Modern Tools and Techniques to Understand Microbes

459 Pages·2017·10.64 MB·English

by  Ajit Varma

Ajit Varma · Arun Kumar Sharma E ditors
Modern Tools
and Techniques
to Understand
Microbes
Modern Tools and Techniques to Understand
Microbes
Ajit Varma (cid:129) Arun Kumar Sharma
Editors
Modern Tools and
Techniques to Understand
Microbes
Editors
AjitVarma ArunKumarSharma
AmityInstituteofMicrobialTechnology ICAR-NationalBureauofAgriculturally
AmityUniversityUttarPradesh ImportantMicroorganisms(NBAIM)
Noida,UttarPradesh Kusmaur,MauNathBhanjan,UttarPradesh
India India
ISBN978-3-319-49195-0 ISBN978-3-319-49197-4 (eBook)
DOI10.1007/978-3-319-49197-4
LibraryofCongressControlNumber:2017934472
©SpringerInternationalPublishingAG2017
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Foreword
Microbiology is one of the leading branches of modern biology and is interdisci-
plinaryinscopeasitinterfaceswithvariousbasicandapplieddisciplinesofscience,
including but not limited to biology, chemistry, physics, agriculture, medicine,
engineering, and environmental/atmospheric sciences. Consequently, the study of
microbiology has been divided into several subspecialties such as agricultural
microbiology, medical microbiology (bacteriology, virology, mycology, and para-
sitology),food/dairymicrobiology,soilmicrobiology,environmentalmicrobiology,
microbial ecology, and microbial biotechnology, among others. The commonality
among these subspecialties is the study of microorganisms, including prokaryotes
(bacteriaandviruses),eukaryotes(yeasts,fungi,algae,andprotozoa),andarchaea,
whichessentiallyrequirestheuseofoverlappingbasictoolsandtechniques.
Microorganisms interact with both living beings (plants, animals, and host
microbes) and non-living matter (natural and engineered materials and environ-
ments including soil, water, and air) in useful or harmful associations. Conse-
quently, microorganisms are studied for understanding their role in causing
infections/diseasesinhumans,animals,andplantsaswellasformediatingvarious
industrial/biotechnological and natural processes important for sustaining and
improving life on this planet. Except for an estimated 1% of microbes which
cause disease, microorganisms contribute positively to improve various facets of
human life. Notably, microbes have been harnessed for development of new
products including medicines (such as antibiotics, probiotics, antioxidants, and
herbal products), agro-fertilizers, agro-pesticides, biocides and biocontrol agents,
biosurfactants,bioplastics,specialtychemicals,andfermentation-basedproductsof
brewery and dairy/food industries. In the modern-day economic scenario, under-
standingandmanipulationofnewgenesandproteinsdominatetheintendedroleof
microorganisms; however, the cellular environment must be optimized for effi-
ciency,practicability,andproductioncostsinthecreationofnewproducts.Inother
contexts, addressing of pressing topics in modern microbiology, such as the esca-
lating concerns on antimicrobial resistance and the need for characterization of
microbial communities and microbiomes, requires the understanding of OMICs
v
vi Foreword
approaches(genomics/metagenomics,proteomics,andmetabolomics)formicroor-
ganisms,theirhosts,andhost–microbeinteractions.
Historically, human survival and sustainability have depended on the unavoid-
able natural resource for mankind, the plant kingdom. Beneficial interactions
between the plants and microbes have been known to play a crucial role in the
establishmentofthisresourceinagivenecosystem.However,thereisincomplete
understanding of the microbial types critical in such symbioses, although those
developing preferentially or exclusively in roots, like rhizospheric and rhizoplane
microbes, and on leaves, such as phyllospheric and phylloplane microbes, must
playakeyrole.
Fortheaforementionedreasons,ithasbecomeofcrucialimportancetoassemble
the ever-improving and newly emerging methods of modern microbiology in a
comprehensive book (such as this compilation) relevant for immediate use in the
laboratory as well as for pursuing the practical applications of microbiology. The
covered topics essentially include cutting-edge techniques of molecular biology,
immunology, microbiology, and structural biology as applied to modern microbi-
ology;thecomplexityofthesystemsinvolvedrangesfromindividualmoleculesto
prokaryoticorganismstoeukaryoticorganisms,withafocusonbacteria,fungi,and
related plant host aspects. Furthermore, this book has been authored by experts
from different subspecialties of modern microbiology to facilitate the extremely
difficult task of compiling the state-of-the-art methods covering diversified topics
of interest to modern microbiologists. The 27 chapters are indeed outstanding
contributions on important topics in the field. The comprehensiveness should
make the volume equally valuable to students, teachers, and enthusiastic
researchers entering or pursuing the field of microbiology, as a “go-to” reference
book as well as a resource to access expert laboratories and scientists in various
specializations.Consideringthestate-of-the-artnatureofthecontents,thisvolume
isexpectedtobeimmenselyusefulforalongtimetocome.
