Research Article Rheology and Microbiology of Sludge from a Thermophilic Aerobic Membrane ...

20 Pages·2017·4.05 MB·English

Hindawi
Journal of Chemistry
Volume 2017, Article ID 8764510, 19 pages
https://doi.org/10.1155/2017/8764510
Research Article
Rheology and Microbiology of Sludge from a Thermophilic
Aerobic Membrane Reactor
AlessandroAbbà,1MariaCristinaCollivignarelli,1SauroManenti,1RobertaPedrazzani,2,3
SaraTodeschini,1andGiorgioBertanza3,4
1DepartmentofCivilandArchitecturalEngineering,UniversityofPavia,ViaFerrata1,27100Pavia,Italy
2DepartmentofMechanicalandIndustrialEngineering,UniversityofBrescia,ViaBranze38,25123Brescia,Italy
3UniversityResearchCenter“IntegratedModelsforPreventionandProtectioninEnvironmentalandOccupationalHealth”
(MISTRAL),DepartmentofMedicalandSurgicalSpecialties,RadiologicalSciences,andPublicHealth,
UniversityofBrescia,VialeEuropa11,25123Brescia,Italy
4DepartmentofCivil,Environmental,ArchitecturalEngineeringandMathematics,UniversityofBrescia,
ViaBranze43,25123Brescia,Italy
CorrespondenceshouldbeaddressedtoGiorgioBertanza;[email protected]
Received 13 April 2017; Revised 21 June 2017; Accepted 27 June 2017; Published 28 August 2017
AcademicEditor:NicolasRoche
Copyright©2017AlessandroAbba`etal. This is an open access article distributed under the Creative Commons Attribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
A thermophilic aerobic membrane reactor (TAMR) treating high-strength COD liquid wastes was submitted to an integrated
investigation,withtheaimofcharacterizingthebiomassanditsrheologicalbehaviour.Theseprocessesarestillscarcelyadopted,
alsobecausetheknowledgeoftheirbiologyaswellasofthephysical-chemicalpropertiesofthesludgeneedstobeimproved.Inthis
paper,samplesofmixedliquorweretakenfromaTAMRandsubmittedtofluorescentinsituhybridizationfortheidentificationand
quantificationofmainbacterialgroups.Measurementswerealsotargetedatflocsfeatures,filamentousbacteria,andmicrofauna,in
ordertocharacterizethesludge.Thestudiedrheologicalpropertieswereselectedastheyinfluencesignificantlytheperformances
ofmembranebioreactors(MBR)and,inparticular,oftheTAMRsystemsthatoperateunderthermophilicconditions(i.e.,around
∘ −1
50C)withhighMLSSconcentrations(upto200gTSL ).Theproperdescriptionoftherheologicalbehaviourofsludgerepresents
ausefulandfundamentalaspectthatallowscharacterizingthehydrodynamicsofsludgesuspensiondevotedtotheoptimization
oftherelatedprocesses.Therefore,inthisstudy,theeffectsonthesludgerheologyproducedbythebiomassconcentration,pH,
temperature,andaerationwereanalysed.
1.Introduction BOD(biologicaloxygendemand),nitrogenandphosphorus
compounds, surfactants, oils and greases, and suspended
Themonitoringofbiologicalwastewatertreatmentprocesses, solids. The biological process is based on the activity of
whatever its final goal is (assessment of compliance with differentpopulationsoforganismssetatgrowinglevelsofthe
effluent legal emission values, evaluation of specific stages detritusfoodweb.Therefore,anytoolsenablingustoidentify
efficiency, calculation of the maximum treatment capacity andmeasuretheactivityoftheseorganismsprovidevaluable
and comparison with the actual treated load, mass balance informationforimprovingandstrengtheningtheoperation
for conventional and emerging pollutants, estimation of ofwastewatertreatmentplants([1–4]amongothers).
energy consumption, etc.), is based on physical, chemical, The removal capacity of specific pollutants can be
andbiologicalanalyses.Guidelinesandtechnicaldocuments assessedbyperformingthemeasurementofmetabolicactiv-
define the main parameters together with the proper ana- ity,forexample,bymeansofammoniauptakerate(AUR)and
lytical methods in case of conventional and/or nonspecific nitrateuptakerate(NUR)tests[5–9]andenzymaticactivity
pollutants,suchastheCOD(chemicaloxygendemand),the quantification[10,11].Besides, themicroscopicobservation
2 JournalofChemistry
of floc characteristics and the identification and quantifica- As the particulate concentration increases, the sludge
tionoffilamentousbacteriaallowustohighlightdisturbances deviates from Newtonian behaviour. For a non-Newtonian
within their community and to foresee the occurrence of liquid,themeasuredviscositybecomesdependentontherate
specificdysfunctions,forexample,sludgefoaming,bulking, of shear, and the so-called apparent viscosity is introduced
andsolidswashout[4,12–15].Ecologicalfeatureslikespecific whichisdefinedastheratiobetweenshearstressandshear
indexes(SludgeBioticIndex(SBI),designedby[1]andSludge rateatagivenpointoftheflowcurve.Theapparentviscosity
Index (SI), proposed by Grupo de Bioindicacio´n de Sevilla allows us to interpret the external and internal interaction
(GBS, Spain) [16–19]) enable plant managers to tune the forcesbetweentheconstituentsofthesludgethatinfluences
operationparameters,suchassludgeage,hydraulicretention itsrheologicalbehaviour[51].Ahighlyconcentratedsludge
time,anddissolvedoxygenconcentration,inordertomain- exhibitsyieldstressthatisthecriticalstresstriggeringplastic
tainoptimumconditionsforthesludgebioticcomponents. deformation of the material continuum. Under the yield
Scientific literature reports hundreds of applications of stress,nosignificantflowcanbedetected.Accordingto[49],
conventional and innovative biological tools, as well as theyieldstresscanberelatedtoreciprocalinteractionsamong
examplesofinvestigationsbasedonecologicalcriteria[9,20– the suspended solid particles opposing to the deformation.
