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Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge
FEMS Microbiology Letters 205 (2001) 253^257
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Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge Shams Tabrez Khan, Akira Hiraishi * Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441-8580, Japan Received 29 July 2001; received in revised form 22 October 2001; accepted 22 October 2001 First published online 13 November 2001
Abstract A new denitrifying chemoorganotrophic bacterium capable of aerobic and anaerobic degradation of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) was isolated from activated sludge. A phylogenetic analysis based on 16S rDNA sequences indicated that the new isolate is a member of the L subclass of the Proteobacteria and represents a distinct line of descent within the family Comamonadaceae. During denitrifying growth with 3-hydroxybutyrate, PHB, or PHBV as the sole carbon source, the isolate reduced nitrate to N2 without appreciable accumulation of nitrite and nitrous oxide as intermediate products. Kinetic analyses of the denitrification with different grades of PHBV indicated that approximately 0.7 g of PHBV was required to reduce 1 g of NO3 3 . A high 31 31 denitrification rate (19 mg N-NO3 removed h g dry wt of cells) was found with PHBV as the electron donor. ß 2001 Federation of 3 European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Poly(3-hydroxybutyrate); Poly(3-hydroxybutyrate-co-hydroxyvalerate); Denitrifying bacterium; Nitrogen removal; Activated sludge
1. Introduction Poly(3-hydroxybutyrate) (PHB), which is intracellular storage material produced by a large variety of bacteria, comprises a class of biodegradable polymers that o¡er an environmentally sustainable alternative for the conventional plastics (for reviews, [1,2]). While aerobic degradation by microorganisms of external PHB and the bioplastic made of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) has been studied extensively, much less information is available on their biodegradation under anaerobic conditions [3]. One of the major concerns in this respect is anaerobic degradation of PHB and PHBV under denitrifying conditions. Although some investigators have reported on denitrifying bacteria that are capable of anaerobic degradation of these polymers [4^6], the available information on the phylogeny and taxonomy of such bacteria is limited. An interesting aspect of PHB-degrading, denitrifying bacteria is involved with possible high denitri-
* Corresponding author. Tel. : +81 (532) 44-6913; Fax: +81 (532) 44-6929. E-mail address : [email protected] (A. Hiraishi).
¢cation with PHB and PHBV as the electron donor. Here we report the phenotypic and phylogenetic characteristics of a new PHB-degrading, denitrifying bacterium isolated from activated sludge. We also report the kinetics of denitri¢cation with concomitant degradation of PHBV by this bacterium. 2. Materials and methods 2.1. Chemicals used Di¡erent grades of PHB and PHBV powders and pellets, all of which were generous gifts from Japan Monsanto Co. (Tokyo, Japan), were used. The powder types of PHBV designated PHBV5, PHBV8, and PHBV12 had an average particle size (in diameter) of 50, 100, and 60 Wm and a co-hydroxyvalerate (HV) content of 5, 8, and 12%, respectively. The PHBV pellets had an average diameter of 2 mm (see Fig. 1) and a co-HV content of 8%. 2.2. Sludge sample and bacterial strains Activated sludge was collected from a sewage treatment
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 8 7 - 6
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plant in Osaka Prefecture, Japan, and used for isolation of PHB-degrading denitri¢ers immediately upon return to the laboratory. A new strain (designated strain NA10B) isolated from this sludge was studied. Strain NA10B has been deposited with the Japan Collection of Microorganisms under accession number JCM 11421. Acidovorax sp. DSM 13225 ( = strain LW1) and Comamonas testosteroni JCM 10170, which have been reported to be a chlorobenzene-degrading bacterium [7] and a PHB degrader [8], respectively, were used as reference organisms. 2.3. Growth media and cultivation A peptone-containing complex medium designated PBY [9] and PBYN medium (PBY+0.2% KNO3 ) were used for preculture. For main culture, mineral base RM2 (pH 7.0) [10] was used as the basal medium. For isolation and cultivation of PHB-degrading denitri¢ers, the basal medium was supplemented with 2 g of PHB powder, 2 g of KNO3 , and 0.1 g of yeast extract (Difco Laboratories) per liter (pH 7.0). This medium, designated PHBN medium, was modi¢ed by replacing PHB with an equal weight of PHBV, 3-hydroxybutyrate (3HB), and other simple organic compounds in some experiments. All media contained 1.8% agar when used as solid media. Cultivation with liquid medium was carried out in 20-ml screw-capped test tubes or 100^500-ml bottles. For anaerobic growth, the
