Occurrence of Manganese-Oxidizing Microorganisms and Manganese Deposition During Bio¢Lm Formation on Stainless Steel in a Brackish Surface Water

FEMS Microbiology Ecology 39 (2002) 41^55 www.fems-microbiology.org

Occurrence of manganese-oxidizing microorganisms and manganese deposition during bio¢lm formation on stainless steel in a brackish surface water

Jan Kielemoes a, Isabelle Bultinck b, Hedwig Storms b, Nico Boon a, Willy Verstraete a;* Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021

a Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium b OCAS N.V., John Kennedylaan 3, B-9060 Zelzate, Belgium

Received 25 May 2001; received in revised form 8 October 2001; accepted 10 October 2001

First published online 5 December 2001

Abstract

Biofilm formation on 316L stainless steel was investigated in a pilotscale flow-through system fed with brackish surface water using an alternating flow/stagnation/flow regime. Microbial community analysis by denaturing gradient gel electrophoresis and sequencing revealed the presence of complex microbial ecosystems consisting of, amongst others, Leptothrix-related manganese-oxidizing bacteria in the adjacent water, and sulfur-oxidizing, sulfate-reducing and slime-producing bacteria in the biofilm. Selective plating of the biofilm indicated the presence of high levels of manganese-oxidizing microorganisms, while microscopic and chemical analyses of the biofilm confirmed the presence of filamentous manganese-precipitating microorganisms, most probably Leptothrix species. Strong accumulation of iron and manganese occurred in the biofilm relative to the adjacent water. No evidence of selective colonization of the steel surface or biocorrosion was found over the experimental period. The overall results of this study highlight the potential formation of complex microbial biofilm communities in flow-through systems thriving on minor concentrations of manganese. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Bio¢lm; Pilotscale simulation; Stainless steel; Manganese-oxidizing microorganism; Biocorrosion

1. Introduction potential of the steel (Ecorr or corrosion potential) in a positive or negative direction, depending on the nature Biocorrosion of stainless steel can be catalyzed by a of the deposit. In principle, the more positive the poten- series of metabolic activities of a wide range of microor- tial, the better metal ions are capable to leave the metal ganisms. In the past, sulfate-reducing bacteria (SRB) were surface. Pitting corrosion of stainless steel occurs when considered to be the most common catalysts of biocorro- this open-circuit potential becomes equal to or more pos- sion, whereas the role of other bacteria has been seriously itive than the pitting potential of the steel (Ep). Manganese neglected [1]. Moreover, aerobic bacteria generally have oxide biodeposition on stainless steel surfaces for example much higher growth rates than anaerobes, and their meta- can force a shift of Ecorr in the positive direction, making bolic activity is much higher, potentially leading to higher some stainless steels more vulnerable to pitting and crevice corrosion rates. Consequently, other microorganisms than corrosion [2,3]. On the other hand, bioprecipitated sul¢des SRB are currently suspected to be related to biocorrosion can lead to a lower potential of the steel. Mineral disso- in industrial systems. Microorganisms performing miner- lution reactions can remove protective passive layers (e.g. alization reactions for instance can in£uence corrosion by Cr and Fe oxide layers in the case of stainless steel) or both forming and dissolving mineral deposits [2]. Mineral force mineral replacement reactions that lead to further deposition on a metal surface can shift the open-circuit metal dissolution: e.g. the metal-reducing bacterium She- wanella putrefaciens can reduce solid Fe3 oxide to soluble Fe2 oxide [4]. According to the mixed potential theory * Corresponding author. Tel.: +32 9 264-59-76; Fax: +32 9 264-62-48. this cathodic reaction consumes electrons produced by the E-mail address: [email protected] (W. Verstraete). anodic dissolution of the underlying metal, thus increasing

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart 42 J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 the corrosion rate. Interrelationships and cooperation be- were used in the experiments. The £ow/stagnation/£ow tween for instance manganese- and iron-oxidizing bacteria regime was used to simulate the hydrostatic testing prac- and SRB can lead to a more severe corrosion than can be tice. Water systems are subjected to a hydrotest before expected from each individual member of such a complex putting them into service. At least one failure occurred microbial community. A model for these interactions was after a hydrotest leaving the brackish surface water in already proposed by Dickinson and Lewandowski [5]: the piping system for several weeks before the start-up.

MnO2 depolarization shifts Ecorr to values exceeding Ep The bio¢lm was extensively characterized and the e¡ect at local sites of diminished redox potential (SRB niche). of bio¢lm development and its reaction products on the In laboratory bio¢lm and/or corrosion experiments pure corrosion of the stainless steel was investigated. cultures are often used to obtain a better understanding of the mechanisms of bacterial adhesion, bio¢lm formation and corrosion in more complex industrial systems. The 2. Materials and methods dynamic interactions of di¡erent organisms together with Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 a constantly changing environment as in the case of nat- 2.1. Pilotscale £ow-through system ural waters cannot be simulated in laboratory experiments only. The processes of bio¢lm formation and corrosion are Fig. 1 represents the design of the pilotscale £ow- interrelated and observed corrosion rates in practice rarely through system. Five transparent, disconnectable PVC correlated with pure-culture simulations. Detailed back- tubes (inner diameter = 36 mm and length = 1 m) were ground information and the application of the appropriate coupled to a water reservoir of about 0.6 m3 and mounted analytical techniques to determine this multiplicity of pa- in a closed wooden construction to control the develop- rameters are vital in corrosion failure analyses or during ment of photosynthetic bacteria and algae. Stainless steel simulation experiments in order to reach reliable conclu- coupons (2.9U6.9 cm) were tungsten inert gas welded sions. grade AISI 316L sheets (2 mm thick) with a smooth 2B The aim of the present work was to study in situ natural surface ¢nish obtained from stainless steel producer ALZ bio¢lm adhesion and biocorrosion simulation experiments (Genk, Belgium). Five welded stainless steel coupons to- on welded stainless steels in a brackish surface water using gether with one microscope glass slide as reference materi- a pilotscale £ow-through system with an alternating £ow/ al were mounted in Ertalon1 rails with the weld perpen- stagnation/£ow regime. In the past, the application of this dicular to the £ow direction, electrically insulated from brackish surface water in cooling water systems resulted in one another to prevent galvanic e¡ects. The £ow-through numerous corrosion failures of type 316L stainless steel medium was natural brackish surface water (canal water tubes, some of which have been tentatively attributed to Ghent^Terneuzen, Belgium), indicated further as `canal microbial in£uences. As the corrosion was predominantly water'. The chemical composition of the water in the res- appearing at the circumferential unpickled welds, welded ervoir of the pilot plant system (sample point D, Fig. 1) stainless steel coupons without removal of the heat tints measured at the end of the experiment (representative for

Fig. 1. Design of the pilotscale £ow-through installation. Sampling points: (A) fresh canal water, (B) water in the reservoir after manually mixing and stirring the content, (C) sedimented sludge at the bottom of the tank, (D) water at the upper part of the reservoir (mixing zone of incoming fresh canal water and the water present in the tank) and (E) water at the end of the over£ow tubing.

