biofilm colonizing the nam theun 2 power plant penstock ... · biofilm colonizing the nam theun 2...

Hydroécol. Appl.© EDF, 2014DOI: 10.1051/hydro/2014008

Biofilm colonizing the Nam Theun 2 power plantPenstock (Lao PDR) - mechanism and potentialevolution

Biofilm colonisant la conduite forcée de l’aménagementde Nam Theun 2 (RDP du Laos) - mécanisme et évolutionpotentielle

S. Pécastaings (1), A. Godon (2), C. Roques (1)

(1) Université de Toulouse, UPS, Laboratoire de Génie Chimique, BioSym dpt UMR 5503,Faculté de Pharmacie, 35 chemin des Maraîchers, F-31062 Toulouse cedex [email protected]

(2) Nam Theun 2 Power Company Limited (NTPC), Environment & Social Division – Water Qualityand Biodiversity Dept. – Gnommalath Office, PO Box 5862, Vientiane, Lao PDR

Abstract – Biofilms are the most common bacterial life mode on Earth. These tri-dimensionbacterial structures occur at a substratum-liquid interface. Due to their intrinsic properties(niche for pathogens, resistance to biocide treatments, etc.), they cause major problems invarious industries. In water systems, the physical and chemical characteristics of biofilms(viscoelastic behavior, roughness) may lead to the lowering of flow velocity. A rough biofilmhas developed in the Penstock of the Nam Theun 2 hydropower plant (Khammouane Prov-ince, Lao PDR). This biofilm is thought to lead to additional head losses and to slightly affectthe power production. The mineral, chemical and microbiological compositions of the biofilmwere investigated in order to propose solutions to reduce its effect. Samples were taken dur-ing two water drainages in 2011 and 2012. In order to complete the knowledge from the waterquality monitoring, major elements, trace elements and rare earth element (REE) contentsin samples were measured using ICP-AES and ICP-MS. Crystalline phases were identifiedand quantified by X-ray diffraction (XRD). The microbial composition of the biofilm was firstassessed by culture (2011) and then monitored according to the location and the time afterwater drainage by molecular biology methods (2012). Results show that the chemical com-position of the biofilm is dominated by ferric iron Fe3+ and its mineralogy is mostly constitutedof lepidocrocite and magnesioferrite. The bacterial population was dominated by beta-Pro-teobacteria but population profiles varied strongly according to the layer of the biofilm, thenature of the substratum and the time during which the biofilm was subjected to the condi-tions of the water drainage. These observations are concordant with the modification of thebiofilm properties and the reduction in head losses when returning to functioning regimes inthe Penstock.

Key words – biofilm, head loss, penstock, biofouling, water drainage

Article publié par EDP Sciences

http://publications.edpsciences.org

http://dx.doi.org/10.1051/hydro/2014008

2 S. Pécastaings et al.

Résumé – Les biofilms représentent la forme de vie bactérienne la plus répandue sur Terre.Ce sont des structures bactériennes tridimensionnelles qui se forment la plupart du tempsaux interfaces substrat-liquide. Du fait de leurs propriétés (niche pour micro-organismespathogènes, résistance aux traitements biocides, etc.), les biofilms causent des problèmesmajeurs dans diverses industries. Dans les réseaux d’eau, les caractéristiques physico-chimiques des biofilms (comportement viscoélastique, rugosité) peuvent conduire à unediminution du débit. Un biofilm rugueux s’est développé au sein de la conduite forcée del’aménagement de Nam Theun 2 (Province de Khammouane, Laos). Ce biofilm est suspectéd’engendrer des pertes de charges supplémentaires et de réduire la production d’énergie demanière marginale. Les propriétés minérales, chimiques et microbiologiques du biofilm ontdonc été étudiées afin d’envisager des solutions pour diminuer ses effets. Des échantillonsont été prélevés durant deux vidanges en 2011 et 2012. Pour compléter les connaissancesprovenant du suivi de la qualité de l’eau, les concentrations en éléments majeurs, en traceset en terres rares dans les échantillons globaux ont été mesurées par ICP-AES et ICP-MS.Les phases cristallines ont été identifiées et quantifiées par XRD. La composition micro-bienne du biofilm a d’abord été analysée par culture (2011), puis, en 2012, par biologie molé-culaire à différents sites et en fonction du temps après vidange. Les résultats montrent quela composition chimique du biofilm est dominée par l’ion ferrique Fe3+ et que sa minéralogieest principalement constituée de lépidocrocite et de magnésioferrite. La population bacté-rienne du biofilm est dominée par les béta-Protéobactéries, cependant, les profils de floresvarient fortement en fonction de la couche de biofilm prélevée, de la nature du substrat et dela durée pendant laquelle celui-ci a été soumis aux conditions de vidange de la conduite for-cée. Ces observations concordent avec la modification des propriétés du biofilm et la dimi-nution des pertes de charge suite au retour à des conditions de fonctionnement normalesdans la conduite forcée.

Mots-clés – biofilm, pertes de charges, conduite forcée, bioencrassement, vidange

1 INTRODUCTION

The development of biofilms on theinternal walls of hydraulic pipes is awell-described phenomenon. In spiteof this, the impact of biofouling onhydraulic performances and overallproduction efficiency is not welldescribed because of the large diver-sity of installations. The Nam Theun 2(NT2) hydropower plant unit (Lao PDR)presents unique specificities to betaken into account: (i) the tropicallocalization with specific environmen-tal parameters (temperature, relativehumidity, etc.; Descloux et al., sameissue) influencing the microbiological

development and (ii) the large pipe sizewith seasonal variations of hydraulicparameters. The literature on this topicis dated and mostly qualitative. Morerecently, Barton et al. (2008) describedthe biofilm composition on the pipewalls of a hydroelectric plant unit inTasmania. The predominance of bac-teria, and especially alpha-Proteobac-teria, corresponds to biofilm character-istics in water systems in temperatezones. Most importantly, authors high-light the role of roughness on thedecrease of hydraulic performances.Even though the literature is poor,some reports show the effect of bio-films on near-wall flow (Stoodley et al.,

Biofilm colonizing the Nam Theun 2 power plant Penstock (Lao PDR) 3

1998; Schultz & Swain, 2000) and alsolink the roughness to the viscoelasticnature of the biofilm. In spite of this,head losses observed in large pipesneed further studies. In order to explainthe implication of biofilms on industrialsites, the present work consisted inanalysing the physical, chemical andmicrobiological properties of the biofilmformed on the Penstock of the NT2hydropower plant, where head lossesare measurable.

