Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge

System. Appl. Microbiol. 17,405-417 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge MICHAEL WAGNER 1 , RUDOLF AMANN 1 *, PETER KAMPFER2 , BERNHARD ASSMUS 3 , ANTON HARTMANN 3 , PETER HUTZLER\ NINA SPRINGER!, and KARL-HEINZ SCHLEIFER1 Lehrstuhl fur Mikrobiologie, Technische Universitiit Miinchen, 80290 Miinchen, Germany Fachgebiet Hygiene, Technische Universitiit Berlin, 13353 Berlin, Germany 3 GSF -:- Forschungszentrum fiir Umwelt und Gesundheit, Institut fiir Bodenokologie, 85758 Oberschlei~heim, Germany 4 GSF - Forschungszentrum fiir Umwelt und Gesundheit, Institut fur Pathologie, Biomedizinische Bildanalyse, 85758 Oberschlei~­ heim, Germany 1

2

Received May 5, 1994

Abstract Gram-negative filamentous bacteria are commonly observed in activated sludge and contribute to poor settlement of activated sludge flocs in secondary sedimentation tanks, a problem referred to as activated sludge bulking. However, the standard morphological identification system is of limited value for a high resolution, rapid monitoring of these bacteria. Therefore, specific 16S rRNA-targeted oligonucleotide probes were developed for Haliscomenobacter spp., Sphaerotilus spp., Leptothrix spp., Thiothrix spp., Leucothrix mucor and bacteria of the Eikelboom type 021N. Probe specificities were evaluated by nonisotopic dot blot hybridization to 145 reference strains representing a diverse collection of taxa. In situ hybridization with fluorescent probe derivatives was combined with scanning confocal laser microscopy (SCLM) for analyzing the three dimensional localization of the filaments inside the sludge flocs. Filaments could be localized even in the center of fixed flocs at a high resolution undisturbed by problems like autofluorescence.

Key words: In situ hybridization - Oligonucleotide - 16 SrRNA - Filamentous Bacteria - Activated Sludge - Bulking - SCLM Introduction The term sludge bulking describes a quite common malfunction of wastewater treatment plants in which the biomass shows poor settling properties in the secondary sedimentation tank. As consequences sludge recycling becomes difficult and the quality of the secondary effluent decreases. From time to time, filamentous bulking is a serious problem in 40-50% of all activated sludge plants (e.g. Blackbeard et al., 1986). Plants dealing with industrial wastewater are even more affected by bulking. The principle reason for this solid seperation problem is the presence of high numbers of filamentous bacteria in the mixed liquor (e.g. Pipes, 1967; Eikelboom, 1975; Strom and Jenkins, 1984). By using morphological characters and simple staining reactions, Eikelboom (1975) distinguished twenty-six types of filamentous bacteria in acti* Corresponding author

vated sludge samples. However, this identification key has general limitations. Firstly, morphology and staining reactions of microbial cells can vary in a broad range depending upon environmental conditions. Nonfilamentous growth forms have been described for the filamentous bacteria Haliscomenobacter hydrossis (van Veen et aI., 1973), Sphaerotilus natans (Waitz and Lackey, 1959; Mulder and Deinema, 1992) and Microthrix parvicella .(Foot et aI., 1992). For Leptothrix discophora, it was suggested that sheath forming capacity is encoded on easily lost genetic elements like plasmids (Emerson and Ghiorse, 1992). Certain filamentous organisms, e.g. Microthrix parvicella (Foot et ai. 1992) and Eikelboom type 1863 (Wagner et aI., 1994) can even show variable gram stain reactions. Secondly, the filamentous sulfur bacteria Thiothrix spp., Eikelboom type 021N, and Leucothrix mucor are hardly distinguishable by their morphology (Nielsen" 1984;

