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BLBS011-Khanal 9780813823461 July 11, 2008 16:41

Chapter

6

Molecular Techniques in Anaerobic

Biotechnology: Application in

Bioenergy Generation

Srisuda Dhamwichukorn

6.1 Background

Anaerobic microorganisms can serve as cell factories for converting biomass tobiofuel and bioenergy, and to optimize this function, it is necessary to closelyexamine the microbial communities responsible for bioenergy production. Tradi-tional methods of microbial analysis include culture-type techniques such as platingand counting, microscopic enumeration, biochemical analysis, and immunolabel-ing. These techniques, however, cannot provide detailed information about thestructure, function, dynamics, and diversity of microbial communities. Moleculartechniques that can measure cell DNA (deoxyribonucleic acid) and RNA (ribonu-cleic acid) are more direct and robust. Not including viruses, all cells contain DNAthat can be monitored at any point in an organism’s existence, and in fact evenafter the cell has expired. Molecular techniques can characterize genes, proteins(enzymes), and metabolic products associated with bioenergy production at ex-tremely low concentrations without difficulties of culturability. Molecular analysisof microbial cells and communities can, therefore, furnish useful information aboutstructure (who they are), function (what they do), and dynamics (how they changethrough space and time). This chapter focuses on the application of moleculartechniques for microbial bioenergy conversion within anaerobic biotechnology.

6.2 Molecular Techniques in Anaerobic Biotechnology

As molecular techniques evolved in recent decades, applications for anaerobicbiotechnology and bioenergy production (e.g., methane, hydrogen, butanol, and

115Anaerobic Biotechnology for Bioenergy Production: Principles and Applications Samir Kumar Khanal © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82346-1


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116 Anaerobic Biotechnology for Bioenergy Production

Polymers

Proteins

Sugars

Nitrogenase Hydrogenase

Methyl CoM reductase

Butanol dehydrogenase

Pyruvate decarboxylase

Alcohol dehydrogenase

Lipase enzyme Triglycerol hydrolase Triacylglycerol lipase Acylglycerol lipase Hydrolase

Propionate, butyrate, acetate, formate

Biohydrogen

Rhodopseudomonas palsutris Rhodobacter sphaeroides

nifH, nifD, nifK

Methanobacterium themoautotrophicum

mcr

Clostridium acetobutylium bdgA, bdhB

Sacchromyces serevisiae Ethnogenic bacteriaZymomonas mobilis Pdc, adh

Arthrobacter sp,Psudomonas sp. Staphylococcus sp. Chromobacterium sp.

Biomethane Biobutanol Bioethanol Biodiesel

Fatty acid

TriglyceridesAlcohols

Amino acids Pyruvate, citric acid

cycleMetabolism

Polysaccharides Lipids(I) Hydrolysis

(II) Fermentation

Fig. 6.1. Integrated bioconversion system for bioenergy production.

ethanol) have been developed to identify and quantify the responsible microbialcommunities.

All aspects of the biomass-to-bioenergy conversion process illustrated in Fig. 6.1can be evaluated using molecular techniques. Anaerobes including hydrogen pro-ducers, clostridia, methanogens, sulfate reducers, and yeasts carry out the nonox-idative metabolism. Many bioenergy production systems using a nonoxidativemetabolism similar to that shown in Fig. 6.1 are not functionally stable and oftenresult in low yields. Numerous anaerobic studies using molecular techniques havedemonstrated that characterization of the microbial community structure, func-tion, and dynamics aids in better system design, process control, and stability.Successful systems also are those that successfully eliminate inhibiting microbialspecies while maintaining productive microbial species.

Concepts of using molecular techniques in anaerobic biotechnology and itsapplication in bioenergy production, methane, hydrogen, butanol, and ethanol,are described in the following sections.