The study was conducted duringtwo missions occurring during two con-secutive cool-dry seasons in 2011 and2012. During these periods, outageswere performed allowing biofilm obser-vations and samplings. In 2011, thecampaign consisted in a physical andchemical characterization of the Intakewater and the mineralization of the bio-film. Classical microbiological analysesof the biofilm (culture) were done andemphasized the importance of improv-ing the microbial profiles analysis inorder to better characterize the biofilm.This was achieved in 2012, taking intoaccount (i) the different parts of thePenstock and the nature of the pipewalls and (ii) the impact of outages andwater drainages. These observationswere correlated with the physical andchemical characterization performed in2011.

The main part of biofilm control liter-ature describes chemical or physicaltreatments that are unrealistic in thepresent context. The low flexibility ofthe system (difficult access and size ofthe Penstock, release of water in theenvironment) prevents the use ofchemical and abrasive treatments toremove the biofilm. The final aim of thisproject was to evaluate and suggest

solutions to monitor and limit biofilmdevelopment in order to avoid in-creases of head losses. A focus wasmade on the impact of water drainageon the biofilm removing/stabilization.

2 MATERIALS AND METHODS

2.1 Sampling sites

Inside the Penstock, two samplingsites for chemical, mineralogy andmicrobiology studies were chosen dur-ing the first Penstock water drainage,from January 22nd to January 29th,2011. Supplemental samples for micro-biology studies were taken during thesecond Penstock water drainage, fromJanuary 14th to January 16th, 2012 atthese two sites. The first site, HR, refersto the Headrace section. This section ismade of reinforced concrete. The Pen-stock is 9.2 m in diameter with a slopeof 0.5%. The pressure is at 11.5 barsand the water velocity is 4.7 m s-1. Thesecond site, HP, refers to the HighPressure section (Fig. 1). The junctionbetween concrete and galvanized steelpipes, coated with an anti-corrosivepaint (protecting coat), is accessible atthis site. The steel pipe was coated toprevent corrosion. The paint consists in2 layers: an epoxy primer enriched inzinc (Epicon Zinc HB-2, Toa Chugoku,Japan) and an epoxy top coat (BisconHB, Toa Chugoku, Japan), both dilutedwith CMP-31 thinner. The Penstock is7.15 m in diameter with a slope of1.1%. The pressure is at 34 bars andthe water velocity is 7.8 m s-1. Minera-logical and microbiological samplingswere taken within a length of less than5 m, a negligible distance compared to


the length of Penstock (more than2.8 km).

The physical and chemical proper-ties of the water have been monitoredsince 2009 by the NT2 Aquatic Environ-ment Laboratory (AE Lab, NTPC) at theIntake (site RES9 in the HeadraceChannel of the NT2 Reservoir) and atthe outlet of the Power House in theTailrace Channel (site TRC1). Ex-tended details of this monitoring can befound in Chanudet et al. (same issue).Thus, RES9 was also chosen for micro-biological samplings.

2.2 Sampling methods

2.2.1 Mineralogical samplings

One sample was collected on eachsite using a pre-cleaned large spatula.

Fig. 1. Sampling sites.

Fig. 1. Points de prélèvements.

Cleaning was achieved using fumingHNO3 and rinsing with pure water(electrical conductivity measured at0.055 µS cm-1) obtained from a brandnew batch of filtered and deionized dis-tilled water (Schott Water DistillingApparatus Typ 82000 combined withan Elga PureLab Ultra Analytic MK2equipped with two ion exchange car-tridges, a UV lamp and a micron filter).One kilogram of sludge/biofilm materialwas collected and preserved in a largemouth 1 L pre-cleaned Nalgene bottle.Since the sludge was thin at both sites(from 1 to 5 mm), each sample corre-sponds to a large surface: 50 x 50 cmat the HR site and 60 x 300 cm at HPsite, respectively. Samples in Nalgenebottles were stored in darkness atapproximately 10 °C and transported toALS Minerals service laboratory inVientiane (Lao PDR).


2.2.2 Microbiological samplings

Two samples were taken in dupli-cate during the 2011 sampling cam-paign at both HR and HP sites. Duringthe 2012 water drainage, nine sampleswere made in duplicate (Tab. I). Afterthe water drainage, two water sampleswere collected at the Intake RES9).Biofilm was sampled with a sterile spat-ula and transferred immediately in ster-ile bottles.

2.3 Physical, chemical andmineralogical analysis

Density measurements were donewith a pycnometer on a 3 g aliquot. Themoisture percentage was determinedby a gravimetric procedure after dehy-dration of another aliquot at 105 °C.Finally, few aliquots of at least 1 g of

Table I. Samples taken during the 2012 water drainTableau I. Échantillons prélevés pendant la vidange

Date Site

14th of January HR Conc

14th of January HR Conc

15th of January, morning HP Conc

15th of January, morning HP Conc

15th of January, morning HP Galva

15th of January, afternoon HR Conc

15th of January, afternoon HR Conc

16th of January HP Conc

16th of January HP Galva

21st of JanuaryIntake,RES9

21st of JanuaryIntake,RES9

sample were dried and the solid resi-due was crushed into fine powder.

Aliquots of powders were then sentto ALS Minerals service laboratory inBrisbane (Australia) for chemical com-position and mineralogical analyses.Total Carbon and Total Sulfur contentswere individually analysed with a Lecofurnace. The total volatile constituentsportion of the samples was also meas-ured using the fraction lost on ignition(LOI data) after combustion at 1000 °C.An aliquot of powder (0.5 g) was pre-pared by digestion with strong acidsand analysed either by ICP (InductivelyCoupled Plasma)-AES (Atomic Emis-sion Spectrometry) or ICP-MS (MassSpectrometry), both with ultra-lowdetection limits, to obtain some majorand minor elements (Al, Fe, Ca, Mg,Na, K, Cr, Ti, Mn and P) and anextended overview on REE (RareEarth Element) and trace elements

age.de 2012.

Surface Sample’s description

rete Top part of biofilm

rete Complete biofilm



nized Steel Complete biofilm




nized steel Complete biofilm

-100 mL, bottom of the watercolumn at the Intake

-100 mL, middle of the watercolumn at the Intake


(focusing on heavy metals). Anotherpowder sample (2 g) was used for lith-ium borate fusion to prepare the sam-ple for a whole rock analysis performedby ICP-AES, which provided major andminor constituents, reported as oxides(SiO2, Al2O3, FeO, Fe2O3, CaO, MgO,Na2O, K2O, Cr2O3, TiO2, MnO, P2O5,SrO, BaO and completed to 100%using LOI) according to a theoreticalsequence of crystallization. Mineralog-ical phases were identified using pow-der X-ray diffraction (XRD) on pellets(PANalytical vertical diffractometer,copper K radiation). XRD results arenominal (Rietveld technique with thesoftware SiroQuant V3, Sietronics PtyLtd). Non-diffracting or unidentifiedphase was discarded (Jade V9.0,Materials Data Inc.).

