MICROBIOLOGY APPLIED TO REMEDIATION OF THE ENVIRONMENT

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MICROBIOLOGY APPLIED TO REMEDIATION OF THE ENVIRONMENT

VÍT MATĚJŮROBIN KYCLTENVISAN-GEM, a.s.Biotechnologická

divize, Radiová

7,102 31 PRAHA [email protected]

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BASICS

When we apply microbiology for whatever purpose, we must remember that:

•

Microorganisms do not work for us, that we want, but because, they have some advantages for themselves from this work. They gain energy for multiplication, biomass formation and for vital physiological processes.

•

When we improve environmental conditions for microorganisms, they

work better.

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NO COMMENTS

Correct

name:

DAVID PERLMAN

UNIVERSITY

OF WISCONSIN

Died

in the

year

1980

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LAWS OF APPLIED MICROBIOLOGY

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MICROORGANISMS MICROORGANISMS capable

to degrade

or

biotransform

pollutants:

- Archaea-

bacteria

- yeasts-

molds

-

fungi

Mostly

used

are bacteria.Archea

are used

very

little

(experience

is

missing) but

their

utilization

in remediation

is

growingUtilization

of yeast

is

very

specific –

for reduction

of phenols

from

industrial

wastewaters

only. Fungi

(both

brow

rot and white rot): there

was

huge

interest

in

their

utilization

for remediation of contaminated

soil

in the late 80ties. The results

were

not too

conclusive

and their

practical

use

in remediation projects

very

quickly

died

away.

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ARCHAEAArchaea

were

first

identified

by Carl

Woese,

PhD, of

the

University of

Illinois

in 1975. It

took twenty

years

for

the

scientific

community

to accept

his discovery. Archaea

are now

the

most studied microorganism

for

their

value

in medicine, industry

and

bioremediation.

Archaea

are not Bacteria Archaea

and

bacteria

are similar

in size

and

shape,

however

Archaea

are not bacteria. Neither

have

a cell nucleus, or

other

organelles

within

their

cells.

They

differ

in that

bacteria

cell walls

have

a fatty, lipid core

while

Archaea

has a glycerol cell wall.

Additionally, Archaea

have

a complex

gene control system

using

histones

and

RNA similar

to plants

and

animals.

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ARCHAEA - BACTERIAWhat

are bacteria?

-Prokaryotic -Cell walls

of

peptidoglycan

-Plasma membranes

similar

to eukaryotes -Distinct

ribosomes

-RNA polymeraseWhat

are Archaea?

-Prokaryotic -Unicellular -Cell walls

of

polysaccharides

-Ribosomes

and

RNA polymerase

similar

to eukaryotes

-No archaea

cause disease

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ARCHAEA

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ARCHAEAVery

often

Archaea

could

be

find

in

extreme

environments

(high

or

low temperatures, pH, high

concentration

of

inorganic

salts

etc

= extremophiles)

SulfolobusHalococcus

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MICROORGANISMS

•

More detailed

information on yeats

and fungi use in reduction

of pollutants

will

be given

later.

Yeast cell (Saccharomyces cerevisiae) with

scars

after

budding. The number

of scars

is equivalent

to number

of newly

produced

cells.

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CELLS RELATIVE SIZES

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CELLS RELATIVE SIZES

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MICROORGANISMS

•

Eukaryotic

organisms

–

having

cell nucleus

– yeasts, fungi, algea,…………

•

Prokaryotic

organisms

–

having

no nucleus

- bacteria

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EUCARYOTIC AND PROCARYOTIC COMMONS

What

Prokaryotic

and Eukaryotic

Cells

Have

in Common

1.

Both

have

DNA as their

genetic

material

(it’s

DNA that

tells

cells

what

kind

of cells

they

should

be).

2.

Both

are covered

by a cell membrane.3.

Both

contain

RNA.

4.

Both

are made

from

the same

basic

chemicals: carbohydrates, proteins, nucleic

acid, minerals, fats

and vitamins.5.

Both

have

ribosomes

(the structures

on which

proteins

are made).

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EUCARYOTIC AND PROCARYOTIC COMMONS

6.

Both

regulate

the

flow

of the

nutrients

and wastes

that

enter

and leave

them.

7.

Both

have

similar

basic

metabolism

(life processes) like

photosynthesis

and

reproduction.8.

Both

require

a supply

of energy.

9.

Both

are highly

regulated

by elaborate sensing

systems

("chemical noses”) that

make

them

aware

of the

reactions

within

them

and the

environment around

them.

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EUCARYOTIC AND PROCARYOTIC DIFFERENCES

Age

Differences Scientists

believe

that

prokaryotic

cells

(in the form

of bacteria) were

the first

life forms

on earth. They

are considered

“primitive”

and originated

about

3.5 billion

years

ago. That's 2 billion

years

earlier

than

eukaryotic

cells

and billions

of years

before

our

earliest ancestors, the hominids.

A brief

timeline

of the development

of life on Earth: 4.6 billion

years

ago the Earth

was

formed

3.5 billion

years

ago the first

life arose: prokaryotic

bacteria

1.5 billion

years

ago eukaryotic

cells

arose

0.5 billion

years

ago the Cambrian

explosion

–

multi-celled eukaryotes

arose

3 million

years

ago our

earliest

ancestors, the hominids, appeared

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Structural

Differences Eukaryotic

cells

contain

two

important

things

that

prokaryotic

cells

do not: a nucleus

and organelles

(little organs) with

membranes

around

them.

