Advance in

environmental
microbiology
College of Environment, Hohai University
Association Prof.:Deqiang Chen
Email:hjycdq@hhu.edu.cn
Cell phone:13584018783
Office: B324, Hydraulic Tower

Content

II.

Metagenomics (Microbial environmental

genomics)
Immobilized enzyme system

III.

Microbial flocculant

I.

.Metagenomics (Microbial environmental

genomics)

Definition
Metagenomics is the study of microbial
meta-genomes directly from
environmental samples, which is
independent on the ability to cultivate
microbes in the laboratory.

Background

Conventional sequencing begins with a culture of

identical cells as a source of DNA. However, there
are probably large groups of microorganisms in
many environments that cannot be cultured and
thus cannot be sequenced.
Early environmental gene sequencing cloned
specific genes (often the 16S rRNA gene) to
produce a profile of diversity in a natural sample.
Such work revealed that the vast majority of
microbial biodiversity had been missed by
cultivation-based methods.
The cultivation based methods find less than 1% of
the bacterial and archaeal species in a sample.

Background

Because of its ability to reveal the previously

hidden diversity of microscopic life,
metagenomics offers a powerful lens for
viewing the microbial world that has the
potential to revolutionize understanding of
the entire living world.
Metagenomics allows microbial ecology to
be investigated at a much greater scale and
detail than before.

Project

The total DNA of all microbial species (metagenomes ) in a specific environment, not the
total DNA of particular microorganism or its
cell.
Objectives

Discover the composition and diversity of

microbial communities of special
environmental habitats (e.g., polluted river).

Methods

Flow diagram of a typical metagenome project

Key

technology-DNA sequencing

First-generation sequencing technology

2. Next-generation sequencing technology
3. Third-generation sequencing technology
1.

chemical
degradation
method by
MaxamGilbert
method
(1977)

1950

1960

Chemical
degradation
method by
Whitfield
(1954)

1970

1980

Development
of Sanger
Sequencing
(1977)

Invention of
Heliscope
single
molecular
sequencer

1990

Invention of
Single
molecule real
time(SMRT)
DNA
sequencing

2000

2010

Invention of
Capillary
Sequencer
(1996)
Invention of
Automated
Fluorescent
Sequencer
(1985)

Invention of
454 GS 20
Sequencer
(2005)

Invention of
Nanopore
single
molecular
sequencing
(Oxford
Nanopore
corporation)

Invention of
Applied Biosystems
Solid System
(2007)
Invention of
Illumina
Genome
Analyzer
System
(2006)

Sequencing Technology Development

1. First-generation sequencing technique

Chemical degradation method

Dideoxy chain termination method
Automated sequencing

Chemical degradation method (Maxam and

Gilbert, 1977)
Principle: the sequence of a doublestranded DNA molecule is determined by
treatment with chemicals that cut the
molecule at specific nucleotide positions.

Procedure
(i) Label the 3'ends of DNA with 32p;
(ii) Separate two strands, both labelled at 3'ends;
(iii) Divide the mixture in four samples, each treated with a
different reagent having the property of destroying either
only G, or only C, or 'A and G' or 'T and C '. The
concentration of reagent is so adjusted that 50% of target
base is destroyed, so that fragments of different sizes
having 32p are produced.
(iv) Electrophoreses each of the four samples in four
different lanes of the gel.

Chemical
degradat
ion
method
(Maxam
and
Gilbert,
1977)

Advantages/disadvantages of Chemical
degradation method
Requires

lots of purified DNA, and many intermediate

purification steps;
Relatively short readings;
Automation not available (sequencers);
Remaining use for footprinting (partial protection against
DNA modification when proteins bind to specific regions,
and that produce holes in the sequence ladder).
In contrast, the Sanger sequencing method requires
little if any DNA purification, no restriction digests, and
no labeling of the DNA sequencing template.

 Dideoxy chain termination method (Sanger

et al, 1977)

Principle: the sequence of a singlestranded DNA molecule is determined by

enzymatic synthesis of complementary
polynucleotide chains, these chains
terminating at specific nucleotide positions.