406

M. Wagner et al.

Brock, 1992). Thiothrix filaments without sulfur granules cannot be distinguished from Leucothrix (Brock, 1992). Discrimination berween Thiothrix spp.l021N and Leucothrix mucor can be achieved by a sulfur storage test (Nielsen, 1984) which is quite time consuming. Given the scope of the problem and the fact that no reasonable monitoring is possible by cultivation techniques (Wagner et al., 1993), new methods of in situ detection of filamentous bacteria in activated sludge samples need to be developed. Fluorescent-antibody technique (Bohlool and Schmidt, 1980) was carried out to identify Sphaerotilus natans (Howgrave-Graham and Steyn, 1988), Acinetobacter spp. (Cloete and Steyn, 1988) and Nocardia amarae (Hernandez et al., 1993) in activated sludge. However, the applicability of the immunofluorescence approach is limited by antibody penetration problems through extracellular polymeric substances (Szwerinski et al., 1985) which are components of the activated sludge flocs, and by unspecific binding of antibodies to detritus particles and fungal spores leading to high levels of background fluorescence. A further, more recent development is the use of fluorescently labeled, ribosomal RNA (rRNA) targeted oligonucleotides for high resolution and rapid monitoring of defined bacterial populations in activated sludge samples and trickling filters (Wagner et al., 1993; Manz et al., 1994; Wagner et al., 1994; Ramsing et al., 1993). Haliscomenobacter spp., Sphaerotilus spp., Leptothrix spp., Thiothrix spp., Leucothrix mucor and the unnamed organism of the Eikelboom type 021N are widely reported to represent an important part of the gram-negative filamentous bacteria in activated sludge (Farquhar and Boyle, 1971; van Veen, 1973; Eikelboom, 1975; Nielsen 1984; Williams and Unz 1985; Jenkins, 1986; Mulder and Deinema, 1992). In the present study, specific oligonucleotide probes were developed for in situ identification of these filamentous bacteria. They were applied to activated sludge flocs of ten German wastewater plants. Two techniques were compared for their potential to visualize specifically stained cells: standard epifluorescence microscopy and scanning confocal laser microscopy (SCLM). When imaging a fluorescent biological specimen with SCLM, the object is illuminated by a focussed laser beam and the fluorescent signals are detected by a photomultiplier. The confocal pinhole allows only those signals emanating from the focussed plane to be detected. SCLM therefore offers the possibility to obtain nondestructive optical sections of a sample and avoids disturbing effects of out-of-focus fluorescence (Caldwell et al., 1992; for reviews see Shotton et al., 1989; White et al., 1987). This technique should therefore be perfectly suited for studying the localization of filamentous bacteria in intact fixed activated sludge flocs.

Materials and Methods Organisms and growth conditions. The organisms investigated in this study are listed in Table 1. All strains of Sphaerotilus spp., Leptothrix spp. and of the Eikelboom type 1701 were grown

aerobically at 25°C in modified Rouf and Stokes medium (Rouf and Stokes, 1964) which contained the following ingredients per liter: Peptone 5.0 g; MgS0 4 • 7H 2 0, 0.2 g; Fe(NH 4) (S04b 0.15 g; sodium citrate, 0.1 g; CaCI 2 , 0.05 g; MnS04' H 2 0, 0.05 g; FeCI 3 · 6 H 2 0, 0.01 g. After autoclaving at 121°C for 15 min the medium was supplemented with 1 ml of filter-sterilized (0.2 !Am; Millipore, Eschborn, Germany) vitamin stock solution containing per liter 5 mg vitamin B12 (Merck, Darmstadt, Germany); 4mg thiamin (Merck, Darmstadt, Germany) and 4mg biotin (Merck, Darmstadt, Germany). Strains of the Eikelboom type 021N were cultured aerobically at 25°C on fructose supplemented modified MSV medium (Williams and Unz, 1985) which contained the following ingredients per liter: TRIS, 0.5 g; NaHC0 3 , 0.42 g; NH 4Cl, 0.36 g; MgS0 4 • 7 H 2 0, 0.15 g; K2 HP0 4 , 0.11 g; CaCI 2 · 2 H 2 0, 0.1 g; KH 2 P0 4 , 0.08 g; FeCI 3 • 6H2 0, 0.003 g; EDTA, 0.003 g. The autoclaved medium was supplemented with 10 ml filter-sterilized vitamin solution (Eikelboom, 1975) and 10 ml of filter-sterilized fructose (50 gil) solution. The pH of all media was adjusted to 7.2-7.4. Solid versions of media contained 15 glliter Bacto-Agar (Difco). All other strains were cultured as described in the catalogues of the respective strain collections (Table 1). Sampling. Grab samples of mixed liquor were collected from the aeration stages of the municipal wastewater treatment plants Berlin-Ruhleben (Germany, 1.3 million population equivalents [PE = 60 g biological oxygen demand d-\ Imhoff and Imhoff, 1985]), Miinchen I (GroBlappen, Germany; 1 million PEl, MJnchen II (Gut Marienhof, Germany; stage 1 and stage 2, 1 million PEl, Bamberg (Germany; 200.000 PEl, Gersthofen (Germany; 45.000 PEl and Hirblingen (Germany; 30.000 PEl. In addition, samples were collected from plants treating industrial wastewater (Hoechst, Augsburg, Germany; 20.000 PEl, wastewater from an animal waste processing (Kraftesried, Germany; 6.000 PEl, and municipal wastewater mixed with sewage of a brewery and a galvanization factory (Aldersbach, Germany; 27.000 PEl. Furthermore, a sample of a sequencing batch reactor treating dairysewage (Aretsried, Germany; 13.000 PEl was collected. For in situ hybridization activated sludge samples were fixed with paraformaldehyde solution (4%) immediately after the samples were taken (Amann et aI., 1990a). Staining techniques and sulfur storage test. Lipophilic granules have been stained with Nile blue A according to the method of Ostle and Holt (1982). For identification of Thiothrix spp. and type 021N in activated sludge, a sulfur storage test with thiosulfate was performed as described by Nielsen (1984). Sulfur granules were distinguished from other granules by their removal with methanol. Oligonucleotide probes. The following oligonucleotides were used: (i) HHY, complementary to a region of the 16S rRNA of Haliscomenobacter hydrossis; (ii) LDI, complementary to a region of the 16S rRNA of Leptothrix discophora, (iii) LMU, complementary to a region of the 16S rRNA of Leucothrix mucor; (iv) SNA, complementary to a region of the 16S rRNA of Sphaerotilus natans; (v) TNI, complementary to a region of the 16S rRNA of Thiothrix nivea; (vi) 21N, complementary to a region of the 16S rRNA of the filamentous bacterium of the Eikelboom type 021N. Sequences and target sites are shown in Table 2. In addition the following rRNA targeted oligonucleotides were used: (i) ACA, complementary to a region of the 16S rRNA of Acinetobacter calcoaceticus (Wagner et aI., 1994), (ii) CF, complementary to a region of the 16S rRNA of members of the cytophaga-flavobacterium cluster (Manz et aI., in preparation); (iii) CTE, complementary to a region of the 16S rRNA of Comamonas testosteroni, Bradyomonas denitrificans, RhodocycIus purpureus and Leptothrix discophora (Schleifer et aI., 1992), (iv) BET and GAM complementary to a region of 23S rRNA conserved in the beta and gamma subclasses of the class Pro-

