Enzyme and Ethanol production using water hyacinth biomass

Rajeev K Sukumaran, PhDScientist, Biotechnology Division

National Institute for Interdisciplinary Science and TechnologyTrivandrum

Council of Scientific and Industrial Research (Govt. of India)

Enzyme and Bio-ethanol production using Water Hyacinth Biomass

The Potential and Prospects

National Institute for Interdisciplinary Science and Technology

Water Hyacinth Facts Botanical Name: Eichornia crassipes

Common Name : Common Water Hyacinth (One of the seven species of the genus Eichornia)

Free floating perennial aquatic plants native to South America

Introduced in other parts of the world as ornamental plants and later became one of the worst aquatic weeds

Reproduces either through runners (stolons) which eventually form daughter plants or through seeds

Water Hyacinth is one of the most productive plants on earth

It can double in biomass in as little as 6-15 days

An acre of water hyacinth can weigh more than 200 tons

When not controlled, water hyacinth will cover lakes and ponds entirely blocking waterways and hampering all aquatic activity

This can impact water flow, block sunlight from reaching native aquatic plants, and starves the water of oxygen often leading to fish kills.

The plants also create a prime habitat for mosquitoes and a species of snail known to host a parasitic flatworm which causes schistosomiasis (snail fever)


WATER HYACINTH FACTS

Water ways infested with water hyacinth

The problems caused by excessive growth of the weed have attained devastating proportions in Kerala where inland water bodies are many

Massive invasion of paddy fields elicits a serious threat to paddy cultivation in Kuttanad area and in the districts of Ernakulam, Kottayam and Alappuzha

Many waterways in the state has become unserviceable due to the water hyacinth menace

Excessive infestation by the weed can severely constrain other activities like irrigation and fishing and prevent access to water navigation

The plant multiples rapidly in stagnant waters and sometimes can occupy the entire water surface from bank to bank stretching over 2-4 km along water channels


WATER HYACINTH MENACE IN KERALA

VietnamLake Victoria

Kenya

Florida

Philippines

WATER HYACINTH IS A GLOBAL PROBLEM !


CONTROL OF WATER HYACINTH

Chemical Control

Aquatic herbicides: 2,4-D, Diquat (6,7-dihydrodipyridol) and Glyphosate (low toxicity and rapid decomposition in water)

Methods are less expensive but serious ecological problem and human health hazard

Mechanical control

Choppers and shredders, mechanical harvesting, manual harvesting

Low efficiency, extremely expensive, energy intensive, perpetual practice needed. Probably suited only for small scale operations

Biological Control

Natural enemies of the plants employed (eg: Mite- Orthogalumna terebrantis Moth- Acigona infusella, Weevils- Neochetina eichhorniae, Neochetina bruchi, Fungi: Fusarium sp, Trichoderma sp

None of the methods are fully effective in controlling the weed !!


INTEGRATED CONTROL

No single method is effective in control of water hyacinth

An integrated control combines the different available methods chosen based on the region, type and level of infestation


INTEGRATED CONTROL WITH GENERATION OF ECONOMY

CONCEPT

Control measures should involve active participation from public and end benefactors

Active participation can come only through financial incentives

Remedial of water hyacinth should generate income and employment opportunities

Can the water hyacinth biomass removed from water bodies be in-fact beneficial ?

Isolated cases of benefits - utilization

Water hyacinth as animal feed/fodder

Generation of biogas

Production of compost/fertilizer

Water hyacinth as raw material (furniture, handicrafts, enzymes, alcohol)


ENZYME PRODUCTION FROM WH – A CASE FOR UTILIZATION

Water Hyacinth, as any other plant material contains cellulose, hemicellulose and lignin

Hemicellulose (40-50%)

Cellulose (12-18%)

Lignin (3-5%)

Cellulose is a polymer of glucose (a hexose sugar)

Hemicellulose is a polymer mainly containing arabinoxylan (Arabinose and xylose –pentose sugars)

Also contain Tannins

Microorganisms which can utilize these polymers for growth produces proteins which can degrade these polymers

The proteins which hydrolyze (breakdown) these polymers are enzymes

Cellulases , Xylanses, Tannases


ENZYME PRODUCTION FROM WH

Microorganisms –(Bacteria/Fungi) can grow on plant biomass and secrete these enzymes

Cellulases are a class of enzymes which find applications in

Textiles

Paper and pulp Industry

Food Industry

Laundry and Detergents

Biomass conversion and bio-ethanol production

World market estimated at 500 million US$

Xylanses : Applications in

Paper and pulp industry

Hemicellulose bioconversion and bioethanol production


ENZYME PRODUCTION FROM WH

Fermentative production of enzymes

Water Hyacinth biomass is sun-dried, chopped and pulverized to a particle size of about 1-2mm, and is used as substrate for growth of microorganisms

Mineral salt medium containing necessary salts and nitrogen sources is supplemented with the biomass and sterilized.

Inoculated with spores of the cellulase producing organism (Trichoderma reesei , or Aspergillus niger) and cultivated in a bioreactor.