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 43

Table 1 chloride (CTC)/4,6-diamidino-2-phenylindole (DAPI) (see Chemical composition of the water in the reservoir of the pilot plant below). Procedure B was intended to analyze the chemical system (sample point D, Fig. 1) measured at the end of the experiment composition of the adhered material. The bio¢lm at both (expressed in mg l31, except pH) sides of the coupon, except from the welded area and the Parameter Value Parameter Value heat-a¡ected zone (HAZ), was removed by swabbing with pH 7.7 Kj-N 5.0 cotton wool sticks and transferred into 30-ml sterile Milli- Suspended solids 3 NO3-N+NO3-N 6.5 3 2 Q water. These solutions were vortexed intensively during Chemical oxygen demand 24 Cl3 453 5 min to break up particulate matter and some important Biological oxygen demand 6 Natotal 148 Dry residue 1550 Ktotal 17 chemical parameters were determined (see below). Proce- Ash residue 1180 Mgtotal 25 dure C was almost identical to procedure B, with the ex- SO23-S 55 Ca 133 4 filtrate total ception that the coupons were swabbed completely at both Ptotal 0.3 Fetotal 1.70 sides. This is done in order to evaluate the e¡ect of weld- NH-N 0 Mn 0.23 4 total ing on the chemical/biological composition of the bio¢lm. Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 In procedure D one side of the coupons was sampled according to procedure C. The other side with the intact the whole experiment) is described in Table 1. A centrifu- bio¢lm was subjected to an electron microscopical inves- gal pump recycled the canal water from the reservoir tigation (electron probe micro analyzer (EPMA)/EDX, see through the system. The testing period was about 12 below). weeks. In a ¢rst phase the canal water was pumped through the system during 9 days at an average £ow 2.3. Biological characterization of the medium and rate of 0.16 m s31. In a second phase, stagnancy condi- the bio¢lm tions were applied during about 8 weeks (53 days) simu- lating the period after hydrostatic testing without draining 2.3.1. Light microscopy the tubes. In a third period, two tubes were disconnected Visualization of the bio¢lm material was performed us- and kept at room temperature until sampling (indicated as ing a Reichert^Yung Polyvar light microscope with No- `stagnant'), while in the other tubes a second £ow-through marski di¡erential interference contrast. The microscope period was applied with an average £ow rate of 0.11 m s31 was equipped with a Peltier cooled single chip digital color during about 3 weeks (19 days) allowing the bio¢lm thick- CCD camera (Hamamatsu Orca IIIm) connected to a PC ness to increase (indicated as `£ow'). The reservoir re- to obtain digital images. ceived fresh canal water at a rate of 0.15 m3 h31 during the whole experimental period, thus the content of the 2.3.2. Epi£uorescence microscopy tank was replaced about six times daily. During the experi- Putative viable and non-viable bacteria of the scraped ment the pH in the tank was about 7.7, the redox potential bio¢lm (procedure A) and the canal water were stained 0 (Eh) about +455 mV, the temperature between 10 and with a £uorescent bacterial viability stain LIVE/DEAD 15³C and the chloride content around 450 mg l31. BacLight1 Bacterial Viability kit L-13152 (Molecular Probes) [6]. The stained microorganisms on the polycar- 2.2. Sampling procedures bonate ¢lters were visualized using a Leitz Orthoplan epi- £uorescence microscope. Digital photographs from 10 spa- Each sampling procedure described below was applied tially distinct, randomly chosen spots of each coupon for on one coupon from each tube, unless otherwise indicated. each time interval were analyzed by digital image analysis. Procedure A was performed under sterile conditions to In situ coloration of intact bio¢lms present on the stainless visualize the microorganisms adhered to the surfaces by steel coupons and glass slides with CTC (staining respira- means of ex situ and in situ techniques. For this, half of tory active cells) (Polysciences Inc.) and DAPI (staining all the coupon at both sides was sampled: the adhered mate- cells) (Sigma-Aldrich) and the visualization thereof were rial was scraped and swabbed o¡ by sterile cotton wool performed according to the method described elsewhere sticks, transferred aseptically in 5 ml physiological solu- [7]. tion (8.5 g NaCl l31), containing 0.1% (w/v) sodiumthio- glycolate to obtain reducing circumstances and to avoid 2.3.3. Standard microbiological and speci¢c isolation oxygen stress towards anaerobic microorganisms. These techniques mixtures were vortexed to disaggregate clusters of micro- Spread plating of the scraped bio¢lm suspensions (pro- organisms and clumps of inorganic or organic material. cedure A) on nutrient agar (NA) and anaerobic agar (AA) The LIVE/DEAD0 coloration technique and standard mi- (Difco) was performed. A 10-fold dilution series was ap- crobiological plating techniques were applied on these sus- plied and the 1033^1037 dilution suspensions were used pensions (see below). The intact part of the bio¢lm still initially. The aqueous £ow-through medium was also present at each side of the steel coupons and glass slide sampled aseptically, 10-fold diluted and plated onto NA was colored in situ with 5-cyano-2,3-ditolyl-tetrazolium- and AA. These plates were incubated respectively under

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart 44 J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 aerobic and anaerobic conditions at 28³C for 48 h. Anaer- of the DGGE patterns on this DGGE gel was done with obic incubation of plates was performed in jars with a the GelCompar software 4.1 (Applied Maths). The calcu- controlled atmosphere containing 84% N2,8%H2 and lation of the matrix of similarities is based on the Pearson 8% CO2, adjusted by the Anoxomat 8000 system (Mart). product-moment correlation coe¤cient. The clustering al- Aerobic and anaerobic colony-forming units (log10 CFU gorithm of Ward was used to calculate dendrograms. A ml31) were determined. In the case of scraped bio¢lm second attempt to indicate the presence of Leptothrix sp. suspensions CFU were recalculated to values in log10 in the samples was performed: a DGGE gel was run with cm32. The aqueous media containing the scraped bio¢lms 16S rDNA fragments from the samples and from L. cho- were also plated onto speci¢c growth media: manganese lodnii LMG 8142 and L. mobilis DSM 10617, ampli¢ed agar no. 2 [8], a Mn2 containing growth medium de- with primer set P338f and P518r. In a third attempt to scribed as a selective medium for sheath-forming manga- characterize the microbial population of the black sludge nese- and iron-oxidizing bacteria type Leptothrix species found in the pilot system (sample C, Fig. 1), a bacterial (indicated further as Mn agar). Another speci¢c medium clone library was created. Therefore, 16S rDNA gene frag- Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 was CGY agar (casitone glycerol yeast extract agar; [8]), ments were ampli¢ed by using universal primers R1n and which is used for the enumeration of ¢lamentous iron- U2 as described previously [13]. The speci¢c 16S rDNA oxidizing bacteria type Sphaerotilus species. The same gene fragments were cloned by using the TOPO TA clon- techniques were applied on a representative sample of ing kit (Invitrogen) according to the manufacturer's in- the canal water. The most prevailing single colonies grow- structions. DNA sequencing of the clones was carried ing on the plates were picked up, restreaked and puri¢ed out by IIT Biotech ^ Bioservice (Bielefeld, Germany). by plating on selective media to obtain pure cultures. Analysis of the DNA sequences and homology searches These colonies were Gram-stained and studied microscopi- were completed with standard DNA sequencing programs cally. Selective coloration of manganese-oxidizing capacity and the BLAST server of the National Center for Biotech- by using a leuco-crystal violet solution bu¡ered at pH 4 nology Information using the BLAST algorithm [14] for coloring the Mn4 purple as described by Kessick et al. [9] the comparison of a nucleotide query sequence against a was applied to evaluate the presence of mineral encrusta- nucleotide sequence database (blastn). Nucleotide sequen- tion. Visual assessment of the presence of manganese- ces for fragments of the di¡erent libraries have been de- oxidizing bacteria in the bio¢lm was performed by light posited in the GenBank database. microscopy after coloration of the scraped bio¢lm sample itself and of isolated colonies on streak-plated inocula on 2.4. Chemical characterization of the medium and Mn agar. the bio¢lm

