Environmental

Biotechnology

DCV, FCT/UNL 21th and 28th March 2012

Margarida Moreira dos Santos

Ideal situation
Population
Industry
Farming

Waste

absortion and breakdown by:

Atomsferic systems
Terrestrial systems
Aquatic systems

In reality
Large accumulation of WASTE in
dump sites, soil and water systems

Reduce, reuse, recycle

3Rs not enought!

Environmental
Biotechnology
Application of biological agents, along with
chemical, physical and engineering processes
to maintain, protect or restore environment

Environmental
Biotechnology
Biomass utilization (waste and by-product)
Treatment of liquid and solid waste from
domestic
municipal
agricultural
industrial sources
Bioremediation (removal of toxic waste
from the environment)

Biomass utilization
Biomass:
source material for production of economically important products
material of biological origin, excluding biological fossils like coal,
natural gas or oil
by-products of the food and agricultural industries (discarded in
the past) (=waste)
e.g. starch, lignocellulose, whey, meat flour, part of municipal
waste, activated sluge, etc.

 Bioenergy: Electricity or heat obtained from

biomass
Biofuel: liquid or gas fuel for the transportation
sector, produced from biomass

Biofuel
Bioethanol and biobutanol
Biodiesel
Biomethanol
Biohydrogen
EU Directive for Biofuel (2003/30/CE) set goals for energy
obtained from waste (not food) for the transportation sector:
2005 2%
2010 5,75%
2020 10%

Biofuel
Additives/substitutes to oil derivatives

Bioethanol
may be produced directly from sugar-rich plants (sugar cane, sugar beet)
starches from foodstuffs (corn, wheat, sorghum, grain) require a hydrolysis reaction
(saccharification), to convert the starch into sugars, either enzymatically (amylases) or
chemically
lignocelluloses and hemicelluloses (straw, wood) usually are subjected to chemical
hydrolysis
hydrolyzed wastes (household, other) may also be used

Bioethanol
Sacharomyces cerevisiae
Zymomonas mobilis
high specific productivity (g ethanol g-1 cell h-1)
high ethanol yield (g ethanol g-1 substrate)
high alcohol tolerance
narrow substrate range, difficult GE
Improving tolerance of the fermentation micro-organisms to ethanol
Improving the stoichiometric yield of ethanol per molecule of sugar
Improving the ability of the organism to utilise lignocellulose

Lignocellulose waste
Very abundant and non-edible, but very challenging
Cellulose: composed of glucose residues; 33 % of plant biomass
Hemicellulose: xylose, mannose, galactose, rhamnose, and arabinose
- sugars not readily fermented; 20 % of plant biomass
Lignin: phenylpropane polymer; 30 % of all non-fossil organic carbon

Engineering
xylose
utilization
for ethanol
production

Heterologous
enzymes
Overexpression

Bioethanol and biobutanol

Biological, chemical and thermal processes
biological fermentation
thermal gasification followed by ethanol/butanol synthesis
(syngas: H2, CO, CO2)
thermal gasification followed by biological fermentation

Biobutanol
Clostridium acetobutylicum + starch
Acetone, butanol and ethanol (3:6:1), hydrogen and other by-products
Higher energy density
Ease of blending with conventional fuels
Higher toxicity of butanol to the producing organism
Production from sugar is three fold less efficient
than for ethanol production
Chemical and thermal routes for production are dependent on
a clean source of syngas (CO + H2) from biomass gasification

Acetone-Butanol
Fermentation
Enzymes are indicated by letters as follows:
(A) glyceraldehyde 3-phosphate dehydrogenase
(B) pyruvate-ferredoxin oxidoreductase
(C) NADH-ferredoxin oxidoreductase
(D) NADPH-ferredoxin oxidoreductase
(E)NADH rubredoxin oxidoreductase
(F) Hydrogenase
(G) phosphate acetyltransferase (phosphotransacetylase)
(H) acetate kinase
(i) Thiolase (acetyl-CoA acetyltransferase)
(J) 3-hydroxybutyryl-CoA dehydrogenase
(K) Crotonase
(L) butyryl-CoA dehydrogenase
(M) phosphate butyltransferase (phosphotransbutyrylase)
(N) butyrate kinase
(0) acetaldehyde dehydrogenase
(P) ethanol dehydrogenase
(Q) butyraldehyde dehydrogenase
(R) butanol dehydrogenase
(S) acetoacetyl-CoA:acetate/butyrate:CoA transferase
(T) acetoacetate decarboxylase
(U) phosphoglucomutase
(V) ADP-glucose pyrophosphorylase
(W) granulose (glycogen) synthase
(X) granulose phosphorylase
Micribiologcal Reviews 1986, 50: 484-524