DNA arrangementAlthough

both

eukaryotic

and prokaryotic

cells

contain

DNA, the DNA in eukaryotic

cells

is

held

within

the nucleus. In prokaryotic

cells, the DNA floats

freely

around

in a unorganized

manner.Presence of organellesThe organelles

in eukaryotic

cells

allow

them

to perform

more complex

functions

than

prokaryotic

cells, which don’t

have

these little

organs.

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Some

of the

organelles

in eukaryotic

cells are:

The

Nucleus

–

the

“brain”

or

control

center of the cell. It

contains

DNA, which

makes

up genes.

That

DNA gets

transcribed, or

copied

onto messenger

RNA. That

messenger

carries

a copy

of the

genes

orders

for certain

protein production. These orders

go

to the

protein

factories.

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Structural

DifferencesRibosomes

–

These are the protein factories. They

follow

instructions

from

messenger

RNA (remember

that

the messenger

RNA got

its

orders

from

the DNA). The

instructions

tell

the ribosomes

to make

specific proteins. Note, this

particular

organelle

is

found

in

prokaryotes

too!

Endoplasmatic

reticulum

(ER) –

structures

that

modify proteins

produced

in the ribosomes. Not all

of the

proteins

made

by the ribosomes

need

changing, but those

that

do get

“altered”

here.

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Golgi

Apparatus

–

This

structure

will

make

even

more changes

to the

proteins

that

already

got

changed

when

they

were

in the

E.R. Remember

those

proteins

were made

in the

ribosomes, changed

once

in the

E.R. and

will

be changed

again

in the

Golgi

Apparatus. The Golgi

also

acts

as a post office

by packaging

and

shipping

proteins

to other

parts

of the

cell or

out

of the cell.

Mitochondria

–

structures

which

produce

the

cell’s energy, a.k.a. powerhouses

of the

cell.

Chloroplasts

–

structures

which

allow

plants

to trap sunlight

and carry

out

photosynthesis

ONLY PLANTS

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Size Eukaryotic

cells

are, on average, ten times

larger

than

prokaryotic

cells.Cell Wall

Differences

Prokaryotic

cells

have

a cell wall

composed

of peptidoglycan

(amino acid and sugar). Some

eukaryotic

cells

also

have

cells

walls, but

none

that

are made

of peptidoglycan.

Flagella

ArrangementThe flagella

in eukaryotic

cells

are different

from

the

flagella

in prokaryotic

cells. Flagella

are the structures

that help cells

move

(scientists

call it motility). The flagella

in

eukaryotic

cells

are composed

of several

filaments

and are far more complex

than

the flagella

in prokaryotic

cells.

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All

cells

have

their

genes

arranged

in linear chains

called

chromosomes. But

eukaryotic

cells

contain

two

(or

more) copies

of every

gene. During

reproduction, the

chromosomes

of eukaryotic

cells

undergo

an

organized

process of duplication

called

mitosis.

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PLASMIDS•

Plasmid: an

extrachromosomal

genetic

element that

is

not essential

for growth and has no extracellular

form

(Madigan

et al. 2000)•

Many genetic

systems

associated

with

organic

pollutant

biodegradation

areplasmid-born

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PLASMIDSA plasmid

is

a DNA molecule

that

is

separate

from, and

can

replicate

independently

of, the chromosomal

DNA. They

are double-stranded

and, in many cases, circular.

Plasmids

usually

occur

naturally

in bacteria, but

are sometimes

found

in eukaryotic

organisms

(e.g., the 2-

micrometre ring

in yeast Saccharomyces

cerevisiae).

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PLASMIDSPlasmids

are very

important

from

the point

of view

of the remediation of contaminated

sites.

Plasmids

may

carry

genes

that

provide resistance

to naturally

occurring

antibiotics

in a competitive environmental niche, or

the proteins

produced

may

act

as toxins

under

similar circumstances. Plasmids

can

also

provide

bacteria

with

the ability

to fix elemental nitrogen.

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PLASMIDSMost important

is

(from

remediation

point

of view) that

plasmids

provide

bacteria with

the

ability

to degrade

recalcitrant

organic

compounds

= pollutants

that provide

an

advantage

when

nutrients

are

scarce.

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PLASMIDSBecause

plasmids

are

capable

of replicating

autonomously

within

a suitable

host it is

necessary

to perform

cultivation

of biodegrading

bacteria

under

selection

pressure

to preserve

their

biodegradation capabality.

The reason

is

that

when the selection

pressure

(presence of pollutant) is

not present, the capabality

is

diluted

and finally

is

completely

missed.

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PLASMIDS

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PLASMIDS

The number

of plasmids

in one

bacterial cell differs substantially

and could

be between

one

and several

thousand.

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MICROBIOLOGYBasicaly

plasmids

encode

many gene for enzymes

which

are used

in a degradative

pathway

of pollutants. Their production

is

inductive

mostly. It means

that

they

are

not produced

when substrate

is

not present

in the enviroment. This

is

the reason, why

presence of

selection

pressure

during

multiplication

cultivation

of degrading

bacteria

is

the must.