Gram-negative Filamentous Bacteria

teobacteria, respectively (Manz et aI., 1992); and (v) EUB, complementary to a conserved region of bacterial 16S rRNA molecules (Amann et aI., 1990a). All probe sequences are given in Table2. Oligonucleotides were synthesized with a C6-TFA amino linker [6- (trifluoroacetylamino )-hexyl-(2-cyanoethyl)- (N ,N-diisopropyl)phophoramidite1 at the 5' -end (MWG Biotech, Ebersberg, Germany). Labeling with tetramethylrhodamine-5-isothiocyanate (Molecular probes, Eugene, Oreg.) or 5(6)-carboxyfluorescein-N-hydroxysuccinimide-ester (Boehringer GmbH, Mannheim, Germny) and purification of the oligonucleotide-dye conjugates were done as described by Amann et al. (1990b). Probes EUB, HHY, LDI, LMU, SNA, TNI and 21N were also labeled with digoxigenin as described previously (Zarda et aI., 1991). In situ hybridization. Optimal hybridization conditions were determined for probes HHY, LDI, LMU, SNA, TNI and 21N using the hybridization buffer and procedure described by Manz et al. (1992). Optimal hybridization stringency required the addition of formamide to a final concentration of 20% (probe HHY), 35% (probes 21N, LDI, LMU) or 45% (probes TNI and SNA). The stringency of the washing step was adjusted at a given temperature of 48°C by lowering the sodium chloride concentration to guarantee sufficient stringency (Manz et aI., 1992). Simultaneous hybridization with probes requiring different stringencies were realized by a successive hybridization procedure: hybridization and washing procedures for the probe requiring high stringency were conducted; subsequently hybridization and washing procedures for the probe requiring lower stringency were performed. Microscopy. Slides were examined with an Axioplan microscope (Zeiss, Oberkochen, Germany) with filter sets 09 and 15. Black-and-white photomicrographs were taken with Kodak Tmax 400 film. Exposure times were 0.06 s for phase contrast micrographs and 30 s for epifluorescence micrographs. Color photomicrographs were made on Kodak Ektachrom P1600 color reversal film. Exposure times were 0.01 s for phase contrast micrographs and 8 to 15 s for epifluorescence micrographs. A Zeiss LSM 410 scanning confocal laser microscope (Zeiss, Oberkochen, Germany) equipped with an Ar-ion laser (488 nm) and a HeNe-laser (543 nm) was used to record optical sections. Image processing, depth profiles and three dimensional reconstructions were performed with the standard software package delivered with the instrument. Dot blot hybridization. The specificities of the oligonucleotide probes HHY, LDI, LMU, SNA, TNI and 21N were evaluated by dot blot hybridizations of digoxigenin-Iabeled derivates with nucleic acids isolated from 147 reference organisms listed in Table 1 (Manz et aI., 1992). Reference nucleic acids were isolated as described by Manz et al. (1992). Gram-positive cells were mechanically disrupted with glass beads (0.17 mm in diameter) in combination with sonication (2 min). Optimal hybridization stringency required the addition of formamide to a final concentration of 25% (probe HHY), 35% (probe 2IN, LMU) and 45% (probes LDI and TNI). Specific hybridization with probe SNA required the addition of formamide to a final concentration of 50% and addition of an equimolar amount of unlabeled probe CTE as an competitor oligonucleotide (Manz et aI., 1992),

Results and Discussion Specificity of the probes

The oligonucleotide probes HHY, TNI, LMU, 21N, LDI and 5NA were designed complementary to diagnostic regions of the 165 rRNA sequences of Haliscomenobacter hydrossis (Gherna and Woese, 1992), Thiothrix nivea

