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MicrobiologyOpen. 2017;e487.
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https://doi.org/10.1002/mbo3.487
www.MicrobiologyOpen.com
Received:3January2017
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Revised:13March2017
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Accepted:21March2017
DOI: 10.1002/mbo3.487
ORIGINAL RESEARCH
Characterizing bacterial communities in paper production—
troublemakers revealed
Anita Zumsteg | Simon K. Urwyler | Joachim Glaubitz
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,
provided the original work is properly cited.
©2017TheAuthors.MicrobiologyOpenpublishedbyJohnWiley&SonsLtd.
OmyaInternationalAG,R&DMicrobiology,
Oftringen,Switzerland
Correspondence
AnitaZumsteg,OmyaInternationalAG,
Oftringen,Switzerland.
Email:anita.zumsteg@omya.com
Abstract
Biofilm formation is a major cause of reduced paper quality and increased down time
during paper manufacturing. This study uses Illumina next- generation sequencing to
identify the microbial populations causing quality issues due to their presence in bio-
films and slimes. The paper defects investigated contained traces of the films and/or
slimeofmainlytwogenera,Tepidimonas and Chryseobacterium. The Tepidimonas spp.
found contributed on average 68% to the total bacterial population. Both genera have
been described previously to be associated with biofilms in paper mills. There was in-
dication that Tepidimonas spp. were present as compact biofilm in the head box of one
paper machine and was filtered out by the paper web during production. On the other
hand Tepidimonas spp. were also present to a large extent in the press and white wa-
tersoftwononproblematicpapermachines.Therefore,themerepresenceofaknown
biofilm producer alone is not sufficient to cause slimes and therefore paper defects
andothercriticalfactorsareadditionallyatplay.Forinstance,weidentifiedAcidovorax
sp.,whichisanearlycolonizerofpapermachines,exhibitingtheabilitytoformextra-
cellularDNAmatricesforattachmentandbiofilmformation.
KEYWORDS
biofilms,diversity,indicators,metagenomics,microbialcontamination
1 | INTRODUCTION
Papermanufacturing requiresa largevolume ofwater,which, today,
is permanently recycled at the various stages during the production
process.Assuch,bacterialgrowthand biofilmformationinthe paper
machines are inevitable. These recycled waters are a main cause of
slime production related to the presence of bacteria which leads to
smell,discoloration,andirregularitiesinthepaperformationandweb
breaks(Blanco, Negro,Gaspar, &Tijero, 1996;Kolari,2007).Tomit-
igate these effects the microbial population is continuously treated
with biocides (Blanco, Negro, Monte, Fuente, & Tijero, 2004). But
when bacterial colonization is out ofcontrol, the consequences are
variablepaperquality,increasingdowntime,andhighermaintenance
costs(Kolari,Nuutinen,Rainey,&Salkinoja-Salonen,2003).
Various bacterial species may be responsible for biofilm formation
in paper machines. Deinococcus geothermalisisaprimarycolonizerlead-
ingtothick, synergistic biofilms withdifferentbacilli species (Kolari,
Nuutinen,&Salkinoja-Salonen,2001).Furthermore,Tepidimonasspp.,
belonging to the Betaproteobacteria,were identified directly in the
paperprocessalreadyatthe earlystage ofbiofilmformation(Tiirola,
Lahtinen,Vuento,&Oker-Blom,2009).Several bacterial classesand
genera are known to populate the waters and raw products in paper
machines.Vaisanenetal.(1998)analyzed390cultivableaerobicbac-
teria from process steps and raw materials and demonstrated a vast
bacterialdiversity.Athoroughphylogeneticanalysisof404cloned16S
rRNAgene amplicons was performed by Granhalletal. (2010), who
analyzedtwodifferentpapermillsthatshowedsimilaroverallprofiles
but still unique individual populations. Bacteroidetes (including the
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ZUMSTEG ET al.
genus Chryseobacterium)predominated,butseveralotherPhylawere
identified such as members of the Firmicutes (including Clostridium
sp.),Alpha—andGammaproteobacteria,butnotBetaproteobacteria.
Most of the published research focuses on cultivable bacteria from
smoothlyrunningpapermachines.However,in thisstudyweuse,to
ourknowledgeforthefirsttime,Illuminanext-generationsequencing
to analyze the total bacterial community,including the uncultivable
bacteria,to comparethecommunities presentin the processwaters
of four paper machines at the same mill. The exemplified paper mill in
this report experienced recurring problems in one of the four paper
machines. We identified and compared the bacterial population found
directly in the irregularities on the paper sheets consistently produced
by this machine. Such a thorough process analysis allows us to identify
process steps harboring the problematic microbialpopulations, and
thus,inprinciple,enablingamoreefficient strategytobefollowedin
the future for their control.