Yield increase using Metabolic Engineering

Algae to fuels

Algal fuel factories can absorb waste carbon dioxide emissions from
coal power plants or other polluting entities

Certain algae in a high carbon dioxide environment will produce a viable
fuel similar to diesel

Biodiesel
First engine invented run on
groundnut oil (Rudolf Diesel)
Diesel, fraction of crude oil composed
mainly of saturated hydrocarbons
Use of vegetable oils for making biodiesel, less polluting and renewable nature
Biodiesel mixture of methyl esters, which is made from the transesterification
of plant oils
Modern diesel engine can not run solely on vegetable oil due to high viscosity,
deposit formation on injection systems and poor cold-start properties
World biodiesel production (2006) about 5-6 million tonnes, with 4.9 million
tonnes processed in Europe (Germany, Italy, Austria, France and Sweden)
Biodiesel is currently limited to 5% in diesel in Europe due to concerns over
engine warranties, materials, cold weather performance and compatibility
Germany is the major biodiesel producer (2499 x106 L, 2006)

Biodiesel
Feedstock derived from renewable sources, mainly vegetable plant oil
Type and yield of fatty acids accumulated vary on crop, but all contain triacylglycerols
Soybean (USA and South America), rapeseed (Europe), sunflower, palm, coconut,
peanut, corn, castor, cottonseed, radish, rice bran, hemp, canola, sorghum
Jatropha, jojoba
Algae
Wastes, as recycled cooking oil, waste animal grease
Oleaginous microorganisms (including genetically modified)
Fatty acid

Soybean

Sunflower

Palm

Rapeseed

Palmitic (C16:0)

11

42.8

Stearic (C18:0)

4.5

Oleic (C18:1)

23

18

40.5

11

Linoleic (C18:2)

54

69

10.1

12

Linolenic (C18:3)

0.2

Erucic (C22:1)

52

Biodiesel
Transesterification - reduces the viscosity of the oil, improves its consistency and miscibility
with diesel, as well as improving other properties, such as its viscosity when cold.
Fatty acids reacts with low molecular weigh alcohol, usually methanol, under acid or
alkaline catalysis (0.5% NaOH or KOH)
Enzymes can also be used as catalyst
Supercritical methanol technology without catalyst
The ester of the fatty acid is formed together with glycerol (10:1, w/w)

100 000 ton/y, Marl, Germany

Energetics of Biofuel

Pathways to fuel

Reductive acetyl-CoA pathway (Wood-Ljungdahl)

Methanogenic and acetogenic (acetate producing) bacteria

Much room for improvement

Plsticos
Polmeros mais usados
Polipropileno - PP
Petrleo

Poli(tereftalato de etileno) - PET

Polietileno - PE

Totalmente
NO
BIODEGRADVEIS

Poliestireno - PS
Problemas ambientais

Adaptado de: Vtor Alves, CEER Centro de Engenharia dos Biossistemas ISA/UTL

 90 m

Polmeros
biodegradveis

Bioplsticos

Plsticos em que 100% do seu

carbono tem origem em fontes
renovveis (p.e. agricultura e
floresta)

tais

como

amido,

Podem ou no ter
origem em

fontes

renovveis

protenas e celulose

Bioplsticos: No so uma nica classe de polmeros, mas sim uma famlia de

produtos com caractersticas fsico-qumicas que podem ser bastante distintas.

Biodegrabilidade
Decomposio em dixido de carbono, gua, metano, compostos
inorgnicos ou biomassa, por aco enzimtica de
microorganismos presentes no ambiente, num perodo de tempo
definido.