407

(Distel et aI., 1988), Leucothrix mucor (Springer, unpublished), Eikelboom type 021N strain II-26 (Wagner, unpublished), Leptothrix discophora and Sphaerotilus natans (Corstjens and Muyzer, 1993). Computer assisted comparison with other accessible 165 rRNA sequences (1,500 complete or almost complete sequences [Neefs et aI., 1993; Larsen et aI., 1993]) revealed that all probes have at least one mismatch to 165 rRNA sequences of nontargeted bacteria. Probe specificities were further checked by dot blot analyses with nucleic acids isolated from 147 reference strains (for sources and strain numbers see Table 1). Under stringent hybridization conditions probes HHY (25% formamide, 46°C), TNI (45% formamide, 46°C), LMU (35% formamide, 46°C) and 21N (35% formamide, 46°C) showed the expected probe specificities (Table 1). Probe HHY hybridized only to nucleic acids isolated from Haliscomenobacter hydrossis (strain D5M 1100T) and from isolates of filamentous bacteria tentatively identified as Haliscomenobacter spp. (strains VI-40, VI-42, VI-46; Kampfer et aI., in preparation) due to morphological and physiological characters. Probes TNI and LMU hybridized to nucleic acids derived from Thiothrix nivea strain D5M 5205 and Leucothrix mucor strain 2I57T, respectively (Table 1). Probe 2IN detected nucleic acids derived solely from pure cultures of filamentous bacteria identified as Eikelboom type 021N (strains II-4, II-7, II-ll, II-26; Kampfer et aI., in preparation) due to morphological and physiological characters (Table 1). Under stringent hybridization conditions probe LDI (45 % formamide, 46°C) hybridized to nucleic acids isolated from "Leptothrix discophora" strains LMG 8141, ATCC 43182 and [AquaspirillumJ metamorphum strain D5M 1837T (Table 1). Using 165 rDNA sequencing data Corstjens and Muyzer (1993) proved that" 1. discophora" ATCC 43182 belongs to the beta I-subdivision of the beta subclass of Proteobacteria. [A.] metamorphum is phylogenetically not an authentic aquaspirillum (Pot et aI., 1992), it forms a separate rRNA branch in the family Comamonadaceae (Willems et aI., I99Ia; b; c) and is therefore related to members of the beta I-subdivision of the beta subclass of Proteobacteria. Whereas probe LDI is unable to distinguish between the closely related strains of "1. discophora" ATCC 43182 and [A.] metamorphum strain D5M I837 T it did not hybridize to nucleic acids of other tested members of the beta I-subdivision, e.g., Sphaerotilus natans strain D5M 565 and Comamonas testosteroni strain LMG 1800T. Nucleic acids derived from "1. discophora" strains LMG 8142 and 8143 as well as from 1. cholodnii strain LMG 9467 were not detected by hybridization with probe LDI. This result indicates differences between these strains and "1. discophora" LMG 8141 and ATCC 43182. "1. discophora" strain LMG 8143 had been isolated from a culture of strain LMG 8142 as a spontaneous nonsheathed mutant (Emerson and Ghiorse, 1992). Willems et a1. (I991b) reported almost identical protein patterns for these two strains which were different from protein patterns of "1. discophora" strain LMG 1841. In the same study, 1. cholodnii strain LMG 9467 produced a unique protein banding pattern which was different from that of "1. discophora" strain LMG

408

M. Wagner et al.

Table 1. Listing of studied strains and results of dot blot hybridization with oligonucleotide probes Hybridization with probe: Taxon

Strain a

EUB

ATCC 23308 T DSM 2787 T DSM I690 T DSM 3675 T DSM 30131 T DSM 6361 T DSM 65 T DSM 1635 DSM 30135 T DSM 1710T DSM 123 T DSM 107 DSM 700 T WS 1610

+ + + + + + + + + + + + + +

LMG 5943 T DSM 531 T ATCC 8750T DSM 1837T DSM 50181 LMG 1222T DSM 30191 T LMG 1800T LMG 9467 LMG 8141 LMG 8142 LMG 8143 ATCC 43182 DSM 565 LMG 7172 Ttl ATCC 13338 T ATCC 15291 ATCC 13925 ATCC 13923 ATCC 13503 ATCC 13508 ATCC 13921 ATCC 13917 ATCC 13926 ATCC 13918 ATCC 13929 TUB ATCC 25935

+ + + + + + + + + + + + + + + + + + + + + + + + + + + +

LMG 1041 T LMG 1046T LMG 996 T LMG 998 T LMG 999 T LMG 1029T LMG 10613 T WS 1406 TUB II-26 TUB II-4 TUB II-7 TUB II-ll

+ + + + + + + + + + + +

HHY

LDI

SNA

TN!