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MICROBIOLOGYCompounds

for which

plasmid-born

degradative

genetic/enzyme systems

exist:–

Alkyl benzyl sufonates

–

Monoaromatics

(e.g. Benzoate, Phenol)–

Chlorinated

monoaromatics

(e.g. Chlorobenzoates,

Chlorophenols)–

Toluene and Benzene

–

Chlorobenzenes–

Alkanes

and Alkenes

–

Chlorinated

alkanes

and alkenes– PAHs– PCBs

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MICROBIOLOGY OF BIOREMEDIATION

BACTERIAL STRAINS SELECTION1.

For bioremediation purposes

the indigenous

bacterial strains

are mostly

used. The main

aim

is to improve

environmental conditions

for

indigenous

bacteria

-

autochtonous

population2.

It is

possible

to employ

strains

selected

in

laboratory. They

are prepared

by the cultivation under

the selection

pressure. These strains

possesses

requested

characteristics

-

allochtonous strains.

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MICROBIOLOGY OF BIOREMEDIATION

3.When

allochtonous

strains

are applied

on a contaminated

site, the

process is

called

bioaugmentation. This

means

amendment

with selected

strains. The

term bioaugmentation

could

be replaced

by the

term inoculation.4.

Very

questionable

is

the

time of survival

of

inoculated

selected

bacteria

in the contaminated

soil

or

groundwater

after

amendment. This

survival

time depends

upon many factors

like

environmental conditions

and antagonism

of indigenous

bacteria.

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BACTERIAL SURVIVAL

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WHAT BACTERIA CAN DO?

•

BIOTRANSFORMATIONBiotransformation

is

the chemical modification

(or

modifications)

made

by an

organism

on a chemical compound.

•

BIODEGRADATIONA process by which

microbial

organisms

transform

or

alter

(through

metabolic

or

enzymatic

action) the structure

of chemicals introduced

into

the environment." -

U.S. Environmental Protection

Agency, 2009

•

MINERALIZATIONBiotransformation

is

the chemical modification

(or

modifications)

made

by an

organism

on a chemical compound. If

this modification

ends

in mineral

compounds

like

CO2

, NH4+, or

H2

O, the biotransformation

is

called

mineralization.

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WHAT BACTERIA CAN DO?MINERALIZATIONIs

the most prefererd

process for remediation projects.

According

to the definition

no rest materials can

be produced. It means

that

the only

products are CO2

, water, energy and biomass

under

aerobic conditions.

When mineralization

proceeds

under

anaerobic conditions, the final

products should

be NH4

+, CH4

, water, CO2

and very

small

amount

of energy as well

as biomass.

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BASIC MECHANISMS FOR POLLUTANT UTILIZATION

METABOLISM

is

the

sum of

all

of

the chemical

activities

cells

undergo

throughout

their

lives. It

is

composed

of two

main

subdivisions:

CATABOLISMANABOLISM

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NOT BACEAUSE WE WANT

POLLUTANT

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•

CATABOLISM•

The process involving

a series

of degradative

chemical reactions

that

break

down

complex

molecules

into

smaller

units, usually releasing

energy in the process.

•

A destructive

type of metabolism.•

For instance, large

molecules

such as polysaccharides, fats

and

proteins

are broken

down

into

smaller

units

such as monosaccharides, fatty

acids

and amino acids, respectively.

ENERGY PRODUCING PROCESS!!

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•

ANABOLISM•


a sequence

of chemical

reactions

that

constructs

or

synthesizes

molecules from

smaller

units, usually

requiring

input

of energy

(ATP) in the process.•

A constructive

type of metabolism.

ENERGY CONSUMING PROCESS !!!

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•

METABOLISM•


a set of chemical reactions

that

modifies

a molecule

into

another

for storage, or

for immediate

use in another

reaction or

as a by product.

•

Metabolism

includes

processes

for cell growth, reproduction, response to environment, survival

mechanisms, sustenance, and maintenance

of cell structure

and integrity. It is

made

up of two

categories: catabolism

and anabolism.

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•

COMETABOLISM•

A process in which

compounds

not utilized

for growth

or

energy are transformed

to other

products by microorganisms.

•

Non-specific enzymes

for given

substrate

are capable

to utilize

another

compounds, which

are structurally

similar

(an

example

of such an

enzyme is monooxygenase)

•

Co-metabolism

is

defined

as the simultaneous degradation of two

compounds, in which

the

degradation of the second

compound

(the secondary substrate) depends

on the presence of the first

compound

(the primary substrate).

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•

COMETABOLISM –

CONTINUE•

A process in which

a substance may

be biodegraded

only

in the presence of a secondary

source of carbon

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•

COMETABOLISM –

CONTINUE•

Fortuitous transformation of a compound by a microbe relying on some other primary substrate

•

Generally a slow process -

Chlorinated solvents don’t provide much energy to the microbe

•

Most oxidation is of primary substrate, with only a few percent of the electron donor consumption going toward dechlorination

of the contaminant

•

Not all chlorinated solvents susceptible to cometabolism

(e.g., PCE,

and carbon tetrachloride)

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COMETABOLISMPollutants

that

can

be degraded

cometabolicaly: Trichloroethylene

(aerobic)

Trinitrotoluene1,1,1-trichloroethaneMTBEChloroform2,4-dichlorophenoxyacetic

acid

PCBPAHs and many more …..