2.4 Microbiological analysis

2.4.1 Culture of biofilm samples andisolates description – 2011 campaign

The 2011 campaign was a prelimi-nary study to design the future molecu-lar biology study. The objective was tohave an idea of the level of bacteriapresent in the biofilm. Hence, only onegeneric growth medium (Trypcase SoyAgar - TSA) was used.

Frozen biofilm samples supple-mented with glycerol (30% v/v) weresent to the Laboratory of Paul SabatierUniversity (Toulouse, France) formicrobiological analyses, since thoseanalyses were not feasible in the Laoslaboratory. Once gently thawed, vol-umes of 0.1 mL and 1 mL were spreadon TSA (bioMérieux, Marcy-l’Étoile,France) and incubated at 22.5, 30.0,

37.0 °C under aerobic condition and37.0 °C under anaerobic condition.After 48 h, the macroscopic descriptionof the colonies was used to determinemajor bacterial types on each plate.These colonies were subcultured on afresh TSA plate. After 48 h of incuba-tion in the same condition as previouslyused, a Gram staining, an oxidase testand catalase test were performed.

2.4.2 Microscopic observations –2012 campaign

Microscopic observations weremade at the Nam Theun 2 PowerCompany (NTPC) Laboratory (AELab) immediately after sampling dur-ing the 2012 campaign. Samples werediluted in Trypan blue, a vital dye thatstains only dead cells. The counting ofviable and dead cells was performedusing a Malassez cell, using micro-scope equipped with a ×100 objective.The quantification limit of this methodis 106 cells per grams (g) of biofilm.

2.4.3 Quantification of main bacterialgroups and establishment of bacterialprofiles – 2012 campaign

2.4.3.1 DNA extraction

DNA was extracted in duplicatefrom each biofilm and water samplefrom the 2012 campaign, with the Ultra-Clean Soil DNA isolation kit (Mo-Bio,Carlsbad, CA, USA). 250 mg of biofilmsamples were transferred in micro-tubes containing the lysis buffer andmicrobeads provided by the manufac-turer. A negative extraction samplewas made with 200 µL of sterile water.60 µL of S1 solution and 200 µL of IRS


solution were added in each tubebefore vortexing at maximum speed for10 min. After centrifugation (10000 g,30 s), 250 µL of S2 solution wereadded to the supernatant and the mixwas incubated 5 min at 4 °C. After cen-trifugation (10000 g, 1 min), 450 µL ofsupernatant were mixed with 900 µL ofS3 solution and this mixture was trans-fered on a silica column to bind theDNA. 300 µL of S4 solution were usedto wash the column and purify theDNA. Finally, the DNA was elutedusing 200 µL of S5 solution and centrif-ugating the columns at 10000 g for30 s. DNA samples were immediatelyfrozen (-20 °C). They were sent to theLaboratory (Paul Sabatier University,Toulouse, France) under this condition.One sample (a duplicate of the HRcomplete biofilm taken on the 15th ofJanuary 2012) was lost during transferfrom Lao PDR to France.

Water samples (100 mL) werethawed and filtered on polycarbonatemembranes (porosity = 0.2 µm) to col-lect bacteria from the samples. Mem-branes were transfered in microtubescontaining the lysis buffer and mi-crobeads provided by the manufac-turer. The same extraction protocolewas applied as for biofilm samples.

2.4.3.2 Quantification of main bacterialgroups by qPCR

The qPCR was run on MyiQ RealTime PCR Detection (BioRad, Marnes-la-Coquette, France), in 96-well micro-plates. In this study, the proportion ofBacteria, alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacte-ria, Bacteroidetes, Firmicutes, Actino-bacteria, Acidobacteria was quantified

in biofilm and water samples. Onlyeukaryotic fungal cells (Fungi) werequantified, and not total Eukaryoticcells (Algae or Protozoa). A sequenceof the 16S rRNA gene (for Bacteria) or18S rRNA gene (for Fungi), specific ofeach group and already described inthe literature, was targeted (Tab. II).

The qPCR mixture (25 µL per well)contained 12.5 µL of iQ SYBR GreenSupermix (2X, BioRad), 350 nmol L-1

of each forward and reverse primers(Life Technologies, Villebon-sur-Yvette,France), 5 µL of sample (or standard)DNA. Each sample was analysed intriplicate. Two negative controls weremade for each qPCR run: one with 5 µLof PCR grade sterile water instead ofDNA sample and one with the negativeextraction control. A positive controlwas made for each sample by adding1 µL of internal inhibition control intothe mixture to monitor qPCR inhibition.The amplification was achieved after a10 min activation step at 95 °C and theamplification cycle (15 s at 95 °C, 30 sat hybridization temperature and 30 sat 72 °C) was repeated 40 times. SYBRGreen Fluorescence was monitored inthe wells after each amplification cycle.

Standard DNA curves were obtainedfor each target group after purification ofgenomic DNA from reference strains(Tab. III). DNA was quantified by meas-uring absorbance at 260 nm, assumingthat 1 absorbance unit correspondsto a concentration of 50 ng µL-1.Genomic DNA concentration wasdeduced according to the genome sizeof bacterial species used for the studyand considering that 1 base pair =660 g mol-1.

Serial dilutions of standard genomicDNAs were made with elution buffer


Table II. Primers used for qPCR.Tableau II. Amorces utilisées pour la qPCR.

Domain Target group Primer Sequence (5'-3')HT*(°C)

Reference

Bacteria Bacteria 926FAAA CTC AAA KGA

ATT GAC GG60.0

(Bacchetti DeGregoris et al., 2011)

1062RCTC ACR RCA CGA

GCT GAC

Alpha- α682FCIA GTG TAG AGG

TGA AAT T60.0


proteobacteria 908αRCCC CGT CAA TTC

CTT TGA GTT

Beta- Eub338FACT CCT ACG GGA

GGC AGC AG60.0 (Fierer et al., 2005)

proteobacteria Bet680RTCA CTG CTA CAC

GYG

Gamma- 1080γFTCG TCA GCT CGT

GTY GTG A60.0


proteobacteria γ1202RCGT AAG GGC CAT

GAT G

Bacteroidetes 798cfbFCRA ACA GGA TTA

GAT ACC CT61.5


cfb967RGGT AAG GTT CCT

CGC GTA T

Firmicutes 928F-FirmTGA AAC TYA AAG

GAA TTG ACG61.5


1040FirmRACC ATG CAC CAC

CTG TC

Actinobacteria Act920F3TAC GGC CGC AAG

GCT A61.5


Act1200RTCR TCC CCA CCT

TCC TCC G(Bacchetti De

Gregoris et al., 2011)