2.3.4. Community analysis by denaturing gradient gel 2.4.1. Chemical determination electrophoresis (DGGE) and sequencing The ash and dry residue, and the total amount of Fe, Characterization of the principal members of the micro- Mn, Ni and Cr were determined by £ame atomic absorp- bial communities of the adjacent water at di¡erent sam- tion spectrometry (Perkin-Elmer AAS 3110) on the pling points (Fig. 1) was performed with DGGE and iden- scraped bio¢lm solutions (procedures B and C) and on ti¢cation via sequencing. Total DNA was extracted from the £ow-through medium at the end of the experiment the samples and from Leptothrix cholodnii LMG 8142 according to standard methods [15]. (=Leptothrix discophora SP-6(s), ATCC 51168) and Lep- tothrix mobilis DSM 10617 based on the protocols de- 2.4.2. Electron microscopy scribed by Boon et al. [10]. PCR conditions were applied Surface analysis of the adhered bio¢lm was performed as described by Boon et al. [10], by using P63f or P338f as with an EPMA type Jeol JXA 8800L on coupons obtained forward primers and P518r as the reverse primer [11,12]. from sampling procedure D. Morphological and chemical DGGE based on the protocol of Muyzer et al. [11] and information of the bio¢lm was collected by recording sec- Boon et al. [10] was performed using the Bio-Rad D Gene ondary electron images, EDX spectra and elemental dis- System. The polyacrylamide gels were made with denatur- tribution mappings. ing gradient ranging from 45 to 60%, 6% acrylamide (primer set P63f and P518r) and 50^65%, 8% acrylamide 2.4.3. X-ray di¡raction (XRD) measurements (primer set P338f and P518r) (where 100% denaturant A Siemens D 5000 X-ray di¡ractometer was used for contains 7 M urea and 40% formamide). The electropho- the identi¢cation of crystalline phases in the precipitated resis was run for 16 h at 60³C and 50 V (primer set P63f material on the stainless steel surfaces. and P518r) or 40 V (primer set P338f and P518r). In a ¢rst attempt to identify the bacteria present in the samples, 2.5. Labscale electrochemical measurements abundant bands of the DGGE with primer set P63f and P518r were excised, PCR-ampli¢ed and sequenced by The open-circuit potential of the stainless steel coupons Eurogentec (Lie©ge, Belgium). The statistical comparison with and without bio¢lm was measured ex situ in a syn-

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 45 thetic brackish water solution. The electrochemical mea- pilot plant installation at irregular moments in time, but surements were performed using a Potentiostat EGpG some increase by growth in the reactor cannot be ex- Princeton Applied Research 273A, Model KO235 cell cluded. with an Ag/AgCl reference electrode. 3.2. Biological characterization of the medium and 2.6. Statistical analyses the bio¢lm

Multivariate analysis of variance (ANOVA) was per- 3.2.1. Light microscopy formed on the bacterial counts and chemical determina- The predominant microorganisms found in the bio¢lm tions with the statistical data analysis software SPSS 9.0. adhered to the stainless steel coupons, the glass slides and A P value 90.05 was considered signi¢cant. the sidewalls of the PVC tubes, and in the black sludge layer at the bottom of the reservoir are depicted in Fig. 2. They showed a striking resemblance with sheathed iron- Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 3. Results and manganese-oxidizing bacteria (L. discophora) according to Bergey's Manual of Systematic Bacteriology [16], 3.1. Visual observation of bio¢lm formation based on some visual characteristics such as the presence of ¢laments, the diameter of the ¢laments, the occurrence Due to the transparency of the PVC tubes, a ¢rst visual of metal encrustation (irregular, rusty, inorganic deposits inspection of biofouling on the surfaces (glass, stainless at the outer layer of the ¢laments), and the presence of steel and PVC) could be performed. After the ¢rst period empty sheaths (arrow in Fig. 2). (9 days) of laminar £ow, a thin brownish and spotty layer was observed to develop or adhere on all surfaces. During 3.2.2. Selective coloration the stagnancy period a uniform black layer developed, DIC microscopy of a leuco-crystal violet-colored covering the complete surface of the stainless coupons scraped sample of a stainless steel bio¢lm revealed the and glass surfaces. In the second period of laminar £ow, presence of manganese mineral encrustation (purple-col- a lot of brown/black suspended solids were observed to ored ¢laments), with obviously also empty sheaths (no deposit within a few days on the PVC tube walls and coloration). When coloring isolated colonies on streak- onto the coupons, resulting in a thick and loosely adhering plated inocula of scraped bio¢lm suspensions onto Mn bio¢lm. It was also noticed that a comparable brown/ agar, most colonies were coloring dark purple, indicating black sludge accumulated on the bottom and on the walls the presence of manganese-oxidizing capacity of the bac- of the water reservoir. This kind of material entered the teria originating from the bio¢lm (results not shown).

Fig. 2. Di¡erential interference contrast microscopy of the ¢lamentous growth in the pilot plant installation (500U magni¢cation). The arrow indicates an empty sheath. The diameter of the ¢laments was 3^5 Wm. Metal encrustation was shown by selective coloration with crystal violet.