GROWTH SUBSTRATE

PRODUCTS OF RESPIRATION OR FERMENTATION

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Classic

example: conversion

of 2,4-D to 2,4dichlorophenol and 3,5 dichlorocatechol, discovered

during

search

for intermediates

in 2,4-

D degradation, 2,4-D is

is

an

herbicide and secondarily

a plant

growth

regulator

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COMETABOLISM

CometabolicDehalogenation TCE under

aerobic

conditions

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COMETABOLISM

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Substrates:–

Trichlorethylene and

tetrachloroethylene–

Chlorophenols

–

Halobenzoates–

Nitrobenzenes

–

Various

chlorinatedpesticides

Enzymes:–

Oxygenases

–

Dehalogenases–

Hydroxylases

–

Phosphatases–

Dehydrogenases

– Deaminases

COMETABOLISM

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•

Interaction

in which

two

or

more microorganismscarry

out

a pollutant

transformation

that

neither

of

which

can

perform

alone; •

Biodegradation

carried

out

by a multispecies

mixture

is

more rapid than

the

sums

of the

rates

of reactionsthat

could

be effected

by the

separate

species

•

Can

involve(i) multiple

organisms

all

gaining

energy for growth

from

partial

metabolism

of compounds; or(ii) combination

of cometabolism

and energygenerating

biodegradation

SYNERGISM

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SYNERGISM(iii) There

exists

synergism

between

bacteria

and

Archaea

in contaminated

environment

very

often

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SYNERGISM COUPLED TO COMETABOLISM

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BACTERIA USED IN BIOREMEDIATION

•

Pseudomonas sp.•

Burkholderia

sp.

•

Xanthomonas sp.•

Acinetobacter sp.

•

Micrococcus sp.•

Rhodococcus sp.

•

Ralstonia metallidurans•

Deinococcus

radiodurans

•

Arthrobacter sp.•

Sphingomonas aromaticivorans

•

Nocardia sp.•

Comamonas

sp.

•

Dehalococcoides

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BACTERIA USED IN BIOREMEDIATION•

Alcaligenes sp.

•

Nitrosomonas

sp.•

Nitrobacter

sp.,

•

Paracoccus sp.•

Thiobacillus

sp.

•

Seratia

marcenscens•

Shewanella sp.

•

Bacillus sp.•

Citrobacter sp.

•

Desulfomonile sp.•

Dehalobacter sp.

•

Desulfitobacterium sp.•

Desulfuromonas sp.

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BACTERIA USED IN BIOREMEDIATIONBactera

of genera Xanthomonas sp., Burkholderia sp., and

Pseudomonas sp. are able to utilize huge number of chemical compounds as a growth substrate (> 100)

Rhodococcus sp. –

this bacterium possesses lipids in the cell wall, which helps to transport non-polar pollutants into the cell. They produce biosurfactants

and are

capable to employ various metabolisms. They

are able to utilize

many various

substrates.

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BACTERIA USED IN BIOREMEDIATION

Rhodococci

are aerobic actinomycetes

showingconsiderable

morphological

diversity. A certain

group of these bacteria

possess

mycolic

acids

at the external

surface

of the cell. These compounds

are unusual

long-chain alcohols

and fatty

acids, esterified

to the peptidoglycan

of the cell wall.

Probably, these lipophilic

cell structures

have

a significance

for the affinity

of rhodococci

to

lipophilic

pollutants.In general, rhodococci

have

high and diverse

metabolic

activities

and are able

to synthesizebiosurfactants.

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BACTERIA USED IN BIOREMEDIATIONBacteria

responsible

for biodegradation of oil

hydrocarbons

(Aliphatic

and Aromatic Hydrocarbons, Polycyclic

Aromatic

Hydrocarbons)Pseudomonas sp., Nocardia sp.,Acinetobacter sp., Mycobacterium sp.,Alcaligenes sp. Corynebacterium sp.,Flavobacterium/ Arthrobacter sp./Cytophaga

group, Xanthomonas sp., Bacillus sp.,

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BACTERIA USED IN BIOREMEDIATIONChorinated

ethenes

(PCE, TCE, DCE, VC)

Desulfomonile sp., Dehalobacter sp., Desulfitobacterium sp., Desulfuromonas sp., Dehalospirillum sp.,

Bacteria

responsible

for dechlorination

of TCE and PCE to cis-1,2-DCE. They

are not capable

to further

dechlorinate cis-1,2-DCE to VC and ethene.

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BACTERIA USED IN BIOREMEDIATIONChorinated

ethenes

(PCE, TCE, DCE, VC)

Dehalococcoides sp.

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MICROBIOLOGY

Biological remediation of soil

and/or groundwater

is

perfomed

by bacterial

consortia

everytime. Use of one

bacterial strain is

not possible

due

to non-sterile

nature

of soil

and/or

groundwater.

Microbial

consortiumcapable

of dechlorination

of tetrachloroethane

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MICROBIOLOGY

Rhodococcus

BURKHOLDERIA

Xanthomonas

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CONDITIONS FOR BIOREMEDIATION

•

A microorganism

must

exist

which

has the

necessary metabolic

capacity to bring

about

biodegradation

(“Principle

of Microbial

Infallibility”)•

Biological

degradation of a pollutant

is

possible

only

when microorganisms

contain

enzymes, which

can catalyze

degradation or

biotransformation

reactions

of a

pollutant. •

Physiological

processes

may

not be inhibited

by

present

pollutants

(enzymes

inhibition).•

Polluted

environment may

not be toxic

for

microorganisms.•

There

must

be enough

of macrobiotic

elements

(nitrogen, phosphorus) for non-limited growth

of bacteria.