Acidobacteria Acid31FGATCCTGGCTCAG

AATC60.0 (Fierer et al., 2005)

Eub518RATTACCGCGGCTG

CTGG

Eucarya Fungi ITS1FTCC GTA GGT GAA

CCT GCG G59.5 (Fierer et al., 2005)

5.8sRCGC TGC GTT CTT

CAT CG

* HT = hybridization temperature


(from Ultraclean Soil DNA extractionkit) to prepare standard ranges of DNAfrom 5 to 50000 gu (genome units) perµL. Each run of qPCR was conductedwith standard DNA (5 points in tripli-cate), 2 negative PCR controls and 2negative extraction controls (DNAextraction of sterile water) and sam-ples. After completion of qPCR, thestandard curves were constructed byplotting threshold cycles (Ct) valuesversus the log quantity of standardDNA in a well. Results are an averageof qPCR triplicate results, expressed inlog gu per gram of biofilm (or per 100 mLof water in case of liquid samples).

Table III. Reference strains.Tableau III. Souches de références.

Domain Target group

Bacteria Bacteria Escher

(CIP54

Alpha- Brevun

proteobacteria (CIP 10

Beta- Burkho

proteobacteria (ATCC

Gamma- Escher

proteobacteria (CIP54

Bacteroidetes Bactero

(ATCC2

Firmicutes Staphy

(CIP68

Actinobacteria Microcc

(ATCC

Acidobacteria Acidoba

(DSM1

Eucarya Fungi Saccha

Since the quantification limit is 5 gu in25 µL of PCR mix, the quantificationlimit in biofilms is 1350 gu g-1 of biofilmand 220 gu 100 mL-1 of water. Resultsare expressed as percentages of eachgroup amongst total Bacteria in eachsample duplicate. Proportion averageswere calculated and plotted in graphsthat represent the bacterial populationprofiles.

It is important to note here thatqPCR gives information on the pres-ence of microorganisms in a sample,but does not give indication about theirviability (dead or alive). Viability studieswere made by microscopic analysis.

Strain

ichia coli

.8T)

dimonas diminuta

3020)

lderia cepacia

24416)

ichia coli

.8T)

ides fragilis

5285)

lococcus epidermidis

21)

us luteus

9341)

cterium capsulatum

1244)

romyces cerevisiae subspecies boulardii


3 RESULTS

3.1 Macroscopic field observations –2011 & 2012 campaigns

The biofilm covering the Penstockin the HR section appeared to be uni-form. The deposit contained nodularconcretion of a few millimetres in size.Its thickness reached a maximum of5 mm. Colour varied from black to redand yellowish (Fig. 2) possibly indicat-ing the presence of iron oxides. Thissludge material was sticky but notstrongly fixed onto the inner walls of theTunnel. It was easy to detach by patch

Fig. 2. Biofilm on the concrete HR Tunnel. The whit

Fig. 2. Biofilm formé sur le Tunnel HR. La flèche bdu biofilm.

from the concrete. No difference wasobserved according to the curve of theTunnel. The deposit was similar every-where in the HR section.

On the HP section, the biofilm wasdifferent from the HR site. The depositlooked also like a nodular concretionbut contained smaller nodules (maxi-mum of 1 to 2 mm). On the HP section,the junction between the concrete andgalvanized material (with anti-corrosivepainting) is accessible. A macroscopicdifference between the biofilm formedon both surfaces was clearly noted(Fig. 3): the biofilm was thinner (2 to3 mm) on the galvanized steel and less

e arrow indicates biofilm removal after sampling.

lanche indique le détachement après prélèvement


rough. Colour varying from black to redand yellowish was also observed. Sim-ilarly to the HR site, this sludge materialwas very wet. It also appeared that theconcrete biofilm was constituted of2 layers. The upper layer was highlyhydrated, viscous and easily detachedand suspended in water. The bottomlayer was mineralized, rougher andalso more difficult to detach than theupper layer.

3.2 Intake water physicaland chemical properties

The water quality monitoring hasbeen extensively described in Chanudetet al. (same issue). Reservoir water is

Fig. 3. Biofilm on the galvanized steel (left) and the cshows the separation between both zones.

Fig. 3. Biofilm formé sur les parties en acier galvaniau niveau du site HP. La flèche blanche indique la s

carbonated and shows low content ofSi, in coherence with the regional geo-logic sedimentary setting. Iron contentis in the range of 0 to 3 mg L-1 and pre-dominantly occurs as dissolved ferrousiron Fe2+, with only 10% as Fe3+. Sincethe beginning of test operation inMarch 2010, the water column at RES9has been homogeneous (Tab. IV).

RES9 and TRC1 are close in termsof chemical composition and it lookslike nothing much happened inbetween (i.e.: in the Penstock and allthe plumbing system of the PowerHouse) besides the increase of pres-sure up to 34 bars (which may changethe thermodynamics of the chemicalequilibriums) and its fast return toatmospheric pressure.

oncrete part (right) of the HP site. The white arrow

sé (à gauche) et en béton (à droite) de la conduiteéparation entre les deux zones.


3.3 Chemical compositionand mineralogy of the sludge

Results on both samples are sum-marized in Table V. The moisture per-centage reveals that both samples arehighly hydrated, and that sample fromHP site is richer in water than the onefrom HR site. This is consistent withfield observations (see Sect. 3.1.).

The sludge is dense, with a specificgravity near 3. Even though the sam-ples could have been shaken and com-pacted during their transportation toVientiane, it means that the dry matrixis highly dense, in agreement withheavy metals loads.

LOI is too high (~ 20%) to be solelyexplained by volatile constituentsfrom the organic matter, the carbon-ated phases or salts, since the sum ofC and S (~ 7% maximum) is far fromreaching the LOI content. Moreover,Ca and Mg concentrations are toolow to involve a significant quantity of

Table IV. Typical chemical composition of the waterTable IV. Composition chimique typique de l’eau à R

Parameter (Unit) RES9 Surface RE

Depth (m) 0.2

Si (mg L-1) 2.35

Fe2+ (mg L-1) 0.14

Fe3+ (mg L-1) 0.17

TOC (mg C L-1) 1.78

Ca (mg L-1) 3.68

Mg (mg L-1) 0.90

Na (mg L-1) 0.89

K (mg L-1) 0.89

SO42- (mg S L-1) 0.59

PO43- (mg P L-1) <0.03

carbonated constituents in the sludge.HCl test performed on a small aliquot ofsludge at the laboratory right after thesampling did not show any efferves-cence. Nevertheless, high LOI can beexplained by the presence of manyhydroxides.