27]. Almost all the biological processes have been explored Therearetwotypesofyieldstress:staticanddynamic.Static
intermsofbiomasscomposition(bacteria,protozoa,meta- yieldstresscanbemeasuredinanundisturbedsample,while
zoan,andfungi)andactivity:conventionalactivatedsludge, dynamic yield stress refers to a perturbed sample and is
attached biomass, membrane reactors, and treating either extrapolated from its equilibrium flow curve as shear rates
municipal [27–34] or industrial wastewater [35–39]. Like- approach zero. It has been reported that the static yield
wise, identification and quantification of microorganisms stress was not observed at a mixed liquor suspended solid
operating under different conditions (aerobic, anoxic, and (MLSS) concentration equal to 16g/L, probably because at
anaerobic)havebeenreported[25,40–46]. this concentration the static yield stress almost equals the
Rheological properties are crucial for activated sludge dynamicyieldstress[51].
applications in wastewater treatment plants, since they Therheologicalcharacterizationofnon-Newtonianfluids
severely impact the flow behaviour and many aspects that leads to practical difficulties; different measurement proto-
interferewithprocessperformanceandenergyconsumption, cols and devices (i.e., capillary rheometers as in [66] and
for example, sludge pumping, bioreactor hydrodynamics, rotationalrheometersasin[67])havebeenadoptedtostudy
mass transfer efficiency of aeration systems, sludge-water therheologicalpropertiesandawidevarietyofmodelshave
separation via settling, and filtration [47–54]. Due to the been proposed. Widely used non-Newtonian relationships
observed significant impact of rheology on different pro- betweenshearstressandshearrateforactivatedsludgearethe
cesses,agoodknowledgeoftheactivatedsludgerheological Ostwald de Vaele model, the Bingham model, the Herschel
behaviourisofgreatimportanceinbothoptimizingdesign andBulkleymodel,andtheCassonmodel[49,68].Allthese
and operation of sewage treatment plants [55]. Appropriate modelsbelongtothecategoryoftime-independentrheolog-
treatment of wastewater as a result of efficient design and icalmodelsforwhichtherelationbetweenstressandapplied
operationwouldhelpinenvironmentprotectionandpreser- shearratedoesnotdependuponthedurationofshearing.
vation[56].Thissignificancemotivatestherecentexperimen- The properties of activated sludge, such as MLSS con-
tal investigations and mathematical modelling on activated centration, particle size distribution and shape, interaction
sludgerheologyandontheimpactofrheologicalproperties among particles, flocculation ability, and surface physico-
on operating parameters for conventional activated sludge chemical characteristics, all have effects on rheology and,
plants (e.g., [57–59]) and for membrane bioreactors MBRs thus, on the kind of model most efficient in describing the
(e.g.,[51,60,61]). sludgerheologicalprofileandonthevaluesofthepreferable
Basically, rheology of a sludge is defined by its viscous modelparameters[49,69].
characteristics,whichcanbedeterminedbytherelationship A number of studies demonstrate that also temper-
between shear rate and shear stress, obtained through a ature significantly affects the rheological characteristics:
rheological measurement (which imposes either shear rate sludgebecomesprogressivelymorefluidasthetemperature
or shear stress [62–64] and measures alternatively these increasesduetothermal[65,70,71].Thesestudiesalsoprove
parameters). The observed relationship is strictly related thattemperatureirreversiblymodifiesthesludgestructure.
to the characteristics of the sludge, such as the relative Thispaperreportstheresultsofanintegratedstudyper-
concentration of water and suspended matter due to the formedonthesludgederivingfromarealscaleTAMR,sup-
natureofthewastewaterandtothetreatmentprocessthatis pliedwithpureoxygen(beingtheaveragedissolvedoxygen
−1
subjectedto[65]. concentrationofthemixedliquorequalto2.3mgL ,while
−1
Atlowerconcentrationofthesuspendedmatter,sludge’s minimum and maximum values equal 0.2 and 15.0mgL ,
behaviour can be reasonably approximated to a Newtonian resp.)locatedatafacilityforthetreatmentofsolidandhigh-
fluid characterized by a linear relation between shear stress strengthliquidwastes.Thesludgehasbeencharacterizedin
andrateofdeformation,withtheproportionalitycoefficient terms of microbiological and rheological features. Actually,
being the fluid viscosity. The measured viscosity is rather the authors found, previously, links between the amount of
independentfromshearrate,atgivenvaluesoftemperature filamentousbacteriaandzoogleaclustersandviscosityvalues
and pressure. The flow curve is therefore a straight line ofmixedliquorsfromconventionalactivatedplantstreating
throughtheoriginofthecoordinateaxes. municipalwastewater,thussuggestingtheuseofrheological
JournalofChemistry 3
Chemical-physical Disposal
Aqueous
waste treatment
NF concentrate
TAMR
Thermophilic Ultrafiltration Nanofiltration
biological reactor (UF) unit (NF) unit
UF concentrate
NF permeate
Adsorption on Discharge
Ammonia
Sludge activated carbon in public
stripping
treatment (if necessary) sewer
Water line
Sludge line
Figure1:Processschemeofthestudiedfacility.