A biodegrabilidade no est directamente relacionada com a origem das matriasprimas, mas sim com a natureza qumica do polmero e com as propriedades finais
da embalagem/produto
Existem polmeros derivados do petrleo que so biodegradveis

Existem bioplsticos que no so biodegradveis em ambientes marinhos

Polmeros Biodegradveis
Biomassa
vegetal/animal/algas

Polissacridos
Amido

Protenas

Hemicelulose
Alginato
Carragenato
Pectina
Quitosano

Monmeros da
petroqumica

Polihidroxialcanoatos

Poli(cido lctico)

(PHAs)

(PLA)

Policaprolactona
(PCL)

Polissacridos
De soja

(batata)
Celulose

Monmeros
microbianos

De soro de leite

(milho)
(trigo)

Origem
microbiana

Polieteramidas
(PEA)

(gelano)

Gelatina

(xantano)

(bovinos)
(peixe)

(celulose
bacteriana)

Glten

(alginato
bacteriano)
(pululano)

Polisters alifticos
(p.e. PBSA)
Polisters aromticos
(p.e. PBAT)

Polihidroxialcanoatos (PHAs)
Produzidos por bactrias
Acumulados dentro das clulas em grnulos
Reservas de energia
Fontes de carbono: acares, cidos orgnicos volteis,
glicerol (de preferncia
presentes em resduos e subprodutos)

Poliester

Propriedades semelhantes
ao polipropileno
Hidrofbico
 Barreira a CO2 e O2
 Barreira vapor de gua

Poli(cido lctico) - PLA

Produzido por polimerizao do cido lctico

cido lctico obtido for via microbiana a partir de resduos agroindustriais contendo hidratos de carbono (p.e. milho e melao)
Polister aliftico termoplstico e biodegradvel
Pode ser processado com o equipamento usado para os
plsticos convencionais
Hidrofbico
 Barreira a CO2 e O2
 Barreira vapor de gua

Lactic Acid
Lactic acid bacteria
Lactobacillus, Sporolactobacillus, Enterococcus, Lactococcus,
Bacillus, Streptococcus, Pediococcus, Leuconostoc
Two different pathways of lactic fermentation exist:
homolactic - lactic acid is the only product
Heterolactic produces lactic acid plus various co-products such as ethanol, carbon
dioxide and acetate

Bifidobacteria
They have a unique hexose metabolism
(catabolize sugars via frutose-6-phosphate phosphoketolase),
Are exclusively anaerobic (others are generally microaerophilic)

Lactic Acid
Used
as a preservative and iron chelator in food
in cosmetics
in pharmaceuticals
Produced by
Lactic acid bacteria, that require:
complex media, pH control

Can we use Saccharomyces cerevisiae?

acid-tolerant, grows in synthetic media, anaerobic growth

Engineering lactic acid production

by Saccharomyces:

No growth under anaerobic

conditions since lactate
export requires ATP,
unlike ethanol

Unsuccessful!
Abbott, DA. FEMS Yeast Res 9 (2009) 11231136

Single Cell Protein

Microorganims have always been a small part of human diet:
Mushrooms
Cheese
Vinegar
Yeast
Algae
Use of biomass, by-products and waste:
Waste of cellulose processing
Coffee production
Stach production
Food processing
Alkanes, methane, methanol

Single Cell Protein

Easy to obtain supplement to conventional food (and feed)
Non expensive source of protein is it?

Crueger & Crueger 1894, Biotechnology

Vitamins, essential aminoacids, high protein content, lipid content

High nucleic acid content, production of toxins, absorption of carcinogenic
or toxic compounds, slow digestion, allergies

Alkanes (- oil)

Candida tropicalis
Candida oleophila
Saccharomycopsis lipolytica
Alkanes are insoluble, but cells produce emulsifying substances and are enriched in
lipids in order to consume droplets by passive diffusion

Methane
(not all is used as fuel)
Obligate methylotrophs
Methylomonas methanica
Methylococcus capsulatus
Methylovibrio soehngenii
Methanomonas margaritae

CH4

+ O2 + XH2

HCHO
Methanol
(inhibitor)

