Advance in environmental microbiology College of Environment, Hohai UniversityAssociation Prof.:Deqiang ChenEmail:hjycdq@hhu.edu.cnCell phone:13584018783Office: B324, Hydraulic Tower

ContentMetagenomics (Microbial environmental genomics)Immobilized enzyme systemMicrobial flocculant

.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.

BackgroundConventional 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.

BackgroundBecause 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.

ProjectThe total DNA of all microbial species (meta-genomes ) in a specific environment, not the total DNA of particular microorganism or its cell. ObjectivesDiscover the compositionanddiversityof microbialcommunities of special environmental habitats (e.g., polluted river).

MethodsFlow diagram of a typical metagenome project

Key technology-DNA sequencingFirst-generation sequencing technology Next-generation sequencing technologyThird-generation sequencing technology

1950196019701980199020002010Sequencing Technology Development

1. First-generationsequencing techniqueChemical degradation methodDideoxy chainterminationmethod Automated sequencing

Chemical degradation method (Maxam and Gilbert, 1977) Principle: the sequence of a double-stranded 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 degradation method(Maxam and Gilbert, 1977)

Advantages/disadvantages of Chemical degradation methodRequires 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 chainterminationmethod (Sanger et al, 1977) Principle: the sequence of a single-stranded 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) MethodDNA purification of test sample;A small amount of a dideoxynucleotide (ddNTP); DNA polymerase for chain teminationsequencing (Sequenase)T7 DNA polymerase ; high processivitynegligible or zero 53exonuclease activitynegligible or zero 35 exonuclease activity Single-stranded DNA templateDenaturation after be cloned in plasmidM13 vectorPhagemid vector PCR.

Dideoxy nucleotideNo hydroxyl group at 3 end prevents strand extensionCH2OOPPP53BASE

Sanger Method Sequencing Gel

Sample Output

ComparisonSanger MethodEnzymaticRequires DNA synthesisTermination of chain elongationMaxam Gilbert MethodChemicalRequires DNARequires long stretches of DNABreaks DNA at different nucleotides

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

Thermal cycle sequencingTwo advantagesIt uses double-stranded rather than single-stranded DNA as the starting material;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 electrophoresisHigh separation efficiency;High speed;Very small sample volume needed1-50 nanoliter.

Applied Biosystems3730 DNA AnalyzerHigh-throughput 48-capillary analyzer dramatically increases productivity, while providing application flexibility and significantly lower project costs. Easily upgradable to 96 capillaries.

Next-generationsequencing technology1High-throughputsequencingtechnologyRoche/454FLX Illumina/Solexa Genome Analyzer Applied Biosystems SOLID system 2DNAchiptechnology

1High-throughputsequencingtechnology Roche/454FLXPrinciple Sequencing by SynthesisBy 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/454FLX pyrosequencing

Work flowLibrary construction

Emulsion PCR

Pyrosequencing

Illumina/Solexa Genome Analyzer Principle Sequencing by SynthesisOn 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 flowLibrary preparation;Cluster generation;Sequencing;Data analyzing.

Applied Biosystems SOLID system Principle Sequencing by LigationThe 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 flowLibrary 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.

Genomic DNAFragment libraryMate-paired library1. Prepare library of DNA fragments

2. Emulsion PCR +Templates Enzyme + dNTPsP1-coupled DNA beads~ 100,000 P1 sites per bead

Start with 2 Billion beads per emulsionPolymerase

Mix PCR aqueous phase into a water-in-oilemulsion and carry out emulsion PCRReactor with template, bead and PCR reagentsMineral oil + surfactants

Beads collected following emulsion PCR:Beads with amplified product (~40K PCR products per bead)Beads with no product

3. Beads enrichmentCentrifuge using a Glycerol GradientCaptured beads (+ templates) in supernatantUncaptured beads (no template) in pellet

4. Deposite beads

5. SOLiD 4-color ligation reaction

5. SOLiD 4-color ligation reaction

A56. SOLiD 4-color ligation visualization

C20T15G25A5T 107. SOLiD 4-color ligation Reset

8. SOLiD 4-color ligation (1st cycle after reset)p5

Consequences of 2 Base Pair Encoding

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

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 AExample :

Comparison of three next-generation sequencing technology SOLiD Solexa RocheBases/Run 3GB 1GB 0.1GBCost/Run ~$3000 ~$3000 ~$10000+Lg. Mate Pairs Yes No Yes*QC Yes No NoSlides/Run 2 1 1Samples/Run 16 8 162 base read Yes No No

High throughput enables new applicationsAlternatively, more samples can be run for much lower cost

2DNAchiptechnologyPrinciple Sequencing by hybridizationSequencing by hybridization is a non-enzymatic method that uses a DNA microarray(DNAchip ). 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-generationsequencing techniqueCharacteristic: Single molecular sequencingHeliscope 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 microorganismpreparation.