After growth for about 4-6 days, the culture medium is filtered and processed to recover enzyme

Alternatively, the sterilized biomass is spread on trays and inoculated with spores.

Cultivation is carried out in special sterile rooms/cabinets called koji rooms (Solid State fermentation)

After growth, the enzyme is harvested by extraction with citrate buffer


SCHEMATIC FOR SUBMERGED FERMENTATION

Centrifuge/FilterCulture supernatantCentrifuge/Filter

Pulverized WH

Water Hyacinth

Chopping

Pulverization

Bioreactor

Medium

AcetoneCellulase

Solid mass

Bio gas

Organic Manure Spore

inoculum


SCHEMATIC FOR SOLID STATE FERMENTATION


Pulverized WH

Water Hyacinth

Chopping

Pulverization

Koji RoomTray Fermenter

Medium

Centrifuge/FilterCulture supernatant

Acetone

Centrifuge/Filter

Cellulase

Solid mass

Bio gas

Organic Manure Spore

inoculum

Fermented WHB

Citrate buffer

OTHER ENZYMES

Phytase : Food and Feed Applications

Tannase : Feed Applications

Beta-glucosidase : Synthesis, Bio-ethanol


ETHANOL FROM WATER HYACINTH BIOMASS

Ethanol from lignocellulosic biomass is projected as a potential transportation fuel which is renewable

Ethanol is produced by microbial fermentation of sugars

Both hexose sugars (glucose) and pentose sugars (xylose) can be fermented to ethanol by appropriate organisms (Saccharomyces cerevisiae , Pichia stipitis)

Water hyacinth contains cellulose (polymer of glucose) and hemicellulose (rich in xylose) both of which can yield sugars that can be fermented to alcohol


ETHANOL FROM WATER HYACINTH BIOMASS: SCHEMATIC

Pulverized WHB

Cellulases

Xylanases

Acid

Pentose sugarsHexose sugars

Fermentation with pentose fermenting microbes (eg Pichia stipitis)

Fermentation with hexose fermenting microbes (eg Saccharomyces cerevisiae)

Fermentation with C5 and C6 co- fermenting microbes (eg recombninant E coli)

Ethanol

Absolute Alcohol

Distillation and Dehydration


HEMICELLULOSE UTILIZATION

Water hyacinth is rich in hemicellulose (40-50%)

Any value addition to the water hyacinth should consider the utilization of hemicellulose sugars derived from the biomass


Xylitol : a natural food sweetener, a dental caries reducer, and a sugar substitute for diabetics

Can be produced from fermentation of xylose using special yeasts (Candida guillermondii, Candida tropicalis, Debaryomyces hansenii)

2,3-Butanediol: Solvent, liquid fuel, and as a precursor of many synthetic polymers and resins.

Dehydration of 2,3-BD yields the industrial solvent methyl ethyl ketone,which is much more suited as a fuel because of its much lower boiling point. Further dehydration yields 1,3- butanediene, which is the starting material for synthetic rubber and is also an important monomer in the polymer industry

Can be produced by fermentation of xylose using Bacillus polymyxa, Aerobacter aerogenes, Serratia marcescens

Lactic acid : Used in the food, pharmaceutical, and cosmetic industries. It is a component of biodegradable plastic polylactate, the market for which is expected to grow significantly

Can be produced by fermentation of xylose using Lactobacilli

VALUE ADDED PRODUCTS FROM WH-HEMICELLULOSE


TECHNICAL CHALLENGES

Large Scale Harvesting and Logistics of WHB is the major problem in utilization of this resource for any application including biogasification, ethanol or enzyme production, or even composting

The biomass contains almost 95% of water and the density is as low as 0.3 g/cm3

All methods of collection and pretreatment utilize high quality energy and economy of the processes are poor whether it is gasification or production of enzyme/ethanol

All methods of utilization can only add value to a program where the primary objective is remediation

Positive economic balances can be achieved only through an integrated utilization plan working on a bio-refinery concept


BIOREFINERIES FOR WATER HYACINTH PROCESSING

Direct Physical utilization plans with minimal processing

Crafts , Furniture

Composting

Biogas

Needing elaborate processing

Value added compounds

Enzymes

Ethanol

Chemicals

Ethanol, Chemicals

Chemicals

Ethanol

Biogas, Compost Energy



Primary Product

Primary Byproducts

Secondary Byproducts


DE-CENTRALIZED UTILIZATION PLANS

Large Scale Plants for bio-processing may not be feasible in certain cases owing to geographical location, large capital investment, sustainable availability of raw material

Centralized Plants are capital intensive, needs more elaborate infrastructure, are technically intensive, and needs skilled manpower

Decentralized operations can be less capital intensive, needs less infrastructure and are less intensive on technical skills

They can generate local employment, income

Financial incentives to end benefactor of remediation programs can attract more people for utilization plans (Collection centres for WH biomass?)

Can we think of co-operatives with public involvement?


Enzyme and Ethanol production using water hyacinth biomass

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