tools to control dysfunctions caused by the proliferation of Beforeenteringthebiologicaltank,metalsareremovedby
specificmicroorganism[72]. meansofachemical-physicaltreatmentwhichleadstotheir
∘
TAMRsoperateat45 Candareprofitablyappliedforthe precipitationasammoniumsalts,hydroxides,andphosphates
treatment of high-strength wastewater deriving from food (pHequalto11units).TheTAMRsurfaceis267m2wide;net
processing, pulp, and paper and pharmaceutical factories volumeisabout1000m3.
[73–76]. Recently, their extension to sludge treatment has Pure oxygen is supplied through static mixers, yielding
beenexplored[77].Ineffect,anaerobicthermophilicbiomass oversaturation of the sewage, which is recirculated within
ischaracterizedbyaloweryield,henceadecreaseinspecific them. Ultrafiltration unit includes two parallel lines con-
sludge production (down to 0.02kg SSV/kg CODremoved). sisting in a feeding pump, a recirculation pump, and three
Furthermore, other valuable advantages consist in faster channelsofceramicmembranes(eachcontaining99tubular
chemical reaction rates, increased organics solubility, high membraneshaving25channels).Poressizeallowsretaining
process stability, that is, the prompt activity recovery after molecules larger than 0.3 microns and with a molar mass
operational changes, capability of degrading high salinity higher than 300kDa. Minimum and maximum operation
andbiorecalcitrantwastewater,andpossibilityofintegrating pressurevaluesare3and5bar,respectively.
the thermophilic stage, as a pretreatment into a combined The average concentrations of pollutants inlet to the
scheme, thus improving the removal extent of organic sub- TAMR are 25,000mgCODL−1, 1200mgTNL−1, and
stances[76,78,79].Nevertheless,despitetheaforementioned −1
700mgTPL . The TAMR performances, in terms of
advantages,thermophilicprocessesarestillrarelyadoptedat
COD, TN, and TP removal yields, are 78%, 80%, and 90%,
therealscale[80,81].
respectively. The high removal yields for TP, as reported by
This research was aimed, therefore, at exploring the
[79],areduetothechemicalprecipitation:thisphenomenon
rheologicalandmicrobiologicalfeaturesofTAMRbiomass,
couldbeduetothedosageoflime(inthechemical-physical
which remain almost obscure, in spite of the increasing
treatmentbeforetheTAMR),butthemostimportantaspect
knowledgegainedaboutitsexcellentperformances.
is ascribed to the aeration of reactor that promotes the
phosphoruscrystallization.
2.MaterialsandMethods
2.2. Characterization of Sludge. Samples taken from the
Asyntheticdescriptionofthestudiedwastetreatmentfacility, TAMRweresubmittedtoinvestigationsaimedtodefinetheir
together with sampling and analytical procedures, is pre- microbialprofilesandrheologicalbehaviour.
sented.
2.2.1.MicrobialCommunityInvestigation. Mixedliquorsam-
2.1. The Waste Treatment Facility. The facility is located in ples were taken from TAMR and freighted immediately to
∘
NorthernItalyandtreats60,000t/yofsolidandliquidhigh- thelaboratoryat4 C.Afteraddingabsoluteethanol(Sigma
strength COD wastes (except mutagens and carcinogens); Aldrich), (1:1) mixed liquor was pipetted onto a glass slide
∘
sincefifteenyearsago,thistechnologyhasbeeninvestigated and allowed to dry at 46 C for 15 minutes; afterwards,
indetailby[80]. samples were submitted to fluorescent in situ hybridization
Figure 1 depicts the scheme of the unit fed with liquid (FISH), according to prescriptions of VIT(cid:2) (Vermicon AG,
wastes: the main phase consists in the TAMR (see [80] for Munich, Germany) gene probe technology, based on fluo-
acompletedescription). rescent labelled DNA probes. Only viable microorganisms
4 JournalofChemistry
(a) (b)
Figure2:Experimentalapparatus:coaxialcylinderCC25DIN(a)andrheometerRC20(b).
are identified, quantified, and even visualized in their envi- radius: 12.5mm; internal radius of the measuring cylinder:
ronment. The major advantage of this method is that the 13.56mm.
analysis is based on the stable genetic material of the cells Theworkingprincipleoftheinstrumentisbasedonthis
and not dependent on phenotypic features which may be aspect: the sliding of the sludge in the cavity between the
quitevariableamongbacteria.Moreover,asthetargetofthe coaxial cylinders, due to the spindle rotation with a fixed
geneprobesistherRNAofthecells,onlyviablebacteriacan share rate, while the external cylinder is held, requires a
be detected. As fluorescent signal intensities are correlated torquethatismeasuredbytheinstrument.Severalrotational
with the rRNA content within the cells, a further result tests with different controlled shear rate (CSR) were per-
on the physiological activities of the detected cells can be formedtoobtaintherheologicalequation:
−1
obtained. The detection limit is about 1000 cellsmL . A
processofimageanalysisenabledthequantificationofsingle 𝜏=𝑓(𝛾)̇ , (1)
populations,bycomparingthesignalsofeachgroupwiththe
fluorescenceemittedbythealltheviablemicroorganisms. where𝜏istheshearstress[Pa]and𝛾̇isthesharerate[s−1].