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CONDITIONS FOR BIOREMEDIATION

•

The polluted

material

to be biologically

treated must

have

suitable

pH, temperature, redox

potential. There

must

be enough

of moisture

in a soil

and enough

of final

electron

acceptors

in

the environment. Environmental conditions must

be conducive

to proliferation

of

biodegrading

microorganisms

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LIMITATION OF BIODEGRADATIONBiodegradation can be limited by a number of

factors including:-

The absence of appropriate degrading genes in

the indigenous microbial community.-

Toxicity of the organic contaminant which

inhibits cellular metabolism.-

Low bioavailability of pollutants

which is the

combined effect of limited water solubility and sorption to soil surfaces. Low bioavailability will effectively reduce uptake of the contaminant by a microbial cell.

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LIMITATION OF BIODEGRADATION-

Contaminant structure including both steric

and

electronic effects. Steric

effects are imposed by substituent groups on a pollutant

molecule

which hinder recognition or access of the degrading enzyme to the active site on the contaminant molecule. Electronic effects are imposed by substituent groups that alter the energy required to break critical bonds in the molecule.

-

Biodegradation can be limited by low numbers of microbes in the environment.

-

Biodegradation can be limited by insufficient oxygen.

IMETE - 2013

WHAT´S WRONG?

IMETE - 2013

MICROBIOLOGY•

When some

pollutants

are present

in mixture,

they

cannot

be biodegraded

simultaneously. The reason

is

that

production

of some

enzymes

inhibits

production

of another

ones.

An

example

are toluene and

chlorobenzene.

TOLUENE CHLOROBENZENE

IMETE - 2013

MICROBIOLOGY

BACTERIA –

OPTIMAL CONDITIONS FOR WORK•

pH 4,5 to 8,2

•

Moisture

content

at

least

30 % of soil

water

holding capacity

•

Minimum concentration of N-NH4

1.0 mg.kg-1

•

Minimum concentration of P-PO4

0.1mg.kg-1

•

Temperature > 4 oC•

Aerobic degradation: concentration of dissolved oxygen in groundwater

> 0.5 mg.L-1

IMETE - 2013

MOST IMPORTANT ENZYMES

The most important

enzymes

for aerobic degradation of organic

pollutants:

-

Oxygenases-

Monooxygenases

-

Dioxygenases-

Hydrolases

-

Peroxidases-

Carboxylases

IMETE - 2013

BIOLOGICAL DEGRADATION OF POLLUTANTS

The most important things for biological degradation of pollutants are mentioned bellow. All of them must be fulfilled to succeed in bioremediation in the field:

1.

Molecules of pollutants must be transported into the cell. Bacteria do not possess exo-enzymes, i.e. they are not capable to excrete enzymes outside cell and attack pollutant in the environment.

2.

Molecules of pollutants must be bioavailable

to bacteria.

3.

Environmental conditions (pH, redox potential, oxygen concentration, moisture content, temperature, nutrients, ….) must be in an optimum range for microbial activities.

IMETE - 2013


4.

Bacteria

should

be capable

to degrade

targeted

pollutant, it means

bacteria

must

possess

enzymes

for pollutant

degradation.5.

There

should

be enough

of final

electron

acceptors:

Aerobic degradation: oxygenAnaerobic degradation: NO3

-, Fe3+, Mn4+, SO42-, CO2

IMETE - 2013


AEROBIC –

in oxygen presence (aliphatic

and aromatic

hydrocarbons, some

chlorinated

ethylenes, phenols, organic

acids

and many more….).Final

electron

acceptor

is

oxygen

ANAEROBIC

–

without

oxygen (pentachlorophenol, tetrachloroethylene, aromatic

hydrocarbons, organic

acids,

nitroaromatic

compounds

–

TNT, RDX, HRX) Final

elektron acceptors

are nitrate, iron (3+),

sulfate, carbon

dioxide.

(Sometime

they

are called

alternative

electron

acceptors.)

IMETE - 2013

BACTERIAL STRAINS FOR BIOREMEDIATION ACCORDING TO THEIR ORIGIN

•

AUTOCHTHONOUS (INDIGENOUS):

bacteria

living in soil

or

groundwater. On polluted

sites they

are very

often

adapted

to present

pollutants

and could

be used for bioremediation.

•

ALLOCHTONNOUS: bacteria

prepared

in laboratory, having

specific capabilities

according

to real conditions

on a contaminated

site. These bacteria

are multiplied by cultivation

under

selective

pressure

(pollutant) to be

adapted

to existing pollutants

on the site. The cultivation

must

be performed

under

selective

pressure

because

of plasmids

retention

in bacteria.When allochtonous

bacteria

are applied to a

contaminated

site, the activity

is

called bioaugmentation.

IMETE - 2013

BACTERIAL STRAINS FOR BIOREMEDIATION ACCORDING TO SUBSTRATE NATURE

AUTOTROPHIC BACTERIAThese are bacteria

which

are able

to

synthesize

their

own

organic

food

from inorganic

substances. They

use carbon

dioxide

for

obtaining

carbon

and

utilise hydrogen

sulphide

(H2

S) or

ammonia (NH3

) or

hydrogen

(H2

) as the

source

of hydrogen

to reduce

carbon.