6.3 Fundamentals of Molecular Techniques

Molecular techniques are often used to examine genes–DNA sequences or DNAfragments encoded for certain cell functions such as cell structure and cell functionfor converting biomass to bioenergy. Culture-independent techniques that are


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Chapter 6 Molecular Techniques in Anaerobic Biotechnology 117

commonly used include polymerase chain reaction (PCR), reverse transcriptionPCR (RT-PCR), denaturing gradient gel electrophoresis (DGGE), and clone-sequencing to target 16S rRNA structural and functional genes. Sequence andphylogenic analyses are often associated with the aforementioned techniques. PCRcan be used as a tool for DNA replication and to selectively amplify a target genesequence relating to bioenergy production. It is possible to amplify genes or DNAof the bacteria and archaeabacteria, and enhance key functional groups that areneeded to assess changes in microbial community structure and abundance, priorto further analysis or characterization. Information is extracted from cell DNAby amplifying (PCR) and characterizing the DNA fragments. After amplificationand characterization, the microbial community structure within the samples canbe identified. Likewise, to obtain information from mRNA or protein, the mRNAfragments are amplified and characterized or the activity of the protein is monitored.The information gained should reveal the microbial community function anddynamics, for example, what genes are turned on or functioning (gene expression).

Replication or amplification of DNA by PCR requires the following:

1. DNA with target sequence2. Two DNA primers3. Nucleotide bases (A, T, G, C)4. DNA polymerase enzyme5. Salts, buffers, and a thermocycler

Amplification of specific sequences from complex mixtures traditionally has re-quired the use of two specific and conserved primer sequences that correspond tostructural (16s rRNA) or functional (e.g., nitrogenase (nif ) for hydrogen produc-tion or methyl CoM reductase (mcr) for methane production) genes.

Using 16S rRNA gene sequences containing portions of both conserved andvariable regions demonstrate the potential benefits for microbial diversity andcommunity analysis. The highly conservative regions facilitate PCR amplificationusing only one universal primer set to amplify the 16S rRNA gene sequences for alleubacteria from environmental samples. The variable regions are different for eachbacterial species, allowing accurate bacterial detection and identification. Fromthese characteristics, the 16S rRNA genes help to determine the genetic diversityof a microbial community as well as to assist in identifying the phylogeneticaffiliation of its members.

6.4 Phylogenetic Analysis

Phylogenetic tree analyses are used to taxonomically classify microbial gene se-quences. These compare the DNA sequences or related 16S rDNA sequences ofselected microbial groups documented in the National Center for BiotechnologyInformation nucleotide sequence databases and in Ribosomal Database Project by


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Table 6.1. Enzymes and genes associated with bioenergy production.

Types of Bioenergy Process Involved Enzymes Genes Organisms

1. Methane (hydrogenas an intermediate)

Methanogenesis Methyl CoMreductase

mcr Anaerobic methanogens

2. Methane (acetate asan intermediate)


mcr Acetoclasticmethanogens

3. Methane (formate asan intermediate)


mcr Methanogens

4. Methane (methanolas an intermediate)


mcr Methanogens

5. Hydrogen (acetic acidas an intermediate)

Dark fermentation Hydrogenase hya, hup,hox

Anaerobic bacteria, e.g.,Enterobactericeae,Bacillus, andClostridium

6. Hydrogen (butylicacid as anintermediate)

Dark fermentation Hydrogenase hya, hup,hox

Anaerobic bacteria, e.g.,Enterobacteria, Bacillus,and Clostridium

7. Hydrogen Photo fermentation Nitrogenase nifH,nifD,nifK

Nonsulfurphotosynthetic bacteria,Cyanobacteria

8. Butanol Fermentation Butanoldehydrogenase

bdh Clostridiumacetobutylicum andClostridium beijerinckii

9. Ethanol Fermentation Pyruvatedecarboxylase,alcoholdehydrogenase

pdc, adh Saccharomyce sp.,Kluyveromyce sp.,Zymomonas mobilis,Clostridium sp.,Thermoanaerobacterethanolicus, Zymobacterpalmae

neighbor-joining and unweighted pair group method with arithmetic mean (UP-GMA) (average linkage clustering) methods contained in the PHYLIP 3.5c softwarepackage (Altschul et al. 1990; Felsenstein 1989; Zhang and Madden 1997).

The enzymes and genes associated with nonoxidative metabolic bioenergy pro-duction are listed in Table 6.1.

