Characterisation of efficient denitrifying bacteria strains isolated from activated sludge by 16S-rDNA analysis

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War. Sci. Tech. Vol. 38, No. 8-9, pp. 63-68,1998. IAWQ © 1998 Published by Elsevier Science Ltd. Printed in Great Britain. All rights reserved 0273-1223/98 $ 19'()() + o·()()

CHARACTERISATION OF EFFICIENT DENITRIFYING BACTERIA STRAINS ISOLATED FROM ACTIVATED SLUDGE BY 16S-rDNA ANALYSIS Gunnar Magnusson, Helena Edin and Gunnel Dalhammar Department ojBiochemistry and Biotechnology, Royal Institute oj Technology, KTH, 100 44 Stockholm, Sweden

ABSTRACT In order to investigate the diversity of the most efficient denitrifying bacterial species in activated sludge more than ISOO bacterial strains were isolated from five different municipal wastewater treatment plants. The 24 most efficient denitrifying bacteria strains were selected and submitted for partial 16S rRNA analyses. Result indicated that five strains were members of the Rhodobacter group. 16 of Rubrivivax subgroup, one of Pseudomonas subgroup and two unidentified though showing high similarity to certain members of the Brucella assemblage. This indicates efficient denitrifying bacterial strains in activated sludge are members of a restricted number of bacterial taxa dominated by Rubrivivax subgroup. © 1998 Published by Elsevier Science Ltd. All rights reserved

KEYWORDS Activated sludge; denitrifying bacteria; wastewater; 16S rONA. INTRODUCTION The nitrogen content in wastewater is reduced by nitrification and denitrification processes in wastewater treatment plants. Nitrification is oxidation of ammonia to nitrate and denitrification is the reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide. It is well known that Nitrosomonas and Nitrobacter are responsible for the nitrification process. The limited number of species makes the process vulnerable when exposed to toxic substances. In general denitrifying bacteria are present in several bacterial genera. During investigations of activated sludge in municipal waste water treatment plants occurrence of Pseudomonas, Alcaligenes, Hyphomicrobium, Paracoccus, Bacillus and Methylobacterium have been reported (Schmider and Ottow, 1986; Vedenina and Govorukhina, 1988; Knowles, 1982, Sperl and Hoare, 1971; Attwood and Harder, 1972; Nyberg et at., 1992; Timmermans and Haute, 1983), though, it is still not clear whether the most important or efficient denitrifiers in activated sludge have been identified. The aim of this study was to investigate the diversity of the most efficient denitrifying strains in activated sludge. Provided these denitrifiers comprise a homogeneous bacterial group, the denitrification process is likely to be more sensitive than if the denitrifying bacteria are more diverse. This includes both variations in process parameters, changes in carbon sources and exposure to toxic compounds. 63

