Determination of the microbial diversity of anaerobic-aerobic activated sludge by a novel molecular biological technique

8)

Pergamon

W"t. Sci. T~ch. Vol. 37. No. 3.1'1'.417-422.1998 © 1998 IA wQ. Pubhshc<.l by EI"'~lcr SCIence Lid

PH: S0273-1223(98)OO141-3

Ponted on Great Bn.am. 0273-1223198 S19·00 + 0-00

DETERMINATION OF THE MICROBIAL DIVERSITY OF ANAEROBIC-AEROBIC ACTIVATED SLUDGE BY A NOVEL MOLECULAR BIOLOGICAL TECHNIQUE Wen-Tso Liu, Terence L. Marsh and Larry J. Forney Centerfor Microbial Ecology Michigan State University, East Lansing. M148824. USA

ABSTRACf Sequential anacroblc-aerobic batch reactors were maintained on acetate/peptone and lwo different P/total organic carhon ratios lhat select for microbial communities enriched for either glycogcn-accumulating organisms (GAO) or polyphosphate.aeeumulating organism (PAD). The communily profiles of the euhaclerial population and gram-posllive high G-C hacteria (HGC) were charactenzed and compared hy determining the Ierminal restriction fragment length polymorphisms (T·RFLP) of 165 rONA. The Hhal+Rsal dlgcsled 5' T-RFLP pallcms of the eubactenal 16S rONA amplified from the GAO- and PAO• cnnched communities were made up with 12 and 14 rank-abundant fragments (I.e.• nootypes), respectively. Among Ihose ribotypes detected in the GAO-enriched commuRlty. only seven were found in the PAO• ennched communily. The HGC group could only account for no more than 6% and 17% of the eubactenal 16S rONA amplIfied from the GAO- and PAO-enriched communities, respectively. Within the HGC communily. at leasl 16 and 10 rank-ahundanl ribotypes were observed In Ihe Mspl digested T-RFLP pallems of GAO- and PAO-enriched commuOllies. respectively. Among those HGC ribotypes ohserved in hoth communnies. only five were in common. 1llese indicate that the enrichment proces~ leading to the cstahllshmcRl of GAO- and PAD-specific communities ca~d the dramat,c dlfferencc and compleXIty In Ihe mlcrohial populalion. © 1998 IAWQ. Publishcd by Elsevlcr SCience LId

KEYWORDS Activated sludge; biological phosphorus removal; microbial diversity; polyphosphate; 16S rDNA; restriction fragment length polymorphism. INTRODUCfION Wastewater treatment process with a configuration of an anaerobic zone followed by an aerobic zone and a final settling stage has been extensively used for removal of nutrients. Its unique physical configuration has created an environment or 'niche' to a microbial community. This community can take up and store organic substrates a~ carbon reserves in the anaerobic zone, and subsequently use the carbon reserve for growth and other energy requiring processes in the aerobic zone (Comeau el al., 1986; Mino et al., 1987). Recent studies suggest that this microbial community may be composed of two sub-groups differentiated in the use of internally stored energy sources for anaerobic substrate uptake (Cech and HartmaR, 1993; Liu et al., 1994, 1997a). One is the polyphosphate-accumulating organism (PAO) that mediate the enhanced biological 417