6.5 Molecular Techniques for Microbial CommunityStructure Analysis: DNA Fingerprinting, Clone Library,and Fluorescent In Situ Hybridization

Bacterial abundance and diversity in anaerobic processes have been studied usingvarious microbiological approaches. Traditional culturing approaches using count-ing techniques only provide a precursory estimate of bacterial diversity, disregardingthe vast majority of unculturable bacteria because of limitations inherent to the


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Table 6.2. Applications of molecular techniques for microbial community analysis inbioenergy production processes.

Types ofBioenergy Molecular Techniques Utilized Major Findings References

Methane PCR-DGGE and FISH Microbial community changeswith operational time

Diaz et al. (2006) andZheng et al. (2006)

Methane SSCP, qPCR, and FISH Significant effect of volatile fattyacids concentrations onmethanogenic community

Hori et al. (2006)

Hydrogen PCR-DGGE, PCR-RISA,cloning, and T-RFLP

Identify dominant species andcontaminant

O-thong et al. (2007)

Butanol Genomic sequencing,recombinant DNA technology

Improvement in productivity Ezeji et al. (2007)

Ethanol Recombinant DNAtechnology, DNA microarrays

Improvement in productivity Rogers et al. (2007) andSedlak et al. (2003)

growth media. In contrast, molecular methods have the potential to provide a morecomplete assessment of microbial diversity because they provide a means to detectnucleic acids in microbial samples that may not be culturable.

6.5.1 DNA Fingerprinting

DNA fingerprinting techniques include denaturing gradient gel electrophoresis(DGGE), temperature gradient gel electrophoresis (TGGE), and terminal restric-tion fragment length polymorphism (T-RFLP). Examples of microbial communitystructure obtained from various molecular techniques are presented in Table 6.2.These data clearly show that PCR-DGGE has been employed more often thanother fingerprinting techniques (Table 6.3), and this chapter subsequently focuseson DGGE.

6.5.1.1 Denaturing Gradient Gel Electrophoresis Analyses

DGGE is becoming the most widely employed molecular fingerprinting techniquefor investigating microbial diversity in anaerobic studies. The DGGE techniqueis usually accompanied by PCR-amplified partial 16S rDNA fragments, whichrequires conventional DNA extraction and PCR amplification of 16S rRNA gene.

DNA fragments of the same length but with different sequences can be separatedusing DGGE based on partial melting of double-stranded DNA molecules ina polyacrylamide gel containing a linear gradient of denaturant (a mixture ofurea and formamide). As the DNA molecule begins to melt, its molecular volumeincreases, impeding its migration into the gel. Two molecules of DNA of the samelength but differing sequences melt at different concentrations of denaturant. Themolecule melting at a lower denaturant concentration does not migrate as far,thereby electrophoretically separating the molecules.


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By using DGGE, 50% of the sequence variants can be detected in DNA frag-ments up to 500 base pair (bp). Detection can be increased to nearly 100% by theattachment of a GC-rich region (a GC-clamp) to one side of the DNA fragment.This is accomplished by adding a sequence of guanine and cytosine bases to the5′ end of one of the PCR primers. The GC-rich region of the amplified DNAmolecules acts as a high denaturant melting domain, preventing dissociation ofDNA into single strands (Muyzer et al. 1993).

Since broad groups of microorganisms (i.e., multiple genera) are often targetedwith each primer pair, substantial sequence variation in the amplified 16S rDNAis observed. The GC-clamp technology is used to improve separation of sequencevariants, making it possible to effectively compare the microbial communities.

Major bands in the DGGE gels are excised, DNA extracted and reamplifiedby PCR, and rerun on a denaturing gradient gel to verify the purity of the PCRreamplification product. Usually, only those bands accounting for over 5% ofthe total fluorescent intensity of a lane are sequenced. Although the theoreticalmaximum number of bands sequenced from each lane is 20, the DGGE analysisof soil microbial communities has sequenced 5–10 bands, which generally producesthe necessary fluorescence. PCR reamplification products are purified with a PCRpurification kit. Sequencing is performed using an express DNA sequencer. DNAfragments with unlike sequences migrate differently in a DGGE gel, forming bandswith unique positions relative to the loading wells. All major bands occupyingunique positions are then sequenced. Positions and intensities of DGGE gel bandsare recorded using an electrophoresis documentation and analysis system (Jenaet al. 2006).

