A: Pulp collecting vessel; B: Solenoid valve; C: Pulp suspension outlet; D: Sieve.

Blower (i), single nozzle (ii) and Nozzles unit (iii) used in the flotation system
Foam scraping unit used in flotation process
Note: A: Scraper; B: Foam drain; C: Foam/ink particle outlet; D: Water level sensor; E: Water inlet; F: Thermocouple; G: Scraper carrier

: Schematic diagram of diaphragm pump used in the deinking process
Note: A: Diaphragm pump; B: Solenoid valve; C: pulp suspension from bioreactor; D: pulp suspension to flotation vessel; E: Blower; F: Nozzles unit.
Circulation of water (control temperature) around bioreactor and heater.
Note: A: Heater; B: Metering pump; C: Stopper; D: Bioreactor vessel.
Heater used in deinking process.

Mixed Office wastes (MOW)
Old newspapers (ONP)

MARKET POTENTIAL (SIZE)
Global market
The application of enzymes in pulp and paper industries large : Capacity
Indonesia : 10 million tonnes per annum
Thailand : 4.5 million tonnes per annum
Malaysia
Capacity : 1.0 million tonnes, Import : 1.2 million tonnes, Export :waste paper ; 50,000 tonnes (Year 2000), < 5 % recycled by chemicals
(Malaysia is working towards self sufficiency for papers, reduce import and encourage foreign capital inflow)
2.2 million tonnes of papers available for enzymatic
deinking per year
Enzymatic processes cover large market potential
~ RM 4.5 billion
Environmental issues
and sustainable
development
Advancement of
Industrial/Env
Biotechnology and bioengineering
10-20 kg enzyme
per tonne

INVESTMENT COMPARISON
Enzymatic deinking
Installation and setting up of fermentation unit : RM40,000.00
Installation and setting of deinking system : RM 60,000.00
Enzyme production cost (1000kg substrate) - 150 kg crude enzyme : RM7,200.00
(With 150 kg enzymes, a total of 7.5 tonnes of pulp can be deinked)
Energy cost and maintenance : RM7,000.00
Cost of Pulp and pulp processing (7.5 tonnes) : RM3,750.00
Total cost: RM 117,950.00
Chemical deinking (for 7.5 tonnes pulp)
Installation of flotation chemical system/facilities : RM120,000.00
Cost of pulp and pulp processing : RM3,750.00
Cost of chemicals/bleaching agents : RM13,500.00
Processing of finished product : RM22,500.00
Energy cost, operation and maintenance of system : RM18,500.00
Effluent treatment of chemicals and facilities : RM30,000.00
Total cost : RM 208,250.00
Operational cost: (for 7.5 tonnes pulp per first run)
Enzymatic deinking : RM 117,950.00
Chemical deinking : RM 208,250.00 ++++

INOCULUM DEVELOPMENT FOR ENVIRONMENTAL
MANAGEMENT
Bioremediation – hydrocarbon degradation
Organic domestic waste decomposition
Degradation of dyes from batik (textiles) efffluent
PRODUCTION OF BIOPRODUCTS FROM WASTES
Bioplastics
Fermentable sugars (Ethanol)

Inoculum development for hydrocarbon bioremediation
Isolation of hydrocarbon degrading microorganisms:
Oil contaminated soil/water (Penang, Kedah)
Soils at oil refineries (Melaka and Kerteh, Terengganu)
Potential isolates for
consortia development
Slow degradation capability of inoculum preparation
Improvement via enriching with N and P content of soils
BIOSURFACTANTS

Development of inoculum for domestic wastes decomposition
Microbial isolates Inoculum in rice husk as binder
Prototype of domestic waste
decomposer
Fermentation chamber

Decolourisation of dyes from effluent of batik industries
Decolourisation of wastes with the addition of enzyme preparations of LiP and MnP
Enzyme added
Without enzyme
Direct cultivation of Phanerochaete chrysosporium in the medium containing waste water
A. In the presence of waste water, B: absence of waste water.
Enzymes involved: Lignin peroxidase (LiP)
manganese peroxidase (MnP)
Source: Phanerochaete chrysosporium.
Waste water: batik effluent
MnP
LiP
Decolourisation
COD
Decolour
Time profiles of decoulorisation using lignin degrading enzymes

Wastewater from textile
factories
I: Tubular filtration/absorber
system
I
Wastewater tank +
Fungal growth medium
Physical Method
II
Decolourisation process by biological method
Release of water to river/recycle
Natural absorber
Dye degrading fungi
II : Continuous loop
bioreactor system
(Pneumatic type)
Aeration
Pump system
Filtration system
SSF of absorber
materials

Oils
Fermentation
PHA in bacterial cells
Fabrication
Extraction
Ecologically sustainable
‘green’ processes

Biodegradation Test (30 days)

Solid agrowastes
STAGE I Physical treatment
Solid waste intermediates
STAGE II Enzymatic hydrolysis and
microbial fermentation
Volumetric scale up Fermentable sugars
Microbial fermentation
via submerged systems
STAGE III Valuable products
Evaluation of productivities and process economics

Valuable products from microorganisms via submerged fermentation processes
from fermentable sugars
Ethanol fermentation
(Biofuel/Bioenergy)
Saccharomyces cerevisiae
30oC, optimized medium
20%(v/v) ethanol concentration
Membrane system for
separation of solvent
Evaluation as energy source
Enzyme production
Lipase (Mucor sp, Pseudomonas sp)
Xylanase (Aspergillus sp.)
Cellulase (Aspergillus sp.)
Protease (Bacillus sp.)
Wide applications
Yeast biomass
Candida utilis
Saccharomyces cerevisiae
Xanthophyllomyces
dendrorhous
30oC, aerobic
Applications in aquaculture industries as feeds
Microbial fermentation
using single culture of Aspergillus niger USM AI 1

Ethanol production as fuel
Agrowastes
Fermentable sugars
Yeast fermentation
Ethanol fermentation
Separation/concentration/distillation
ETHANOL 10-12% (v/v)
Delignification by fungi
Solid state fermentation (SSF)
Optimization of SSF
Optimization of
ethanol fermentation
Development of separation system
Phanerochaete
chrysosporium
Aspergillus niger
Aspergillus sp.
Saccharomyces cerevisiae

Conclusion
Environmental Issues : Global problem
Multidisciplinary strategies : Biotechnology, Industrial Chemistry, bio-engineering, environmental engineering, bioremediation, biosorption, microbial degradation of wastes
Development of innovative bio-technologies for environmental management
Industrial applications for large scale waste management
Future direction : Biological approaches supported by innovative technologies and engineering
Sustainable development with minimum impact on environment by wastes