2 | MATERIALS AND METHODS
2.1 | Sampling and enumeration of cultivable
bacteria
Allsamples were providedfrom a northernGerman paper manufac-
turer(undisclosed)andarelisted in Table S1. Defective paper sam-
pleswere derivedfrom papermachine1 (PM1).Additionally, waters
(press water, white water, and clear filtrate) were sampled from all
fourpapermachines(PM1,PM2,PM3,andPM4)locatedatthesame
site. Figure 1 represents a simplified scheme of the process and water
circulation, and illustrates the three types of water (press water,
whitewater,andclearfiltrate)sampled.The totalviable count(TVC)
of water samples was determined by plating 0.1 ml of a 10- fold dilu-
tionin phosphatebufferedsaline (PBS)(pH7.4, Sigma-Aldrich)onto
TrypticSoy Broth Agar(TSA) (Sigma-Aldrich).Plateswere incubated
for48h at 30°C priortoenumeration of colony-forming units(cfu).
Counts with 1–9cfu/plate and 10–99cfu/plate were reported as
>102 cfu.ml−1 and >103 cfu.ml−1, respectively. Higher counts were
reported as >104 cfu.ml−1 when colonies remained separated or
>105 cfu.ml−1 when colonies fused to bacterial lawns. No bacterial
viable count was done for paper samples.
2.2 | Propidium monoazide treatment and
DNA extraction
For better accessibility of bacteria in slurries, bacteria were sepa-
rated from turbid insoluble compounds, such as minerals and pig-
ments, using density gradient centrifugation. For this, 1-ml water
samples were overlaid onto 0.3 ml of 1.6- mol.lHistodenz™ (Sigma-
Aldrich) in 2-ml microcentrifuge tubes and centrifuged at 10 000
rcf(relativecentrifugalforce;1rcf=9.81m.s−2)for6minwithslow
deceleration. The upper phase, including the interphase was pel-
letedinanewtubeat 10,000 rcf for 3min. For propidium mono-
azide(PMA)treatment(Nocker,Cheung, &Camper, 2006),thepel-
letwasresuspendedin0.5-mlsterilePBSandPMAaddedtoafinal
concentration of 0.05 mmol.l,placedoniceandexposedtoa500W
halogenlight source for 4mintocross-link the PMAwiththe free
DNA.Thisensures thatDNAfrom deadcellsisnotamplifiedinthe
following PCR reaction. The PMA-treated samples were then pel-
leted again. From these final pellets, DNA was isolated using the
DNeasyBlood&TissueKit(QIAGEN,Hilden,Germany)accordingto
the manufacturer’s instructions.
Toidentifythecausativebacterialcommunityforthedefectpaper,
wealso analyzedthe bacterialpopulation presentat thedefect sites
on the paper sheets. For these paper samples, DNA was isolated
using the PowerSoil®DNA IsolationKit(MOBIOLaboratories,Inc.,
Carlsbad,USA)alsoaccordingtothemanufacturer’sinstructions.
2.3 | Bacterial DNA quantification
DNAwasquantifiedbyreal-timePCRtargetingthe16SrRNAgeneas
describedpreviously(Cliffordetal.,2012).Briefly,ina25-μl final reac-
tionvolumetheprimerpairrtPCR_f(ACTCCTACGGGAGGCAGCAGT)
andrtPCR_r(TATTACCGCGGCTGCTGGC)wereused(Cliffordetal.,
2012) at 500nmol.l, 10% (v/v) of template DNA, and FastStart
SYBR Green Master Mix (Roche cat. No. 4673484001). Using the
ThermocyclerRotorGene(Qiagen) andthesequential thermalprofile
(1)10minat95°C followed by (2) 45 cycles of 20sat95°C,56°C,
and 72°C, the concentration of bacterial DNA was quantified rela-
tivetoaDNAstandardcurveconsistingofaknownconcentrationof
Escherichia coliK1genomes(approx.300016srRNAcopiesperμl).
2.4 | 16S rRNA amplicon sequencing and
data analysis
For library generation the V3 and V4 region of 16S rRNA region
wasamplified by PCR with30cycles from the extractedDNA.PCR
protocol, primer, and library generation were performed exactly as
describedby(Illumina(2013)usingMiSeqReagentKitv3600-cycles
(Illumina, San Diego CA., Cat. No. S102-3003). Data were acquired
using the MiSeqDx System MiSeq and metagenomic analysis of
the raw data was performed using the in-system software MiSeq
Reporter.Fortaxonomicclassification,theGreengenesDatabasefiles
wereused(Mc Donaldetal.,2012).InGreengenesanOTUrefers to
the terminal level at which the sequence is classified.