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MATERIALS AND METHODS ISQlatiQn and selection of efficient denitrifyin~ strains MQre than 1500 bacterial strains were isolated from five municipal wastewater treatment plants. Four Qf the plants were situated in Sweden (Gustavsberg, Kappala, Bromma and Porsmossen) and one in Tjecekia (Pisec). Isolations were made, both aerobically and anaerobically, on nutrient agar incubated fQr tWQ days at 30°C. Possible denitrifiers were detected by gas productiQn when grQwn in broth supplemented with 0.1 % KN0 3 and 0.17% agar The mQst efficient denitrifying strains were selected by their s~eed Qf cQnsumi~g nitrate. Strain I 10, 123 and k7g were iSQlated from Gustavsberg. Strain No. 110 IS the same stram investigated in Gumaelius et al. (1996). Strain 2:99g, 5:38g, 5: 107g, 2: 12g and 2:38r were iSQlated from Kappala. Strain R28g, F89g, F25g, F40g, F66g, R113g, F51g and 2:38r were isolated from Br~mma. Strain lOOg, 25g, 80g and 18g were iSQlated from PorsmQssen. Strain bp 17, bp62, bp64 and bp2 were ISQlated frQm Piseco BiQchemical characterisation The strains were characterised by cell and cQIQny mQrphQIQgy, Gram staining, cytochrome Qxidase activity, motility and API 20 NE (Api internatiQnal s.a., Geneve, Schweiz). In vitro amplification of 16S rRNA gene Amplification Qf the 16S rRNA gene was performed with primers complementary to the regions clQse to the 5' and 3' termini Qf the gene. Freshly cultivated strain was added tQ the PCR mix and amplified with the primers 605 and 621 (Pettersson, 1997). The PCR products were subjected to a semi-nested amplificatiQn using primer 605 and 614B. Biotinylated PCR products, suitable for solid-phase DNA sequencing, were generated with 5 pmQI of each primer, and the follQwing thermQcycling prQfile was used. Denaturation at 96°C for 15 sec, primer annealing at 65°C fQr 30 sec and extensiQn at 72°C for 2 min were repeated 30 times. A final extension at 72°C fQr 10 min was alsQ used. The semi-nested PCR with one primer biotinylated is earlier described by Pettersson et ai. (1996). Direct solid-phase DNA sequencing ImmobilisatiQn Qf the biQtinylated PCR prQducts, followed by strand separatiQn and template preparatiQn, was performed with super paramagnetic beads (Dynabeads M-280 Streptavidin; Dynal, OsIQ, NQrway). The nucleotide sequences Qf the 16S rRNA genes were determined in bQth directiQns by autQmated solid-phase sequencing (Hultman et ai., 1989; Hultman et ai., 1991; PetterssQn et ai., 1994; Wahlberg et ai., 1992) with the automated laser f1uQrescence system (Pharmacia Biotech, Uppsala, Sweden). Used primers were USP and 622F (Pettersson, 1997). Genotypic and comparative analysis The partial sequences were first analysed using the BLAST server at NCBI (National Centre Qf Biotechnology InfQrmatiQn). Then, the partial sequences were cQmpared tQ sequences representing the domain bacteria. Reference sequences were Qbtained frQm the RDP (The RibQsomal Database Project) (Maidak et ai., 1994). The variable positiQns 69-100 and 181-219 were excluded and a similarity matrix were constructed, using Jukes and Cantors parameter mQdel (Jukes and CantQr, 1969). Then a distance tree was cQnstructed using the neighbQr-jQining methQd (SaitQu and Nei, 1987) in PHYLIP 3.51c (Felsenstein, 1993). The results from the BLAST server and this first distance tree were then used fQr construction Qf further similarity matrixes and distance trees cQntaining representatives of all taxa in the phylum indicated. This time nQ pQsitiQns were excluded. In order tQ estimate relative SUPPQrt for branches inferred frQm genetic distance analyses, bQQtstrap (100) resampling was emp1Qyed.

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65

RESULTS AND DISCUSSION Biochemical characterisation The strains were all Gram negative, oxidase positive, aerobic or facultative motile rods. The API 20 NE test indicated that all strains belonged to the proteobacteria phylum. Table 1. BLAST server results Strain lOOg 25g 80g R28g F89g F25g F40g 110 123 2:99g bpl7 5:38g 5:107g 2:12g 2:38r F66g 5:55g bp62 bp64 RI13g F51g bp2 18g k7g

Best-BLAST-hit id 032238 032238 032238 032238 032238 X84630 012794 MI1224 M1l224 M11224 M1l224 M11224 MI1224 X84470 X84470 X84470 X84519 X84519 X84519 X84519 X84479 X84479 X84479 U63944

Paracoccus alkaliphi/us Paracoccus a/kaliphi/us Paracoccus a/kaliphilus Paracoccus a/kaliphilus Paracoccus a/kaliphi/us Unknown organism Ochrobacterium anthropi Pseudomonas testosteroni Pseudomonas testosteroni Pseudomonas testosteroni Pseudomonas testosteroni Pseudomonas testosteroni Pseudomonas testosteroni Unknown organism Unknown organism Unknown organism Unknown organism Unknown organism Unknown organism Unknown organism Unknown organism Unknown organism Unknown organism Pseudomonas sp.

Best-soecies-BLAST-hit id 032238 Paracoccus a/ka/iphilus 032238 Paracoccus a/ka/iphilus 032238 Paracoccus alkaliphi/us 032238 Paracoccus alkaliphi/us Paracoccus alkaliphi/us 032238 014255 Zoog/ea ramigera Dl2794 Ochrobacterium anthropi MI1224 Pseudomonas testosteroni Pseudomonas testosteroni M11224 M1l224 Pseudomonas testosteroni M1l224 Pseudomonas testosteroni MI1224 Pseudomonas testosteroni M11224 Pseudomonas testosteroni MI1224 Pseudomonas testosteroni Pseudomonas testosteroni MI1224 Pseudomonas testosteroni M11224 Pseudomonas testosteroni MI1224 Pseudomonas testosteroni MI1224 Pseudomonas testosteroni MI1224 Pseudomonas testosteroni MI1224 Pseudomonas testosteroni M11224 Pseudomonas testosteroni MI1224 Pseudomonas testosteroni MI1224 Pseudomonas fluorescens U63901

