9/21/2010
1
BIOREMEDIATIONANDBIOMASS UTILIZATION
Iman Rusmana
Department of Biology
Bogor Agricultural University
Bioremediation• Bioremediation is defined as the process whereby
organic wastes are biologically degraded undercontrolled conditions to an innocuous state, or to levelsbelow concentration limits established by regulatoryauthorities.
• Bioremediation uses naturally occurring bacteria andfungi or plants to degrade or detoxify substanceshazardous to human health and/or the environment.
• The microorganisms may be indigenous to acontaminated area and stimulated in activity(biostimulation) or they may be isolated from elsewhereand brought to the contaminated site (bioaugmentation).
9/21/2010
2
Problems withbioremediation• Work in vitro, may not work in large scale. Work well
in the laboratory with simulation, may not work in thefield. Engineering approach is needed.
• Alternatively, select adapted species on site(indigenous species) to remediate similar damage.
• Most sites are historically contaminated, as a resultsof the production, transport, storage or dumping ofwaste. They have different characteristics andrequirements.
• Those chemicals are persistent or recalcitrant tomicrobial breakdown.
Use of bacteria in bioremediation
• Greatly affected by unstable climatic andenvironmental factors from moisture to temperature.
• For examples, pH in soil is slightly acidic; petroleumhydrocarbon degrading bacteria do not work well <10 C.
• These microbes are usually thermophilic anaerobic.• Fertilizers are needed. Seeding or bioaugmentation
could be useful too.• They contain monooxygenases and dehydrogenases
to break down organic matters including most toxicsubstances.
3
9/21/2010
Biomass Utilization
RenewableBioresourceFeedstock
• Plants– crops– trees– algae
• Animals, fish
• Microorganisms
• Organic residues– municipal– industrial– agricultural– forestry–aquaculture
IndustrialBioproducts
• Bioenergy and Biofuels
• Manufactured products:
– biochemicals
– biosolvents
– bioplastics
– ‘smart’ biomaterials
– biolubricants
– biosurfactants
– bioadhesives
– biocatalysts
– biosensors
BioprocessTechnology
Biocatalysis (Enzymes)
Fermentation (Microorganisms)
Physical – ChemicalProcess Technology
Extraction
Pyrolysis
Gasification
The diffusion of biotechnology andgreen chemistry across theeconomy
Additional‘Sectors’
•Informatics
•Genomics
•Nanotechnology
•Security
•Services
Health andMedicine
•Biopharmaceuticals
•Vaccines
•Diagnostic kits
•Artificial organs
•Gene therapies
First Wave
Agricultureand Food
•Disease anddrought resistantcrops
•Functional foods
•Nutraceuticals
•Biopesticides
Second Wave
IndustrialBioproducts•Biofuels and bioenergy•Manufactured products
•Biochemicals•Bioplastics•Biolubricants•Biocatalysts•Biosensors
Third Wave
4
9/21/2010
Advantages of Biomass Utilizationfeedstock waste Energy
Fossil Carbon Economy -carbon is used and discarded
Fossil Carbon
CarbonCycle inNature
Carbon
Cycle inIndustry
Bioproducts Economy -carbon is recycled
Carbon
Cycle inIndustry
RenewableCarbonCycle inNature
Petroleum
Mat
Rain coat
Sandals Shoes
Plastics, Fabrics,
Synthetic rubber, etc.
Hut
Umbrella
Rope
Fertilizer
Chemicals
• Waste is a feedstock opportunity• Growth from increasing value added
(1989~ 2010 )
Gas ovenCars
Electric oven +
Waste
Oven
ENERGY
Compost
CO2
Sandals Shoes
Mat
Rope
Biomass Utilization
Rain hut
Rain coat
Rice straw
• Waste is a disposal problem• Growth from increasing production volume
( 1603 ~ 1867 )
Future
( 2010 ~ 2020 )
Biomass
BiomassRefinary
Bioplastics
Biogass
Alcohol
Recycle
composting
9/21/2010
5
Why BIOMASS ?