2IN

LMU

ND

ND

ND

ND

ND

ND

alpha subclass of Proteobacteria: Agrobacterium tumefaciens Azospirillum amazonenese Azospirillum brasilense Azospirillum halopraeferens Bradyrhizobium japonicum Magnetospirillum gryphiswaldense Paracoccus denitrificans "Pseudomonas" diminuta Rhizobium meliloti Rhodobacter capsulatus Rhodopseudomonas palustris Rhodospirillum rubrum Thiobacillus acidophilus Zoogloea ramigera

beta subclass of Proteobacteria: Acidovorax delafieldii Alcaligenes entrophus Alcaligenes faecalis "Aquaspirillum" metamorphum Burkholderia cepacia Burkholderia cepacia Chromobacterium violaceum Comamonas testosteroni Leptothrix cholodnii "Leptothrix discophora" "Leptothrix discophora" "Leptothrix discophora" "Leptothrix discophora" Sphaerotilus natans Sphaerotilus natans Sphaerotilus natans Sphaerotilus natans Sphaerotilus sp. Sphaerotilus sp. Sphaerotilus sp. Sphaerotilus sp. Sphaerotilus sp. Sphaerotilus sp. Sphaerotilus sp. Sphaerotilus sp. Sphaerotilus sp. 24 strains of Eikelboom type 1701 Zoogloea ramigera

+

+ +

+ + + + + + + + + + + + + + + + +

gamma subclass of Proteobacteria: Acinetobacter baumannii Acinetobacter calcoaceticus Acinetobacter haemolyticus Acinetobacter junii Acinetobacter johnsonii Acinetobacter lwoffii Acinetobacter radioresistens Aeromonas hydrophila Eikelboom type 021N Eikelboom type 021N Eikelboom type 021N Eikelboom type 021N

+ + + +

Gram-negative Filamentous Bacteria

409

Table 1. Continued Hybridization with probe: Taxon

Strain'

EUB

Enterobacter aerogenes Enterobacter cloacae Erwinia carotovora Escherichia coli Leucothrix mucor Moraxella bovis Moraxella osloensis Proteus vulgaris Pseudomonas aeruginosa Pseudomonas alcaligenes Pseudomonas fluorescens Pseudomonas pseudoalcaligenes Pseudomonas putida Serratia marcescens Shewanella putrefaciens Thiothrix nivea Vibrio anguillarum

WS 1292 WS 1293 WS 1394 DSM 30083 T DSM 2157T LMG 986 T LMG 5131 T WS 1356 DSM 50071 T DSM 50342 T DSM 10090 LMG 1225 T DSM 291 T WS 1359 DSM 50426 DSM 5205 NCIMB 2129

+ + + + + + + + + + + + + + + + +

GBF Mx-f2 GBF Mx-v4

+ +

LMG 4024 T LMG 1339 GBF Cy jl LMG 1341 T LMG 4011 T LMG 4012 LMG 4021 T LMG 8334 LMG 8336 LMG 8337 T LMG 1233 T LMG 4028 LMG 3809 T LMG 8379 T DSM 1100T TUB VI-40 TUB VI-42 TUB VI-46 LMG 11054T LMG 11602 LMG 10407T LMG 8340 T LMG 8347 T LMG 8348 LMG 8393 T LMG 8349 LMG 8350 LMG 8351 T lMG 8352

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +

DSM 20425 T DSM 20165 DSM 20300 T

+ + +

HHY

LDI

SNA

TNI

21N

LMU

+

+

delta subclass of Proteobacteria: Myxococcus fulvus Myxococcus virescens

cytophaga-flavobacterium cluster: Cytophaga heparina Cytophaga hutchinsonii Cytophaga ;ohnsonae Cytophaga ;ohnsonae Flavobacterium breve Flavobacterium breve Flavobacterium ferrugineum Flavobacterium gleum Flavobacterium indologenes Flavobacterium indologenes Flavobacterium odoratum Flavobacterium odoratum Flavobacterium uliginosum Flexithrix dorotheae Haliscomenobacter hydrossis Haliscomenobacter sp. Haliscomenobacter sp. Haliscomenobacter sp. Moraxella anatipestifer Moraxella anatipestifer Saprospira grandis Sphingobacterium mizutae Sphingobacterium spiritivorum Sphingobacterium spiritivorum Sporocytophaga myxococcoides Weeksella virosa Weeksella virosa Weeksella zoohelcum Weeksella zoohelcum

gram-positive bacteria with a high G+C content of DNA: Brevibacterium linens Brevibacterium sp. Corynebacterium glutamicum 27 System. Appl. Microbial. Vol. 17/3

+ + + +

ND ND

ND ND

ND ND

ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND

ND ND ND ND ND ND ND

ND ND ND ND

410

M. Wagner et a1.

Table 1. Continued Hybridization with probe: Taxon

Straina

EUB

Micrococcus luteus Nocardoides simplex Propionibacterium freudenreichii Rhodococcus rhodochrous

CCM 169 T D5M 20130 T D5M 20271 T D5M 43008

+ +

D5M 31 T ATCC 6633 NCIMB 8052 T NCIMB 11754T D5M 20478 T DSM 20477T LMG 9091 DSM 20069 T DSM 20481 T DSM 20465 DSM 20231 T DSM 20560 T

+ + + + + + + + + + + +

HHY

LDI

SNA

TNI

21N

LMU

+ +

gram-positive bacteria with a low G+C content of DNA: Bacillus cereus Bacillus subtilis Clostridium acetobutylicum Clostridium stercorarium Enterococcus faecalis Enterococcus faecium Lactobacillus casei Lactococcus lactis subsp. cremoris Lactococcus lactis subsp. lactis Pectinatus frisingensis Staphylococcus au reus Streptococcus salivarius a