Besides, observation of floc features and identification Thetemperaturewascontrolledbytheuseofwaterina
and abundance assessment of filamentous bacteria were beakerplacedonaheatingmagneticstirrer(Figure2(b)).
performed according to [12]. In particular, the analyses InTable1,thedetailsofrheologicaltestaresummarized
were performed by using a Zeiss Axiostar Plus microscope (the pH and temperature values represent the average data
underphasecontrastillumination.Rawsampleswerefirstly recordedduringthetests).Thetestswerecarriedoutontwo
examined:afterwards,duetotheirconspicuousdensity,they samples of thermophilic biomass with different concentra-
weredilutedwiththesupernatant(1:10and1:100)inorder tionsoftotalsolids(TS).ThepHvaluesarebetween6.6and
toenablethevisualizationofsingleflocs.Theirmorphology 9.1(sulphuricacidandsodawereused)andthetemperature
∘
wasregistered(accordingto[12]),byconsideringtheshape iscloseto45 C.
(irregular, round), the firmness, and the mean diameter. The biomass samples withdrawn from the TAMR were
For this purpose, 10 microscopy fields were considered as delivered to the laboratory within 120min after sampling.
∘
replicates; flocs diameter was measured by employing a They were stored (for 180min) at 45 C under different
graduated slide as a reference. This tool was used also for conditions:afirstsubsamplewasnotsubmittedtoaeration;
measuring the length of filamentous bacteria. Likewise, the a second subsample was kept in aeration conditions by an
−1
effectsoffilamentsonflocstructure,theirabundance(with aircompressoratlabscale(1Lairmin );onlyforthetestson
respect to each floc), and the presence of other important sampleB,athirdsubsamplewasaeratedwithanairflowof
−1
factors, such as free cells in suspension, inorganic/organic 0.5Lairmin .
particles,andzoogleas,wererecorded. Each rheological test was performed with fixed shear
Mixedliquorsampleswerealsosubmittedtotheanalysis rates that were maintained for 300 seconds; shear rate was
of the microfauna for the calculation of the Sludge Biotic increasedstepbystepasreportedinTable1.Shearstressand
Index(SBI)accordingto[1](microscopicobservationunder apparentviscositywererecordedevery30s(tendatasetsfor
directillumination;magnification:100x). eachsharerate).
BoththesludgesamplespresentedinTable1weretested
−1
2.2.2. Rheological Tests. The rheological experiments were starting from an imposed shear rate of 100s with a step
−1 −1
carriedoutusingrheometerRC20(RheoTec)withaconfigu- increment of 100s (or 50s in some cases). Anyway,
ration CC25DIN of coaxial cylinders (Figure 2(a)): spindle the adopted rheometer was unable to resolve the shear
JournalofChemistry 5
Table1:Rheologicaltestsperformed:operativeconditions.
Operativeconditions Shearrate
Test#
AveragepH Averagetemperature[∘C] Aeration [s−1]
1 6.7 44.5 N 700,800,900,1000
2 6.7 45.2 Y 700,800,900,1000
SampleA 3 7.7 45.1 N 700,800,900,1000
−1
(150gTSL ) 4 8.3 45.3 Y 750,800,900,1000
5 9.0 45.1 N 750,800,850,900,1000
6 9.1 44.9 Y 800,850,900,1000
7 7.0 45.4 N 500,600,750,900,1000
8 6.7 45.3 1/2 500,600,750,900,1000
9 6.6 45.0 Y 500,600,750,900,1000
10 7.8 44.9 N 500,600,750,900,1000
SampleB
(190gTSL−1) 11 8.3 45.2 1/2 500,600,750,900,1000
12 8.4 45.3 Y 500,600,750,900,1000
13 8.9 45.0 N 500,600,750,900,1000
14 9.0 45.3 1/2 500,600,750,900,1000
15 9.1 45.0 Y 500,600,750,900,1000
N:noaeration;1/2:limitedaeration(0.5Lairmin−1);Y:fullaeration(1Lairmin−1).
−1
stress for values below 700s for the lower concentration
−1 −1
(150gTSL ) and, respectively, for values below 500s for
−1
thehigherconcentration(190gTSL ).
It is worth noting that in the present study a limited
numberofshearrateshasbeeninvestigated(i.e.,fourvalues
fortests1–4and6,andfiveincaseoftests7–15).Thisaspect,
inprinciple,mayrepresentaninherentlimitationofthework
that,however,hasthemerittoprovideafirstinsightintothe
rheologicalbehaviourofahighlyconcentratedthermophilic
sludge. Furthermore, the preliminary findings (see Sec-
tion3.2)showthatthetrendoftheflowcurvesexhibitsacer-
taindegreeofreliability,asconfirmedbytheregressioncoef-
ficientdisplayedinTable3.Anyway,amorerefinedinvesti- Figure3:Micrographoftherawsample(magnification:100x).
gationbyincreasingtherangeofexploredshearratesandthe
amountofsampleswillbeusefultoclarifysomeissuescon-
cerningtheeffects ofaerationonthe rheologicalbehaviour
andwillbeincludedinthenextstepsoftheresearch.
and diffuse. Filamentous bacteria abundance belonged to
classI(few,whichmeansthatfilamentsareobservedonlyin
3.ResultsandDiscussion
anoccasionalfloc),accordingtothedistinctionproposedby
[12],whoidentifiedsixdifferentcases,basedontheaverage
This chapter gathers the information obtained from the
number of filamentous bacteria located in a single floc.
microbiological and the rheological analyses and sets them
Neither zoogleas nor spirochaetes were observed. Likewise,
inthescientificliteraturecontext.