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phosphorus removal (EBPR) activity, and the other is the glycogen-accumulating organism (GAO) that are not responsible for EBPR, but may compete with PAB for anaerobic substrate uptake. The microbial diversity of PAD and GAO and their relatedness are not well known. Light microcopy indicates the two communities are quite different, each having up to three to four unique morphotypes (Liu et al.. 1996. 1997). Conventional culture technique (Jenkins and Tandoi, 1990) consistently identified members of the genus Acinetnhacter as a dominant population in the community. However, non-culture• dependent techniques using 16S rRNA probe specific for Acinetobacter sp. suggested that this group was not a major bacterial population in the community (Wagner et ai., 1994). Not surprisingly. this was attributed to the unculturability of dominant bacteria in various microbial ecosystems (Amann et aI., 1995; Barn et al.• 1994; Ward et al., 1990). Hence the application of non-culture-dependent methodologies to the analysis of the microbial communities of PAD and GAO is warranted. We have recently developed a quantitative technique to characterize the microbial diversity and compare community relatedness among microbial community. This technique examines the terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rDNA (Liu et ai., 1997). In brief, small subunit rDNAs were obtained from microbial communities by PCR amplification using universal primers and one of the two primers was tluorescently-Iabeled at the 5' end. The amplified rDNA products were then digested with restriction enzymes with four-base recognition sites, and analyzed using an automated DNA sequencer. Since the PCR products are labeled at the termini, only the terminal restriction fragments are detected. Computer analysis of the T-RFLP pattern for 686 amplifiable sequences from 1102 165 rRNA sequences of the Ribosomal Database Project (RDP) indicated that those sequences could be classified into 233 different terminal restriction fragments (i.e., ribotypes) (Liu et al., 1997). This indicates that T-RFLP is a sensitive and rapid way to assess the microbial diverSity and obtain a distinctive fingerprint for each microbial community. Here, we have applied this technique to the analysis and comparison of the bacterial community structures of activated sludge samples taken from bioreactors enriched with PAS and GAB. MATERIALS AND METHODS Enrjched activated slud&e Activated sludge was cultivated in sequential batch reactors fed with a mixture of acetate and peptone under alternating anaerobic and aerobic conditions as previously described (Liu el ai., 1994; Liu el ai., 1997). Two different P/tolal organic carbon (TOC) ratios (wt/wt), 2/100 and 20/100, were used in the medium. As a result of Ihe low PITOC feeding ratio applied. the growth of PAD was suppressed and GAO enriched. ACl.:ordingly. a PITOC feedmg ratio of 20/100 was used to promote the growth of PAD over GAO. At the steady ,laIC of each reactor sludge samples were taken and immediately Iyopholized prior to the extraction of IOIaI community DNA. and the analysis of T-RFLP. PCR ampllfjcation and TRFLP of 16S rONA Total community DNA of activated sludge was obtained after cell lysis, phenol-chloroform extraction. and eth,mol preCipitation using a protocol previously described (Liu et ai., 1997). Two diffcrent primer pairs (Fig. I), which target the 16S rDNA of the bacterial population and the gram• po,itivc hl!!h G-C h,lctena, were used in the PCR. Both primer pairs had the same forward primer. Sf-Hex. whil.:h wa' laheled with a phosphoramidite dye. HEX (5-hexachloro-f1uorescein), at the 5'-end. As a l:nnselluenl.:c. two different 16S rDNA regions with an identical terminal end were amplified from the total wmmunity DNA of activated sludge samples. Reaction mixtures and thermoprogram for PCR were wnductcd according to a protocol previously described (Liu et al.. 1997). Fluorescently laggcd PeR products (100 Ill) were then purified using WIZARD PCR purification columns (Promega, MadIson. WI) and eluted in a final volume of 50 III H 20. Six III aliquots of purified PCR products

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were digested with 20 U of BstUI, HhaI, MspI, or HhaI+MspI (BMB). The precise length of the terminal restriction fragments from the amplified rONA products were determined by electrophoresis using an ABI Automated Sequencer (AS I 373A Applied Biosystems Instruments, Forster City, CAl as previously described (Liu et al., 1997). Microbial diversity and community relatedness The species richness of a community is estimated by determining the number of unique terminal restriction fragments or 'ribotypes' observed in digests of 16S rDNAs amplified from different communities. This wa~ a significant underestimate of diversity, since phylogenetically different 16S rONA may have conserved the position of a restriction site. The 'community fingerprint' is defined as the observed pattern of fragments including both the lengths and relative abundance of each peak detected in a community. The relatedness of communities was estimated by visually comparing the electropherograms or by numerically analyzing the pattern of T-RFLPs (Liu et al., 1997). RESULTS AND DISCUSSION The microbial community diversity of GAO and PAD was determined by T-RFLP. PCR, wherein the forward primer (8f-HEX) was labeled with a fluorescent dye, was used to amplify an internal region of 16S rONA from the extracted total community DNA. The PCR products were digested with four different restriction enzymes (BstUI, HhaI, MspI and HhaI+RsaI), and the f1uorescently labeled terminal restriction fragments were separated and sized on an automated DNA sequencer. Figure 2 presents electropherograms from individual lanes of the sequencing gel. The data indicate that about 19-24 different numerically dominant ribotypes (19-24 different terminal restriction fragments) were observed in the samples from PAD and GAO, respectively. The least abundant terminal restriction fragment detected in each community represents less than I % of the total rONA amplified. The diversity of dominant amplifiable ribotypes observed in both populations wa~ much greater than the microscopic observations from our previous studies (Liu et al., 1994, 1997), the cloning library approach (Bond et af., 1994), or culture-dependent methods (see review in Jenkins and Tandoi, 1991) have suggested. Since phylogenetically distinct bacteria may have the same terminal restriction fragment length for a specific restriction enzyme, the actual bacterial diversity in enriched GAB and PAB samples may be greater than observed. (A)