Numerous microbial diversity studies that have used DGGE to assess anaerobicbioenergy production have been conducted, with the outcome of an improvedunderstanding of microbial diversity being the result (Muyzer et al. 1993).

6.5.1.2 Temperature Gradient Gel Electrophoresis Analyses

TGGE is very similar to DGGE, using the same concepts of DNA separation bydifferential melting of GC-rich segments in the amplified DNA molecules. UnlikeDGGE, in which DNA is melted by urea salt, TGGE melts DNA with elevatedtemperature, as the name implies (Gilbride et al. 2006).

6.5.1.3 Terminal Restriction Fragment Length Polymorphism Analyses

T-RFLP is a useful and rapid method of comparing rDNA (rRNA genes) frombacterial community structures. The process begins with extraction of DNA fromthe microbial communities. Before DNA amplification, fluorescent tags are addedto one or both primers to label the PCR products (amplicons). The PCR prod-ucts are then digested by a restriction enzyme, and the fragments are separated by


BLBS011-Khanal 9780813823461 July 11, 2008 16:41


sequencing gel, a DNA sequencer or capillary electrophoresis (automated DNAsequencing instrument). Only the labeled terminal fragments are detected by theinstrument and generate profiles unique to the microbial community (Liu et al.1997, 1998). T-RFLP has been successfully used in community analysis of anaer-obic methane and hydrogen production systems.

6.5.2 Clone Library

This technique follows extraction of DNA from the microbial community withPCR amplification of 16s rRNA genes. The PCR products of the 16s rRNA genesundergo a cloning reaction that inserts gene sequences into the synthetic plasmids(circular DNA containing specific DNA). The 16s rRNA genes of various specieswithin plasmids are transferred into Escherichia coli cells. The colonies containingthe plasmid DNA (i.e., 16s rRNA gene sequences) are then plated, selected, andanalyzed. The plasmid DNA is then sequenced. The obtained DNA sequencesare identified and characterized using phylogenetic analysis through the use ofsoftware and existing databases. Clone library identification of 16S rRNA geneshas been used to determine the predominant microorganisms in biohydrogen andbiomethane production (Sanz and Kochling 2007).

6.5.3 Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) is a useful molecular technique to quan-titatively identify a microbial community. A fluorescence probe specifically hy-bridizes its complementary target nucleic acid sequence and determines the micro-bial population within the community. FISH with 16S ribosomal RNA-targetedoligonucleotide probes can be used to observe the spatial distribution of archaealand bacterial cells in biological systems (Sanz and Kochling 2007).

Examples of the various molecular techniques used for microbial communityanalysis in bioenergy production (e.g., methane, hydrogen, butanol, and ethanol)through nonoxidative metabolism are illustrated in Table 6.2.

6.6 Molecular Techniques for Functional Analysis

Using molecular techniques to understand and characterize the dominant speciesin a microbial community facilitates control of a microbial-based bioenergy pro-duction process. Equally important to defining the community structure is anunderstanding of function within the microbial community. It is important to ex-amine temporal and spatial factors (detection of the expression of genes) and howthe detected genes work (gene expression). It is possible to investigate the functional


BLBS011-Khanal 9780813823461 July 11, 2008 16:41


ability of genes through the detection of mRNA, in the forms of cDNA, which canbe obtained from the reverse transcription-polymerase chain reaction (RT-PCR)of mRNA. For global gene expression or gene expression profiling, microarraytechniques are employed.

6.6.1 Quantitative Real-Time PCR

Quantitative real-time PCR (qPCR) is used to quantify genes, which assists in theexamination of microbial structure (using 16S rRNA genes) and function (usingfunctional genes encoded for enzymes or functions). This technique has been usedto examine microbial-mediated processes, for example, methane oxidation, sulfatereduction, and ammonium oxidation, to quantify gene expression in environmentalsamples.

6.6.2 Stable Isotope Probing

Stable isotope probing (SIP) has recently been developed and applied to recoverthe labeled DNA fraction from methane and methanol assimilation, ammoniaoxidation, and anaerobic sludges. The technique allows for functional detectionof microbial groups. In addition, microautoradiography (MAR), based on micro-scopic detection of microorganisms consuming radiolabeled biomass or substrates,can be used to measure the metabolic activity of members of diverse and complexmicrobial communities. MAR can be utilized concurrently with FISH (Lee et al.1999; Wagner and Loy 2002).