+ H2O

Methanol

Bacteria:
Rapid growth
Higher protein content
Better yields
Simpler culture medium requirements

Catabolism of:
Methanol
Methyamine
Formaldeyde
Formate

Crueger & Crueger 1894, Biotechnology

CH3OH

HCHO

HCOOH

CO2

One-carbon
assimilation pathways

Serine Pathway

Serine pathway
formaldehyde
Quayle cycle
Transketolase and transaldolase
Wood-Ljungdahl pathway
CO2
Calvin Cycle (ribulose diphosphate, plants)

Quayle Cycle

EMP

ED pathway

2 ATP
2 NADH

1 ATP
1 NADH, 1 NADPH

One-carbon
assimilation pathways

Quayle cycle
(Ribulose monophosphate cycle):
most efficient, selected for use

Crueger & Crueger 1894, Biotechnology

Reductive acetyl-CoA pathway (Wood-Ljungdahl)

Methanogenic and acetogenic (acetate producing) bacteria

WasteWaste-water treatment
Increased need due to:
Increase in world population
Agriculture dependence on fertilizers and pesticides
Man-made chemicals
Necessary due to:
The need to reclaim wastewater
Preventing contamination of drinking water
Preventing entry in the food chain
Should treat (amongst others):
Compounds with high organic load (BOC)
Pathogenic organisms and viruses (cholera, hepatitis, thyphoid,
parathyphoid)
Man-made chemicals

Sewage, industrial effluents, agricultural run-off, storm water and urban runoff

Tratamento de efluentes lquidos

ESTRATGIA GERAL

reciclagem, valorizao, deposio

slidos
suspensos

lquidos
no aquosos

solutos
perigosos

correces
fsicas (temperatura)
qumicas (pH, bio-nutrientes)

EFLUENTE
AQUOSO

elevao
tanque de
igualizao
agentes
slidos/lquidos precipitantes /
floculantes
flutuantes

colector
municipal
elevao
slidos
suspensos

Gilda Carvalho, IBET

grosseiros
densos

tanque de
igualizao

resduo slido

TRATAMENTO PRINCIPAL EM
ETAR PRPRIA /
PARQUE INDUSTRIAL

DESCARGA
REUTILIZAO
PRELIMINAR /
PRIMRIO

slidos
suspensos
leves

TERCIRIO
SECUNDRIO (AFINAO)

CBO
turbidez
(carncia
poluentes diludos
bioq. de O2) patogneos
N, P

tratamento / valorizao / deposio

Tratamento de guas residuais

reciclagem

CAUDAIS A TRATAR

UNIDADES INDUSTRIAIS

Balanos mssicos
Higiene de equipamentos e instalaes
gua da rede potvel

??? %

GUAS RESIDUAIS

guas pluviais

MUNICPIOS
infiltrao
consumos de gua

constante (p.ex., consensados)

directa (pluvial)
em condutas

domstico
industrial
(na malha urbana)

UTILIZAO

60-85%

GUAS RESIDUAIS

pblico
rede de saneamento
no contabilizado
(perdas; erros de medida)
Gilda Carvalho, IBET

+
rede pluvial
(sarjetas)

rede
unitria

Tratamento de
guas residuais
ETAR Terras de Santa Maria

Matria Orgnica
Carncia Qumica de Oxignio (CQO) mede o teor de matria orgnica presente numa gua
residual, por oxidao qumica, a quente, em meio cido.
Agente oxidante utilizado: Dicromato de potssio, K2Cr2O7
Agente acidificante: H2SO4 concentrado
Temperatura da digesto qumica: 160C
Tempo da digesto qumica: 110 min
Carncia Bioqumica de Oxignio CBO5(20) permite medir a matria orgnica presente
numa gua residual, atravs de oxidao biolgica, a 20C, durante cinco dias
Agente oxidante utilizado: microrganismos presentes na gua residual, ou uma populao
de microrganismos (inculo) previamente adaptada gua residual
Temperatura da digesto biolgica (incubao): 20C
Tempo da digesto biolgica (incubao): 5 dias
Luminosidade: no escuro, para reduzir a actividade de produo de oxignio pelos
organismos fotossintticos que possam estar presentes na gua residual
No Mtodo Respiromtrico, ao longo dos 5 dias de incubao, procede-se determinao
do oxignio consumido ao fim de cada perodo de 24 horas
Agitao: permanente para facilitar as trocas gasosas entre a amostra e a atmosfera
existente no interior do Respirmetro
e ainda slidos totais, slidos totais em suspenso, C total, C orgnico total, carncia
total de oxignio