The chemical composition is domi-nated by the iron content, which isthe most important element (almost50 wt.%), and occurs mainly as ferriciron Fe3+. Other elements are lower inconcentrations, except oxygen, whichis the element bounding minerals andthus was not analysed by these tech-niques. Si, Al, Ca, Mg, Na, K, Cr, Ti, Mnand P are present in minor amounts(Tab. V). Trace elements and REE arenegligible. Such a chemical composi-tion differs from any precipitation ofpreviously dissolved rocks, because itdoes not fit the geologic setting domi-nated by sandstones and carbonatedsedimentary rocks. The source of theHR Tunnel sludge is unlikely to be the

at RES9 and TRC1 in January 2011.ES9 et TRC1 en janvier 2011.

S9 Middle RES9 Bottom TRC1

8.0 16.0 0.2

2.28 2.63 2.36

0.30 0.24 0.20

<0.10 0.48 <0.10

- 1.30 1.75

3.40 3.42 3.40

0.85 0.85 0.75

0.88 0.86 0.93

0.85 0.81 0.96

0.54 0.59 0.73

<0.03 <0.03 <0.03


Table V. Chemical and phase compositions of the sludge at HR and HP sites.Table V. Composition chimique et minéralogique du biofilm des sites HR et HP.

Parameter(unit)

HR HP

SiO2 (wt.%) 3.68 10.40

Al2O3 (wt.%) 1.13 3.62

Al (wt.%) 0.46 0.87

FeO (wt.%) 0.50 2.42

Fe2O3 (wt.%) 76.6 59.9

Fe (wt.%) 48.4 41.1

CaO (wt.%) 0.77 0.82

Ca (wt.%) 0.47 0.56

MgO (wt.%) 0.21 0.59

Mg (wt.%) 0.09 0.18

Na2O (wt.%) 0.05 0.08

Na (wt.%) 0.02 0.03

K2O (wt.%) 0.09 0.43

K (wt.%) 0.03 0.08

Cr2O3 (wt.%) <0.01 <0.01

Cr (ppm) 20 30

TiO2 (wt.%) 0.03 0.14

Ti (ppm) 130 90

MnO (wt.%) 0.59 1.84

Mn (ppm) 3730 13550

SrO (wt.%) 0.01 0.01

Sr (ppm) 52 45

BaO (wt.%) 0.02 0.03

Ba (ppm) 182 310

P2O5 (wt.%) 0.07 0.14

P (ppm) 130 340

S (wt.%) 0.06 0.11

C (wt.%) 3.00 7.09

LOI (wt.%) 16.65 21.80

Moisture (wt.%) 72.3 90.4

Density 2.97 3.03

Phase Lepidocrocite (100%)Lepidocrocite (64%)

Magnesioferrite (23%)Quartz (9%)


lining of the Headrace Channel, since ithas a different chemical composition(mainly Quartz SiO2; AE Lab. unpub-lished data, 2010). The whole rockanalysis revealed that the most impor-tant group of minerals is iron oxides andhydroxides, because Fe2O3 and LOIare very high.

These results are consistent withXRD patterns and phase occurrences(Tab. V). Lepidocrocite, an iron oxide-hydroxide (γ-FeO.OH composed ofFe3+; density 4.09), scales easily andshows red to yellowish to blackishbrown macroscopic natural colour. Itoriginates mainly from the weatheringof primary iron minerals or rust fromsteel water pipes. Magnesioferrite isan iron oxide (MgFe2O4 also com-posed of Fe3+; density 4.52) occurringas massive or granular of well-formedfine sized crystals, brownish black toblack in colour. Finally, Quartz (SiO2;density 2.65) is ubiquitous (Deeret al., 2013). These results are in goodagreement with field observations.Background level of the powder XRDpatterns are quite high and suggestthat the samples are both disordered,the sludge from HP site being relativelymore crystallized than the one from HRsite. Since the chemical composition ofthe sludge fits its mineralogy, it is pos-sible that the non-crystalline material isan amorphous material analogous ofthe phases identified. This disorderedpart of the sludge could also be verydense.

3.4 Bacterial population profiles

Bacterial profiles were used as atool to characterize the biofilm in the

different parts of the Penstock. Fungiwere analysed in every water and bio-film samples, however they were onlydetected in low quantities, close or infe-rior to the quantification limit. Thisresult was consistent with microscopicobservations (data not shown). SinceBacteria were highly predominant, cal-culating the ratio between Bacteria andFungi was irrelevant. Results pre-sented here are expressed in terms ofbacterial profiles only.

Only the main constitutive groups ofthe Penstock biofilm were analysed.Since the Penstock is fed with reservoirwater, taxa representative of surfacewater and soil were targeted. Proteo-bacteria (Gram-negative) are the widerand most diversified bacterial groups.They include alpha-Proteobacteria,beta-Proteobacteria and gamma-Pro-teobacteria, delta-Proteobacteria andepsilon-Proteobacteria. Delta-Proteo-bacteria and epsilon-Proteobacteriawere not studied here. Actinobacteria(Gram-positive bacteria with high G+Ccontent) are very abundant in soils andhave the capacity to degrade a widevariety of organic compounds. The 2011culture analysis showed the presenceof filamentous Gram-positive bacilliand catalase-positive cocci that couldbelong to that group. Firmicutes (Gram-positive with a low G+C content) havethe capacity to produce endospores andare often free saprophytic bacteria.Bacteroidetes (Gram-negative otherthan Proteobacteria) are anaerobic non-sporulated bacteria isolated mostly inoral cavity and intestinal tracts. Thegroup of Acidobacteria (Gram-negativeother than Proteobacteria) was recentlydiscovered and is still not very wellknown. These microorganisms are


highly present in soil, where they canrepresent up to 25-30% of identified16S sequences. Microscopic observa-tions and Gram determination of colo-nies isolated in 2011 (data not shown)revealed the presence of bacteria thatcould belong to these groups.

3.4.1 Microbial properties of the Intakewater – RES9

Biofilms form at the interface be-tween a substratum and water. In orderto evaluate the relationship betweenthe Intake water and the biofilm compo-sition in the Penstock, bacterial profilesof the Intake water (RES9) were firstdetermined.