ProtozoaandMetazoawerecompletelyabsent(asnoticedby
[78]): therefore, the Sludge Biotic Index (SBI) could not be
3.1. Microbiological Features of Thermophilic Sludge. The calculated.
sludge appeared extremely thick, due to the great sus- Thesefindingstallywiththeoutcomereportedinthesci-
pended solids concentration maintained in the TAMR entificliterature:apoor(orevenlacking)flocculationisusu-
−1
(150–200gL ). Figure 3 is a micrograph (100x magnifica- allyseentooccurunderthermophilicconditions[78,82–84].
tion)ofthemixedliquor,characterizedbyahugeamountof Abouttheamountoffilamentousbacteria,however,[83]
inorganicparticles(clustersofsaltsandhydroxides).Flocsare observedfilamentousabundancesascribabletoclassIV(very
almostabsentandbacterialcellsarefreeintheliquor. common) and class V (abundant), while [85] reported two
Basedonthecharacterizationrulesof[12],themeanfloc cases,wherefilamentsreachclassIV,withtheirgrowthbeing
diameterwas<150𝜇m.Flocscouldbeclassifiedasirregular inverselyproportionaltothehydraulicretentiontime.
6 JournalofChemistry
Thepaper[78]putsforwardseveralhypothesestoexplain
the lack of flocculation: firstly, the absence of floc form-
ing microorganisms (such as zoogleas, not found also in
the present case, as previously mentioned); secondly, the
impossibility to reach the physiological state enabling the
flocculation; finally, the establishment of medium condi-
tions interfering with flocculation and coagulation. Further
assumptions are made by authors in [84] who ascribed
the scarce flocculation to the high shear sensitivity of the
thermophilicfloc,whichbecomespronetoerosion;theyalso
suggestapossibleinfluenceofdecreasedcellhydrophobicity.
Table2presentstheresultsofFISHanalyses,whileFig-
ure4highlightsthepercentagesofdetectedtaxaoverthetotal
bacterialpopulation.Onlythreemajorbacteriagroupswere
found with higher shares (Betaproteobacteria, Gammapro-
teobacteria,andCytophaga-Flexibacter).Twofurthermajor
groups(DeltaproteobacteriaandChloroflexi)werequantified Alphaproteobacteria Chloroflexi
with very low shares and the other groups were com- Deltaproteobacteria Gammaproteobacteria
pletelyabsent,indicatingverylimitingconditionsfortypical Betaproteobacteria Cytophaga-Flexibacter
wastewater treatment plant bacteria. The FISH analyses
Figure4:Piechartofthedetectedtaxashares.
confirmed the nearly total absence of filamentous bacteria.
Furthermore, physiological groups like nitrifying bacteria
were completely absent. Such a profile is quite unusual
Alphaproteobacteria were detected with a share of only
for a mesophilic activated sludge and can be attributable
1%andappearedcompletelyascoccus-shapeduniformcells.
to temperature conditions and wastewater characteristics,
Shares of up to 20% of this group are quite normal for
whichtriggeraveryspecialcompositionofthepopulation.
industrialactivatedsludge.
Actually, the above-mentioned factors, together with plant
Members of the Chloroflexi genus were measured with
runningstrategies,canbenowadayslinkedwiththegrowth
a very low share of<1% in the sample. The single filaments
of specific bacterial populations and the achievement of a
hadalengthofabout50𝜇m.Inwastewatertreatmentplants,
particular physiological state [86–88]; on the other hand,
10–20%ofthisgrouparequitecommon.Chloroflexibacteria
thisiseven moretrueinthecase study, where temperature
∘ inWWTPsshowedincreasedsharesoverthelastyears;they
is constantly maintained at 45 C, and the influent sewage
cause bulking and foaming events if huge abundances are
consistsinhigh-strengthCODwastes.
reached.
Betaproteobacteria were the dominant group in the
Deltaproteobacteria, including most sulphate-reducing
sludge sample. They were detected with a remarkable high
bacteria,wererepresentedasuniformsinglecells,withashare
share of 77% of the total viable bacteria. No filaments or
of <1%. Values of up to 8% for this group can be found in
ammonium oxidizing bacteria belonging to this class were
themixedliquorofplantstreatingindustrialwastewater.All
observed,asfoundby[78].Almostthecompletepopulation
other main bacteria groups including filamentous bacteria
was formed by members of the 𝛽1-group. All organisms of
werenotdetected.
this group showed bright fluorescence signals indicating a
High values for total cell counts (dead and alive) were
good physiological status of the cells. Levels up to 25% of determined,with2.5⋅1010mL−1 andtotalviablecellcounts
thisgroupareconsideredasnormalinindustrialwastewater with5.0⋅109mL−1,whichislargerthanthetotalviablecell
treatmentplants.Gammaproteobacteriaweremeasuredwith
countsofupto1–3⋅109mL−1 usuallydetectedinindustrial
an unusual high share of 15% in comparison to typical
wastewateractivatedsludgetreatmentplants.