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart 46 J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55

3.2.3. Epi£uorescence microscopy ences were found between glass and steel surfaces on the Viable counts of bacteria of the scraped bio¢lm are in- amount of microorganisms (Table 1). The aqueous £ow- dicated in Table 2. The non-viable bacteria ( 6 5 log10 cells through medium sampled at point D (Fig. 1), i.e. the water cm32, data not shown) were outnumbered by the amount at the upper part of the reservoir (mixing zone with in- 32 31 of bacteria with intact membranes (7^9 log10 cells cm ). coming fresh canal water), resulted in 4.3 log10 CFU ml 31 31 In the the canal water 4.48 log10 viable bacteria ml and and 6.3 log10 CFU ml respectively under aerobic and 31 3.37 log10 non-viable bacteria ml were present, indicat- anaerobic incubation conditions. Plating of the aqueous ing another ratio of viable/non-viable bacteria compared £ow-through medium sampled at point D on speci¢c me- 31 with the bio¢lm. Additional £ow-through during 19 days dia resulted in 5.0 log10 CFU ml (CGY agar) and 5.5 31 resulted in a pronounced and statistically relevant increase log10 CFU ml (Mn agar), revealing a moderate general (about one exponential unit) of viable bacteria on both microbial activity and a relatively high manganese-oxidiz- stainless steel coupons and glass surfaces. There was no ing microbial activity in this kind of surface water. Only sign of selective adhesion of the bacteria on the glass re- Gram-positive bacteria (cocci, grain-shaped rods and long Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 spectively on stainless steel surfaces. The microorganisms rods) were found directly after the stagnancy period, while also heavily colonized the PVC tube walls. The in situ Gram-negative rods and cocci were established after an coloration of the intact bio¢lm on the stainless steel cou- additional period of £ow (results not shown). The most pons and glass slides with CTC and DAPI was di¤cult to predominant on agar cultivable microorganisms after the visualize because the bio¢lm was too thick, especially after stagnancy period and after an additional £ow-through pe- the additional exposure period to canal water. Metal-en- riod were obviously di¡erent, showing a shift in the micro- crusted sheaths, as well as ¢ne ¢laments and isolated cells bial population. were abundant over the entire coupon surface. No striking di¡erences in colonization were found between the base 3.2.5. Community analysis by DGGE and sequencing steel surface and the HAZ or welded area. Analysis of Characterization of the microbial communities of the bio¢lm development and the types of microorganisms water at di¡erent sampling points (Fig. 1) with DGGE present showed only minor di¡erences between stainless is shown in Fig. 3. This ¢gure indicates that after sedimen- steel coupons and glass slides. tation of the canal water (lanes D and E), microbial communities were di¡erent from the mixed canal water (lanes 3.2.4. Standard microbiological and speci¢c isolation A and B), while the latter compared well with the com- techniques munity of the sludge at the bottom of the tank (lane C). Results of spread plating of scraped bio¢lm suspensions Some of the most important members of the microbial on non-speci¢c (NA and AA) and on speci¢c growth me- community of the canal water were further identi¢ed via dia for sheath-forming manganese- or iron-oxidizing bac- excising and sequencing. The closest relatives resulting teria type Leptothrix species (Mn agar) and Sphaerotilus from homology searches are reported, with indication of species (CGY agar) are presented in Table 2. The biomass the amount of identical base pairs as well as the percent- adhered to the stainless steel and glass surfaces consisted age of similarity (Table 3). Nucleotide sequences for frag- of about the same numbers of anaerobic and aerobic bac- ments of the di¡erent libraries have been deposited in the teria, even after a period of 8 weeks of stagnancy. Gen- GenBank database and accession numbers are indicated. erally, more bacteria adhered to the stainless steel surface A large part of the excised and sequenced DGGE bands after the supplementary exposure to £owing natural water. referred to uncultured or unidenti¢ed clones from environ- ANOVA analyses revealed a signi¢cant in£uence of the mental samples, while, except from some bands, similar- additional £ow-through period on microbial colonization ities below 90% were found, possibly indicating the pres- of the stainless steel coupons and glass surfaces, except for ence of some new types of microorganisms. CA8 showed the results of plating on Mn agar. No signi¢cant di¡er- some similarity to L. mobilis (L-proteobacterium, 382/414

Table 2 Microbiological ex situ determinations (48 h incubation at 28³C) of the scraped bio¢lm adhered to 316L stainless steel coupons (SS) respectively glass slides (glass) after a stagnancy period (stagnant) and an additional £ow period (£ow): epi£uorescence microscopic count of LIVE bacteria found in the 32 32 scraped bio¢lm samples (log10 cells cm þsx) and plating on non-speci¢c and speci¢c media (log10 CFU cm þsx)(n =4) Sample LIVE Plating NA AA CGY agar Mn agar SS (stagnant) 7.19 þ 0.67a 7.78 þ 0.62a 7.61 þ 0.89a 8.26 þ 1.53a 8.57 þ 1.49 SS (£ow) 8.75 þ 0.42a 8.88 þ 0.04a 8.51 þ 0.50a 10.24 þ 0.09a 10.43 þ 0.23 Glass (stagnant) 7.49 þ 1.22a 8.27 þ 0.09a 8.20 þ 0.04a 10.08 þ 0.06a 10.74 þ 0.68 Glass (£ow) 8.30 þ 0.28a 8.79 þ 0.01a 8.79 þ 0.04a 9.83 þ 0.39a 10.11 þ 0.16 ANOVA analyses were performed on the results of each speci¢c microbial count (columns). aANOVA: signi¢cantly di¡erent (P90.05) within a column when the kind of material is the constant factor.

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 47

Fig. 3. Digitized scheme of the DGGE pro¢le of 16S rDNA fragments (ampli¢ed with primer set P63f and P518r) with indication of the percentage of similarity (A: incoming canal water; B: canal water after mixing of the sedimented sludge; C: sludge at the bottom of the sedimentation tank; D: canal water in the tank after sedimentation; E: over£ow canal water). Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 and 21/21, 92% and 100%, X97071) or Leptothrix SL23 and SL33), chemolithothrophic sulfur-oxidizing bac- MBIC3364 (L-proteobacterium, 384/414, 92%, teria (Q-proteobacteria) (SL6, SL24, SL26 and SL28), and AB015048) and a denitrifying Fe-oxidizing proteobacte- slime-producing bacteria type Pseudomonas sp. and Sphin- rium (410/459, 89%, U51102). Aeromicrobium sp. (CA10 gomonas sp. (SL11, SL23 and SL37). Clones SL12, SL16 and CA11) is an actinomycete. CA1, CA2 and CA3 be- and SL22 belong to the Actinobacteria. Clone SL25 (My- long to the Cytophaga^Flavobacteria^Bacterioides (CFB) cobacterium) is an actinomycete (Actinobacteria), while group. SL30 and SL35 are planctomycetes. Ferromicrobium Another DGGE with the two Leptothrix sp. as reference acidophilum is a heterotrophic iron-oxidizing acidophile. strains (Fig. 4) indicated that L. mobilis DSM 10617 SL8 belongs to the CFB group of bacteria and SL19 is showed up at about the same height as an important a L-proteobacterium. An interesting result of this sequenc- band of the canal water (indicated by the arrows). How- ing reaction is the relation of SL14 and SL18 with uncul- ever, no unequivocal conclusions could be drawn about tured Green Bay ferromanganous micronodule bacteria the identity of this speci¢c band. MNF4 (Rhodoplanes) or MND8 (Thiobacillus) [17], as A clone library was created of the bacterial species in the presence of such kind of micronodules was detected the black sludge found at the bottom of the reservoir on stainless steel surfaces with EPMA (see further). No (sample C, Fig. 1), which also appeared to adhere easily direct indication of the presence of Leptothrix sp. in this to all kinds of surfaces (glass slides, stainless steel coupons sludge was found via these techniques. and PVC sidewalls of the tubes). Forty distinct colonies were randomly selected, of which ¢nally 22 visually di¡er- 3.3. Chemical characterization of the medium and ent positioned DGGE bands were further sequenced. Re- the bio¢lm sults of the sequencing of these clones are shown in Table 4, indicating rather low similarities (between 80 and 95, 3.3.1. Standard chemical determinations except from SL25), but with rather high amounts of iden- The chemical composition (dry residue, ash residue, Fe, tical base pairs. The black sludge at the bottom of the Mn, Ni and Cr content) of the bio¢lm sampled according reservoir consisted of a very complex microbial commun- to procedures B and C was determined on scraped bio¢lm ity comprising: SRB (mostly N-proteobacteria) related to suspensions after the stagnancy period and after the addi- Desulfovibrio sp., Desulfobulbus sp. or Geobacter sp. (SL5, tional £ow period. No remarkable di¡erences or selectivity