IMETE - 2013


HETEROTROPHIC BACTERIAThese are bacteria

which

are unable

to manufacture

their

own

organic

food

and

hence are dependent

on external source. These bacteria

can

be

distinguished

into

three

groups

as follows: i) Saprophytic

Bacteria:

These bacteria

obtain

their

nutritional

requirements

from

dead

organic

matter. They

breakdown

the

complex

organic

matter

into

simple

soluble

form

by secreting

exogenous

enzymes. Subsequently

they

absorb

the

simple

nutrients

and

assimilate

them, during

which

they

release

energy. These bacteria

have

a significant

role in the

ecosystem,

functioning

as decomposers

IMETE - 2013


ii) The

aerobic breakdown

of

organic

matter

is

called

as decay

or

decomposition. It

is

usually

complete

and

not

accompanied

by the

release

of

foul

gases. iii) Anaerobic

breakdown

of

organic

matter

is

called

fermentation. It

is

usually

incomplete

and

is

always accompanied

by the

release

of

foul

gases.

NO OXYGEN

IMETE - 2013

FINAL ELECTRON ACCEPTORS

Anaerobic

Aerobic

IMETE - 2013


Redox situation

in a contaminated

aquifer

IMETE - 2013

AEROBIC METABOLISM

CO2

+ H2

O + biomasa

IMETE - 2013

AEROBIC METABOLISM OF HYDROCARBONS

General

principles

of hydrocarbon

biodegradation

under

aerobic condiotions.Process is

connected

with

a bacterial growth.

A NEXT SLIDE

IMETE - 2013

AEROBIC METABOLISMAEROBIC DEGRADATION OF n-ALKANES

A

IMETE - 2013

STOICHIOMETRY OF AEROBIC DEGRADATION OF HYDROCARBONS

•

Oxidation:

C6

H6

+ 12 H2

O → 6 CO2

+ 30 H+

+ 30 e-

•

Reduction:

7.5 O2

+ 30 H+

+ 30 e-

→ 15 H2

O

•

1 g C6

H6

consumes

for

total

oxidation

3 g O2

IMETE - 2013

AEROBIC METABOLISM

1.

There exist several aerobic metabolic processes, which help bacterial cell to survive in contaminated environment. For example bacteria are capable to synthesize biosurfactants, which enhances transport of non-polar

pollutant molecules into the cell.

Pollutant molecules has to be transported into a bacterial cell, because bacteria do not possess degradative

enzymes, which are excreted

from

bacterial cell into

the environment (exo-enzymes).

IMETE - 2013

CELL

CELL WALL

IMETE - 2013

AEROBIC METABOLISM

2.

Initial

steps

in oxidation

of an

organic substrate

(in illustrated

case

it is

an

n-alkane)

starts

with

activation

of oxygen and its incorporation

into

the molecule

of pollutant.

The most important

enzymes

catalyzing oxidation

are oxidases

and peroxidases

in

case

of aliphaticmolecules.

IMETE - 2013

AEROBIC METABOLISM

3. Degradation pathway

changes

an

organic pollutant

molecule

step by step to

intermediatiates

of a central

metabolism, for example

to a cycle

of tricarboxylic acids.

4. Biomass

synthesis

proceeds

from

precursors generated

in a central

metabolism

like

acetylCoA, succinate, pyruvate

etc. Sugars

are synthetized

de novo in the cell (e.g. synthesis

of glucose

by gluconeogenesis).

IMETE - 2013

AEROBIC METABOLISM

Monooxygenase

incorporatesto substrate

molecule

1 atom

of oxygen. The secondOxygen atom is

reduced

to H2

O

IMETE - 2013

AEROBIC METABOLISM

Dioxygenase

incorporates

into

a substrate

molecule2 molecules

of oxygen

IMETE - 2013

AEROBIC METABOLISM OF POLYAROMATIC HYDROCARBONS

First

step of oxidation

of aromatic

hydrocarbons

is

catalyzed

by a dioxygenase

and consists

of transfer of 2 oxygen atoms

to a molecule

of naphthalene

followed

by an

aromatic

ring cleavage.

PAHs having

more aromatic

rings

do not follow

exactly

the same

pathway. The reason

is

that

thermodynamic

properties

of both

molecules

differ

substantially.

Bacterial catabolic

degradation of naphthalene

IMETE - 2013

AEROBIC METABOLISM OF PAHs

DEAD END PRODUCTS OF BIOLOGICAL OXYDATION OF PAHs

DIOLS !!!

IMETE - 2013

AEROBIC DEGRADATION OF BTEX HYDROCARBONS

CATECHOL

IMETE - 2013

IMETE - 2013

AEROBIC DEGRADATION

CLEAVAGE OF CATECHOLUNDER AEROBIC CONDITIONS IS MOST COMMON PATHWAY OF DEGRADATION OF MONOAROMATIC HYDROCARBONS

The two

alternative

pathwaysof aerobic degradation of aromaticcompounds: o- and m-cleavage,1. phenol

monoxygenase,2. catechol1,2-dioxygenase,3. muconate

lactonizing

enzyme, 4. muconolactone

isomerase,5. oxoadipate

enol-lactone

hydrolase,6. oxoadipate

succinyl-CoA

transferase,7. catechol

2,3-dioxygenase,8. hydroxymuconic

semialdehyde

hydrolase,

9. 2-oxopent-4-enoic

acid hydrolase,10. 4-hydroxy-2-oxovalerate

aldolase.