wastewatersludgevalues.Ineffect,normally,thisgroupdoes
Figures 5–7 clearly show the structure of the sludge,
notexceedashareof10%.Here,theyappearedmainlyasthin
consistingbasicallyinclustersofsaltsandhydroxidesandfree
long rod-shaped bacteria with middle fluorescence signals
cells.
indicatingamoderatephysiologicalactivityofthecells.No
characteristicfilamentsofthisgrouplikeThiothrixorEikel-
boomtype021Nwerefound.Theauthorsin[88]postulated 3.2. Rheological and Statistical Analysis of Activated Sludge.
thatthesecoccipossessweakflocculationproperties(atleast Several analytical models have been proposed to mimic
asubgroupofGammaproteobacteria):alsotheirabundance the rheological behaviour of biological sludge, each with
mightberelatedtothepresenceofsmallflocs. differentdegreeofcomplexitydependingonthenumberof
TheshareofmembersoftheCytophaga-Flexibactersub- theparameterscontained[68].Mostofthesemodelscanbe
phylumwas4%ofthetotalviableflora,whichcorresponds mathematicallydescribedbyapower-lowcurveincludingan
moreorlesstotypicalvaluesinactivatedsludge,whichcan offsettoaccountforyieldstress.
reachapercentageof10%.Thethinrod-shapedcellsshowed In the paper [51], the rheological characteristics of an
low fluorescence signals. Filaments like Haliscomenobacter activatedsludgesampledinapilotMBRsystem,withaMLSS
−1 −1
hydrossiswerenotdetected. concentrationvarying between 2.74gL and 16gL , were
JournalofChemistry 7
Table2:ResultsoftheapplicationoftheVITgeneprobes.
Analysedtarget Shareofeachgroupinrelationtothe
Physiologicalfeatures
microorganism(s) overallbacteriapopulation(%)
Species Group
Alphaproteobacteria Groupwithhighamountofheterotrophicorganisms(e.g.,
1
(amountoffilamentous generaParacoccus,Sphingomonas,Rhizobium,Caulobacter,and
(n.d.)
bacteria) Rhodospirillum)
GroupconsistingoffilamentousbacteriasimilartoNostocoida
Alysiosphaera limicolaII,withtypicalchainstructures.Responsiblefor n.d.
bulkingsludgeinindustrialwastewatertreatmentplants
Groupconsistingofthe𝛽1-group(manyfilamentousbacteria)
Betaproteobacteria andofthe𝛽2-group(mostammoniaoxidizingbacteria).In
77
(amountoffilamentous municipalWWTPs,mostlythedominatinggroup(e.g.,genera
(n.d.)
bacteria) Burkholderia,Sphaerotilus,Alcaligenes,Thiobacillus,and
Nitrosomonas)
𝛽1-groupofthe
Betaproteobacteria GroupconsistingofflocformingbacteriaandS.natansrelated 76
(amountoffilamentous filamentousbacteria (n.d.)
bacteria)
Gammaproteobacteria Groupconsistingofheterotrophic,mixotrophic,and
15
(amountoffilamentous autotrophicmicroorganisms(e.g.,generaAcinetobacter,
(n.d.)
bacteria) Aeromonas,Pseudomonas,Vibrio,andThiothrix)
Filamentousandnonfilamentousbacteria.Commonin
Acinetobacter municipalandindustrialWWTPswitharelativelyhighsludge n.d.
age.Favouredbyscarcedissolvedoxygenconcentration
Filamentoussulphurbacterium,favouredbynutrientdeficiency.
Thiothrix n.d.
Adaptedtolowdissolvedoxygenconcentration
Filamentoussulphurbacteriumcommoninmunicipaland
Eikelboomtype021N industrialWWTPs.favouredbynutrientdeficiency.Adaptedto n.d.
lowdissolvedoxygenconcentration
Deltaproteobacteria Groupincludingmostsulphatereducingbacteria(e.g.,families <1
(amountoffilamentous Desulfobacteraceae,Desulfobulbaceae,and
(n.d.)
bacteria) Desulfovibrionaceae)
Groupoffilamentousorganismswithincreasingimpacton
Chloroflexi municipalandindustrialWWTPs,duetotheircontributionto <1
(amountoffilamentous foamingandbulkingevents,verystableagainstmechanical
(n.d.)
bacteria) stress(genusHerpetosiphon,type1851,type0803,andtype0092
andmanyotherfilamentsstillunknown)
Filamentousbacterium;contributiontosludgebulking.
Eikelboomtype1851 n.d.
Favouredbynutrientdeficiency
Herpetosiphonaurantiacus Filamentousbacterium.Aerobicandanaerobicconditions n.d.
Cytophaga-Flexibacter
Groupcontainingfilamentousandflocformingbacteria.Mainly
subphylum 4
inWWTPswithnutrientsremoval(e.g.,generaCytophaga,
(amountoffilamentous (n.d.)
Flavobacterium,andFlexibacter),
bacteria)
Haliscomenobacter Filamentousbacterium;contributiontosludgebulking.
hydrossis Favouredbyhighsludgeage,lowdissolvedoxygen n.d.
concentration,andhighammoniaconcentration
Group,mainlyinmunicipalWWTPs;membersofthisgroup
Planctomycetes
areinvolvedintheANAMMOXprocess.Adaptedtolow
(amountoffilamentous n.d.
dissolvedoxygenconcentration(e.g.,generaPlanctomyces,
bacteria)
Pirellula,Isosphaera,andScalindua)
CandidatusNostocoida
Filamentousbacterium;contributiontosludgebulking n.d.
limicolatypeIII
8 JournalofChemistry
Table2:Continued.