Overall,thisbookisanexcellentcompendiumonmoderntoolsandtechniques
inthemostusableformonthefrontlinetopicsinmicrobiology.Iwishtoextendmy
congratulationsandbestcomplimentstotheeditorsofthebookfortheiroutstand-
ing efforts in obtaining valuable contributions from a team of global experts. The
book is a timely and cutting-edge compilation on modern aspects of a subject
(microbiology)whichhaswide-rangingmajorimplicationsacrosstheglobeinclud-
ing, but not limited to, the importance for food security worldwide as well as for
ameliorating the socioeconomic condition of communities affected by climate
changeatthegrassrootsofhumansociety.
DepartmentofEnvironmentalHealth JagjitS.Yadav
MicrobialPathogenesisand
ToxicogenomicsLaboratory
UniversityofCincinnatiCollegeof
Medicine
Cincinnati,OH
USA
Preface
It is our consensus that many new methods and approaches are now available to
gain access to the microorganisms residing in an ecosystem that allow better
assessments of microbial diversity. Species diversity consists of species richness,
the total number of species present, species evenness, and the distribution of
species. Methods to measure microbial diversity in any ecosystem can be catego-
rized into morphological/biochemical (phonetic) and molecular (genetic) based
techniques.Someofthemoleculartechniquesprovidecharacteristicsthatareuseful
to discriminate among communities without providing any information about the
types composing the community, e.g., DNA hybridization, percent G+C, DNA
reassociation,restrictionfragmentlengthpolymorphism(RFLP),andcommunity-
levelphysiologicalprofiles(CLPP).AvarietyofDNAfingerprintingtechniquesby
employing polymerase chain reaction (PCR) can be used to rapidly differentiate
closely related environmental stain, viz., arbitrarily primed (AP-PCR), repetitive
(REP-PCR), denaturing/temperature-gradient gel electrophoresis (DGGE/TGGE),
terminal-restriction length polymorphism (T-RFLP), amplified ribosomal
DNA-restriction analysis (ARDRA), etc. Researchers argued that measures of
diversity are pointless because the dynamic nature of the microbial world meant
that communities did not have a characteristic diversity but changed as the envi-
ronmentchanged.
The relationship between structure and function in a community can only be
understood, predicted, and engineered through an understanding of the source of
diversity from which the community is drawn. Based on polyphasic approaches,
prokaryotictaxonomyisgenerallytakenasasynonymofsystematicsorbiosyste-
maticsandistraditionallydividedintothreeparts:(i)Classification,i.e.,theorderly
arrangement of organisms into taxonomic groups on the basis of similarity;
(ii)Nomenclature,i.e.,thelabelingoftheunitsdefinedin(i),and(iii)Identification
of unknown organisms, i.e., the process of determining whether an organism
belongstooneoftheunitsdefinedin(i)andlabeledin(ii).
Species diversity consists of species richness, the total number of species
present, species evenness, and the distribution of species. Methods to measure
vii
viii Preface
microbial diversity can be categorized into two approaches: biochemical-based
techniques and molecular-based techniques. Typically, diversity studies include
the relative diversities of communities across a gradient of stress, disturbance, or
other biotic or abiotic differences. It is difficult with current techniques to study
diversitysincewedonotknowwhatispresentandwehavenowayofdetermining
theaccuracyofourextractionordetectionmethods.Anattempthasthereforebeen
madetoreducetheinformationgatheredbydiversitystudiesintodiscrete,numer-
icalmeasurementssuchasdiversityindices.
Traditionally, diversity was assessed using selective plating and direct viable
counts.Thesemethodsarefastandinexpensiveandcanprovideinformationonthe
active, heterotrophic component of the population. Limitations include the diffi-
culty in dislodging bacteria or spores from soil particles or biofilms, growth
mediumselections,growthconditions(temperature,pH,light),inabilitytoculture
a large number of bacterial and fungal species with current techniques, and the
potentialforcolony–colonyinhibitionorofcolonyspread.Inaddition,plategrowth
favors those microorganisms with fast growth rates and those fungi that produce
largenumberofspores.Alloftheselimitationscaninfluencetheapparentdiversity
ofthemicrobialcommunity.