Organic Matter

Tratamento de guas residuais

Primary treatment:
Solid removal

Secondary treatment
(biological, with
activated sluge microbial consortia):
Decrease BOD

Aggregation
Adsorption
(Polysaccharides,
metallothioneins)

Biological treatment
Aerobic (activated sluge)
surface aeration
OR
trickling filter
(with sand and stones)

Anaerobic digestion
on sediment sludge from aerobic process
occasionally on raw sewage

Restricted process control

Uncontrolled population of microorganisms
Odor emission
Low efficiency of O2 transfer, large amount of space
Pure O2 (4.8 x more O2 than air):
less sludge is produced, lower cost, but higher investment and more complicated

TRATAMENTO AERBICO
- LAMAS ACTIVADAS
Consrcio microbiano

OBJECTIVO: mineralizao mxima de CBO e nutrientes

ADSORO

CBO

CBO

BIODEGRADAO (crescimento celular)

CO2 + H2O + NH3 + PO43+ biomassa

CBO + O2
NUTRIENTES
azotados
fosfatados

CBO residual

solvel
coloidal
suspenso

O2

CO2 + H2O
NO3 -

NITRIFICAO (crescimento celular)

NH3

NO2
O2

NO3 -

O2

MANUTENO, MORTE E LISE CELULAR

PREDAO
protozorios
rotferos

OUTROS
(p. ex. nutrientes
residuais)

bactrias, fungos

BIOMASSA
(separvel da
gua tratada)

Denitrification
(anoxic or anaerobic)

O2

O2

Remoo de Fsforo de
guas Residuais
Tratamento Qumico
remoo: precipitao com sais de ferro ou alumnio (cloreto frrico, almen)
pros: eficiente, de baixo custo de investimento e relativamente simples
cons: elevados custos (produtos qumicos utilizados, volume acrescido de
lamas), maior contedo em sais no efluente final; quantidade de
precipitante qumico aumenta exponencialmente na razo inversa da
quantidade de fosfato a obter no efluente
Tratamento Biolgico
remoo: crescimento microbiano (consrcio); acumulao intracelular
(polifosfato)
pros: efluente com muito baixa concentrao de fsforo, sem custos
adicionais, maior flexibilidade (conjugada com a remoo biolgica de
azoto); lamas utilizadas como fertelizante aps estabilizao
cons: eficincia e estabilidade do processo (reversibilidade)

Anaerobiose/Aerobiose
Efluente

REACTOR
ANAEROBIO

Efluente
(baixa [P])

REACTOR
AEROBIO

Purga
Biomassa
Recircula o de lamas

Anaer

Aerob
PHA

Glicognio
Crescim.

Carbono

Fosforo
Tempo

Microorganismos: PAOs e GAOs

Bactrias Acumuladoras de Fosfato (PAOs)
X Acinetobacter
X Microlunatus phosphovorus
Candidatus Accumulibacter phosphatis (-Proteobacteria);
Rhodocyclus-related bacteria
? Actinobacteria (Tetrasphaera spp.)

Bactrias Acumuladoras de Glicognio (GAOs)

? Amaricoccus kaplicensis (- Proteobacteria)
? Micropruina glycogenica e
Kineosphaera limosa (Actinobacteria)
? Quatrionicoccus sp. (-Proteobacteria)
? Sphingomonodales (- Proteobacteria)
Candidatus Competibacter phosphatis (-Proteobacteria)
Defluvicoccus vanus cluster 1 e 2 (- Proteobacteria)
Paulo Lemos, REQUIMTE