1. To substitute or complement drying uppetroleum.
2. To reduce the amount of CO2 emission.
3. To decrease (urban/agricultural /forestry)organic wastes.
4. To re-activate rural life and economy.
Special waste bioprocesing
-- Degradation of oil in spills
-- Degradation of xenobiotics (chlorinated insecticides,herbicides and fungicides)
-- Degradation of polymers (plastics)
-- Transformation of
toxic metal species
into less or non-toxic forms.
9/21/2010
6
Pseudomonas
• Genetically engineered bacteria(Pseudomonas) with plasmid producingenzymes to degrade octane and manydifferent organic compounds from crudeoil.
• However, crude oil contains thousands ofchemicals which could not have onemicrobe to degrade them all.
• Controversial as GE materials involved.
Degradable organic compoundsin the environment
1) Biodegradable: undergoes a biological transformation.Complete degradation results in CO2 and some inorganic
compounds
2) Persistent: do not biodegrade in certain environments
3) Recalcitrant: resist biodegradation in most environments
Not always beneficial process:1) non toxic compound toxic metabolite,
2) toxic chemical more toxic chemical(mercury waste transformed into very toxic methyl mercury compounds).
9/21/2010
7
Microbes have the ability to consumenatural or synthetic organic substancesas nutrient and energy sources
Pseudomonas group – digest more than 100 organic compounds
Microbes have an extensive setof enzymes that can serve as araw material for furthermutagenesis
Modified enzymes = Digesting new substrates, working inaltered environment or having improved kinetics
“Catabolic plasmids” ofPseudomonas
1) Encode enzymes for waste-degradation pathway
2) Could be transferred between species
3) Transfer across the communityincreases in response to novel nutrient use,or exposure to a potentially toxic compound.
Enzymes of catabolic plasmidsable to degrade :
octane, naphthalene, toluene,benzene, pesticides, herbicides...
9/21/2010
Examples of naturally occurring“catabolic plasmids” capable tointerspecies transfer
Compound
Biphenyl
Plasmid
pBS241
Size
195 kb
Bacterium
P. putida
Camphor pPG1(CAM) Near 500 kb Pseudomonassp.
Octane
Chlorobiphenyl
OCT
pSS50
Near 500 kb
53.2 kb
P. oleovorans
Alcaligenes spp.
From Microbial Ecology, 19:1-20, 1990
Ananda Chakrabarty’s“Super-Pseudomonas”
1974 Chakrabarty created super-Pseudomonasdegrading oil in spills
Plasmids CAM (camphor degradation), OCT (octane degradation),NAH (naphtaline degradation) and XYL (Xylol degradation)were combined in the same cell
8
9/21/2010
Creation of Super-Pseudomonas
XYLCAM NAH
Conjugation
XYL
NAH
CAM/OCT
XYL NAH
OCT
Conjugation
CAM/OCT
Conjugation
CAM (camphor degradation),OCT (octane degradation),
NAH (naphtaline degradation)XYL (Xylol degradation)
Pseudomonas putida mt-2 (pWWO)very good bacteria
capable of decontaminating organic substancesincluding solvents, such as toluene,one of the components of gasoline.
very common in soil and plant rhizosphere
It promotes plant growth,is a biocontrol agent for plant pathogens
9
catabolic plasmid
10
H
OH
OH
OH
Ortho-cleavage
Benzene
Ortho-cleavage pathway
(chromosomal)
Meta-cleavage pathway(TOL catabolic plasmid),
activated when
Acetyl CoASuccinate
chromosomal pathwayoverloaded
AcetaldehydePyruvate
Degradation of benzene by pseudomonads (cited in Galzer and Nikaido,Microbial Biotechnology, Freeman and Company, NY)
Different benzenederivatives induce
different degradativepathways;
TOL cleavage supplyP. putida with energy
9/21/2010
1,2-dihydro-
TOL 1,2-dihydroxybenzene Meta-cleavageH
CatecholOH
2,4,5-trichlorophenoxyaceticacid (2,4,5-T)
Burkholderia cepacia
2,4,5-trichlorophenoxyacetic acid
Succinate+
AcetateDioxygenase
WIDELY USED HERBICIDE
Cl
Makes moleculedifficult to degrade
Burkholderia cepacia
9/21/2010
Most biodegradation-performingbacteria are mesophilic(20-40 C is their growth optimum)
Rivers or soils that need to be cleaned are at 0-20 Care difficult to clean !!!