Table 3 Sequence similarities of the excised bands of some important members of the microbial communities from the incoming canal water (A) (no. CA1^ CA11) and from the over£ow of the reservoir (E) (CA15) of the excised DGGE bands shown in Fig. 3 Sequence Nearest homology results (accession no.) Identical base pairs (similarity, %) Accession no. Canal water CA1 Uncultured bacterium SY4-2 (AF107525) 351/409 (85) AY034832 CA2 Uncultured Cytophagales clone CRE-FL39 (AF141460) 352/408 (86) AY034833 CA3 Uncultured Cytophagales clone CRE-PA7 (AF141497) 342/415 (82) AY034834 CA4 Uncultured eubacterium clone WR124 (AJ233553) 372/419 (88) AY034835 CA6 L-Proteobacterium SCB53 (AF026392) 320/398 (80) AY034836 CA7 Uncultured Q-proteobacterium clone CRE-FL8 (AF141439) 293/353 (83) AY034837 CA8 L-Proteobacterium SCB52 (AF026391) 456/459 (99) AY034838 CA10 Aeromicrobium erythreum (AF005021) 410/428 (95) AY034839 CA11 A. erythreum (AF005021) 389/434 (89) AY034840 Canal water at the over£ow of the reservoir CA15 Uncultured Q-proteobacterium clone CRE-PA88 (AF141555) 349/391 (89) AY034844

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart 48 J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55

were found between the bio¢lm chemical composition on the welded area, the HAZ or the stainless steel base material (results not shown). On the other hand, signi¢cant di¡erences in bio¢lm chemical composition were detected between the stainless steel and the glass surface. Average values of the ash and dry residue in mg per cm2 are given in Table 5. After 8 weeks of stagnancy, dry residue values of the bio¢lm adhered to the stainless steel coupons doubled those of the bio¢lm on glass surfaces, while the ash residues were the same (Table 5). This means that the bio¢lm on the stainless steel surface contained more organic material. After an additional £ow-through period, the di¡erences between glass and steel surfaces became Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 minimal. A lot of black material was observed to deposit within a few days during this £ow period. It resulted in the case of stainless steel surfaces in equally increased values of dry or ash residues, while it resulted in the case of glass surfaces in distinct higher values of the dry residue (two to three times higher). In all scraped bio¢lms adhered to stainless steel, fairly low concentrations of Ni and Cr (the principal alloying elements of austenitic stainless steels) were found, while no Ni or Cr was found in bio- ¢lms on glass surfaces (results not shown). Average values of the total amount of Fe and Mn present in the bio¢lm are given in Table 5. The Fe and Mn concentrations in the bio¢lm increased signi¢cantly when subjected to an additional £ow-through period. Values of about 3 and 2 wt% of the dry residue, respectively for Fe and Mn, were found. Further characterization of the sludge at the bottom of the reservoir revealed the following values: dry 31 Fig. 4. DGGE pro¢le of 16S rDNA fragments (ampli¢ed with primer residue: 98.5 mg g sludge (wet weight); all other param- 31 23 set P338f and P518r) with two pure cultures of Leptothrix species as eters are expressed in mg g sludge (dry weight): SO4 - reference strains (LMG 8142 and DSM 10617) (A: incoming canal 33 Stotal : 17.1; PO4 -Ptotal : 6.4; Natotal : 33.1; Ktotal : 1.3; water; C: sludge at the bottom of the sedimentation tank). The arrows Catotal : 73.2; Mgtotal : 7.5; Nitotal : 0.3; Crtotal : 0.8; indicate the band in lane A that shows up at spatially the same place as Fe : 32.4; Mn : 47.3. Iron and manganese accumu- DSM 10617. total total

Table 4 Sequence similarities of the clones originating from the black sludge Clone Nearest homology results (accession no.) Identical base pairs (similarity, %) Accession no. SL5 Metal-contaminated soil clone K20-12 (AF145815) 417/477+59/65 (87+90) AF379684 SL6 Sulfur-oxidizing bacterium OAII2 (AF170423) 554/604 (91) AF379688 SL8 Uncultured bacterium PHOS-HE79 (CFB) (AF314434) 566/616 (91) AF379685 SL11 Sphingomonas subartica strain NKF1 (X94104) 648/686 (94) AF379686 SL12 F. acidophilum (AF251436) 551/621 (88) AF379687 SL14 Uncultured bacterium MNF4 (AF292996) 570/617 (92) AF379689 SL16 Unidenti¢ed planctomycete OM190 (U70712) 485/572 (84) AF379690 SL18 Uncultured bacterium MND8 (AF292999) 546/611 (89) AF379691 SL19 Azoarcus sp. BH72 (AF011344) 562/614 (91) AF379692 SL22 Uncultured bacterium ARFS-13 (AJ277692) 522/559+32/37 (93+86) AF379693 SL23 Desulfobulbus sp. McCal 25m (AF132868) 306/351+40/42 (87+95) AF379694 SL24 Ochrobactrum intermedium isolate OiC8a (AJ242583) 545/634 (85) AF379695 SL25 Mycobacterium sp. IFO16251 (AB023372) 599/622 (96) AF379696 SL26 Uncultured Q-proteobacterium Sva0304 (AJ240971) 633/698 (90) AF379697 SL28 Sulfur-oxidizing bacterium ODIII6 (AF170422) 683/746 (91) AF379698 SL30 Uncultered planctomycete clone DSP08 (AJ290171) 668/701 (95) AF379699 SL33 Myxococcus xanthus strain DSM 435 (Mx-x1) (AJ233929) 506/610 (82) AF379700 SL35 Uncultered bacterium 0319-7F4 (AF234144) 385/431 (89) AF379701 SL37 Unidenti¢ed pseudomonad ps.4 (AF006505) 530/595 (89) AF379702