6.6.3 Microarrays

DNA microarrays have been used almost exclusively for the study of genome-wideexpression or transcriptional profiling within various organisms. Yet, due to theirability to utilize biological reactions in order to detect specific analytical targets(based on nucleic acid hybridization or recognition), microarrays can also be usedas biosensors. Biosensors are fast, sensitive, and selective devices that use biologicalrecognition to detect target analytes that provide either qualitative or quantitativeresults. The exploitation of microarrays as biosensors for bacterial detection andidentification purposes, targeting either structural (e.g., 16S rRNA) or functionalgenes, has begun to produce noteworthy results. Successful studies have focusedon cyanobacteria (Burja et al. 2003), methanogens, and many others. Microarrayshave been developed to enhance understanding of microbial communities in regardto structure, function, and dynamics to enhance bioenergy production (Burja et al.2003; Dhamwichukorn et al. 2004; Liu and Zhu 2005; Zhou 2003).


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6.7 Nucleic Acid Extraction of Anaerobic Cells/Isolates and Sludge

Nucleic acid from anaerobic cells, biomass, or sludge can be extracted for furtherPCR amplification (DNA) or RT-PCR (mRNA), similar to soil and sedimentsamples. DNA/RNA extraction is a critical step in the sample preparation of allmolecular techniques. Different extraction methods provide variable results duringmolecular analysis. There are many commercial kits available such as BIO101, Qia-gen DNA Stool Mini DNA Extraction Kit, MoBio Ultraclean Soil DNA extractionKit, MoBio Power Soil DNA extraction Kit, Epicentre, and the QBiogene FastDNASpin Kit for Soil. Anaerobic sludge and cell DNA and RNA can be extracted byusing one of these kits, the best choice being dependent on sample characteristics.

6.8 Molecular Techniques for Structure and Function Analysis

Concurrent utilization of multiple molecular techniques is a common practice forstudying microbial community structure, function, and dynamics. For example,a combination of FISH and microautoradiography (FISH-MAR), FISH-SIP, andmicroarrays can be used to evaluate microbial structure and function. Figure 6.2illustrates how different molecular techniques can be used to analyze microbial com-munity structure, function, and metabolic transformation separately or together.FISH, DGGE, ribosomal intergenic spacer analysis (RISA), T-RFLP, clone libraries,DGGE, and T-RFLP are the monitoring techniques that have been used most fre-quently for microbial community structure analysis, with DGGE and T-RFLPbeing the procedures most commonly used concurrently with other techniques.

Table 6.3 summarizes information generated by molecular techniques formethanogens, fermentative hydrogen producers, butanol producers, and ethanolproducers through nonoxidative metabolisms.

Table 6.4 presents a summary of currently available molecular approaches, in-cluding microarrays for microbial diversity and community analysis. Among the

Clone library

DGGE

T-RFLP RISA RAPD MAR FISH + microelectrode

FISH SIP FISH + SIP

Microarrays

FISH + MAR

Metaproteomics (FISH + 2D PAGE)

Structure, function, and metabolic transformation

Microbial structure and function

Microbial functionMicrobial structure

Fig. 6.2. Conceptualization of the role of currently available molecular biology and

postgenomic techniques for analysis of microbial community structure, function, and metabolic

transformation.


BLBS011-Khanal 9780813823461 July 11, 2008 16:41

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chSa

ccha

rom

yces

yeas

tsD

NA

mic

roar

rays

Sed

lak

etal

.(2

00

3)

125


BLBS011-Khanal 9780813823461 July 11, 2008 16:41

Tab

le6.

4.S

um

ma

ryo

fm

ole

cu

lar

tech

no

log

ies

for

mic

rob

iald

ive

rsit

ya

nd

co

mm

un

ity

an

aly

sis

.