It clearly appears that Proteobacte-ria is the predominant bacterial group,and among them, beta-Proteobacteriarepresents 88.1±3.4% of all Proteobac-teria in the Intake water (Fig. 4). Eventhough only 2 water samples weremade, results show that profiles in both

Fig. 4. Bacterial profiles at the bottom of the Intake

Fig. 4. Profils bactériens au plus profond et au milie

locations are different, i.e. Actinobacte-ria were only detected in the “Middle”Intake sample (9.8% in “Middle Intake”versus 0.0% in the “Bottom Intake”sample).

3.4.2 Biofilm compositionin the different parts of the Penstockand evolution according to time

Microscopic observations revealedthat biofilms contain amorphous parti-cles and crystals. The level of free bac-teria was 1010 to 1011 bacteria g-1. Thelevel of eukaryotic cells was inferior to106 cells g-1, mostly dead (stained byTrypan Blue). A clear loss of viabilitywas also observed for bacteria duringthe water drainage (more than 50% onJanuary the 16th).

The semi-quantification method (bymicroscopy) and qPCR results did notgive the same results: a difference of2-log bacteria per gram was observed.However, microscopic observations

water and in the middle of the Intake water.

u de la prise d’eau.


may have overestimated bacterialconcentrations because of the pres-ence of a complex and dense mineralmatrix (see Sect. 3.3). Secondly, eventhough qPCR was made according tocontrolled and recognized methods,its yield is affected by the DNA extrac-tion step. DNA extraction includes alysis step to break bacterial cells andallow access to the DNA. This stepcan be altered by the presence ofextracellular matrix components and/or differences in cell wall propertiesaccording to bacterial species. How-ever, as previously observed by micro-scopic observations, total bacterialconcentrations measured by qPCR didnot vary significantly among biofilmsamples during the water drainage.

3.4.2.1 In the HR section

The structure of the biofilm pre-sented 2 layers. Considering these dif-ferences, bacterial profiles were ana-lysed in the upper layer and complete

Fig. 5. Variation with time of bacterial groups in the

Fig. 5. Variation au cours du temps des grands grotion HR.

biofilm and according to the time of thewater drainage.

First, results show that bacterial pro-files differed in the upper and thecomplete biofilm on the initial samplingday. Beta-Proteobacteria was the majorbacterial group. It represented 51.3% ofthe bacterial population in the completebiofilm (Fig. 5) and 86.7% in the upperbiofilm (Fig.6); aproportion that ishighlysimilar to that of the intake water(Fig. 4). The proportion of alpha-Pro-teobacteria, gamma-Proteobacteria,Actinobacteria and Acidobacteria wasalso higher in the complete biofilm thanin the upper biofilm, indicating a lowerbiodiversity in the upper biofilm layer.

On the second day of water drain-age, bacterial profiles were significantlydifferent than the previous day. Theproportion of beta-Proteobacteria de-creased by half in the complete and up-per biofilm (51.3% versus 26.3% in thecomplete biofilm; 86.7% versus 43.6%in the upper biofilm). On the contrary,the proportion of alpha-Proteobacteria,

complete biofilm of the HR section.

upes bactériens dans le biofilm complet de la sec-


Actinobacteria and Acidobacteria in-creased in the biofilm, resulting in a highbiodiversity in both layers of the biofilmafter 24 h of water drainage.

It is also interesting to note that thewater drainage not only affected bac-terial profiles, but also the viability.During the sampling campaign, a lossof viability was observed in the HRsection according to time, as indicatedby the increase of blue cells observedmicroscopically (no dead bacteriadetected in samples in HR on the14th of January; 25 to 50% of deadbacteria 24 h after the beginning ofwater drainage).

3.4.2.2 In HP section

3.4.2.3 Upper versus complete biofilmin the concrete HP section

As for the HR section, samples ofthe upper biofilm and the completebiofilm were analysed on the first sam-pling day (Fig. 7).


Fig. 6. Variation au cours du temps des grands groude la section HR.

Interestingly, profiles of the upperlayer and complete biofilms were simi-lar. Indeed, the proportions of alpha-and beta-Proteobacteria (the majorgroups, representing 57% of the bacte-rial population) varied by less than15%. It is interesting to note that theproportion of beta-Proteobacteria wassignificantly different to that of theintake water and this group was notpredominant in HP biofilm, contrary HRbiofilms.

3.4.2.4 Impact of the material on biofilmcomposition

The HP sampling site consists of2 types of materials: concrete and gal-vanized steel coated with an anti-corro-sive paint. Samples were taken on bothmaterials in order to analyse the impacton the biofilm.

On the concrete HP part, bacterialproportions changed with time (Fig. 8):the beta-Proteobacteria proportionincreased significantly (+16%), whereas

upper biofilm of the HR section.

pes bactériens dans la partie supérieure du biofilm


the Actinobacteria proportions de-creased from the first to the second day(-18%). On the contrary, all bacterialgroups were stable in the biofilmformed on the galvanized steel (less

Fig. 7. Bacterial profiles in the concrete part of the H

Fig. 7. Profils bactériens dans la partie supérieure e


Fig. 8. Variations au cours du temps des grands grode la partie HP.

than 5% of variation in every group;Fig. 9).

In parallel, on both types of material,the percentage of dead cells increasedfrom 25 to 50% (data not shown).

P Tunnel in the upper and complete biofilm.

t le biofilm complet sur le béton du Tunnel HP.

biofilm formed on concrete of the HP section.

upes bactériens dans le biofilm formé sur le béton


4 DISCUSSION

4.1 Relationship betweenthe mineral and the microbiologicalparts of the biofilm

In the Penstock, the only knownsource of iron comes from the Intake.High concentrations of iron (reservoiraverage up to 11.5 mgFe L-1, predom-inantly occurring as Fe2+) have beenreported in Chanudet et al. (sameissue) in the bottom of the reservoirduring periods of thermal stratification(March to October). In fact, reservoirthermal stratification and anoxia in thebottom waters of the reservoir promotethe reduction of insoluble iron (Fe3+) inthe sediment/soil particulate phase tosoluble iron (Fe2+). Then, the dissolvedFe2+ moves into the reservoir bottomwaters and increases their iron con-centrations. This may be the source of

Fig. 9. Variation with time of bacterial groups in tsection.