. Immobilized Enzyme SystemsDefinitionThe 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.

1. Advantages of Immobilized EnzymesAdvantages:(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 EnzymesImmobilized enzyme are employed in many fields.

3. Methods of ImmobilizationImmobilized Soluble Enzymes

EntrapmentThe physical enclosure of enzymes in a small space.

Membrane entrapmentMembrane: nylon, cellulose, polysulfone and polyacrylate.In this technique, microscopic hollow spheres are formed. The spheres contain the enzyme solution, while the sphere is enclosed with a porous membrane.

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

BoundSupport materials:

Covalent BindingIt 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.

SUMMARYThe most suitable support material and immobilization method vary depending on the enzyme and particular application.Binding capacity, which is a function of charge density, functional groups, porosity, and hydrophobicity;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

CharacteristicCarrier Binding MethodsCross-linking MethodEntrapping MethodPhysical adsorptionIonic Binding Covalent BindingPreparationEasyEasyDifficultDifficultDifficultEnzyme ActivityLowHighHighModerateHighSubstrate SpecificityUnchangeableUnchangeableChangeableChangeableUnchangeableBinding ForceWeakModerateStrongStrongStrongRegenerationPossiblePossibleImpossibleImpossibleImpossibleGeneral ApplicationLowModerateModerateLowHighCost of ImmobilizationLowLowHighModerateLow

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. Microwave irradiation

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

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.Photo-immobilization technology

To avoid the harsh immobilization process and partial denaturation of enzyme proteinAs 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, LLQG-tag) at their C-terminus and immobilized onto the casein-coated polystyrene surface. Enzymatic immobilization of enzyme

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. Multi-step immobilization

. Microbial flocculants1.DefinitionMicrobial flocculants is a new type water treatment agent which was extracted and purified from microbes or its secretion and can be natural degradation.

2.TypeMicrobial cells;Microbial cell wall extract;Microbial cells metabolites;products from clones.

3. IngredientsGlycoprotein;Polysaccharide;Cellulose;Fat;Proteins DNA, etc

4.AdvantageCompared to the inorganic polymeric flocculants and synthetic organic polymeric flocculants, the bio-flocculants 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 mechanismsuspended particle

Microbial flocculantsBridge connectionPrecipitation Ionic bond, hydrogen bond And Van der Waals force

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 wastewaterdairy industry wastewater.(2)Dye wastewater decolorization;(3)High concentration inorganic suspended wastewater;pottery wastewatercoal ash wastewater;(4)Heavy metal wastewater treatment;electroplating wastewater(5)Sludge dewatering;(6) Feed water treatment.

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5**After ligation, there is a capping step (with phosphatase) which will remove the 5 phosphate from any un-extended primer. Removing the 5phosphate from strands not extended in the previous sequencing round will render them inactive in the next ligation reaction. This capping step therefore reduces dephasing of template strands. **Wash out unligated probes, and image array using a powerful xenon light source (no lasers). Each bead will light up in 1 of 4 colors (color corresponds to either A, C, G or T). Bead images are recorded with the color of each defined at each cycle. **This is the result of a reset: the extended template strand is melted off and the sequencing template re-exposed as a fresh, clean template and devoid of any noise generated by previous sequencing cycles. In this method, noise is generated by the attrition of strands at each cycle (uncompleted extensions) that serve to reduce template number (and therefore, fluorescence intensity) on each bead, and increases the noise-to-signal ratio of each bead.

The ability to reset is one of the major benefits of this chemistry. A major problem that limits read length of all NGS systems is that as the read gets longer, the signal falls and noise rises, until you can no longer accurately call a base. By resetting the system every 5 cycles we remove all the accumulated noise at each sequencing round.*****

05**This slide explains how to decode a sequence. All you need to decode a base sequence is to know IN this instance we will say we know the first base is an A [important it does not need to be the first base as long as you are certain of one of the bases then the decoding is automatic]Remember that the 2nd base in each decoded pair is the first base of the next pair

In this example we know the first base is an A using the lookup table 1st base A and blue color tells us second base is an ANow moving to the second observed color we know the first base this pair must be an A so if we see 1st base A and green signal 2nd base must be a C and so on .This slide should be self-explanatory. If people want to know how we will get from 2 GB/run -> 3 GB/run, you can say it is partially by going from 25 to 35 bases and partly by increasing the number of beads we place on the slide. The number of beads placed on the slide is only limited by bead recognition software, which we are currently customizing.Carrageenin collagenPorous ceramic diatomaceous earth Polysulfone polyacrylate Desorption Microwave irradiation * Microwave irradiation *