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 49

Table 5 Average values and standard deviation of dry residue, ash residue and of total iron and manganese (with their average wt% of the dry residue between brackets) determined on scraped bio¢lm suspensions of stainless steel coupons (n = 12) (SS) and glass surfaces (n = 4) (glass) after stagnancy (stagnant) or after an extra £ow-through period (19 days) (£ow) Dry residue (mg cm32) Ash residue (mg cm32)Fe(Wgcm32)Mn(Wgcm32) SS (stagnant) 0.94 þ 0.28a;b 0.35 þ 0.12 6.63 þ 3.97 (0.78 þ 0.26)a 2.28 þ 1.16 (0.21 þ 0.08)a SS (£ow) 1.36 þ 0.32a 0.60 þ 0.24 31.26 þ 12.33 (2.27 þ 0.61)a 24.88 þ 8.70 (1.83 þ 0.50)a Glass (stagnant) 0.44 þ 0.18a;b 0.35 þ 0.12 4.65 þ 1.06 (1.19 þ 0.71) 1.45 þ 0.07 (0.35 þ 0.11)a Glass (£ow) 1.14 þ 0.32a 0.47 þ 0.05 20.00 þ 18.53 (1.59 þ 1.17) 19.7 þ 14.99 (1.61 þ 0.87)a aANOVA: signi¢cantly di¡erent (P90.05) when the kind of material is the constant factor. bANOVA: signi¢cantly di¡erent (P90.05) when the kind of £ow regime is the constant factor. lated (compared to the values of less than 0.1 mg Mn g31 3.3.3. XRD measurements Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 water dry residue in the adjacent water) up to 3.2 and The adhered material on a stainless steel coupon from 4.7% respectively, while also the high concentration of the pilotscale system was hardly containing any crystalline calcium is apparent. Colorimetric spot tests using leuco- phase. No manganese oxide containing crystalline phases crystal violet indicated that the black deposits present as were present. Only some small amounts of CaCO3 (calcite) well on the glass surfaces, on the stainless steel coupons as and possibly SiO2 (quartz) could be detected. in the sludge colored purple (results not shown). This suggests the presence of manganese in an oxidized state 3.4. Electrochemical measurements (Mn4).

Measurements of the open-circuit potential Ecorr of a 3.3.2. Electron microscopy stainless steel coupon from the pilotscale system with the Filamentous bacteria were found to colonize the glass adhered bio¢lm were performed. Compared to a cleaned and stainless steel surfaces (Fig. 5A,B). Elemental map- reference 316L coupon (EcorrW3200 mVAg=AgCl), the cou- ping of the surface (Fig. 5C) and EDX spot analysis pon covered with a manganese deposits containing bio¢lm (Fig. 5D) indicated elevated values of Mn, Fe, Ca and O showed hardly any di¡erence in open-circuit potential on the ¢laments and to a lesser extent the presence of Si, (EcorrW3100 mVAg=AgCl). Conclusively, no signi¢cant bio- P, S and Cl. Also other remarkable microbial features logically mediated increase in potential of the steel (so- were found under several operating conditions, e.g. the called ennoblement) could be observed. occurrence of manganese-rich micronodules. These micronodules were about 10 Wm in diameter and had an apparent outlook with a central hole of 1^2 Wm (Fig. 5A, within 4. Discussion rectangle). EPMA analysis indicated that these micronodules were high in Mn, Ca and O. The combined presence Characterization of the undisturbed and disturbed bio- of both manganese encrusted ¢laments and small micro- ¢lm deposited on welded stainless steel coupons and on nodules is shown in Fig. 5A. Apparently, the ¢lamentous reference glass slides present in the pilotscale £ow-through microorganisms were dominating the bio¢lm. Growth of system ¢lled with brackish surface water (`canal water') ¢lamentous bacteria with metal precipitation at the outer was performed. Light and epi£uorescence microscopy re- layer of the ¢laments, entanglement of the bio¢lm and vealed the presence of sheathed ¢lamentous metal-deposit- adhesion of higher organisms (such as diatoms) occurred ing microorganisms resembling Leptothrix sp. [16,18]. The during the stagnancy period. During the additional £ow- latter was also substantiated by crystal violet coloration of through period, these Fe- and Mn-rich ¢laments further Mn4 on the sheath of the ¢lamentous growing bacteria. developed together with deposition and precipitation lead- The same ¢laments colored by in situ CTC/DAPI were ing to a thick ( s 1 mm) black sludge layer, which was observed to play a role in the entanglement of the bio¢lm visually observed. The longitudinal axis of most ¢laments and the cross-linking of separated microcolonies. Determi- coincided with the £ow direction. Detailed EPMA analysis nation of the amount of microorganisms present in the showed that these ¢lamentous microorganisms were not scraped bio¢lm adhered to surfaces by epi£uorescence mi- selectively colonizing speci¢c surface features of the stain- croscopy and plating (selective or non-selective) revealed less steel, like scratches, pits or other microheterogeneities no di¡erences between the glass and stainless steel surfa- of the surface. Moreover, they were evenly distributed on ces. Surface heterogeneities (either chemical or physical) the whole coupon surface, thus also in the weld area (both played no major role after a few weeks of incubation, as weld root and HAZ). In the HAZ a crumbling oxide layer no di¡erences between the base metal, the HAZ and the was observed. Filamentous microorganisms developed welded area were observed. Additional £ow-through after under and above these loosely adhering oxides. No corro- a stagnancy period obviously resulted in either growth of sion attack was observed. the already adhered microorganisms and/or additional

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart 50 J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021

Fig. 5. A: Adherence of ¢lamentous bacteria and micronodules on a 316L stainless steel surface (400U magni¢cation); B: entanglement of ¢lamentous bacteria on a 316L stainless steel surface (500U magni¢cation); C: EPMA analysis (with a qualitative color scale on the right) of the surface visualized in the SEM picture on the right (B); D: EDX spectrum of the ¢lamentous bacterium at the arrowhead on the SEM picture on the right (B).