ATCC, American Type Culture Collection, Rockville, MD, USA; Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; GBF, Gesellschaft fiir Biotechnologische Forschung, Braunschweig, Germany (H. Reichenbach); LMG, Laboratorium voor Microbiologie, Universiteit Gent, Ghent, Belgium; NCIMB, National Collection of Industrial and Marine Bacteria, Torry Research Station, Aberdeen, Scotland, U.K.; TUB, Technische Universitat Berlin, Germany (P. Kampfer); WS, Bakteriologisches Institut der Siiddeutschen Versuchs- und Forschungsanstalt fiir Milchwirtschaft, TU Miinchen, Freising-Weihenstephan, Germany.

8141, whereas DNA-rRNA hybridization experiments indicated a strong relationship to "L. discophora". Probe 5NA (50% formamide, 46°C, competitor probe CTE) hybridized to nucleic acids derived from four S. natans strains, nine Sphaerotilus sp. strains, twenty-four different strains of the Eikelboom type 1701, L. discophora strains LMG 8142, 8143 and L. cholodnii strain LMG 9467 (Table 1). 165 rDNA sequence analysis of S. natans showed its affiliation to the beta I-subdivision of Pro-

teobacteria (Corstjens and Muyzer, 1993). Eikelboom (1975) described chains of cylindrical cells enclosed by a sheath as type 1701. The morphology of this filamentous bacterium closely resembles those of Sphaerotilus natans, e.g., false branching of the filaments may occur in both types. Hybridization of probe BET to nucleic acids derived from the twenty-four different strains of the Eikelboom type 1701 supported their affiliation with the beta subclass of Proteobacteria (data not shown). These results indicate

Probe

Probe sequence (5'-3')

Target site a (rRNA positions)

% formamide % formamide dot blot in situ

HHY LDI LMU SNA TNI 21N EUB BET GAM ACA CTE CF

GCCTACCTCAACCTGATT CTCTGCCGCACTCCAGCT CCCCTCTCCCAAACTCTA CATCCCCCTCTACCGTAC CTCCTCTCCCACATTCTA TCCCTCTCCCAAATTCTA GCTGCCTCCCGTAGGAGT GCCTTCCCACTTCGTTT GCCTTCCCACATCGTTT ATCCTCTCCCATACTCTA TTCCATCCCCCTCTGCCG TGGTCCGTGTCTCAGTAC

165, 16S, 16S, 16S, 16S, 16S, 16S, 235, 23S, 165, 16S, 16S,

25 45 35 50 45 35 0

655-672 649-666 652-669 656-673 652-669 652-669 338-355 1027-1043 1027-1043 652-669 659-676 319-336

Escherichia coli numbering (Brosius et aI., 1981). § Used as unlabeled competitor together with probe SNA.

a

§

20 35 35 45 45 35 0 35 35 35 35

Table 2. Probe sequences, target sites, and formamide concentration in the hybridization buffer required for specific dot blot and in situ hybridization

Gram-negative Filamentous Bacteria

a close affiliation of the Eikelboom type 1701 with Sphaerotilus spp. We could demonstrate that all tested strains of Leptothrix spp., Sphaerotilus spp. and Eikelboom type 1701 can be detected and assigned to two clusters by hybridization with probes LDI and SNA. At this time it is not possible to decide whether the two clusters reflect a phylogenetic division into two groups. In situ identification of filamentous bacteria

Fluorescently labeled probes were used for whole cell hybridization. Optimal hybridization stringencies for whole-cell hybridization were evaluated for each probe (Table 2). For probes HHY, LDI, and SNA, formamide concentrations in the hybridization buffer were slightly modified from those used for dot blot hybridization experiments. Application of the probe HHY specific for Haliscomenobacter spp., a member of the cytophaga-flavobacterium cluster (Cherna and Woese, 1992) resulted in the specific visualization of heavily stained thin filamentous bacteria. They commonly occurred inside the sludge flocs (Figure 1A), as described by van Veen et al. (1973). All cells hybridizing with probe HHY could simultaneously be detected with probe CF. Haliscomenobacter spp. was found in six of the ten examined activated sludge samples orginating from different German wastewater treatment plants (Table 3). The number of Haliscomenobacter spp. is underestimated by phase contrast observation, as the thin filaments are almost undetectable in thick sludge flocs without a specific staining reaction. By hybridization of activated sludge samples with fluorescein-labeled probe TNI and rhodamin-labeled probe 21N, two discrete populations of filaments could be visualized simultaneously (Figure 1B). Surprisingly, probe TNI hybridized also to large coccoid cells in the activated sludge samples (Figure 1B). These coccoid cells may either represent a species not included in the 16S rDNA data bases and in the list of the tested reference strains, or

Table 3. In situ identification of filamentous bacteria in ten activated sludge plants in Germany Treatment plant location Berlin Miinchen I Miinchen II Gersthofen Hirblingen Bamberg Hoechst§ Kraftesried Aldersbach§ Aretsried

Probe" HHY

+ + + + +

+

LDI

SNA

+ +

+

+

+ + +

+

+ + + +

+

TNI

2IN

+

+

+

+

+

+

+

" For abbreviations see Material and Methods. § Bulking observed at the time of sampling. + Detectable occurrence of probe target population. - Probe target population not detectable.