IMETE - 2013

AEROBIC METABOLISM

o-cleavage

products

m-cleavage

productssuccinate acetylCoA

acetaldehyde pyruvate

ALL PRODUCTS ENTER KREBS CYCLE

IMETE - 2013

AEROBIC METABOLISM

C2

H4

O (Acetaldehyde) → C2

H4

O2

(Acetic Acid) ++ Acetyl-CoA

+ 3H2

O+2CO2

acetylCoA

acetaldehyde

IMETE - 2013

AEROBIC METABOLISM

KREBS CYCLE (TCA)

SUCCINATE

ACETALDEHYDE

IMETE - 2013

oxygenases

oxygenases

oxygenases

Biodegradation

of aromatic compounds

and n-alkanes

TCA

β-oxidation

loop

IMETE - 2013

AEROBIC METABOLISM POLLUTANTS, WHICH COULD BE BIOLOGICALLY

DEGRADED UNDER AEROBIC CONDITIONS:

•n-alkanes• alkenes• MTBE• organic

acids

•aliphatic

alcohols•catechol• trichloroethene• dichloroethylenes• vinylchloride• monoaromatic

hydrocarbons

(benzene, toluene, ethylbenzene

and xylenes)

•

substituted

alkanes

•

neutral

lipids

•

Cresol

•

phenols

•

aldehydes

•

PAHs

(up to five

condensed

aromatic

rings)

•

Fuel

oil

•

Jet fuel

•

chlorobenzenes

•

anilines

•

substituted

anilines

•

and others

. .

. . . .

IMETE - 2013

ANAEROBIC METABOLISMFinal

electron

acceptors

different

from

oxygen:

nitrate, sulfate, Fe3+, carbon

dioxide, Mn4+…Anaerobic

processes

are substantially

slower

than

aerobic ones.During

anaerobic

metabolism

much more byproducts

is

formed. Some

of them

can

be toxic

or

bad smelling.

IMETE - 2013

ANAEROBIC METABOLISMProduction

of energy and biomass

is

substantially

lower

in comparison

to aerobic degradation.

IMETE - 2013


--2

IMETE - 2013

FINAL ELECTRON ACCEPTORSREDOX POTENTIAL [mV]

IMETE - 2013

ANAEROBIC METABOLISM

Anaerobic biodegradation is used for degradation of substituted hydrocarbons and

chlorinated organic

compounds.Reductive dechlorinationof PCE is frequently used asa remediation technique.Carbon in PCE is in themaximum oxidationstage so that it cannotbe degraded

by

oxidation

IMETE - 2013

REDUCTIVE DECHLORINATION

H2

is

an

electron

donorChlorinated

solvents

are electron

acceptors

An anaerobic process

is referred to as reductive dechlorination

For example:

RCl

+ H + e-

= RH + H+

+ Cl-

•

Can occur via –

Dehalorespiration

(anaerobic)

–

Cometabolism

(aerobic)

IMETE - 2013


Classic

example: reductive

dechlorinationof chlorobenzoate

by Desulfomonile tiedjei

IMETE - 2013


•

More complex

chlorinated

compounds

such as polychlorinated

biphenyls

(PCBs) are also

subject

to reductive

dechlorination•

Loss

of Cl-

groups

can

make

compound

more

succeptible

to biodegradation•

Sequential

aerobic/anaerobic

shifts

may

enhancebiodegradation

of highly recalcitrantpolychlorinated

contaminants;

process may

beresponsible

for degradation

of PCBs

in contaminated

riverine

sediments

IMETE - 2013

DEHALORESPIRATION

•

Certain chlorinated organics can serve as a terminal electron acceptor, rather than as a donor

•

Confirmed only for chlorinated ethenes•

Rapid, compared to cometabolism

•

High percentage of electron donor goes toward dechlorination

•

Dehalorespiring

bacteria depend on hydrogen- producing bacteria to produce H2

, which is the preferred primary substrate

IMETE - 2013

IMETE - 2013


•

Rate

of dehalogenation

under

anaerobic conditions:

PCE > TCE > DCE > VC > ethene

•

Rate

of dehalogenation

under

aerobic conditions:TCE < DCE < VC

+ ++++ +

- ---- -

+ ++++ +

+ ++++ +

- ---- -

- ---- -

AsPb

CrCd

ZnAs

Pb

CrCd

Zn

IMETE - 2013

IMETE - 2013


Anaerobic

processes

are used

as a pretreatment

of soil and groundwater

contaminated

with

polyhalogenated

compounds

like

DDT, polychlorinated

dibenzo dioxins, PCB, chlorinated

pesticides, polybrominated

flame

retardants

or

explosive

TNT, RDX, HRX etc.

DDT

IMETE - 2013


DDT

IMETE - 2013


Dehalococcoides sp.

Tetrachloro

dibenzo dioxin

IMETE - 2013

REDUCTIVE DEHALOGENATION

IMETE - 2013


•

Reductive

dechlorination

decreases

number

of chlorine atoms

in molecule

and simultaneously

the

thermodynamic

properties

are changed. Biodegradability

is

increased

and can

continue

under

aerobic conditions.•

Polychlorinated

compounds

are treated

sequentially. I.

stage

–

anaerobic, II. stage

-

aerobic

IMETE - 2013

ANAEROBIC METABOLISM –

n-ALKANES

•

Anaerobic degradation of n-alkanes

and other aliphatic hydrocarbons is possible, but it is very slow. It is the reason, why the anaerobic biodegradation of n-alkanes

is not used in a full-scale

remediation

projects.•

Cost

of anaerobic

degradation

of n-alkanes

would

be

very

expensive

due

to very

complex

conditions

needed and long

remediation

time.