Methane Production
The main goal is not always energy production, but stabilization of sludge
Only a small fraction of the production is distributed to others
Methane is insoluble thus readily removed from the reaction mixture
but
Its long-distance transport is difficult
1 kg of sludge 600 l of methane (60-70% of produced gas, inc. CO2 and H2S)
There is a risk of explosion

Starting material varies on water content, composition and physical properties:

Activated sludge
Domestic wastewater
Agricultural wastewater
Farming waste
Domestic waste
Agricultural and forestry waste

Methane Production
Methane production: final link in the anaerobe food chain
Microbial consortia are required
Starting material: cellulose, starch, fats and protein - methanogenics dont use!
Methane production depends on anaerobic fermentative bacteria:
Polymer hydrolysis facultative anaerobes
Production of low chain fatty acids (sludge liquefaction phase) other anaerobes
Direct substrates for methanogenesis:
Acetate (75 %), methanol, formate, H2 + CO2 (almost all the rest)
Yield depends on substrate composition and temperature.
Improvement: Multistage process
Temperature optimization (45-60C)
Mixing

Methane Production

Syntrophomonas
Syntrophobacter
Methanobacterium
thermoautotrophicum

Methanosarcina

Composting

Solid organic waste:

Green plant waste
Municipal solid waste (otherwise incinerated or disposed in landfield)

Useful for:
Soil conditioning material
Commercial production of
Agaricus bisporus
Providing rich microflora and
fauna for bioremediation

Composting
Aerobic solid state fermentation
Controlled oxidation of organic matter to
produce a stable and humified product
(it may be necessary to add a N-source in order to obtain a C/N of 30:1)
Microbial consortium depends on substrate composition and temperature
(commercial inoculants also for sale):
1.
2.
3.
4.
5.

Mesophilic bacteria and fungi generate heat

Rise in temperature suppresses their growth and thermophilic bacteria take
over (Bacillus strearothermophilus, Thermus and actinomycetes)
peak heating phase (70-80 C): other thermophilic proliferate
Temperature slowly declines; at 40-60 C thermophilic fungi produce
cellulases and hemicellulases: Aspergillus, Chaetomium and Humicola
After 20-30 days, new mesophilic stage

For mushroom production, phase 4 should be avoided since cellulose is needed

as carbon source; bacteria serve as organic nitrogen source for mushrooms

Composting

Solid waste

Bioremediation
Removal of toxic waste
from the environment
using biological agents

Conversion of xenobiotics (substrates that do

not occur in nature - foreign to life):
herbicides, pesticides, refrigerants, solvents,
pharmaceuticals, household and personal care
products, sunscreens, shampoos, detergents,
deodorants, synthetic chemicals, oil derivatives,
dyes, heavy metals

Bioremediation
Technology used to speed up the natural processes of waste degradation and
recycling
Use of (naturally occurring) microorganisms such as bacteria, fungi, and yeast to
degrade pollutants or hazardous substances in soil, water and air into non-toxic
or less toxic substances
Biologically-based remediation detoxifies hazardous substances instead of
merely transferring contaminants from one environmental medium to another
Bioremediation is generally less disruptive to the environment than excavationbased processes
The cost of treating a hazardous waste site using bioremediation technologies
can be considerably lower than that for conventional treatment methods:
vacuuming, absorbing, burning, dispersing, or moving the material
Bioremedia\on biodeterioration

Xenbiotico
Biodegradvel
Sofre degradao primria, parcial ou mineralizao
Persistente
Degradao muito lenta em certo ambiente
Recalcitrante
No est biodisponvel ou no sofre degradao em
certo ambiente

Biorremediao: recorre a mecanismos biolgicos para destruir,

transformar ou imobilizar contaminates ambientais,
de forma a proteger receptores potenciamente sensveis

Recalcitrant
compounds
Hydrofobic nature

Bioaccumulation in fat tissues

(due to partition coeficient)

Biomagnification or bioconcentration
(organisms higher up the food chain)