Procedure
(i) Denaturation;
(ii) Primer attachment and extension of bases;
(iii) Termination ;
(iv) Gel electrophoresis.

Sequencing of DNA by the Sanger method

Materials for Dideoxy (Sanger) Method

a)
b)
c)

a)
b)
c)
d)

DNA purification of test sample;

A small amount of a dideoxynucleotide (ddNTP);
DNA polymerase for chain teminationsequencing
(Sequenase)T7 DNA polymerase ;
high processivity
negligible or zero 53exonuclease activity
negligible or zero 35 exonuclease activity
Single-stranded DNA template
Denaturation after be cloned in plasmid
M13 vector
Phagemid vector
PCR.

Dideoxy nucleotide
PPP

5
CH2
O

BASE

3
No hydroxyl group at 3 end prevents strand extension

Sanger Method Sequencing Gel

Sample Output

1 lane

Comparison

Maxam Gilbert Method

Chemical
Requires DNA
Requires long stretches of
DNA
Breaks DNA at different
nucleotides

Sanger Method
Enzymatic
Requires DNA
synthesis
Termination of chain
elongation

Automated sequencing
thermal

cycle sequencing ;
Fluorescent primers are the basis of automated
sequence reading;
Capillary Electrophoresis instead of
Polyacrylamide Gel Electrophoresis.

Thermal cycle sequencing

Two

advantages

a)It

uses doublestranded rather than

single-stranded DNA as
the starting material;

b)Very

little template
DNA is needed, so the
DNA does not have to
be cloned before being
sequenced.

Automated DNA
sequencing with
fluorescently
labeled
dideoxynucleotides

Capillary Electrophoresis
Electroosmotic

flow;
The speed of ionic movement is governed by
ionic size and charges;
Less Joule heat produced in electrophoresis

High separation efficiency;

High speed;
Very small sample volume needed1-50 nanoliter.

Applied Biosystems
3730 DNA Analyzer
High-throughput
48-capillary
analyzer
dramatically
increases
productivity, while
providing
application
flexibility and
significantly lower
project costs. Easily
upgradable to 96
capillaries.

Next-generation

sequencing technology

1 High-throughput sequencing technology

Roche/454 FLX
Illumina/Solexa Genome Analyzer

Applied Biosystems SOLID system

2 DNA chip technology

1 High-throughput sequencing technology

Roche/454 FLX
Principle Sequencing by Synthesis

By the action of DNA polymerase, ATP-sulfurylase,

luciferase and apyrase, each dNTP polymerization
on the primer was coupling with the release of a
chemical luminescence signal. So the real-time
detection of DNA sequences was achieved by
detecting the presence of and strength of
chemiluminescence signal.

Principle of Roche/454 FLX pyrosequencing

Work flow

Library construction

Emulsion PCR

Pyrosequencing

Illumina/Solexa Genome Analyzer

Principle Sequencing by Synthesis

On the basis of Sanger sequencing methods,

different color fluorescent was used as tags four
different dNTP. When complementary DNA
polymerase chain was synthesized, each added
dNTP will release different fluorescence. So the
DNA sequence information of test samples was
obtained by detecting the fluorescence signals and
processing it by specific computer software.

Work flow
Library

preparation;
Cluster generation;
Sequencing;
Data analyzing.

Applied Biosystems SOLID system

Principle Sequencing by Ligation

The SOLiD instrument utilizes a series of ligation and

detection round to sequence millions of fragments
simultaneously. There are five primer cycles performed on
the instrument with each cycle staggered by a single base
and including a series of seven or ten ligations for either 35
or 50 base pair sequencing run. Each ligation decodes
two bases and is recorded by fluorescent imaging. By
compling the fluorescent reads in color space for each
fragment, an accurate sequence can be generated.

Work flow

Library preparation;
Emulsion PCR;
Beads Enrichment;
Deposite beads;
SOLiD 4-color ligation reaction;
SOLiD 4-color ligation visualization;
SOLiD 4-color ligation (1st cycle after reset);
Data analyzing.