.

IMETE - 2013

ANAEROBIC METABOLISM –

BTEXMost

well-studied for toluene

under denitrifying or sulfate reducing conditions.

mechanism is nucleophilic attack on the

methyl-carbon by succinyl

CoA, resulting in two

“dead end”compounds: benzylsuccinic

acid, benzyl

fumaric

acid.

IMETE - 2013

ANOXIC STRESS

•

Between

aerobic and anaerobic

metabolism exists

so

called

anoxic

conditions

(concentration

of dissolved

oxygen in goundwater

is

0,1 mg.L-1 to 0,5

mg.L-1).

•

When

concentration

of dissolved

oxygen is such low

in the

environment, it

develops

anoxic

stress in microorganisms. It

is physiological

state, when

increases

specific

consumption

of oxygen for degradation

of unit weight of organic

substrate

(up to 30times).

IMETE - 2013

ANOXIC STRESS

•

Sophisticated

defense mechanisms

against anoxic

stress is

implemented

by altering

the

pattern

of protein expression. These modifications

are reflected

in the

oxygen

utilization

rates

by increasing

the

synthesis

of proteins

involved

in oxygen consumption,

which

are mediated

by transcription

factors. It was

proved that

the

increase

in specific oxygen

uptake

rate

in the

anoxic

condition

is

a response to the

phenomenon

of anoxic

stress.

IMETE - 2013

WHERE TO FIND INFORMATION

http://umbbd.msi.umn.edu/University of Manitoba Biocatalysis/Biodegradation Database -UM - BBD1. 217 metabolic pathways2. 1488 reactions3. 1379 substances4. 984 enzymes5. 534 mikroorganisms6. 250 biotransformation rules7. 50 organic functional groups8. 76 reactions of nafthalene 1,2-diooxigenase9. 109 reactions of toluene diooxigenase

21.1.2013

IMETE - 2013

EUCARYOTIC MICROORGANISMS•

Yeast and fungiFungi and yeasts

may play a role in soil, but

are less important in groundwater

IMETE - 2013

YEASTS•

Yeasts

are employed

only

for

biodegradation

of phenols

in industrial wastewaters

(coke

production, tar

transformation, manufactured

gas)

IMETE - 2013

YEASTS•

Yeasts

may

use n-alkanes

as a growth

substrate.

•

Formerly

there

was

production

of „single cell proteins“

from

n-hexane. The

yeast strain

employed

was

Candida

lipolytica

IMETE - 2013

WOOD-ROTTING FUNGI

white-rot fungiWood-rotting

fungi:

brown-rot fungi

They

possess

extracellular

enzymesDegradation

of pollutants

is

easier

because

they

do not need

transport pollutant

molecules

into cells

IMETE - 2013

WOOD-ROTTING FUNGI

•

Brown-rot fungi

–

enzyme cellulases

–

they degrade

cellulose, lignin is

left

nearly

unchanged•

White-rot fungi

–

enzyme peroxidases

–

they

degrade

lignin –

cellulose

is

left

nearly unchanged. The

white-rot fungi

produce

only

a

few

enzymes

(lignin peroxidase, manganese peroxidase, H2

O2

-generating

enzymes, and laccase) and these generate

strong

oxidants,

which

virtually

“combust”

the

lignin framework

IMETE - 2013

WOOD-ROTTING FUNGI

IMETE - 2013

WOOD-ROTTING FUNGI

IMETE - 2013

WHITE-ROT FUNGI•

Production

of exo-enzymes

–

mostly

oxidizing

enzymes

like

peroxidases, laccases, H2

O2

- generating

enzymes, which

are capable

to

attack

PAHs. PAHs

have

similar

structure

to lignin. No transport to the

cell is

needed

–

an

advantage.•

Problem

with

colonization

of soil

by fungal

mycelium. High consumption

of an

inoculum.•

Inoculum size

needed

is

too

big

–

very

costly

•

Formation

of dead-end

products

IMETE - 2013

LIGNIN

IMETE - 2013

WHITE-ROT FUNGI

BACTERIA DIOXYGENASE

FUNGI PEROXIDASE

IMETE - 2013

WHITE-ROT FUNGI•

Earthfax

Engineering, Dr. Lamar, Utah,

USA•

ATE, French

company, Dr. T.M. Vogel,

Czech

subsidiary

performed

several

full scale projects

–

no possitive

results

achieved

IMETE - 2013

BIOPULPING -

SUCCESSFUL FULL- SCALE APPLICATION OF WHITE-

ROT FUNGI

WHITE-ROT FUNGI + WOOD CHIPS

IMETE - 2013

BIOPULPING

IMETE - 2013

BIOPULPING

IMETE - 2013

BIOPULPING

•

Savings: operational

costs

$

10 per ton•

Reduction

of energy consumption

by

30 %

BEFORE

AFTER

IMETE - 2013

QUESTIONS ?

IMETE - 2013

THANKYOU FOR

YOURATTENTION !

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MICROBIOLOGY APPLIED TO REMEDIATION OF THE ENVIRONMENT

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