Subdivision

Taxon

Alfapurple Alfapurple Alfapurple Alfapurple Alfapurple Alfapurple Alfapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Betapurple Gammapurple

Rhodobacter group Rhodobacter group Rhodobacter group Rhodobacter group Rhodobacter group Brucella assemblage Brucella assemblage Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Psuedomonas sublUOuo

Table 2. Result of distance tree studies Strain l00g

25g

80g R28g F89g F25g

F40g 110

123 2:99g bp17

5:38g

5:107g 2:12g

2:38r F66g

5:55g

bp62 bp64

R1l3g F51g bp2

18g

k7g

Affilatiing taxon

Cluster

Rhodobacter group Rhodobacter group Rhodobacter group Rhodobacter group Rhodobacter group Rubriyiyax subgroup RubriYivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Rubrivivax subgroup Pseudomonas subgroup

Genotypic and comparative analysis The BLAST-server results indicated that 7 strains belonged to alpha, 16 to beta and 1 to gamma subdivision of the purple bacteria phyla. BLAST hits and their taxonomic positions are listed in T~bl~ 1.. On the ,basis of comparative analysis of nucleotides corresponding to 50-312 E. coli (Lan~, 1991), a slmIl~lty matn~ and.a distance tree was constructed with the denitrifying strains and representatIves of the domam Bactena. ThIS

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tree confirmed the initial grouping indicated by the BLAST-server by affiliating the clones to representatives of same subdivisions. of purple bacteria phylum (Table 2). Based on the first distance tree and BLAST results, comparisons were made between the taxa within alpha, beta and gamma purple and the clones affiliating. A similarity matrix was constructed, for eac~ s~bdivision, including representative sequences from all taxa within the subdivision and all sequences wlthm the ~axa affiliating to the clones. The Phyla, subdivisions, groups, assemblages and subgroups were defmed according to the RDP. Grouping was made on the basis of tree topology when the bootstrap values were low «75%) (Zharkikh and Li, 1992). A similar method of grouping was earlier used by Bond et al. (1995). Table 3. Bootstrap result Taxon

Rhodobacter group

Rubrivivax subgroup Pseudomonas subgroup

Bootstrap 52 69 93

Table 4. Sequence similarity between clones identified as Rhodobacter group

HiOg

100.0 100.0 100.0 100.0 96.5

25g 80g R28g F89g 032238

100.0 100.0 100.0 100.0 100.0 100.0 96.5 96.5 96.5 96.4

Table 5. Sequence similarity between unidentified alpha purple affiliating clones F25g X84630 014255 F40g 012794

95.4 95.4 99.6 88.0 88.7 88.2 89.0 90.7 90.2 98.2

Table 6. Sequence identify among clones identified as Rubrivivax subgroup 110 2:99g 5:I07g 123 bpl7 5:38g M1l224 2:12g 2:38r F66g X84470 5:55g bp62 bp64 R113g X84519 F51g bp2 18g X84479

100.0 100.0 99.6 99.6 99.6 97.2 90.4 90.4 90.4 90.4 89.0 89.0 89.0 89.0 89.0 89.5 88.6 89.0 89.0

100.0 99.6 99.6 99.6 97.2 90.4 90.4 90.4 90.4 89.0 89.0 89.0 89.0 89.0 89.5 88.6 89.0 89.0

99.6 99.6 99.6 97.2 90.4 90.4 90.4 90.4 89.0 89.0 89.0 89.0 89.0 89.5 88.6 89.0 89.0

99.2 99.2 97.6 89.9 89.9 89.9 89.9 88.6 88.6 88.6 88.6 88.6 89.0 88.1 88.6 88.6

99.2 97.2 89.9 89.9 89.9 89.9 88.6 88.6 88.6 88.6 88.6 89.0 88.1 88.6 88.6

96.8 90.4 90.4 90.4 90.4 89.0 89.0 89.0 89.0 89.0 89.0 88.1 88.6 88.6

89.5 89.5 89.5 89.5 88.1 88.1 88.1 88.1 88.1 87.7 86.7 87.2 87.2

100.0 100.0 100.0 98.8 98.8 98.8 98.8

100.0 100.0 98.8 98.8 98.8 98.8 ~8.8 98.8 94.7 94.7 94.3 94.3 94.7 94.7 94.7 94.7