Fig. 9. Variations au cours du temps des grands ggalvanisé de la section HP.

iron for the formation of Lepidocrocitein the Headrace Tunnel. As the ironrich, anoxic reservoir bottom waterenters into the Intake, it mixes with oxy-genated surface water. This may bethe source of the oxygen that oxidizesiron into crystallized Lepidocrocite.However, the insoluble iron (Fe3+) mayalso directly originate from the reser-voir water since this iron speciesoccurs, in a very small extent as dis-solved ions. It means that oxygen maynot be needed to get some iron at theright oxidation state in the biofilm. Theorder of magnitude of total mass ofdeposited iron Fe3+ assuming the fewmillimetres thick biofilm is homogene-ously mineralized on the inner cylindri-cal Penstock of 2642 m length (1500 mfor HR plus 1142 m for HP) has beenestimated at 1 ton of Fe3+. This valuerepresents only 0.03% of the total dis-solved iron flux (2900 ton) that flowsthrough the Intake between March

he biofilm formed on galvanized steel of the HP

roupes bactériens dans le biofilm formé sur l’acier


2010 (start of test operation) andJanuary 2011 (first water drainage)([Fe]moy = 0.48 mgFe L-1 and Qmoy =215.5 m3 s-1). Even if the total iron con-centration in the reservoir is likely todecrease with time, the iron stock avail-able for the biofilm development willremain high. It means that preventingsuch a biofilm accumulation may becomplex (drastic change in pH, oxida-tion state and/or iron content in largevolumes of water) considering indus-trial and environmental constraints.

As observed macroscopically, thebiofilm was highly mineralized. Thethermodynamics and kinetics of themineralization process are still unclear.In particular, it is not known whether theadhesion of bacteria triggers/enhancesthe deposition of oxide-hydroxidesand speed up its kinetics. For instance,iron-oxidizing bacteria (IOB), including

Fig. 10. Hypothetical origin of the iron mineralization

Fig. 10. Hypothèse sur l’origine de la minéralisation

Acidovorax sp., Aquabacterium sp. orThermomonas sp. can derive energyfrom the oxidation of Fe2+, usually sol-uble, to Fe3+ (Straub et al., 2004). IOBhave diverse phylogenies, like Firmi-cutes or Nitrospirae, but most of thembelong to the Proteobacteria phylum(Hedrich et al., 2011), which is in agree-ment with our results. Fe3+ is poorly sol-uble and may precipitate as various ironoxi-hydroxides, including Goethite andLepidocrocite, in specific geochemicalconditions (Larese-Casanova et al.,2010). The authors demonstrated thatbiological Fe2+ oxidation is influencedby the pH and phosphate concentration.Figure 10 shows a hypothetical ironcycle that could occur in the NT2 Pen-stock, involving the biofilm.

In a geological context, Magnesiof-errite is representative of hydrother-malism. In the present context of

in the biofilm in the NT2 Penstock.

du fer dans la conduite forcée de NT2.


industrial conditions, it may be due tomineral transformations linked to thepressure increase along the Penstock.The mineralogical transformation ofLepidocrocite into Magnesioferrite withthe pressure increases also remainsan open question.

Further experimental studies willlead to a better understanding of thekinetics and the biological, physicaland chemical thermodynamics condi-tions required to form such a biofilmwith specific mineralogical properties.

4.2 Evolution of the biofilm during the2012 water drainage

Even though this study is based ona low number of samples, due to thelimited access to the Penstock, inter-esting observations could be madeconcerning biofilm behaviour, consid-ering the poor literature on this type ofwater plant versus drinking water sys-tems. This study also shows that bacte-rial profiles, correlated to bacterial via-bility, are good indicators of the state ofthe biofilm inside the NT2 Penstock.

In general, three parametersimpact the biofilm composition: (i) thebiofilm layer, (ii) the surface materialand (iii) the Penstock water drainage.The influence of all parameters issketched in Figure 11, according totheir importance.

Beta-Proteobacteria were the pre-dominant population in water and bio-film samples. These bacteria are con-sistently re-injected in the Penstock viathe water Intake and influence the com-position of the upper biofilm layer. Onecan hypothesize that this population isalso more resistant to operating condi-tions in the Penstock (pressure, shear

stress). They were also an interestingpopulation to study since their propor-tion decreased when the biofilm wasaffected. The dominance of Proteobac-teria has been earlier demonstrated inmany studies on water plants, includingdrinking water distribution systems(Jang et al., 2012). In a study publishedin 2008, Barton et al. also noticed thepredominance of Proteobacteria in thebiofilm of a hydroelectric plant in Tas-mania, and more specifically alpha-Pro-teobacteria and Chloroflexi. In thepresent study, the operating conditions(flow, pipe diameter, water characteris-tics and temperature) in both sites arevery different and may have led to dif-ferences in the biofilm composition.Douterelo et al. (2013) have recentlydescribed the bacterial profiles of waterand biofilm in an experimental drinkingwater distribution system. They demon-strated a predominance of gamma- andbeta-Proteobacteria in the biofilms,while alpha-Proteobacteria were pre-dominant in bulk water samples. Theylinked the biofilm enrichment in gamma-and beta-Proteobacteria to the proper-ties of some bacteria inhabiting the bio-film like Pseudomonas, Zooglea andJanthinobacterium to express extracel-lular polymeric substances and toadhere to surfaces and favour co-aggregation between cells. In our case,it can be hypothesized that some ofthese bacteria are present in the waterIntake and so exist in the biofilm, spe-cifically in the upper layer.

As we demonstrated, the materialaffects biofilm composition and behav-iour. Implication on bacterial attach-ment on the inner surface of the pipesystem is based on surface character-istics and stability (Pedersen, 1990;


Percival et al., 1998; Niquette et al.,2000). The biofilm growth is alsoaffected (Schwartz et al., 1998; Lehtolaet al., 2004), resulting in increased bac-terial concentration, species diversityand corrosion of the pipe (Jang et al.,2012). It is important to note that mostof these previous studies are based onculture-dependent methods. In suchconditions, no or few differences werenoted on microbial quantities betweenthe pipes materials. Molecular tech-niques offer new approaches for thedetermination of biofilm composition,more specifically viable but non-culti-vable microorganisms or VBNC (Neria-Gonzalez et al., 2006; Jang et al.,2012) or microbial activity via metabo-lomics (Beale et al., 2013). This wasconfirmed by our results on culturesperformed during the 2011 campaign

Fig. 11. Parameters impacting the biofilm compositi

Fig. 11. Paramètres influençant la composition duWD = vidange de la conduite.

with no significant difference betweenthe samples and qPCR analysis per-formed during the 2012 campaignunderlying dramatic modification in bio-film contents. The precise identificationof bacteria present in both biofilmswould be the next step to determinehow material can affect the microbialcolonization.