3 | RESULTS AND DISCUSSION
The exemplified paper mill experienced recurring problems in one of
the four paper machines (PM1). The final paper showed defects in
terms of irregular spots and holes of approximately 1 cm diameter
due to slime deposits in the web during continuous line production.
Consequently,themachinehadtobestoppedandcleanedmorefre-
quentlythantheotherpapermachines(PM2,PM3,andPM4)leading
to costly down time and maintenance. Biofilms have been described
as a reason for such slimes and consequently the resulting paper
defects(Lahtinen,Kosonen,Tiirola,Vuento,&Oker-Blom,2006).
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ZUMSTEG ET al.
To identify the causative bacterial community we analyzed the
bacterial population present at the paper defect site.The DNA was
isolated from the paper samples and the amount of bacterial DNA
quantifiedby 16S rDNAPCR (Table1).Allpaper samples containeda
highamount ofbacterialDNAequivalentto approximately105 to 106
Escherichia coli genomes per cm2.UsingthepurifiedDNA,thebacterial
population was further characterized and quantified by Illumina 16S
rRNA metagenomics analysis (Illumina, 2013). Interestingly, all nine
samplesanalyzedshowedtheexactsamegenusdistributionwithtwo
extraordinarily predominant genera; Tepidimonas and Chryseobacterium
(Figure2).Thesetwogenerarepresentedatleast90%(average95%)of
allclassifiedgenerainallpapersamplesanalyzed,wherebyTepidimonas
contributes byfar the majority with at least 60% (average68%). Out
of the more than 80 Chryseobacteriumspeciesthatexist (Parte,2014),
only one Chryseobacterium soli was found here. For Tepidimonas four
differentspeciesoutoffiveknowntodatewerefound(Albuquerque,
Tiago,Veríssimo,&DaCosta,2006).Tepidimonas has been associated
previously with biofilms in different paper mills (Tiirola etal., 2009).
Particularlyatearlystagesofbiofilmformation,thisgenusrepresented
more than 40% of the population as quantified by length- heterogeneity
PCR analysis of 16S rRNA (Tiirola etal., 2009). The other genus,
Chryseobacterium,andrelatedgenerafromtheBacteroideteshavebeen
identifiedbyT-RLFPinbiofilmsofpapermills(Granhalletal.,2010)and
they have been described to form slimes (Oppong, King, & Bowen,
2003). Ourdata point toward Tepidimonas spp. and Chryseobacterium
sp. as causative agents for the defects in the paper sheets. It was very
FIGURE1 Simplifiedschemeofwatercirculationinatypicalpapermachinedisplayingthethreesamplingpoints:clearfiltrate,whitewater,
and press water. Red arrows indicate sites of biocide addition. Remark: waters from the clear filtrate water tanks of all paper machines are used
for pulping
Wire sectionPress/drying section
Head
box
Pressure
screen
Mixing
pump
Mixing
chest
Disc
filter
Water
tank
Water
tank
Stock preparation
Clear
filtrate
White water
Pulp Coating
rejects
PM
rejects
Additives
(e.g.
starch)
Retains
Water
tank
Press water
Processing and tanks
TABLE1 Quantificationofbacterialcontentsinpapersamplesby
16Sreal-timePCRrelativetoastandardconsistingofgenomicDNA
equivalents of E. coliK1
Paper sample no.
Genome equivalents
cm−2
1 2·106
2 5·105
3 5·105
4 1·106
5 4·105
6 7·105
7 6·105
8 5·105
91·105FIGURE2 Bacterialpopulation,identifiedby16SrRNA
metagenomicsanalysis,atsitesofdamageinthefinalpaperofPM1
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ZUMSTEG ET al.
surprising, though, that the bacterial diversity in the samples was
extremelylow,reducedtomainlythesetwogenera.
Astheproblemwithdefectirregularitiesonthepaperwasmainly
on PM1 (as informed bythe paper mill), we assessed the bacterial
communities in the water circulations of all paper machines to com-
pare them and identify differences. The clear filtrates are well filtered
and used to prepare the rawmaterial (e.g. pulp fiber) and, as such,
may enter the circulation of all four paper machines. The two recycled
watersfromthewiresection(whitewater)andfromthepresssection
(presswater)areturbidwatersthatare,forthemajority,recycledcon-
tinuously for wet end fiber stock preparations.