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 51 deposition and adhesion of bacteria originating from the ing is consistent with the EPMA results: manganese-con- adjacent water. After a stagnancy period, especially Gram- taining micronodules of about 10 Wm in diameter are positive microorganisms were found, even on the selective detected in the bio¢lm on the stainless steel surface. This plates, while after the additional £ow-through period only kind of micronodules can cause corrosion problems, espe- Gram-negative bacteria were retrieved. It is known that cially when combined with SRB [5]. These results indicate certain SRB, generally embraced in the genus Desulfoto- that the black sludge (precipitated at the bottom of the maculum (a non-monophyletic group of microorganisms), reservoir or deposited on the glass and stainless steel sur- are Gram-positive [19]. Also other anaerobic or facultative faces) can pose biofouling and, maybe under speci¢c con- anaerobic Gram-positive bacteria exist, e.g. certain Actino- ditions, biocorrosion problems towards the stainless steel myces species. However, Bamarouf et al. [20] stated that underneath. Conclusively, the DGGE and sequencing ap- especially several species of anaerobic bacteria display var- proach revealed the presence of a wide variety of bacteria. iable Gram stain reactions that hamper identi¢cation. For In the context of bio¢lm formation and biocorrosion, this this reason, more precise molecular microbiological deter- approach appeared, however, to be too unfocussed to be Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 minative techniques were used. further implemented in the search for causal colonization DGGE was applied to obtain information about the phenomena. In the future, DGGE analysis with primers composition of the microbial communities found in the for speci¢c groups of bacteria that are of interest in bio- canal water sampled at di¡erent points and in the sludge ¢lm formation or biocorrosion will be used to reveal more precipitated at the bottom of the water reservoir. Analysis detailed information on the kind of microorganisms of the canal water revealed the presence of (Gram-posi- present in the diverse microbial communities. Fluorescent tive) actinomycetes, of which some can have sulfate-reduc- in situ hybridization may represent an additional tool to ing properties [21], and bacteria belonging to the L- and Q- reveal certain corrosion promoting bacterial strains at the proteobacteria. Similarity was found to bacteria from the place of attack or a tool to elucidate selective microorgan- Cytophaga^Flavobacterium cluster of the CFB phylum. In ism^steel interactions. addition, Cythophaga sp. possesses gliding motility, de- Chemical determination of the adhered material on the ¢ned as the movement of a cell on a (solid) surface in coupons revealed a signi¢cant in£uence of the kind of the direction of the longitudinal axis of the cell [22]. Bac- substratum on the dry residue values only in the case of teria from the Cytophaga^Flavobacterium cluster and from stagnant conditions, while no di¡erences in the ash residue the L- and Q-proteobacteria are regularly found to be the content of the bio¢lms were found. Higher dry residue prevailing groups in bio¢lms on surfaces exposed to nat- values in the case of stainless steel and comparable ash ural waters [23,24]. MacDonald and Bro«zel [25] already residue values indicate a higher amount of organics in found that members of the L-proteobacteria were the pre- the bio¢lms adhered to stainless steel surfaces. Because dominant bacteria (50% of the Eubacteria present) in bio- of the fact that no higher bacterial counts were found ¢lms in the industrial cooling water systems they studied, on stainless steel these organics are most probably not while Q-proteobacteria (e.g. Shewanella or Pseudomonas due to higher biomass, but to increased production of sp.) only accounted for 10%. In the case of CA8 (Table extracellular polymers or deposition of other organic mol- 4) also some similarity to Leptothrix sp. and with a deni- ecules such as humic acids. Additional £ow-through led to trifying Fe-oxidizing bacterium was obtained. A further increased values of both organic and inorganic matter of indication of this resemblance was also found by means the bio¢lm. Especially iron and manganese were enriched of DGGE performed on the canal water with two Lepto- i.e. up to 2^3 dry weight percent of the scraped bio¢lm. thrix species as reference strains (Fig. 4). The latter is very Because no signi¢cant in£uence of the substratum was interesting as Leptothrix-like microorganisms were already found on the amount of Fe and Mn, one can conclude found microscopically in the canal water, in the bio¢lms that these Fe and Mn concentrations originated from ac- and in the black sludge at the bottom of the reservoir. cumulation out of the water, and not from the stainless Further cloning and sequencing of a representative sample steel matrix underneath the bio¢lm. Oxidation and precip- of the black sludge revealed initial evidence of a heteroge- itation of dissolved Fe2 and Mn2 is observed regularly neous microbial community with some members already in natural river water, groundwater or drinking water dis- mentioned to be related to biocorrosion of (stainless) steel: tribution systems [34^37]. These transformations may SRB [24,26,27], chemolithothrophic sulfur-oxidizing bac- eventually result in biofouling problems, deterioration of teria (Q-proteobacteria) [21,28,29] and slime-producing or water quality, and even corrosion [38]. Manganese often bio¢lm-related bacteria type Pseudomonas sp. and Sphin- occurs in the same types of oxic^anoxic interfacial envi- gomonas sp. [30^32]. F. acidophilum is a heterotrophic ronments where iron is found, although it is generally iron-oxidizing Gram-positive acidophile [33]. Myxococcus 5^10 times less abundant [39]. Since manganese is not is a gliding bacterium [22]. An interesting ¢nding of this subject to a rapid chemical oxidation, Mn2 may accumu- sequencing reaction is the relation of clones SL14 and late to greater concentrations in more aerobic zones of the SL18 (Table 5) with `uncultured Green Bay ferromanga- water column of marine, brackish and freshwaters than nous micronodule' bacteria MNF4 or MND8. This ¢nd- Fe2 does. In our experiments, an equal amount of iron

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart 52 J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 and manganese accumulation occurred in the bio¢lm. The £uenced corrosion sites on stainless steels. Microprobe fact that the concentrations of Fe and Mn in the sludge at analysis and colorimetric spot tests demonstrated the the bottom of the reservoir were about twice the concen- abundant presence of oxidized manganese (Mn4) on the trations in the deposits on the coupons is most probably sheath structures in the bio¢lm adhered to the stainless due to the fact that during the stagnancy period in the steel coupons and glass surfaces. Thick mature bio¢lms tubes (about 8 weeks), the content of the reservoir was were characterized by entanglement between microcolonies still replaced with fresh canal water. This suggests that by either thick or thin ¢laments and presence of higher Fe- and Mn-depositing microorganisms are also active in organisms. Microbially deposited manganese oxides are the reservoir and that additional £ow-through can lead to generally amorphous MnO2 and sometimes form a crys- gradually increased accumulation of Fe and Mn in the talline black precipitate (Na4Mn14O27W9H2O, birnessite) bio¢lm and on suspended particles. The apparent strong typically found with the oligotrophic Leptothrix sp. [37]. growth of Mn- and Fe-accumulating consortia in the res- The precise form of the oxide/hydroxide may be a¡ected ervoir, together with the fact that in the tubes growth was by the initial Mn2 concentration as well as by the pH, Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 particularly prominent under £ow conditions, clearly sug- redox potential (Eh) and ionic strength of the medium [42]. gests that surface conditions and characteristics were not In our case no crystalline manganese oxides were detected the predominant factor. The high Eh of the reservoir and in the bio¢lm using XRD. Conclusively, the bio¢lm con- the fact that these metal species are in the oxidized state sists of micronodules and mainly ¢lamentous sheath-form- indicate that oxidative conditions are conducive to the ing bacteria precipitating amorphous manganese oxide growth of the causal bacteria. The reaction product of and hydroxide deposits. manganese oxidation, Mn oxides, may decompose com- When the open-circuit potential was measured ex situ plex polymeric (humic) substances (which in general can- on a stainless steel coupon from the pilot plant system not be directly used by microorganisms) to low-molecular with an intact bio¢lm, no signi¢cant ennoblement was mass organic compounds that can be used as substrates measured. An important factor in this context is that man- for microbial growth, e.g. of SRB [40]. Conclusively, such ganese precipitates have to be deposited in direct contact Mn oxide biodeposition can result in enhanced bio¢lm with the stainless steel to exert their electrochemical e¡ects development and microbial turnover reactions ultimately and that precipitated MnO2 may discharge in time [41]. leading to a lowering of the redox potential in the oxygen- This can be the reason why in the case of the pilotscale depleted interior of the cell cluster at the steel surface. coupon the increase of the open-circuit potential was far The combined action of these manganese-oxidizing and below the one caused by a similar manganese oxide con- sulfate-reducing microorganisms together with the electro- taining bio¢lm in a recently studied premature corrosion chemical potential increasing reactions (MnO2 reduction failure of a 316L stainless steel cooling water circuit using to MnOOH) on the stainless steel surface and the preven- the same canal water (EcorrW+300 mVAg=AgCl) or caused tion of repassivation in nucleation sites by MnO2 may by a synthetic MnO2 paste (EcorrW+400 mVAg=AgCl) under mutually increase the risk of pitting corrosion. the same experimental conditions (unpublished results). Characterization of the adhered material by EPMA An increase of the open-circuit potential of stainless steel con¢rmed the presence of di¡erent types of manganese- is considered to represent the ¢rst step of biocorrosion related microorganisms. Both ¢lamentous metal-deposit- [43]. An upward shift in the open-circuit potential exhib- ing sheath-forming microorganisms (e.g. type Leptothrix) ited by microbiologically colonized stainless steel has been and manganese-depositing microorganisms growing in mi- reported for batch labscale experiments with manganese- cronodules (e.g. type Siderocapsa) were found (Fig. 5). depositing pure strains such as L. discophora [3,44^46].