Abiochemicalmethodthatdoesnotrelyonculturingofmicroorganismsisfatty
acid methyl ester (FAME) analysis. This method provides information on the
microbial community composition based on grouping of fatty acids. It has been
used to study microbial community composition and population changes due to
chemicalcontaminantsandagriculturalpractices.PCRtargetingthe16SrDNAhas
been used extensively to study prokaryote diversity and allows identification of
prokaryotes aswell asthe predictionofphylogenetic relationships. These are two
similarmethodsforstudyingmicrobialdiversity.Thesetechniqueswereoriginally
developedtodetectpointmutationsinDNAsequences.DGGEcanseparateDNA
with one bp difference, whereas TGGE uses the same principle as DGGE except
that the gradient is temperature rather than chemical denaturant (formamide and
urea). DGGE/TGGEhas beenusedtoassessthediversityofbacteriaandfungiin
the rhizosphere, caused by changes of nutrient addition and addition of anthropo-
genic chemicals. While the rRNA genes have been the main target of microbial
diversity studies using DGGE, some researchers have targeted catabolic genes,
such as methane monooxygenase for DGGE analysis. Besides above approaches,
there are some other approaches which can be used for the identification of
microbial diversity. Bacterial phylogenetic classification is based on sequence
analysis of the SSU 16S rRNA molecule or its genes. Over 20,000 SSU RNA
genesequenceshavenowbeendepositedinspecialistr-RNAdatabasessuchasthe
rRNADatabaseProject(RDP).
Itisimportanttostudymicrobialdiversitynotonlyforbasicscientificresearch
but also to understand the link between diversity and community structure and
function. Although methods to study diversity (numerical, taxonomic, structural)
are improving for both bacteria and fungi, there is still not a clear association
between diversity and function. Even if an organism is functionally redundant in
onefunction,chancesareitisnotredundantinallfunctionsandwillhavedifferent
Preface ix
susceptibilitiesandtolerancestoabioticandbioticstresses.Itisgenerallythought
that a diverse population of organisms will be more resilient to stress and more
capable of adapting with environmental changes. Methods to understand the link
betweenstructuraldiversityandfunctioningofbelow-andabovegroundecosystem
needtobedevelopedsothatthequestionofhowdiversityinfluencesfunctioncan
beaddressed.
This volume has been designed to enumerate microbial community in an eco-
system by using phenetic and genotypic approaches. The volume comprises
26chaptersdealingwiththemostrecentandmoderntool,techniques,andprotocol.
Itwaspreparedinsuchamannerthatanynovicecanfollowwitheaseandconduct
theexperimentforconclusiveresults.Weareverygratefultocontributorsfortheir
great efforts in the compilation of such informative book that will be useful to
researchersandfurtherdisseminationofknowledge.Theirpatienceandsupportis
highly appreciative. The volume editors are thankful to Dr. Jutta Lindenborn and
Dr.HannaHensler-Fritton.
AjitVarma
ArunKumarSharma
Contents
1 IntroductiontoModernToolsandTechniquestoUnderstand
Microbes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
PankajGoyal,DikshaBhola,andAjitVarma
2 NovelApproachestoIdentifyandCharacteriseMicroorganisms
inFoodIndustry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
GamzeNurKara,MustafaGuzel,andBulentKabak
3 StrategiesofMassCultivationofMicrobes:Sebacinales. . . . . . . . . 45
DikshaBholaandVarmaAjit
4 BiophysicalPhenotypingasanEssentialToolforUnderstanding
Host–MicrobeInteraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
AvinashP.Ingle,DnyaneshwarRathod,MarianBrestic,
HazemM.Kalaji,andMahendraRai
5 TargetedGeneDisruptionToolsforFungalGenomics. . . . . . . . . . 81
RameshN.Pudake,MayaKumari,BinodBihariSahu,andEramSultan
6 PolyphasicApproachestoCharacterizeMushroomSpecies. . . . . . 103
NeerajShrivastava,VivekKumar,ManojKumar,BingganLou,
AjitVarma,andShwetKamal
7 Function-BasedMetagenomicstoRevealRhizosphereMicrobiome:
AGlimpse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
DevendraK.ChoudharyandAjitVarma
8 BiologicalIndicatorsforSoilHealth:PotentialforDevelopment
andUseofOn-FarmTests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
JwadE.Mahdi,LynetteK.Abbott,NatashaPauli,
andZakariaM.Solaiman
9 MycotoxinBiosyntheticPathways:AWindowontheEvolutionary
RelationshipsAmongToxigenicFungi. . . . . . . . . . . . . . . . . . . . . . 135
AntoniaSusca,AntonioMoretti,andAntonioF.Logrieco
xi