100.0 98.8 98.8 98.8 98.8 98.8 94.7 94.3 94.7 94.7

98.8 98.8 98.8 98.8 98.8 94.7 94.3 94.7 94.7

100.0 100.0 100.0 100.0 95.1 95.6 96.0 96.0

100.0 100.0 100.0 95.1 95.6 96.0 96.0

100.0 100.0 95.1 95.5 95.9 95.9

100.0 95.1 95.6 96.0 96.0

95.1 95.6 98.8 96.0 99.2 96.0 99.2

99.6 99.6

100.0

Table 7. Sequence similarity between clones identified as Pseudomonas subgroup

U63901

lag U63944

100.0 100.0 100.0

Most of the clones, 16 out of 24 were identified as Rubrivivax subgroup (Table 2). No other taxa within the beta subdivision were detected. All these clones had Pseudomonas testosteroni (MI1224) as best-species• BLAST-hit. Six of them had Pseudomonas testosteroni (MI1224) as best-BLAST-hit. Remaining had best• BLAST-hit to clones reported in a flora descriptive study by Bond et al. (1995). Three of these had best• BLAST-hit to X84470, four to X84519 and three to X84479 (Table 1). Total sequence identity were found

Characterisation of efficient denitrifying bacteria strains

67

among 110, 2:99g and 5:l07g. Total sequence identity were also found among 2l2g, 238r, F66g and X84470. Clones 555g, bp62, bp64, Rl13g and X845l9 had also total identity as well as clones l8g and X84479 (Table 6). Among t~e cl~nes affiliating to alpha subdivision five clones were identified as Rhodobacter group (Table 2). Total IdentIty was found among all these clones but none had full identity towards reported sequences (Table 4). These clones had Paracoccus aikaliphilus (D32238) as best-BLAST-hit (Table 1.). Paracoccus spp. have earlier been isolated from activated sludge by Vedenina and Govorukhina (1988) and from a denitrifying sand filter by Neef et ai. (1996). It has also been reported that members of the genera Rhodobacter and Paracoccus are able to denitrify using methanol as carbon source (Holt et al., 1994; Sly, 1985; Verseveld and Stouthamer, 1992). The remaining two alpha subdivision affiliating clones are probably members of the Brucella assemblage. Both clones have high similarity to reported clones in this taxon (Table 1), though, it was not possible to get a coherent cluster of this group (Table 2). None of these two clones had full identity towards reported sequences (Table 5). One of these clones had X84630 as best-BLAST-hit and Zooglea ramigera (D14255) as best-species-BLAST-hit and the other had Ochrobacterium anthropi (D12794) as best-BLAST-hit (Table 1). The clone affiliating to gamma purple was identified as members of the Pseudomonas subgroup (Table 2). Best-species-BLAST-hit was Pseudomonas fluorescens (U63901) (Table 1). The clone had full identity to this sequence. The genus Pseudomonas has earlier been observed when denitrifiers were isolated from activated sludge (Schmider and Ottow, 1986). Total identity among some clones and among clones and reported sequences indicates they are closely related, but does not prove they are the same species. In addition sequencing of the entire l6S rRNA gene and DNA reassociation analyses are needed. Sequencing of the entire gene is also needed in order to perform a complete phylogenetic analysis. In this study the aim was to cultivate and select the most efficient denitrifying strains. However, the methodologies that have been used have some limitations. Culture based methods can fail in representing the microbial community structure (Wagner et ai., 1993).There is also a possibility that less efficient but more common species are of greater importance of the total denitrification in the sludge. Still, identical and highly similar sequences were found from the five treatment plants, indicating closely related denitrifying species. Clones identified as Rubrivivax subgroup, were found in all the five plants. This subgroup dominated in this study. The best-species-BLAST-hit for all these clones was Pseudomonas testosteroni. These results indicate that the majority of the most efficient denitrifying bacteria in activated sludge comprise a homogeneous bacterial group. The denitrification process is therefore likely to be more sensitive than might be expected towards variations in process parameters, changes in carbon sources and toxic compounds. The results of this study also gives the opportunity to use selected species as biomarkers investigating the denitrifying status of a process. Pure cultures of these species could also be used for testing toxicity in activated sludge. In some applications, pure cultures could be used as additive to improve existing processes or to inoculate a trickling filter for the development of a biofilm process. CONCLUSIONS This study indicates that the most efficient cultivable denitrifying strains in activated sludge, performing the complete pathway of denitrification, are of a limited number of taxa which includes members of Rubrivivax subgroup, Rhodobacter group and Pseudomonas subgroup, with domination of the Rubrivivax subgroup.

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