1. Prepare library of DNA fragments

Genomic DNA
Mate-paired library
Fragment library

27b
p

27b
p

27b
p

27b
p

2. Emulsion PCR

Templates

P1-coupled DNA beads

~ 100,000 P1 sites per bead
Start with 2 Billion beads per
emulsion

Enzyme + dNTPs
Polymerase

Mix PCR aqueous phase into a water-in-oil

emulsion and carry out emulsion PCR
Reactor
with
template,
bead and
PCR
reagents

Mineral oil
+
surfactants

Beads collected following emulsion PCR:

P2

P1

P2
Beads with no product
Beads with amplified product (~40K PCR
products per bead)

3. Beads enrichment

Centrifuge
using a
Glycerol
Gradient

Captured beads (+ templates) in supernatant

Uncaptured beads (no template) in pellet

4. Deposite beads

Template bead
deposition

3-end
modification
Beads attached to glass
surface in a random array

5. SOLiD 4-color ligation reaction

universal seq primer

3

p5

ligase

A-probe

3
5

nnnnCzzz

nnnnGzzz

nnnnAzzz

C-probe

G-probe

T-probe

nnnnTzzz

universal seq primer

p5

1m 1m
beadbead

Adapter Oligo Sequence

Template Sequence

5. SOLiD 4-color ligation reaction

universal seq primer

3

p5

ligase

A-probe

3
5

nnnnCzzz

nnnnGzzz

nnnnAzzz

C-probe

G-probe

T-probe

nnnnTzzz

ligase

universal seq primer

p5

1m 1m
beadbead

Adapter Oligo Sequence

Template Sequence

6. SOLiD 4-color ligation visualization

universal seq primer

1m 1m
beadbead

Adapter Oligo Sequence

Template Sequence

A
5

7. SOLiD 4-color ligation Reset

1m 1m
beadbead

Adapter Oligo Sequence

Template Sequence

5
10

15

20

25

8. SOLiD 4-color ligation (1st cycle after reset)

universal seq primer n-1

3

ligase

p5

A-probe

3
5

C-probe

nnnnCzzz

nnnnGzzz

nnnnAzzz
3

G-probe

T-probe

nnnnTzzz

ligase

universal seq primer n-1

p5

1m 1m
beadbead

Adapter Oligo Sequence

Template Sequence

Consequences of 2 Base Pair Encoding

2nd Base
A

Detecting a single color

does not indicate a base
Each reading contains
information from two bases
To decode the bases you
must know one of the
bases in the sequence

A
C
1st Base

G
T

2nd Base
A

A
1st Base

Example :

C
G
T

AA
CC
GG
TT

AC
CA
GT
TG

AC
CA
GT
TG

AA
CC
GG
TT

AG
CT
GA
TC

AT
CG
GC
TA

AA
CC
GG
TT

AG
CT
GA
TC

AG
CT
GA
TC

If know first base is an A then immediately it decodes 2nd base. This must be
an A as Blue translates 2nd base A if first base A

2 DNA chip technology

Principle Sequencing by hybridization
Sequencing by hybridization is a non-enzymatic
method that uses a DNA microarray( DNA chip ). A
single pool of DNA whose sequence is to be
determined is fluorescently labeled and hybridized to
an array containing known sequences. Strong
hybridization signals from a given spot on the array
identifies its sequence in the DNA being sequenced.

3. Third-generation sequencing technique

Characteristic: Single molecular sequencing
Heliscope single molecule Sequencing;
Single molecule real time (SMRT)DNA
sequencing;
Nanopore single molecule sequencing.

Application

of metagenomics
Research of environmental microbial
ecology;
Research and development of
microorganism preparation.