103 or 106 higher concentration in

fish eating birds and other animals

Xenobiotics
Bio-degradable
petroleum products (gas, diesel, fuel oil) crude oil compounds (benzene, toluene,
xylene, naphthalene) some pesticides (malathion) some industrial solvents coal
compounds (phenols, cyanide in coal tars and coke waste)
Partially degradable
TCE (trichlorethylene) threat to ground water PCE (perchlorethlene) dry cleaning
solvent PCBs (have been degraded in labs, but not in field work) Arsenic,
Chromium, Selenium
Not degradable / Recalcitrant
Uranium Mercury DDT
In natural communities
Halogenated compounds, especially halogenated aromatic compounds (such as
polychlorinated biphenyls) are often recalcitrant
The presence of halogens in a meta position makes the compound more recalcitrant
Often one stereoisomer of an organic compound will be biodegradable, while another
isomer will be recalcitrant
Specific organisms in an environment may be able to degrade recalcitrant compounds,
at varying rates depending on the conditions

Degradation of xenobiotics

Xenobiotic as sole energy source
may lead to
Mineralisation: conversion of organic to inorganic compounds
(opposite of partial degradation)

Co-metabolised with another substrate (usually when microbial
enzymes synthesized for the degradation of another substrate also
degrade the xenobiotic)
may lead to
Primary biodegradation: disappearance of parent compound
Intermediates can be more toxic and persistent than parent compound
(activation), or may become accessible to further degradation

Physiological conditions
Requirements for biodegradation:
Electron acceptor at a residual level of 1 ppm or more
Essential organic and inorganic nutrients
Microbes and substrate must be in contact
Water

Other relevant conditions : pH, temperature, salinity, type of

contaminant

Usually, microbial consortia are required

Oxidation of xenobiotics

Faster oxidation
when O2 is the
electron acceptor

Fe3+

HCO3SO42-

NO3-

Alvarez, Pedro J. J., Illman, Walter A., Bioremediation and Natural Attenuation, 2006

Pseudomonas degrades more than 100 coumpounds

Degradation enzymes
are plasmid-encoded

Glick and Pasternak, Molecular Biotechnology. 2003

Plasmid-encoded
genes

Glick and Pasternak, Molecular Biotechnology. 2003

Biostimulation
Biodegradation by naturally-occurring organisms may be stimulated by
- adding essential nutrients to the contaminated area
- providing aeration or limiting aeration, depending on whether the
contamination is better degraded under aerobic or anaerobic conditions
- using plants and the microbial communities of their rhizospheres
(phytoremediation)

Bioaugmentation
Adding microbes not normally found in an environment to try to alter or accelerate
the biodegradation process
When the microbes are added without consideration of their normal habitat (e.g.,
just adding a pure culture), there may be short-term improvement but the added
microbe usually fails to establish a stable population
Better results are may be seen when the added organisms microenvironment
(nutrients, oxygen, aeration, etc.) are included in the bioaugmentation strategy

Metal bioremediation
Remediation of heavy metals such as:
lead, chromium, arsenic, and others
in soil and groundwater
frequently uses reduction reactions to lower the solubility of an
oxidized metal (lowers two or three valence states)
Ultimately, the metal is precipitated as a sulfide or in another
immobile and low toxicity form
It may also be retained in the biomass bioabsortion
A soil washing technique may also be coupled to metal reduction

Radioactive bioremediation

Uranium
One of the heaviest elements found in nature, 1.7 times denser than lead
emits alpha, beta, and gamma particles
The half-life of U238 is 4.5 billion years!
By-products (such as Thorium (Th232)) have even longer half-lives,
making uranium an element that gets more radioactive during its own
decay process
Natural uranium is 99.274% U238, 0.720% U235, and 0.0055% U234
Uranium is enriched when its content of U235 (fissible) reaches 3.2-3.6%

Radioactive bioremediation

The legacy of nuclear

weapons and nuclear
energy development has
left ground water and
sediment at hundreds of
sites across the United
States and many more
around the world
contaminated with
uranium (mining and
enriching sites)
There is a need for lowcost treatment systems

Radioactive bioremediation
Uranium contamination:
is a threat to wildlife and water supplies
can cause kidney damage and cancer in humans
takes billions of years to decay radioactively
traditional clean-up methods involving pumping of contaminated water
are useless against uranium because it sticks to soil