The samples taken from the recycled waters from the paper
machinesshowedamoderatebacterialcontamination,asdetermined
byculturingmethods andquantitativePCR oftotalDNA(Table2).In
additiontothetotalDNAextractionfromthewaters,wetreatedthe
samplesprior toDNAextractionwith PMAtoassess thefractionof
DNAarisingfromlivebacteria(Table2).ThePMAtreatmentremoved
thefreeDNAfromdeadcellsandreducedtheDNAvaluesmeasured
inallsamplescomparedtothetotalDNAfraction.
ThetotalandPMA-treated(live)DNAsamplesweresubsequently
used to identify and quantify the genera present in the bacterial
community (Figure3). There were only minor differences apparent
betweenthe genus diversitydetermined usingtotalDNAandPMA-
treatedDNA.Theproportionsofthegeneravariedbetweenthetwo
DNApreparationmethods,butthemaingenerashowupinbothsam-
ples,asshowninFigure3.Thenumberofabundantgenera(i.e.atleast
0.5% of all classified genera) correlates between the live and total
DNAsamplewith linearcorrelationcoefficientofR2 = 0.82. Table S2
displays the number of the abundant genera identified in the different
samples as well as the calculated Shannon’s diversity (Shannon &
Weaver,1946),evenness,andstatisticaldataoftheanalysis.
Eventhoughallsamplingswerefromthesame mill,the bacterial
diversities were, nonetheless, unique for each paper machine and
sample type. This confirms previous observations showing the unique
bacterial population in different papermachines and mills (Granhall
etal.,2010).Nevertheless,severalsimilaritiesbetweenthemachines
and samples became apparent.
The most distinct bacterial population appears in the samples
fromPM2, with membersofthe Gammaproteobacteriapredominat-
ing in all waters where the genera Pseudomonas and Azorhizophilus
aredominating. PM1,PM3, and PM4mainly harbormembers ofthe
Bacteroidetes and Betaproteobacteria. Abundant genera besides
Chryseobacterum, Tepidimonas, and Acidovorax which are discussed
below were Clostridium,Pseudomonas, andSteroidobacter. The genus
Pseudomonas is vast and consists of many environmental bacteria
that can be basically found in everyhabitat (Peix, Ramírez-Bahena,
& Velázquez, 2009). Thegenus Clostridium was mainly found in the
white water of PM3. They are anaerobic and endospore forming and
were found in diverse environments (Rodloff, 2005). Of the genus
Steroidobacter foundin PM1, only one species could be found was
Steroidobacter denitrificans. It was isolated from wastewater of a
wastewatertreatmentplant(Fahrbachetal.,2008).
Interestingly, the two genera Chryseobaterium and Tepidimonas,
identified as causative factors forbad paper quality from PM1, could
also be identified in all other paper machines. Especially in the water
cycle of PM3 and PM4 the two genera represented the majority of all
theclassifiedgenera.InPM1, thesetwo generawerea minorityinthe
two immediate recycled turbidwaters (white water and presswater).
TABLE2 Quantificationofbacterialcountsinwatersamples
ATotal viable count [cfu.cm−3]
PM1 PM2 PM3 PM4
Clear filtrate >103>103>103>104
White water >104>105>104>104
Press water >104>104>105>104
BTotal DNA [genome equivalents cm−3]
PM1 PM2 PM3 PM4
Clear filtrate 2·1034·1036·1045·103
White water 3·1043·1041·1054·104
Press water 3·1031·1045·1035·103
CPMA- treated DNA (live) [genome equivalents cm−3]
PM1 PM2 PM3 PM4
Clear filtrate 1·1033·1036·1035·103
White water 1·1042·1041·1043·103
Press water 8·1021·1043·1032·103
(A)Totalviablecountascolony-formingunits(cfu)percm3.(B)TotalbacterialDNA.(C)DNAfromlivebacteria.Forlivefraction,thesampleswerePMA-
treatedpriorDNAisolationandquantificationtoremoveDNAfromdeadbacteria.BacterialDNAwasquantifiedby16Sreal-timePCRrelativetoastand-
ardconsistingofgenomicDNAequivalentsofE. coliK1.
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ZUMSTEG ET al.
Onthe otherhand, theclearfiltrate,which isheavilyreducedin parti-
cles,andrepresentswaterleaving the PM1tobereused forallpaper
machines, showed predominantlythe two troubling genera. Different
possibilities could account for the seemingly contradicting results.