. Immobilized Enzyme Systems

Definition

The restriction of enzyme mobility in a fixed space is

known as enzyme immobilization. In general,
immobilized enzymes are enzymes that are attached
to, or entrapped within, a macro-scopic support
matrix so that the resulting catalyst can be reused.
Immobilized enzyme
Two opposite concept
Free enzyme

1. Advantages of Immobilized Enzymes

Advantages:
(1)Enzyme reutilization: catalyst reuse;
(2)Elimination of enzyme recovery and purification
processes: product purity , while effluent handling
problems ;
(3)Provide a better environment for enzyme activity;

Disadvantages:
Increase the diffusion resistance, so decreases the
reaction rate.

2. Applications of Immobilized Enzymes

Immobilized enzyme are employed in many fields.
Analytical

devices:

For example: in immobilized enzyme electrodes for

the detection of various chemical substances.
As

catalyst in industrial processes:

Among 2000 types of enzymes found, only 50 ~ 60

have been applied in industry, most of which are
extracellular enzymes.

3. Methods of Immobilization
Immobilized Soluble Enzymes
Entrapped
MatrixEntrapped

Bound

MembraneEntrapped

Adsorbed
(Physical or
Ionic)

CovalentlyBound
To Support

Between
Macroscopic
Membranes

MicroEncapsulated

To Enzyme

 Entrapment
The physical enclosure of enzymes in a small space.

Matrix entrapment
Matrices used for enzyme immobilization are usually:
Ploymeric materials: Ca-alginate, agar, -carrageenin,
polyacrylamide, collagen;

Solid matrices: activated carbon, porous ceramic,

diatomaceous earth.
When immobilized in a polymer matrix, enzyme
solution is mixed with polymer solution before
polymerization takes place.

Membrane entrapment

Membrane: nylon, cellulose, polysulfone and polyacrylate.

In all cases, a semi-permeable membrane
is used to retain high-MW compounds (enzyme)
while allowing small-MW compounds (substrates or products)
access to the enzyme.
Microencapsulation: a special form of mem-brane
entrapment.
In this technique, microscopic hollow spheres are formed.
The spheres contain the enzyme solution, while the sphere
is enclosed with a porous membrane.

Problems:
1. Leakage of enzymes into solution:
Reducing the MW cutoff of membranes or the pore size
of solid matrices;
2. Considerable diffusional resistance emerges:
Reducing the particle size of the matrices and/or
capsules;
3. Reduction of enzyme activity and stability:
Alter the unfavorable microenvironmental conditions;
4. Lack of control of the microenvironment:
A little difficult to handle.

 Bound

Adsorption
A physical attachment of enzyme on the surfaces of support
particles by weak physical forces: van der Waals.
Support materials:
Inorganic materials: alumina, silica, porous glass,
ceramics, diatomaceous earth;
Organic materials: cellulose (CMC, DEAE-cellulose),
starch, activated carbon, ionexchange resin (Amberlite,
Sephadex, Dowex).

Disadvantages:
Desorption is quit common because the binding forces are so
week, especially in the presence of strong hydrodynamic
forces.
Advantages:
Normally, active site is not affected, nearly
all activity is retained after immobilization.

Covalent Binding
It is the retention of enzymes on support
surfaces by covalent bond formation, via certain
functional groups, such as amino, carboxyl,
hydroxyl, and SH group.
The functional groups must not be the active sites.

Cross-Linking
The enzyme molecules can also cross-link with
each other. But this may cause significant
changes in the active site of enzymes, and also
severe diffusion limitations may result.

SUMMARY
The most suitable support material and immobilization
method vary depending on the enzyme and particular
application.

Then how to select support materials ?

a. Binding capacity, which is a function of charge
density, functional groups, porosity, and
hydrophobicity;
b. Stability and retention of the enzyme activity, which
is a function of functional groups on support
material and microenvironmental conditions.

How to select
method of immobilization ?