LMU

411

represent the ovoid structures ("gonidia") described for Thiothrix spp. (Winogradsky, 1888; Larkin and Shinabarger, 1983; Williams et al., 1987). Similar propagules have also been reported for the closly related species Leucothrix mucor (Harold and Stanier, 1955; Brock, 1992). The observation that individual cells in the TNI positive filaments sometimes became spherical (Figure 1B) points toward this second possibility. Occasionally, also the typical formation of true knots and rosettes by Thiothrix spp. (Nielsen, 1984, Williams and Unz, 1985, Shuttleworth and Unz, 1993) was seen. Probe TNI stained two morphotypes of filaments similar to those described by Eikelboom and van Buijsen (1983). Furthermore, the sulfur storage test of Nielsen (1984) resulted in formation of internal sulfur granules in those trichomes that were detected by probes TNI and 21N. The composition of the granules was corroborated by methanol extraction of the sulfur (data not shown). Type 021N bacteria formed long trichoms, sometimes including true knots. In the examined activated sludge samples and in the pure cultures we could not detect rosette formation for type 021N as described by Eikelboom (1975) and also described for certain type 21N varieties by Williams and Unz (1985). In situ hybridization proved the simultaneous presence of Thiothrix spp. and type 21N bacteria in three of the ten examined activated sludge samples (Table 3). In the sample of the industrial wastewater treatment plant (Hoechst, Augsburg, Germany) Thiothrix spp. was found to be a dominant component of the microbial community and the only filamentous bacterium present (Table 3). It is likely responsible for the sludge bulking observed at the time of sampling. Simultaneous application of probe GAM with probes TNI or 21N revealed that all cells hybridizing with probes TNI and 21N belong to the gamma subclass of the class Proteobacteria. Probe LMU specifically stained trichoms of the entirely marine (Brock, 1966) Leucothrix mucor (Figure 2A), but, not surprisingly, no hybridization signals were detectable in the examined activated sludge samples (Table 3). So we could not confirm Leucothrixlike strains in activated sludge (Eikelboom, 1975; Pofte et aI., 1979). In future studies probes TNI, 21N and LMU can be used to analyze the distribution of Thiothrix spp. type 021 bacteria and Leucothrix muca in the natural environment and can help to avoid misidentifications caused by the strong morphological resemblance of these filamentous bacteria. Whole cell hybridization with probe SNA resulted in specific and bright staining of all reference strains assigned to S. natans, Sphaerotilus spp., type 1701, "L. discophora" strains LMG 8142, 8143 and L. cholodnii LMG 9467. The morphologies of the various S. natans and Sphaerotitus spp. strains in pure culture varied from typical sheathed filaments with false branching to small, singlecelled rods (Figure 3). Identification based on morphological characters could therefore result in an underestimation of the number of these bacteria in natural environments. Oligonucleotide probing, on the other hand, allows to monitor the abundance of these organisms independent of their actual morphology. Simultaneous in situ hybridization of activated sludge samples with fluorescein-labeled

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probe LDI and rhodamine-labeled probe SNA was performed (Figure lC). Probe LDI bound to two morphotypes while various .types of trichomes and some rod-shaped single cells showed red fluorescence conferred by probe SNA. In both morphotypes stained with probe LDI the presence of poly-~-hydroxy-butyrate (PHB) was detected by Nile blue A staining. Probe LDI is specific for" L. discophora" strain SS-l and [A.] metamorphum, both of which are able to accumulate PHB. These species were originally isolated from a ferromanganese surface film on a pond (Ghiorse and Chapnik, 1983) and from a putrid infusion from freshwater shellfish, respectively. However, van Veen (1973) isolated manganese oxidizing sheathed bacteria from activated sludge which he considered to be "Sphaerotilus discophorus". Evidence for the affiliation of the LDI-positive cells to "L. discophora", [A.] metamorphum or to species not included in the 16S rDNA data bases and the list of reference organisms (Table 1) could originate from the isolation and subsequent 16S rRNA sequencing of such bacteria from activated sludge in an approach similar to the one described by Kane et al. (1993). Probe SNA stained various morphotypes of filamentous bacteria strongly resembling trichoms of S. natans, L. cholodnii and type 1701. In addition, rodshaped single cells were specifically stained. These cells probably represent swarmer cells of S. natans, L. cholodnii and type 1701, or mutants which have lost their sheath forming ability, as described for L. cholodnii (Mulder and Deinema, 1992). In situ detection of cells hybridizing to probe LDI was possible in six of the ten activated sludge samples (Table 3). In situ hybridization of these activated sludge samples with probe SNA resulted in the specific staining of cells in seven of the ten samples (Table 3). Simultaneous application of probe BET with probes LDI