First,PM1experiencedmorefrequentmaintenanceperiodsduetothe
defective paper sheets. These different frequencies could influence
the bacterial population. The nearly complete absence of Tepidimonas
spp.inthewhiteand presswaterwas,however,verysurprising,as the
defect problems remained after maintenance. Even more surprising is
that although Tepidimonas spp. were the most abundant genera in slime
depositsonthepapersheets of PM1, theywere found tobepresent
inall clearfiltrates usedfor theraw materialpreparation(e.g.,pulping)
and abundantly identified in all waters of the smoothly running paper
machines PM3 and PM4. One explanation could be that Tepidimonas,
together with Chryseobacterium, growas compact biofilms and slimes
in PM1 exclusively due to an unknown trigger. This would then lead to
defectpaperdue todepositoftheslime. Whentheseslimesdislocate,
theyremainin thepaperweb.As such,by farthe majorityofbacterial
cellspresentin the biofilm (i.e., Tepidimonassp.)would be filtered out
by the paper web and not enter the white and press water. Such a trig-
ger for film formation could be the identified species Acidovorax,mainly
identified in PM1 white water. This genus was shown to be an import-
antcolonizerofthe headbox adaptedtothe availablecarbon sources
(Kashama,Prince, Simao-Beaunoir,& Beaulieu,2009)and abundantin
activatedsludge communities (Willems &Gillis,2005). It is known for
itsaggregatingabilitiesduetogenerationofanextracellularDNAmatrix
forattachment(Heijstra,Pichler,Liang,Blaza, &Turner,2009).As such
Acidovoraxsp.contributetoyoungbiofilms(Liuetal.,2012).Itisverywell
possible that Chrysobacterium sp. and Tepidimonas spp. require the extra-
cellular matrix produced by Acidovoraxsp.to generatecompactslimes,
and,as such, causethepaper defects.The bacteriacellsof Acidovorax
sp.,however,arenotpartoftheslime.ThisisconsistentwithKolarietal.
(2001)whoshowedthatBacillus sp. uses Deinococcus geothermalis as an
auxiliaryfactortoformbiofilmsin papermachines. Interestingly,some
Bacillus species then emit heat- stable metabolites in order to inhibit the
growth of Deinococcus geothermalis. This could explain that we did not
find Acidovorax sp. in our samples as it was suppressed by the two later
colonizers.AnotherexplanationisthatAcidovoraxsp.,asaprimarycolo-
nizerispresentinPM1duetothemorefrequentmaintenancesandthat
the trigger for the biofilm formation is due to another factor.
Althoughthis study offersan overviewof thelikelycontributory
bacterial factors in slime formation, besides not investigating repli-
catesamples,avitalremainingfactoralsonotinvestigatedhereisthe
substrate and environment upon which the slime is formed. Surface
morphology,surface chemistry,and physicalconditionssuch as nor-
mally stagnant regions in water flows occasionally exposed to shear
flowand/orpresenceofvibrationencouragingdetachment,aswellas
oxygenationand moisture levels, exposure to biocide concentration
variations,etc.,allcontributetotheimpactofbiofilmandslimeinsen-
sitive processes such as papermaking.
FIGURE3 Bacterialpopulation,
identifiedby16SrRNAmetagenomics
analysis,inprocesswatersofthefour
differentpapermachines(PM1-PM4)
located at the same paper plant. For each
sample,thetotal bacterial population and
thePMA-treatedfraction,representingthe
livebacterialpopulation,werequantified
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ZUMSTEG ET al.
As a conclusion we can saythat as Tepidimonas spp. was found
inallpapermachines, thedevelopmentofproblematicslimesisobvi-
ously not only dependent on the mere presence of given bacteria in a
system.Auxiliaryfactorsgeneratingthe necessaryenvironment,pos-
siblyotherbacterialspecies,canbeasimportant.Athoroughprocess
analysis for the bacterial communities present helps to shed light on
criticalfactorscontrollingslimeformation.Inthepresentcase,target-
ing Chrysobacterium sp. and Tepidimonas spp. would bring little success
astheyarepresentinallpapermachines,andevenintheclearfiltrate.
However,theestablishedbacterialpopulationattheprocessstepsis
indicators for the given environmental conditions. Such differences
asseenbetweenthepaper machines (PM1-4) are recommendedas
the points of action to change the environmental conditions for the
good(e.g.aeration,stirringadaption).Thesuccessofmodificationsto
a favorable microbial community and environment can again be fol-
lowed by population analysis.
ACKNOWLEDGMENTS
We thank the unnamed paper mill for sharing samples and information.
CONFLICT OF INTEREST
No conflict of interest declared.
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How to cite this article:ZumstegA,UrwylerSK,GlaubitzJ.
Characterizingbacterialcommunitiesinpaperproduction—
troublemakers revealed. MicrobiologyOpen. 2017;00:e487.
https://doi.org/10.1002/mbo3.487