Radioactive bioremediation case study

The overall mobility of uranium in the environment is determined by its oxidation state,
which is either hexavalent - U(VI) - or tetravalent - U(IV). In its hexavalent state, uranium is
usually more soluble and travels with water. But in the tetravalent state, uranium is
insoluble and practically immobile. The goal is to reduce uranium and keep it stabilized in
its tetravalent state.
The U(VI)-reduction of uranium present in subsurface environments can be stimulated
with the addition of organic carbon to the groundwater, that serves as an electron donor
(different compounds may enrich different species). This precipitates uranium from the
groundwater preventing its further mobility and concentrates uranium dispersed in a large
volume of groundwater into a discrete zone for subsequent removal. However, there was
a tendency of uranium to reoxidize even when reducing conditions are maintained. Some
working hypothesis:
the oxidation of organic carbon generates a proportional amount of bicarbonate, which
actually brings uranium back into the water, and there is an optimum level of organic
carbon that works best for the overall process
OR
the reoxidation could be due to one of the other predominant elements coexisting in the
contaminated samples - iron or manganese
OR
trying to stabilize uranium through organic carbon reduction is not ideal

Radioactive bioremediation
Sequestring and/or reducing metals
Geobacter metallireducens
reduces uranium
Shewanella oneidensis
produces UO2 with extracellular cyochrome

Fixing nuclear waste in soil for

subsequent clean-up

Deinococcus radiodurans
radiation resistent
Geobacter species have been shown to play important roles in the bioremediation of
groundwater contaminated with petroleum and landfill leachate. Soluble organic
contaminants are oxidized to carbon dioxide with the reduction of iron oxides that are
abundant in most subsurface environments. Molecular analysis of a diversity of
contaminated subsurface environments in which oxidation of pollutants coupled to iron
oxide reduction is important have consistently found that these environments are
enriched in Geobacter species

Petroleum
In addition to pollution through human activities, millions of tons of
petroleum enter the marine environment every year from natural seepages.
Despite its toxicity, a considerable fraction of petroleum oil entering marine
systems is eliminated by the hydrocarbon-degrading activities of microbial
communities, in particular the so-called hydrocarbonoclastic bacteria.
Alcanivorax borkumensis
n-alkane degradation
biosurfactant production
biofilm formation
scavenging of nutrients and cofactors in the oligotrophic saline marine
environment
copes with various habitat-specific stresses

Legislation
Many strains not used due to concerns with:
horizontal gene transfer
persistence of strain in the environment

Stains should have no transmissible mobilizable

transposable genetic elements
Should contain killer genes expressed when pollutant is
depleted

Leaching: dissolution of metals

Copper, sulfur, nickel, lead, zinc, cobalt molybdenum,
cadmium, arsenic, titanium, uranium
When metal content is low, the use of chemical methods
is hindered, and vast quantities of ores are wasted (metal
sufides)
Bioleaching is environmental-friendly, but improper
management causes lasting damage to environment due to
metal content and pH 3

Bioleaching
Thiobacillus, Bacillus, Pseudomonas
Thermofilic: Thiobacillus, Sulfolobus
Many other candidates!
Thiobacillus ferrooxidans and thiooxidans
Chemoautotrophs
Electron donnors: Fe2+ (to Fe3+), molecular and reduced sulfur (to sulphate)
Its important to provide physiological growth conditions

Bioleaching
Copper
2 S + 3 O2 + 2 H2O
2 CuFeS2 + H2SO4 + 8.5 O2
CuS + 2 O2

2 H2SO4
2 Cu(SO4) + Fe2(SO4)3 +H2O
Cu(SO4)

Iron from pyrite

2FeS2 + H2O + 7.5 O2

Fe2(SO4)3 + H2SO4

Fe2(SO4)3 + FeS2

FeSO4 + 2S

Uranium
hexavalent U extracted with hot H2SO4 and Fe3+
UO2 + Fe2(SO4)3

UO2 SO4 + 2 FeSO4

Bioleaching
Field Applications
Laboratory
Recycling cells
Recycling leachate
Use of finely ground ores
Shaking
Temperature control

Good yields
Recovery by
precipitation
organic solvent addition
ion-exchange
microbial methods
Crueger & Crueger 1894. Biotechnology