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or SNA demonstrated that all cells stained with probes LDI or SNA could be assigned to the beta subclass of the class Proteobacteria. Combining oligonucleotide probing with SCLM

Compared to conventional epifluorescence microscopy the visualization of hybridized cells in acitvated sludge could be significantly improved by a modern technology, scanning confocal laser microsocpy (SCLM) (Caldwell et aI., 1992). Hitherto, SCLM had been applied to reveal, e.g., the spatial distribution of rRNA and mRNA in mouse bone marrow cells (Baumann et aI., 1990), and of integrated human papillomavirus DNA in different human cells lines (Lizard et aI., 1993). In both cases biotinylated probes were used and subsequently detected with fluoresceinlabeled streptavidin. In this study whole fixed bacterial cells were examined with SCLM after hybridization with fluorescently labeled, rRNA-targeted oligonucleotides. Due to optical sectioning SCLM gives accurate data on the spatial distribution of fluorescence and the deleterious effects of auto fluorescent background and out-of-focus signals on the overall image quality can be prevented. From a series of optical sections (gallery, not shown) different types of images can be reconstructed. One option allows to show all parts of an extended filament in focus (2-d reconstruction, not shown). Other options yield 3-d reconstructions such as redgreen stereo images or colored depth profiles (Figure 2B and 2C). They offer the possibility to visualize the true arrangement of bacterial populations identified by in situ hybridization, thereby facilitating studies on the microstructure and diversity of immobilized microbial consortia at a so far unreached resolution. For the microbiologist analyzing the wastewater treat-

Fig. 1. In situ identification of filamentous bacteria in an activated sludge sample obtained from the wastewater treatment plant Miinchen 1. For each panel, identical fields were viewed by phase-contrast microscopy (left) and epifluorescence microscopy (right). Bar = 10 11m (panel A, left) and applies to all photomicrographs. (A) In situ hybridization with fluorescein-labeled probe HHY. (B) Simultaneous in situ hybridization with fluorescein-labeled probe TNI and tetramethylrhodamine-Iabeled probe 21N. (C) Simultaneous in situ hybridization with fluorescein-labeled probe LDI and tetramethylrhodamine-Iabeled probe SNA. ~ see page 412 Fig. 2. (A) Whole cell identification of Leucothrix mucor. An artificial mixture of fixed cells of Leucothrix mucor and activated sludge orginating from the treatment plant Miinchen I was simultaneously hybridized with fluorescein-labeled probe LMU and tetramethylrhodamin-labeled probe 21N. An identical field was viewed by phase contrast microscopy (left) and epifluorescence microsocpy (right). Bar = 10 11m. (B) 3-d reconstructions by SCLM-analysis (encompassing 22 optical sections taken at 0.7 11m intervals) of a fixed act.ivated sludge sample (plant Miinchen I) after simultaneous in situ hybridization with fluorescein-labeled probe TNI (left) and tetramethylrhodamine-Iabeled probe 21N (right). Autofluorescence of the fixed floc material is minimized by selection of suitable optical sections and digital contrast enhancement procedures. The red-green reconstructions (anaglyphes) should be viewed through red/green glasses as they can be obtained from Carl Zeiss (Oberkochen, Germany) to recognize an uncolored 3-d distribution. Bar = 25 11m, does also apply to panel C. (C) In the corresponding depth profiles the exact 3-d arrangement of the filaments becomes apparent. Arrangement from top to bottom is indicated by colors changing red to yellow to green and finally to blue. ~ see page 413 Fig. 3. Whole cell identification of some Sphaerotilus spp. with different morphological appearance. For each panel, identical fields were viewed by phase-contrast microscopy (left) and epifluorescence microscopy (right). Bar = 20 11m (patlel A, left) and applies to all photomicrographs. An artifical mixture of fixed cells of Leptothrix discophora strain ATCC 43182 and S. natans strain DSM 565 (panel A), Sphaerotilus sp. strain ATCC 13917 (panel B) and Sphaerotilus sp. strain ATCC 13921 (panel C) was hybridized with fluorescein-labeled probe SNA. ~ see page 414

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ment process in situ hybridization with probes specific for filamentous bacteria is a new tool for a rapid and reliable identification of these bacteria jn activated sludge. Culture-independent studies may be the key to a better understanding of the principles of filamentous bulking.

Acknowledgements. This work was supported by grants from CEC contract BIOT-CT91-0294 to KHS and from the Forschungsverbund Biologische Sicherheit and the DFG (Am 73/2-2) to RA. We thank H. Lemmer (Bayerische Landesanstalt fur Wasserforschung, Munich, Germany) for the helpful discussions, S;bylle Schadhauser for the excellent technical assistance and Liesbeth de Vrind-de jong for "Leptothrix discophora" strain SS1 (ATCC 43182).

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Rudolf Amann, Lehrstuhl fur Mikrobiologie, Technische Universitiit Munchen, Arcisstr. 16, D-80290 Munchen, Germany