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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).

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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.

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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

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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

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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.

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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).

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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...

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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

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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

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catabolic plasmid

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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

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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

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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

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Plant carbohydrates is a valuablematerial for industry

STARCH cellulose

Starch – edible for humans.Cellulose – non-edible for humans

Cellulose microfibrils

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CelluloseLignin

AmorphousRegion

Pretreatment givesenzyme accessible substrate

Pretreatment

CrystallineRegion

Hemicellulose

STARCH

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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.

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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

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Components of plant cell walls

CelluloseFermentable sugars obtainedfrom cellulose in 1819

Lignin

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Uses of non-usable bananastarch

glucose

Straw degradation in flask

Control Inoculated

day 0 day 4

day 8 day 10

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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

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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

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Yields of Ethanol from Corn Stover(Cellulose Ethanol)

From Cellulose: 50 to 55 gal / ton

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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

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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.

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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

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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)

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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 !!!

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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)

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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)

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of p-coumaril alcoholssoftwoods

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The problem of non-degradablelignin remains:

Lignin

Hardwoodssoftwoods

Amorphous co-polymer

hydrocrackingHydro

deoxygenation

Hydrogeno

lysis

Hydrogenolysis

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Lignin derivatives used as fuel additives(to increase octan number)

(Chemical process)

(In stirringautoclave)

Lignin

System of Biomass Utilization

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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

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Future: New SourcesSunlight to Biodiesel?