Analysedtarget Shareofeachgroupinrelationtothe
Physiologicalfeatures
microorganism(s) overallbacteriapopulation(%)
Species Group
GroupofGRAMpositivebacteriawithahighDNAGCcontent.
Actinobacteria
SeveralproblematicfilamentousbacterialikeMicrothrix
(amountoffilamentous n.d.
parvicellaandnocardioforms.InWWTPswithnutrients
bacteria)
removal(alsobiologicalphosphorousremoval)
Filamentousbacterium;inWWTPswithnutrientsremoval.
Microthrixparvicella Contributiontosludgebulking,floating,andfoaming.Strong n.d.
hydrophobiccharacteristics
Groupcontainingfilamentousandflocformingbacteria.
Nocardioforms Contributiontosludgebulking,floating,andfoaming.Strong n.d.
hydrophobiccharacteristics
CandidatusNostocoida Typicalchainstructures.Contributiontosludgebulking.In
n.d.
limicolatypeII municipalWWTPs
Firmicutes GroupofGRAMpositivebacteriawithalowDNAGCcontent.
(amountoffilamentous SeveralfermentativebacterialikethegeneraStreptococcus, n.d.
bacteria) Bacillus,andLactobacillus
CandidatusNostocoida
Filamentousbacterium.Contributiontosludgebulking n.d.
limicolatypeI
Grouprecentlydetected,includingfilamentousbacterialike
CandidatusTM7 n.d.
Eikelboomtype0041
Groupofammonia
Forexample,generaNitrosomonasandNitrosococcus n.d.
oxidizingbacteria(AOB)
Groupofnitriteoxidizing
Forexample,generaNitrobacterandNitrospira n.d.
bacteria(NOB)
Phosphateaccumulating Responsibleforthebiologicalphosphorusremoval(e.g.,
n.d.
organisms(PAO) CandidatusAccumulibacterphosphatis)
Glycogenaccumulating
CandidatusCompetibacterphosphatis n.d.
organisms(GAO)
examined.TheseauthorsshowedthatHerschel-Bulkleymodel In (3), 𝑁 denotes the number of samples included in the
provides suitable fitting of experimental data for shear rate populationofthestochasticvariable𝑦(i.e.,𝑁isthenumber
−1
above25s . ofmeasurescontainedinthedatasetofeachtestcarriedout
Proper fitting of the experimental data analysed in this at a given shear rate, temperature, pH, and aeration), while
work(MLSSconcentrationsintherange150–190gL−1)was 𝑦pre is the corresponding predicted value obtained through
obtained with Herschel-Bulkley model which is recognized thefollowinglinearregressionequation:
to be more efficient in describing the overall rheological 𝑦=𝑎𝑥+𝑏,
behaviourofsludgewithhighsolidconcentration[65]:
𝜏=𝜏 +𝐾𝛾𝑛̇ . 𝑦=log (𝜏−𝜏𝑦),
𝑦 (2)
𝑥=log (𝛾)̇ , (4)
In (2), the two fitting parameters are the consistency coef-
ficient 𝐾 and the flow behaviour index 𝑛. As explained in 𝑎=𝑛,
thefollowing,yieldstress𝜏𝑦 wasnotconsideredasafitting
parameteroftheflowcurve. 𝑏=log (𝐾).
Severalstatisticaldescriptorscanbeadoptedtomeasure
The linearized flow equation for the regression analysis,
the accuracy of prediction of rheological models and the
denotedbythefirstequationof(4),containsthreerheological
reliability of their estimated parameters [65]. In this work,
parametersofthesludgethataretheconsistencycoefficient
athnedctoheeffistcainendtarodfderertoerrmofinthaetieosnti(moratreeg𝜎reestsisnio(n3)cwoeeffirecaiednotp)t𝑅ed2 𝐾be,,tihnegfleonwerbael,hdaivffioeruernitnfdreoxm𝑛,zaenrod.theyieldstress𝜏𝑦thatmay
accordingto[51]
Themethodofordinaryleastsquares(OLS)wasadopted
2 1/2 forlinearregressionforfittingthedependenceofshearstress
𝜎 =[∑𝑁(𝑦𝑖−𝑦pre) ] . (3) versusshearrate.
est 𝑁−2 As well known, only two of the three above-mentioned
[𝑖=1 ] model parameters can be reliably estimated through linear
JournalofChemistry 9
(a) (b)
(c)
Figure5:Micrographs(magnification:1000x)oftheflocsunderphasecontrastandepifluorescence.Comparisonofthesameflocarea.