SAL TOL
PsychrophilicStrain
(cold-liking)
Mesophilicstrain
Conjugation
SAL
TOL
Result: Psychrophilic straindegrading both Salicilate and Toluene
Difficulties with releasing biodegrading“lab” microbes into the environment
Gadd, G.M.,1993,Trends inBiotechnology,v. 11, p 348
11
12
9/21/2010
Plant carbohydrates is a valuablematerial for industry
STARCH cellulose
Starch – edible for humans.Cellulose – non-edible for humans
Cellulose microfibrils
9/21/2010
CelluloseLignin
AmorphousRegion
Pretreatment givesenzyme accessible substrate
Pretreatment
CrystallineRegion
Hemicellulose
STARCH
13
9/21/2010
14
Cellulose
Starch
AmyloseAmylopectin
(100 – 400.000 glucose monomeres)
(10.000 – 4.000.000 glucose monomeres)
corn starchfrom waxy maize
contains only 2% amylose
but that from amylomaizeis about 80% amylose.
9/21/2010
Annual world production of commercialstarchesin millions of metric tons
Botanical Source
Maize
Potato
Tapioca & others
Wheat
Total
1980
11.6
1.4
1.5
0.6
15.1
1990
20
2.4
1.7
1.5
25.6
1993
26.3
2.5
3.2
1.7
33.7
1995/1996
35.2
2.1
3.8
2.8
43.9
ExtractivesAsh
Hemicellulose(need special yeastto convert to ethanol)
Chapple, 2006; Ladisch, 1979
15
Components of plant cell walls
CelluloseFermentable sugars obtainedfrom cellulose in 1819
Lignin
16
9/21/2010
Uses of non-usable bananastarch
glucose
Straw degradation in flask
Control Inoculated
day 0 day 4
day 8 day 10
9/21/2010
17
Major Microorganisms in the consortium
GlucoAmylaseis able to digest branching
points
Is derived from Aspergillusniger;
A. awamori
Alpha-Amylase genesfrom B.amyloliquefaciems;
B.Stearothermophilus
(both expressed in B. subtilis)
Glucose sirup
9/21/2010
Yeast Metabolism: pentose fermentation
Phosphoenolpyruvate
Pyruvate Acetaldehyde
Ethanol
TCA Cycle
Xylose
Xylitol
Xylulose
Xylulose-5-P
NADH NAD+
Glucose
Glucose-6-P
Fructose-6-P
Glyceraldehyde-3-PNAD+
NADH3-Phosphoglycerate
NAD(P)H
NAD(P)+
NAD+
NADH
PPP
Ho et al
From Xylan:
Total:
30 to 35 gal / ton
80 to 85 gal / ton.
Corresponds to about 250,000 tons /yr for20 million gal per year plant
Requires engineered yeast, pretreatmentcellulase enzymes
18
Yields of Ethanol from Corn Stover(Cellulose Ethanol)
From Cellulose: 50 to 55 gal / ton
9/21/2010
Ethanol is renewable energy source(better than oil)
Ethanol vehicles produce:
35% less CO
42% less NOx
43% less NMHc(nonmethane hydrocarbon)
39% less PM(particulate matter)
79% less CO2over life cycle
Previous pictureswere about starch;
It will be niceto transform
cellulose (woods)
Hydrolysed
Hemicelluloseand Lignin
Lignin
into ethanol too…Glucose
Ethanol
19
9/21/2010
Cellulose waste
Lignin
Complex aromatic(organic) polymer
-- no simple repeat unit-- hydrophobic-repels water
-- acts as an adhesive
Cellulose itselfCristalline;amorhous
Hemi-Cellulose
Combinationsof 5 or 6 sugars;
Side groups (branching)
Amorphous
in cell wall-- stiffens wood
Hemicellulose(a mix of many monomeres)
Usually, all of the pentoses are present.The pentoses are also present in ringsthat can be 5-membered or 6-membered.