The water quality, such as tempera-ture, is also of concern in the biofilmdevelopment (Frias et al., 2001; Janget al., 2012). In our conditions, temper-atures are favourable for microbialgrowth. Another important factor in thebiofilm composition is the presence ofnutrients in the water intake, like organiccarbon (Camper et al., 1998; Frias et al.,2001) or the importance of Fe3+. Bac-teria able to use Fe3+ belong to variousgroups including the dominant one

on in the NT2 Penstock. WD = water drainage.

biofilm dans la conduite forcée du barrage NT2.


observed in this study (beta-Proteobac-teria). Jang et al. (2012) have recentlystudied the effect of phosphate additionon biofilm communities on stainlesssteel and ductile cast iron (DCI) pipes.The molecular analysis indicates thedominance of Proteobacteria (50%),followed by Firmicutes (10%) and Actin-obacteria (2%). Differences occur at thegenus level with a dominance ofBradyrhizobium on corroded DCI pipes.The impact of phosphate on the biofilmdiversity was also demonstrated, but inour study, the phosphate level was low.

4.3 Potential biofilm eliminationprocesses

In order to avoid biofilm, it is neces-sary to understand its development.Biofilm development follows a 3-stepcycle: the attachment stage whenplanktonic cells (“swimming” cells ofthe bulk phase) are transferred onto asubstratum; the growth stage, whichcorresponds to cell division and leadsto the formation of microcolonies; thedetachment of bacteria when cells arereleased in the environment. In gen-eral, methods to control the biofilm tar-get one of these steps. For instance,reducing bacterial adhesion can con-sist in selecting or treating materials,lowering the entry of microorganisms ina system by acting on the water quality:the adjunction of residual disinfectantin water (i.e. chlorine <1 mg L-1) ormicrofiltration is a way to limit the bac-terial intake in potable water systems.

The nature of the substratum isknown to influence the biofilm composi-tion. It was also the case in the presentstudy, where results underlined a major

difference in the biofilm compositionand structure between concrete andgalvanized steel coated with a protec-tive paint against corrosion. The litera-ture describes the successful use ofantifouling paints on ship hulls, whereit prevents for formation of biofilmsand surface colonization by higherorganisms that could affects the shipsperformance (Van Mooy et al., 2014).The use of such antifouling paintingsin penstocks appears interesting,however the maintenance needed(reapplication of paint) and the longterm efficacy should be evaluated.Taking into account the limited possi-bility to modify the Penstock at thistime, a surface treatment does notappear to be a relevant solution.

Acting on parameters allowing bac-terial growth can also impair biofilmdevelopment. Norton & LeChevallier(2000) for instance, demonstrated thatit is possible to reduce biofilm prolifera-tion by lowering the AssimilableOrganic Carbon (AOC) concentrationwith biologically active filters. Similarly,the concentration of specific nutrients(phosphorus, nitrogen, etc.) can be low-ered with Fluidized Bed Biofilm Reac-tors. Temperature is also an importantparameter for biofilm proliferation. Tem-peratures comprised between 25 and35 °C are typically the most favourablefor bacterial development. Kusnetsovet al. (1997) showed that lowering thewater temperature in a cooling towerdecreased Legionella pneumophilaconcentration. The warm water temper-atures observed on the NT2 hydro-power plant might favour biofilm prolif-eration. However, temperature is not aparameter that can be modified at theNT2 Penstock and any increase or


decrease would also have importantconsequences for the environment.

Finally, biofilm detachment can beachieved by cleaning and/or disinfec-tion episode(s). According to the “BestPractice Catalogue for Penstocks andTunnels” edited by the MESA Associ-ates, Inc., and Oak Ridge National Lab-oratory, scrubbing and cleaning is agood way to decrease surface rough-ness and limit head losses inPenstocks (Oakridge National Labora-tory, 2011). Barton et al. (2008) alsodemonstrated that the regular cleaningof pipelines is economically beneficialfor hydroelectric plants. However, thissolution can be costly and involvesextensive production stops.

So far, the water drainage of the NT2Penstock seems to be the most conven-ient technique to improve head lossesof the system. Draining the Penstockleads to variation in the biofilm viabilityand population profiles; the long-termanalysis of head losses will bring moreinformation on the efficiency of themethod. In general, it was observed thatwater drainage was a controllableparameter allowing the modification ofthe biofilm composition in the Penstock:two days after the drainage, a diversifi-cation of microbial populations wasobserved in the biofilm (decrease ofbeta-Proteobacteria at the benefit ofActinobacteria, Acidobacteria, alpha-Proteobacteria, gamma-Proteobacte-ria) as well as a loss of biofilm viability.It appears that operating conditions ofthe Penstock are unfavourable for thedevelopment of a number of microor-ganisms. Water drainage allows areturn to atmospheric pressure, higheroxygen availability without any shearstress in the Penstock, which probably

favours the growth of some bacterialspecies and leads to a higher biodiver-sity in the biofilm after two days of drain-age. It is likely that the return to operat-ing conditions in the Penstock is harmfulfor a number of these microorganisms.Most probably, these unfit microorgan-isms are quickly removed from thebiofilm, which leads to a weakening and/or erosion of the biofilm. This phenom-enon could explain the decrease ofhead losses after the Penstock waterdrainage. The study of Douterelo et al.(2013) is very interesting regarding theinfluence of hydraulic regimes on bacte-rial biofilm communities. Using an exper-imental drinking water distribution sys-tem, they observed that flushing alteredthe pipe-wall bacterial community struc-ture without complete removal of bacte-ria from the pipe surfaces, even underhighly varied flow conditions. Knowingthat, water drainage may be consideredas more efficient in biofilm control thanmodification in hydraulic regimes. Oncehead losses caused by the biofilm arestabilized and deemed to be within anacceptable range of values, water drain-age does not appear to be relevant. Astudy of the balance of organic versusmineral composition of the biofilm andbacterial viability (by ATPmetry, viabilityPCR or cytometry) would be interestinginorder togivenew informationabout theimpact of the water drainage.

It is important to note that contrary tothebiofilm formedonreinforcedconcrete,the biofilm formed on galvanized steel inHP was poorly affected by the waterdrainage. However, it was noticed previ-ously that biofilm formed on that part wasless thick and smoother than the biofilmformed on concrete; hence it is probablyless responsible for head losses.


Since hydrodynamic solutions tocontrol biofilm formation (includingWD) are difficult to implement and con-sidering the potential direct linkbetween the microbial profile of waterIntake and those of pipe biofilms, a sur-vey of the water Intake compositionwould be of interest to anticipate biofilmmodification in composition.

ACKNOWLEGEMENTS

We are grateful to Water Qualityand Biodiversity Department (AE Lab)technical staff for their daily efforts tofulfill samplings and analyses and pro-vide a consistent water quality monitor-ing. We also would like to thank EDFand NTPC in providing all logistics,safety requirements, all useful informa-tion/knowledge (Technical Departmentof NTPC), and the time needed to per-form such a study. Their dedication toscience while operating heavy mainte-nance work should be underlined.

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