Table: Comparison of the Characteristics of Different Methods of

Enzyme Immobilization
Carrier Binding Methods
Characteristic

Cross-linking
Method

Entrapping
Method

Physical
adsorption

Ionic Binding

Covalent
Binding

Preparation

Easy

Easy

Difficult

Difficult

Difficult

Enzyme Activity

Low

High

High

Moderate

High

Changeable

Unchangeable

Substrate
Specificity

Unchangeable Unchangeable Changeable

Binding Force

Weak

Moderate

Strong

Strong

Strong

Regeneration

Possible

Possible

Impossible

Impossible

Impossible

General
Application

Low

Moderate

Moderate

Low

High

Cost of
Immobilization

Low

Low

High

Moderate

Low

Microwave irradiation
Enzyme molecules are difficult to distribute
throughout the porous carriers because of
diffusion limitations.
They often remain only on external channels.
Microwave irradiation can help to decrease the time
for immobilization and improve the enzyme loading.

Microwave irradiation

microwave irradiation technology was used to immobilize

papain and penicillin acylase (PA) into MCFs.

Single enzyme nanoparticles

Improve enzyme performance and stability under harsh
conditions to decrease cost.
Kim and Grate (2003) have developed armored singleenzyme nanoparticles (SENs)that surround each enzyme
molecule with a porous composite organic/ inorganic network
of less than a few nanometers thick.

Photo-immobilization technology
When photoreactive polymer and horseradish peroxidase
or glucose oxidase are exposed to ultraviolet (UV) light at
365 nm, the reactive nitrene immobilizes the protein
molecules in 10 to 20 min through covalent bonding.
As nitrene has a property of inserting into C-H bond,
the method may find potential applications for
immobilization of biomolecules irrespective of their
functional groups.
Nahar and Kumar (2007) have also immobilized
horseradish peroxidase (HRP) and glucose oxidase (GOD)
onto the photoreactive cellulose membrane by sunlight.

Enzymatic immobilization of enzyme

To avoid the harsh immobilization process and

partial denaturation of enzyme protein
As model proteins, enhanced green fluorescent
protein (EGFP) and glutathione S-transferase
(GST) were tagged with a neutral Gln-donor
substrate peptide for MTG (Leu-Leu-Gln-Gly, LLQGtag) at their C-terminus and immobilized onto the
casein-coated polystyrene surface.

Multi-step immobilization
This process included:
(i) An initial physical or chemical intermolecular
interaction of the enzyme surface with the new
functional groups introduced on the support surface.
(ii) A subsequent intense intramolecular multipoint
covalent reaction between the nucleophiles of the
already immobilized enzyme and the epoxy groups
of the supports.

. Microbial flocculants
1.Definition

Microbial flocculants is a new type water treatment

agent which was extracted and purified from
microbes or its secretion and can be natural
degradation.

2.Type

a) Microbial cells;
b) Microbial cell wall extract;
c) Microbial cells metabolites;
d) products from clones.

3. Ingredients

Glycoprotein;

Polysaccharide;

Cellulose;

Fat;

Proteins

DNA, etc

4.Advantage
Compared to the inorganic polymeric flocculants and
synthetic organic polymeric flocculants, the bioflocculants have following advantages:
Biodegradable;
Efficient;
Non-toxic;
Non-secondary-pollution

5. Microorganisms with flocculation performance

Bacteria;
Actinomycetes;
Mould;
Saccharomycetes.

6. Flocculating mechanism
(1)Bridge connection

Ionic bond, hydrogen bond

mechanism And Van der Waals force

Precipitation
Microbial
flocculants
suspended particle

Bridge connection

6. Flocculating mechanism
(2)Electrical neutralization mechanism;
(3)Chemical reaction mechanism;
(4) Sweep coagulation mechanism.

7. Factors influencing the flocculation effect

(1)Molecular weight and molecular structure of
flocculants;
(2)Dosage of flocculants;
(3)Temperature;
(4)pH;
(5)Metal ions(Ca2+,Mg2+,Mn2+,Fe3+,Al3+).

8. Application in wastewater treatments

(1)High concentration organic wastewater treatment;
Brewery wastewater dairy industry wastewater.
(2)Dye wastewater decolorization;
(3)High concentration inorganic suspended wastewater;
pottery wastewater coal ash wastewater;
(4)Heavy metal wastewater treatment;
electroplating wastewater
(5)Sludge dewatering;
(6) Feed water treatment.