Xylose is always the sugar present in the largest amount.
20
9/21/2010
21
Problems with commercial glucoseproduction from cellulose waste
1. typical waste cellulose material contains less than half cellulose(lignin and pentosans are non-degradable contaminants).
2. A combination of enzymes is neededto solubilize and degrade cellulose waste.
3. Natural enzymes are comparatively unstable or have low activityagainst complex of the lignin and cellulose.Enzymes are subject to both substrate and product inhibition.
cost of cellulose glucose conversion is excessive
Strach is more expensive to produce than cellulose,but starch glucose is much cheaper
Most pure cellulose sources(most easily transformed to glucose)are most expensive ones
9/21/2010
Cellulose (hydrolysed bycellulases)
Cellulases
Cellulose is associated with lignin and pentosans,so as to resist biodegradation;
dead trees take several years to decay even in tropical rainforests.
Cellulases
cellobiohydrolase (CBH)
hydrolyse the cellulose chainfrom one end
endoglucanase (EG)Cut randomlyInside the cellulose chain.
hydrolyse cellulose cooperatively, i.e. they act in synergy.
endoglucanase
(EG)
22
9/21/2010
23
first, hemicellulases may digest hemicellulose(and open up wood for bleaching, cellulose to beextracted etc…)
Enzyme complex:arabinosidase, mannanase, mannosidase and xylanase.
Enzyme source:Fermentation of a nonpathogenic strain of Aspergillus niger.
Xylose is produced from hemicellulose in large amounts!!!
Xylose is major component of waste. Would be greatto transform it to ethanol somehow !!!
9/21/2010
24
Ideal biocatalyst microorganism:
-- Creates ethanol from glucose (from cellulose)
-- Creates ethanol from xylose (from hemicellulose)
-- Capable to high-yield ethanol production
-- Growth in industrial settings
No “ideal bug”exists yet
Zymomonas mobilis
• Advantages:– Natural fermentative microorganism– Near theoretical ethanol yield from glucose(92%-94% versus 88%-90% for yeast)
– Shorter fermentation time (300%-400% faster than yeast)– No oxygen requirement– Tolerant to inhibitors in hydrolysates– High ethanol tolerance– Fermentation at low pH– Grows at high sugar concentrations– High specific productivity
• Limitations:– Narrow substrate utilization range (only pure glucose)
9/21/2010
25
a strain of Zymomonas mobilisthat produces ethanol from both xylose and glucosewas created
DOE is assisting Arkenol Inc. (California) in building a newbiomass-to-ethanol plant that will provide 8 million gallons peryear of ethanol from rice straw using the Zymomonas mobilisprocess.This project provides an alternative to rice straw burning,
which is being phased out in California for environmental reasons.
Similar result achievedwith genome-integrated copies
of XYL-ARA genes(greater strain stability)
26
of p-coumaril alcoholssoftwoods
9/21/2010
The problem of non-degradablelignin remains:
Lignin
Hardwoodssoftwoods
Amorphous co-polymer
hydrocrackingHydro
deoxygenation
Hydrogeno
lysis
Hydrogenolysis
9/21/2010
Lignin derivatives used as fuel additives(to increase octan number)
(Chemical process)
(In stirringautoclave)
Lignin
System of Biomass Utilization
27
Fallow rice pad
Ethanol Station
Public Education
Town OfficeVolunteer Center
Ethanol car
Ethanol truck
Ranch
Straw/Husk
Rice production
Non-eatable parts
FieldsHorticulture
Industrial crops
Forest
Wood waste
Others
Wastes
AgriculturalProducts
Distribution
Other usage of Ethanol
Ethanol bus
Coperation
Compost CenterWaste Treatment
Local Economy
SightseeingHotelRestrantsBrewery
Center
Local Government
Local EducationHigh School
Biodiesel EthanolBlend
PlantCellulose
CO2
Algae
CO2
E. coli
SugarsBakersYeast
28
9/21/2010
Future: New SourcesSunlight to Biodiesel?
Top Related