Hybridizationtarget:viablebacteriaandBetaproteobacteria.(a)Phasecontrast.Identicalmicroscopicfieldsunderfluorescence:(b)detection
ofallviablebacteriaand(c)analysiswithaspecificprobeforBetaproteobacteria.
regression. Therefore, in the following statistical analysis, it shear-thickening (dilatant) behaviour, with the estimated
wasassumedasfittingparameterstheflowbehaviourindex𝑛 yield𝜏𝑦stressequaltozero.
andtheconsistencycoefficient𝐾thatareincludedinthetwo For all the tests carried out, the apparent viscosity
parameters𝑎and𝑏ofthelinearequation(see(4)).However, increased,atanyshearrate,withthesolidcontent;moreover,
thepropervalueofyieldstressshouldbeestimatedinorder the differences in apparent viscosity measured at different
tofitexperimentaldatainalltestingconditions. shearratesincreasedwithsolidcontent,inaccordancewith
In some works, the yield stress 𝜏𝑦 has been determined [89]. This rheological behaviour was different from the
throughgraphicalextrapolationoftheflowcurve[51].Owing shear-thinning(pseudo-plastic)behaviournoticedby[51]for
totheabove-mentionedlackofexperimentaldatabelowthe MBRsludgesampleswithsuspendedsolidconcentrationnot
shear rate of 500s−1, in the present study, an alternative exceeding16.0gL−1.Inthepresentstudy,however,theMLSS
procedure for the estimate of 𝜏𝑦 was adopted, as explained concentrationwasaboutoneorderofmagnitudehigher.The
below. interactionsbetweenthesludgeparticleswereexpectedtobe
Ineachtest,whencarryingoutthelinearregressionofthe evenmoreintenseandthereforeonemighthaveexpecteda
experimentaldatawithrespect to theunknownparameters nonzeroyieldstress[89].Infact,astheparticleconcentration
𝐾 and 𝑛, the yield stress was considered as a deterministic increases,theybecameprogressivelymoreclosetoeachother,
parameter ranging in the interval [0.0–3.9]Pa at steps of thusleadingtoarapidgrowthofthenumberofinteractions
0.05Pa:foreachofthesevaluesstatisticalestimatesofboth thatopposestodeformation.
𝑎 and 𝑏 were obtained along with the corresponding value Anyway, such a dilatant behaviour may be related to
oftheregressioncoefficient𝑅2.Thevalueoftheyieldstress the fact that in this work the sludge samples were tested at
∘
that, for each test, maximizes the regression coefficient was very high temperature of about 45 C, which is unusual if
assumedasanestimateof𝜏𝑦. oneconsidersthatgenerallythetestingtemperaturedoesnot
∘
Theobtainedresultsofthestatisticalanalysisaresumma- exceed35 C[68].
rizedinTable3forbothconcentrationsofsuspendedsolid. Inordertopointoutpossibletemperatureeffectsonthe
TheresultsobtainedforbothMLSSconcentrationsshow obtained results, the work by [65] can be considered which
thattheflowbehaviourindex𝑛wasgreaterthanone;atthe investigated the rheology of mixed primary and secondary
−1
lowerMLSSconcentrationof150gL ,thesludgeexhibited sludge as a function two critical parameters affecting flow
10 JournalofChemistry
Table3:Summaryofrheologicaltestsandresultsoflinearregressionanalysis.
Test# Average𝑇 AveragepH Aeration MLSS 𝜏𝑦 𝑛 𝐾 𝑅2 𝜎est
[∘C] [—] [gTSL−1] [Pa] [—] [Pasn] [—] [—]
1 44.5 6.7 N 0.0 2.760 1.992𝑒−8 0.997 0.0296
2 45.2 6.7 Y 0.0 2.909 6.805𝑒−9 0.971 0.0944
3 45.1 7.7 N 0.0 2.976 4.474𝑒−9 0.965 0.1066
150
4 45.3 8.3 Y 0.0 3.120 1.642𝑒−9 0.967 0.0905
5 45.1 9.0 N 0.0 2.138 1.125𝑒−6 0.923 0.0711
6 44.9 9.1 Y 0.0 2.925 4.080𝑒−9 0.953 0.0706
7 45.4 7.0 N 2.35 5.307 3.824𝑒−16 0.986 0.2107
8 45.3 6.7 1/2 1.95 3.901 6.532𝑒−12 0.992 0.1146
9 45.0 6.6 Y 2.05 3.508 9.316𝑒−11 0.989 0.1199
10 44.9 7.8 N 1.90 4.433 1.60𝑒−13 0.998 0.0646
11 45.2 8.3 1/2 190 1.65 3.452 1.711𝑒−10 0.994 0.0875
12 45.3 8.4 Y 1.70 3.590 6.253𝑒−11 0.992 0.1091
13 45.0 8.9 N 1.40 4.585 6.285𝑒−14 0.998 0.0651
14 45.3 9.0 1/2 1.60 5.468 1.469𝑒−16 0.998 0.0791
15 45.0 9.1 Y 1.25 5.221 8.227𝑒−16 0.998 0.0852
(a) (b)
(c)
Figure6:Micrographs(magnification:1000x)oftheflocsunderphasecontrastandepifluorescence.Comparisonofthesameflocarea.
Hybridization target: viable bacteria and 𝛽1, Betaproteobacteria. (a) Phase contrast. Identical microscopic fields under fluorescence: (b)
detectionofallviablebacteriaand(c)analysiswithaspecificprobefor𝛽1-groupofBetaproteobacteria.
∘ ∘
behaviour:temperature,rangingbetween25 Cto55 C,and of the temperature. Therefore, an increase of the testing
solid concentration, varying between 4.3% and 9.8% that is temperatureseemsconsistentwitharelevantreductionofthe
quitebelowthetotalsolidcontentinvestigatedinthiswork. yieldstress.
These authors have shown that (for a given solid con- It is worth noting that in all tests with suspended solid
centration)yieldstressdependsexponentiallyontheinverse concentration of MLSS = 150gL−1 when imposing a shear