Pat

tern

Nu

clei

cA

nal

ysis

/T

ech

nolo

gy/

Aci

dF

inge

rA

pp

roac

hE

xtra

ctio

nP

CR

Pri

nti

ng

Ad

van

tage

sD

isad

van

tage

sR

efer

ence

s

Flu

ore

scen

cein

situ

hyb

rid

izat

ion

(FIS

H)

No

No

NA

� Direct

anal

ysis

and

qu

anti

fica

tion

� Nohete

roge

nei

typ

rob

lem

� Suitable

for

targ

etin

gsp

ecifi

cgr

ou

p/s

pec

ies

� Requir

esge

nes

/RN

Aw

ith

hig

hn

um

ber

of

cop

ies

� Limitfo

rto

tald

iver

sity

map

pin

g

San

zan

dK

och

lin

g(2

00

7)

Mic

roar

rays

Yes

No/y

esN

A� Suitab

lefo

rm

app

ing

tota

ld

iver

sity

/pat

tern

anal

ysis

� Further

sequ

enci

ng

not

requ

ired

toid

enti

fygr

ou

p/s

pec

ies

� Highse

nsi

tivi

ty� Fasta

nd

hig

hth

rou

ghp

ut

� Notlab

ori

ou

s

� Donot

reve

alu

nex

pec

ted

spec

ies,

exce

pt

usi

ng

more

pro

bes

Bu

rja

etal

.(2

00

3),

Dah

llof

(20

02

),an

dZ

hou

(20

03

)

Den

atu

rin

ggr

adie

nt

gel

elec

trop

hore

sis

(DG

GE

)Yes

Yes

Mel

tin

g� Suitab

lefo

rp

atte

rnan

alys

isor

mic

rob

ialco

mm

un

ity

div

ersi

ty� Furth

erse

qu

enci

ng

not

requ

ired

,ex

cep

tu

sin

ga

non

het

eroge

neo

us

gen

e

� Canpro

du

cem

ult

iple

ban

ds

from

on

ete

mp

late

wit

hb

ias

PC

Rof

het

eroge

neo

us

gen

es(e

.g.,

16

S)

� Lacksre

solu

tion

� Needs

sequ

enci

ng

for

spec

ies

� Mayhav

eP

CR

bia

sp

rob

lem

s� Cann

ot

actu

ally

ind

icat

esp

ecie

sri

chn

ess

Dah

llof

(20

02

),Je

na

etal

.(2

00

6),

O-T

hon

get

al.(2

00

7),

and

San

zan

dK

och

lin

g(2

00

7)

Tem

per

atu

regr

adie

nt

gel

elec

trop

hore

sis

(TG

GE

)Yes

Yes

Mel

tin

g� Suitab

lefo

rp

atte

rnan

alys

is� Lacks

reso

luti

on

� Needs

furt

her

sequ

enci

ng

� Mayhav

eP

CR

bia

sp

rob

lem

s� Cann

ot

actu

ally

ind

icat

esp

ecie

sri

chn

ess

Dah

llof

(20

02

)

Ter

min

alre

stri

ctio

nfr

agm

ent

len

gth

poly

morp

his

m(T

-RF

LP

)

Yes

Yes

Size

� Suitable

for

pat

tern

anal

ysis

� Oneban

dp

ersp

ecie

s� High

reso

luti

on

� Sensitiv

ed

etec

tion

� Restrict

ion

enzy

me

sele

ctio

nw

ith

un

kn

own

sequ

ence

s� Mayh

ave

PC

Rb

ias

pro

ble

ms

� Cannot

actu

ally

ind

icat

esp

ecie

sri

chn

ess

� Somesp

ecie

sm

aysh

owth

esa

me

len

gth

of

frag

men

ts

Dah

llof

(20

02

)

126


BLBS011-Khanal 9780813823461 July 11, 2008 16:41

Clo

nin

gan

dse

qu

enci

ng

Yes

Yes

NA

� Contain

sla

rger

sequ

ence

s� Provid

esm

ore

posi

tive

ID

� Alarge

nu

mb

erof

clon

esm

ust

be

sequ

ence

dfo

rp

osi

tive

div

ersi

ty� Biasf

rom

PC

R� Seque

nce

sn

eed

toco

mp

are

wit

hea

choth

eran

dli

bra

ries

� Laborio

us

and

tim

eco

nsu

min

g

Dah

llof

(20

02

),G

ilb

rid

eet

al.