test tubes or bottles were completely ¢lled with the same medium. Anaerobic denitrifying growth was also performed in rubber-plugged test tubes (30 ml capacity) containing 25 ml of the medium, in which anoxic conditions were obtained by sparging argon. Because the optimal temperature for growth of our new isolate was 28^30³C, incubation was at 28³C in all cases. 2.4. Measurement of growth Cell growth was monitored spectrophotometrically by measuring the optical density at 660 nm. For strain NA10B, the cell suspension giving an OD660 of 1.0 corresponded to 0.59 mg dry wt of cells per ml. When growth media contained PHB or PHBV, growth was monitored by measuring cell protein because of the interference of the insoluble particles in measuring the optical density. Protein was extracted and measured colorimetrically by Lowry's method [11]. On the basis of the relationship between dry weight of cells and cell protein concurrently determined, 1 mg of cell protein corresponded to 1.7 mg dry wt of cells. 2.5. 16S rDNA sequencing and phylogenetic analysis 16S rDNA fragments (positions 8^1543 of Escherichia coli 16S rRNA [12]) from the crude cell lysate [13] were ampli¢ed by PCR with a pair set of universal primers [14], sequenced directly with SequiTherm Long Read sequencing kit (Epicentre Technologies, Madison, WI, USA), and analyzed with a Pharmacia DNA sequencer. Sequence data were complied with the GENETYX-MAC program (Software Developing Co., Tokyo, Japan) and compared with those available from the DDBJ/EMBL/GenBank databases. The CLUSTAL W program [15] was used for multiple sequence alignments and construction of neighbor-joining [16] phylogenetic trees. The topology of trees was evaluated by bootstrapping with 1000 resamplings [17]. The maximum likelihood inference of phylogeny was carried out using MOLPHY version 2.3 [18]. Alignment positions with gaps were excluded from all calculations. The 16S rDNA sequences determined in this study have been deposited under DDBJ accession numbers AB064317 and AB64318. 2.6. Other analytical techniques.
Fig. 1. PHB degradation by strain NA10B during anaerobic denitrifying growth. a: Growth on PHBN agar. See the clear zone around colonies of strain NA10 (left) in contrast to no growth of the PHB-degrading non-denitrifying bacterium Comamonas testosteroni JCM 10170 used as the reference (right). b: PHBV pellets from the liquid culture before (left) and after 96 h of incubation (right).
The concentration of 3HB in liquid media was monitored by the spectrophotometric method using a L-HBA reagent kit (Sigma, St. Louis, MO, USA). An aliquot of 3HB culture was sampled and centrifuged to remove cells and large debris. The resultant supernatant was ¢ltered through a Millipore Millex ¢lter and subjected to the 3HB dehydrogenase assay in the presence of NAD and hydrazine monohydrate. The increasing concentration of NADH was measured at 340 nm. The amount of PHBV
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degraded in liquid media was estimated by direct measurement of dry weight of them. An aliquot of culture was sampled in a dry pre-weighed centrifuge tube and centrifuged at 15 000Ug for 10 min. The pellet in the tube was washed twice with pure water and dried at 110³C for 24 h. The amount of PHBV was determined by subtracting the dry weight of cells present (measured as noted above) from the total weight of the pellet. The concentrations of nitrate and nitrite were simultaneously determined with a Hitachi L-7110 ion chromatograph equipped with a Hitachi #2740 column in a column oven at 40³C and monitored with a Hitachi L-7470 conductivity detector. Samples were run with 2.3 mM phthalic acid and 2.5 mM Tris as the mobile phase at a £ow rate of 1.5 ml min31 . Dinitrogen gas and nitrous oxide produced during denitri¢cation were measured by gas chromatography as described previously [19]. 3. Results 3.1. Isolation For the enrichment of PHB-degrading denitri¢ers, the sludge sample was diluted serially with 50 mM phosphate bu¡er (pH 7.0) and a 100-Wl aliquot of each dilution was added to 20-ml screw-capped test tubes containing 10 ml of PHBN medium and a Durham tube which were then completely ¢lled with the same medium. After 3^7 days of incubation, most of the PHBN tubes inoculated with the sludge exhibited signi¢cant growth with gas production. These positive cultures were spread onto PHBN agar plates and then incubated anaerobically for 2 weeks using the AnaeroPack system (Mitsubishi Gas Chemicals, Japan). Several positive colonies showing zones of clearance were picked up and subjected to the standard puri¢cation procedure. During this procedure, however, most strains isolated were found to exhibit only weak denitrifying growth in PHBN medium. As a result of the isolation, we selected one strain that well grew anaerobically in PHBN medium and designated it strain NA10B.