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The T-RFLP pattern of a community can also be viewed as a 'community signature' for the assessment of the microbial relatedness between GAO- and PAO-enriched communities. Figure 3 shows the electropherogram of the 5' T-RFLP pattern of the 16S rONA amplified from the GAO- and PAD-enriched communities after HhaI+RsaI digestion (panels A and B). The characteristics of each T-RFLP pattern Including both fragment length and peak area constitute a distinct phylogenetic fingerprint of a community. Common ribotypes (e.g., Fig. 3, peaks 1-7) were observed in both communities. However, the numerically dominant amplifiable ribotypes of the two communities were different. The comparative analysis between these two

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electropherogram\ e\lImated hy uSing the area-sensitive handing coefficient. which takes the relallve peak poslllOn as well as intensllY into account. was about 0.38 (I indicates identical fragment size and Intensity between two T-RFLP pallerns. and 0 sugge\t\ no common fragment between two T-RFLP pallernlo). We can conclude that the two communities enriched with different prroc ratios were composed of very different bacterial populations. When a low prroc feeding ratio was used. growth of PAO was suppressed pre,umably because of the reduction of P feeding concentration. In contrast. higher prroc feeding ratio encouraged the growth of PAO over.GAO. lotal populalion diversily

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RC:l:ently. Gram-pO\lllve bal:terla high G-C (HGC) have been Impltcated In the removal of phosphate In anaerobIc-aerobIc al:tlvated sludge proce\\es (Wagner 1'1 ul.. 1994; Kamper el ul.. 1996; Nakamura el /II .. 1996). In thl\ \tudy. we further Inve\lIgated the possible role of the HGC bacteria In EBPR. and the diverSity of HGC bacteria In both GAO- and PAO-enrlched communities. 165 rONA of HGC bacteria was amplified uSlOg a Ilourescently-labeled eubacterial primer and a HGC-specific primer. Figure 2 (lanes 1-0) \hows the T-RFLP\ of HGC bacteria In both populallons corresponding to the digestion of two different re\trlcllon cnzy mc\. After the digestion of BSIUI, two major ribotypes were observed from each communlly. Of the two obscrwd III the GAO communtty. only one was present m the cognate total bactenal communtty profile (Fig. 2. lane b). and liS Iluorescent IOtc~nlollY represented no more than 61k of the total mtenstly (I.e.. abundam:el of tho,e detectable fragmentlo. In the PAO communtty. at least Sill termmal relotrlctlon fragments v.ere detected (Fig 2. lane mj and only two fragment\ (Fig. 2 lane cl. repre\entlOg no more than 17'7" of the total abundance. wuld be detected m the total bactenal PAO communtty. The digestion of the HGC communtty al1lpllfll:allOnS of GAO and PAO WIth Mspl revealed even greater diverSity (Fig. 4 panel\ A and B) 10 both WlTIl1lunllles. In GAO-ennched communtty. 16 numencally dommant HGC rlbotypes were detected while 10 v. ere detected from the PAO-~nrlched communlly. However. among tho\e dommant HGC

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robotypes observed in both communities, only five were in common. This indicates that the enrichment processel> leading to the establishment of GAO- and PAD-specific communities caused the dramatic difference in the HGC population,

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These results, based on the composition of PCR-amplified bacterial 165 rONA, suggest that HGC bacteria were not the only major component in the total microbial biomass of GAO and PAO. This is consistent with a previous report (Hiraishi et aI., 1988) that the respiratory quinone extracted from activated sludge with a good enrichment of PAO was composed of mainly ubiquinone, a major component of the Gram-negative bacteria, rather than men.lquinone, the major component of the Gram-positive bacteria. The ability to