(20

06

),an

dSa

nz

and

Koch

lin

g(2

00

7)

Am

pli

fied

rDN

Are

stri

ctio

nan

al(A

RD

RA

)Yes

Yes

Size

� Suitable

for

scre

enin

gcl

on

eli

bra

ries

or

isola

tes

bef

ore

sequ

enci

ng

� Notsu

itab

lefo

rp

atte

rnan

alys

is� Produ

ces

mu

ltip

leb

and

sfo

ron

esp

ecie

s� Cann

ot

actu

ally

ind

icat

esp

ecie

sri

chn

ess

Dah

llof

(20

02

)

Res

tric

tion

frag

men

tle

ngt

hp

oly

morp

his

m(R

FL

P)

Yes

Yes

Size

� Suitable

for

scre

enin

gcl

on

eli

bra

ries

or

isola

tes

bef

ore

sequ

enci

ng

� Notsu

itab

lefo

rp

atte

rnan

alys

is� Produ

ces

mu

ltip

leb

and

sfo

ron

esp

ecie

s

Dah

llof

(20

02

)

Sin

gle-

stra

nd

edco

nfo

rma-

tion

poly

morp

his

m(S

SC

P)

Yes

Yes

Size

� Suitable

for

scre

enin

gcl

on

eli

bra

ries

or

isola

tes

bef

ore

sequ

enci

ng

� Suitable

for

pat

tern

anal

ysis

� Produce

sm

ult

iple

ban

ds

for

on

esp

ecie

s

Dah

llof

(20

02

)

127


BLBS011-Khanal 9780813823461 July 11, 2008 16:41


several recent molecular techniques that have been used for microbial identifi-cation and detection, microarray is one of the most advanced and sophisticatedtechniques.

6.9 Postgenomic Approaches for Bioenergy Research

Although the success of the human genome sequencing projects has been mostnewsworthy, many important bacterial genomes related to bioenergy have alsobeen sequenced successfully in recent years. The postgenomic technologies,that is, bioinformatics (computational biology), transcriptional profile studies(microarrays), translational study (proteomics), and metabolic pathway and prod-ucts (metabolomics), and the potential benefits they could produce, have beenacknowledged as cutting-edge technologies for future biofuel production.

Investigation of microbial biocomplexity as it relates to bioenergy production ispossible with powerful new high-throughput monitoring of genes, proteins, andmetabolites (Fig. 6.3). Understanding the complex interactions of organisms andbiological systems can extend beyond the success of the genomic sequencing oforganisms. In order to further expand and accelerate the accomplishments of thesemultidisciplinary research areas, interdisciplinary collaborative research is essential.The bacterial groups mentioned in this chapter are keys to carbon sequestrationand bioenergy production. Examples of the global benefits produced by theseapproaches and the application of bacteria and yeasts are depicted in Fig. 6.4 (theillustration created by the U.S. Department of Energy (DOE) Genome Program’sGenome Management Information System).

Postgenomics technologies

Bioinformatics (computational biology, in silico)

Microarrays (transcriptional profile studies)

Proteomics (translational studies)

Metabolomics (metabolic pathway and products)

The central dogma of molecular biology

DNA RNA ProteinTranscription (RNA synthesis) Translation (protein synthesis)

DNA

Fig. 6.3. Analogy of postgenomics technologies to the central dogma of molecular

biology.


BLBS011-Khanal 9780813823461 July 11, 2008 16:41

Pay

offs

for

the

Nat

ion,

G

rand

Cha

lleng

es fo

r B

iolo

gy

Dev

elop

bio

fuel

s as

a m

ajor

sou

rce

for

this

cen

tury

Dev

elop

bio

logy

sol

utio

ns fo

r in

trac

tabl

e en

viro

nmen

tal p

robl

ems

Und

erst

and

bios

yste

ms

clim

ate

impa

cts

and

asse

ss s

eque

stra

tion

stra

tegi

es

Und

erst

and

eart

h's

natu

ral c

arbo

n cy

cle

and

desi

gn s

trat

egie

s fo

r en

hanc

ed c

arbo

n ca

ptur

e

YG

G-0

1-05

21R

10

Incr

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bio

logi

cal

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els

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e bi

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eth

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dust

ry

in p

lace

Con

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ute

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nerg

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curit

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reac

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Fig.6.4.

DO

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BLBS011-Khanal 9780813823461 July 11, 2008 16:41


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