Epi£uorescence and scanning electron microscopy (SEM) Linhardt [47] stated that even minute amounts of MnO2 of a bio¢lm developed under comparable circumstances by at the surface might have considerable in£uence on the

Dickinson et al. [41] also showed the presence of numer- Ecorr of the stainless steel. In their experiments, Dickinson ous 10^20-Wm diameter Mn-rich annular deposits, associ- et al. [44] found the open-circuit potential to shift from ated clusters of bacterial cells, and abundant sheathed 390 to +340 mVAg=AgCl as a bio¢lm containing 75 nmol of 32 32 bacteria on 316L stainless steel coupons exposed in situ MnOx cm (comparable to 4.1 WgMncm ) formed on to fresh river water for periods of up to 35 days. The most the coupon surface. In our experiments, manganese con- abundant manganese-oxidizing bacteria in natural waters centrations were 10-fold higher: manganese concentra- are Leptothrix sp., Gallionella sp. and Siderocapsa sp. [39]. tions up to 30 WgMncm32 were retrieved after a total Sheathed iron bacteria of the genus Leptothrix are com- exposure time to fresh £owing water of about 4 weeks monly found in aquatic habitats containing aerobic^anaer- (Table 5). In the case of Dickinson et al. [44], however, obic interface zones in which Fe and Mn are cycled be- this open-circuit potential changed little further with con- 32 tween their oxidized (insoluble) and reduced (soluble) tinued MnOx biodeposition up to 15 WgMncm . The forms. Manganese-oxidizing microorganisms are fre- fact that Dickinson et al. [44] investigated the mineral quently found in natural (fresh and marine) waters and deposition of a pure culture made it more probable that manganese-rich deposits are often found at microbial in- these manganese oxides were deposited in direct contact

FEMSEC 1307 26-2-02 Cyaan Magenta Geel Zwart J. Kielemoes et al. / FEMS Microbiology Ecology 39 (2002) 41^55 53 with the steel. In our case also other microorganisms can welded stainless steel type 316L is randomly colonized, i.e. initially adhere to the steel surface or other organic/inor- bio¢lm development is not in£uenced by the presence of ganic deposits may be formed preventing a direct contact welds, by several manganese-related microorganisms, pri- between the manganese oxide and the steel. Moreover, in marily sheathed ¢lamentous metal-depositing bacteria of the failure case mentioned before, the bio¢lm resulting in the Leptothrix type. No signs of corrosion were detected an increase of steel potential contained signi¢cant amounts under the present circumstances (brackish surface water of crystalline birnessite, while the pilotscale bio¢lm con- containing 450 mg l31 chloride circulated in a pilot system tained only amorphous manganese (hydr)oxides. This in- at a temperature of 10^15³C). DGGE analysis seemed to dicated that the type of manganese oxide might also be of be a powerful technique to obtain information about tem- importance in order to cause a electrochemical potential poral or spatial shifts in microbial communities of bio¢lms increase. The temperature in the pilotscale £ow-through and corrosion sites, but it provided no direct information system was low (10^15³C) compared to the failure case about the kind of microorganisms. DGGE with selective (30^35³C). As the microbial activity increases drastically primers, further sequencing of excised bands or hybridiza- Downloaded from https://academic.oup.com/femsec/article/39/1/41/535962 by guest on 29 September 2021 with increasing temperatures within this temperature tion can o¡er a solution to these intrinsic limitations. range, this might have in£uenced the type and the amount In this article extensive research carried out on the de- of the manganese deposits. It is also important to mention posited material on welded stainless steel surfaces in a that full colonization of the stainless steel coupons is not a pilotscale £ow-through installation fed with natural brack- prerequisite for stainless steel ennoblement. Dickinson et ish surface water is described, indicating the presence of al. [48] found that development of increased potential oc- microorganisms involved in the deposition and accumula- curred on surfaces with as little as 3^5% biofouling cover- tion of iron- and manganese oxides in the bio¢lm and on age. Some researchers [41,43,48^50] already performed in suspended particles. Further research should elucidate the situ biological experiments with non-welded stainless steel in£uence of temperature on the processes of microbiolog- coupons where ennoblement was observed, but no real ical adhesion and growth, manganese deposition and alter- evidence of corrosion features (e.g. rust products or pits) ation of the open-circuit potential of the steel as a result of was found on these steel surfaces. In aqueous environ- these phenomena. ments containing moderate amounts of chloride ( 6 1000 mg l31) the open-circuit potential of stainless steel grade 316L is typically far below the pitting potential of this Acknowledgements material, even when ennoblement occurs due to the presence of manganese deposits. Welding and especially the This study was funded by a doctoral fellowship of the presence of heat tints, however, can drastically lower the Flemish Institute for the Improvement of Scienti¢c-Tech- pitting corrosion resistance of stainless steels. In this con- nological Research in the Industry (IWT) and ¢nancially text, Avery et al. [51] found a correlation between the supported by SIDMAR N.V. and OCAS N.V. Chris corrosion of 304 stainless steel piping observed beneath Xho¡er is gratefully acknowledged for his expertise and an adhered manganese^iron deposit and the presence of help concerning the electrochemical experiments, Siska heat tints in the HAZ of circumferential welds (308L ¢ller Maertens for her excellent technical assistance in the mo- metal) in high manganese freshwater. lecular work and Ronald Mortier for the coordination of the design and maintenance of the pilot plant system. Rik Dillen, Kris Van Hege, Kris Pynaert, Elke Vincke, Dave 5. Conclusions Seghers and Inge Van Tomme are indebted for critically reading the manuscript. The brackish surface water of the canal Ghent^Terneu- zen contains microbial communities able to involve in bio- ¢lm formation and biocorrosion in natural waters. 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(2000) Manganese di- large amounts of Fe and Mn biologically enriched from oxide as a potential cathodic reactant in corrosion of stainless steels. Corros. Sci. 42, 211^227. the canal water. Manganese is present in an oxidized state [4] Little, B., Wagner, P., Hart, K., Ray, R., Lavoie, D., Nealson, K. 4 (Mn ) as an amorphous manganese oxide compound, not and Aguilar, C. (1998) The role of biomineralization in microbiolog- resulting in an increase of the stainless steel potential. The ically in£uenced corrosion. Biodegradation 9, 1^10.

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