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accumulate energy-rich compounds under anaerobic conditions may be a highly dispersed trait among phylogenetic groups, unlike other phenotypic traits such as nitrification and methanogenesis which cluster within a single lineage on the phylogenetic tree (Mobarry et aI., 1996: Raskin et a!.. 1994) ACKNOWLEDGEMENT The author thanks Dr. Hans Chen and Mary Hutcheson of the USDA-ARS Avian Disease & Oncology lab for the analysis of T -RFLP samples. REFERENCES Amann. R. I.• Ludqlg. W. and Schleller. H. H. (1995). Phylogenetic identilication and in-situ detection of individual microbial cell> without cultivation. MIcrobial. Rev.• 59. 143-169. Bam,. SM. Fundyga. R. E.• Jeffenes, M. W. and Pace. N. R. (1994). Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc. Nat/. Acad. Sci. USA. 91,1609-1613. Bond, P. L.. Hugcnholtt. P., Keller 1. and Blackall. L. L. (1995). Bactenal community structures of phosphate-removing and non• phm.phate-removmg activated sludge from sequencmg batch reactors. Appl. Environ. Microbiol.. 61.1910-1916. Cech. 1. S. and Hartman, P. (1993) Competition between polyphosphate and polysaccharide accumulating bacteria in enhanced I>lOloglcal phosphate removal system. Wat. Res., 27.1219-1225. Comeau. Y • Hall, K. J.• Hancock. R. E. W. and Oldham. W. K. (1986). Biochemical model for enhanced biological phosphorus removal. Wal. Res.• 20, J511- 1521. Hmushl. A., Masamune, K. and Kitamura, H. (1989). Characterization of the bacterial population structure in an anaerobic• aerol>,c activated loludge system on the basiS of respiratory quinone profiles. Appl. Environ. Mlcrobiol.• 55, 897-901. Jenkmlo, D. and Tandoi. V. (1991). The applied microbIOlogy of enhanced biological phosphate removal-accomplishments and needlo. Will Res.. 25,1471-1478. Kamper. P.. Erhan, R.. Belmfohr, C.. Bohringer, J., Wagner, M. and Amann, R. (1996). Characterization of bacterial communities Irom activated ,Iudgc' culture-dependent bactenal Identilication versus m SitU Identification using group- and genus• lopccIfic rRNA-targeted oligonucleotide probes Microb. Ecol.• 32, 10 1-121. Liu. W.·T, Nakamura. K.. Matlouo. T. and Mmo. T. (I 997a). Internal energy-based competition between polyphosphate- and glycogen-accumulating bactena In biological phosphorus removal reactor-effect of the PIC feeding ratio. Wat. Res.• 31. 1430-14311. Llu. W.- T., Mmo, T.. MatloUO. T. and Nakamura. K. (1996). Glycogen accumulatmg population and its anaerobic substrate uptake m anacroblc-aeroblc activated sludge without biological phosphate removal. Wat. Res.• 30. 75-82. Llu, W.-T.. Mlno. T.. Nlikamura. K. and Matsuo, T. (1994) Role of glycogen in acetate uptake and polyhydroxyalkanoate ,ynthc,,, m ,maeroblc-acroblc activated sludge with a minimized polyphosphate content. J. Ferment. Biotechnol.• 77. ~3~-~40

L,U, W.-T. M'lrloh. T. L.. Chcn, H. and Forney. L. J. (1997b). Characterization of microbial diversity by determining terminal relolncllon fragment Icngth polymorphlsms of 16S ribosomal DNA. Appl. Environ. Mlcrobiol. (in press). Mino. T. Arun. V , Tw/uk,. Y and Malsuo. T (1987). Effect of phosphorus accumulalion on acetate metabolism in the hiologlCal pholophorus removal preceslo. In' Ramadori R. (ed.). Advances in water pollution colltrol: Biological phosphate reiI/OI'"lfrolll "'"",'ell'lI/as. Pergamon Press, Oxford. pp. 27-38. Mobarry. B. K. Wagner. M. Urbam, V .. Rillmann. B. E. and Stahl. D. A. (1996). Phylogenetic probes for analyzing abundance ,lOd ,pallal orgamtaliOn of mtnlying baclena. Appl. EnVIron. Microbiol., 72.2156-2162. MUYlcr. G . Teskc, A. ,md Mlrloen. C. 0 (1995). Phylogenetic relalJonships of Thiomlcrospira species and their identification in decp-,cll hydrothermal vent samplcs by denatunng gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol.• 164, 16~-172 Nakamur.l. K.. Hlra"hl. A.. Yoshunl. Y., Kawaharasakl. M.. Masuda. K.• and Kamagala. Y. (l995b). Mlcrolunatus phosphovorus gen nllv.. ,p nov.. a new gram-pO"lJVC polyphosphate-accumulallng bacterium Isolated from aclivated sludge. Int. J. S\\I. 8m Il'I'lIJl.. 45. 17-22. Ra,~,". L . Slromcly. J M., Rillmann. B. E and Stahl, D. A. (1994) Group-lopccllic 16S rRNA hybridization probes to describe n:llural commumllelo ormclbanogcnlo. Appl. Environ Microbiol., 60. 1232-1240. Wagner. M.. Erhal1. R.. Mant. W.. Amann, R.. Lcmmer. H.• Wedi, D. and Schleifer. K. H. (1994). Development of an rRNA• targeled ollgonucleotldc prohc lopccitic for thc genus Acinetobaclcr and its application for," situ monitoring in activated ,Iudge. AI'pl. £1/1'/1'1111. MI( rob",I., 60. 792-800. Ward, D 11.1 .. Wcller. Rand Batcloun. M. M. (1990) 16S-rRNA sequcnces reveal numerous uncultured inhabitants in a natural <.tlmmunlly. Nt/I",." (Londun) 345. 63-65