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denitrifying growth of strain NA10B with PHBV pellets as the sole carbon source resulted in reducing the average weight of the pellets to 33% after 96 h of incubation (Fig. 1b). The new isolate was able to grow on a wide variety of simple organic compounds as well as PHB and PHBV as the sole carbon and energy source under both aerobic conditions and anaerobic denitrifying conditions. However, the isolate was unable to grow with sugars as the carbon source under any growth conditions. Nearly complete sequences of the 16S rRNA gene of strains NA10B and JCM 10170 were determined and compared with those available from the DNA databases. Results of homology searches indicated that the 16S rDNA sequence of the new isolate was most similar to the sequence of an unidenti¢ed bacterium, strain LW1 [7] (99.9% similarity). A neighbor-joining phylogenetic tree based on 16S rDNA sequences showed that strain NA10B was a member of the L subclass of the Proteobacteria and formed a tight distinct cluster together with strain LW1 within the family Comamonadaceae (Fig. 2). Although this cluster grouped with Aquaspirillum sinosum as its nearest phylogenetic neighbor, the bootstrap value for this node was low. A maximum likelihood analysis gave a similar topography of the tree showing that strains
3.2. Phenotypic and phylogenetic analyses Strain NA10B was a Gram-negative, motile, rodshaped, chemoorganotrophic bacterium that had a strictly respiratory type of metabolism with oxygen or nitrate as the terminal electron acceptor. Aerobic growth on 3HB and PBY agar media was relatively fast, and 36 h incubation was enough to get visible colonies. In the case of anaerobic growth on PHBN agar plates, however, 48 h was required to produce visible colonies, and the clear zone of PHB degradation started appearing after 48^60 h of incubation (Fig. 1a). Under this condition, the PHB-degrading bacterium C. testosteroni JCM 10170 used as a reference formed no visible colonies. Anaerobic
Fig. 2. Neighbor-joining distance matrix tree showing the phylogenetic position of strain NA10B based on 16S rDNA sequences. The 16S rDNA sequence of Paucimonas lemoignei was used as an outgroup to root the tree. Bootstrap values are given at branching points of interest. Scale = 10% nucleotide substitution (Knuc ).
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NA10B and LW1 formed a distinct line of descent within the family Comamonadaceae (data not shown). Because of the high phylogenetic similarity between our isolate and strain LW1, strain DSM 13225 ( = strain LW1) was tested for PHB degradation and denitri¢cation. This bacterium did not degrade PHB but grew anaerobically under denitrifying conditions with simple organic compounds as the electron donor and carbon sources. 3.3. Denitri¢cation with 3HB, PHB, and PHBV Anaerobic denitrifying growth of strain NA10B was studied with 3HB, PHB, and di¡erent grades of PHBV as the electron donor and carbon sources. As 3HB is the monomer of PHB and is completely soluble in water, it was most readily utilized by the isolate. Under anoxic denitrifying growth conditions with 3HB, strain NA10B exhibited a doubling time of 3.5 h and a maximum cell yield of 0.59 mg dry wt ml31 , and removed nitrate in the 31 medium (0.28 mg N-NO3 3 ml ) completely within 24 h of incubation with concomitant consumption of 1.3 mg 3HB ml31 . During this denitrifying growth, nitrite and nitrous oxide as intermediate products were not detected at any growth stage, and the amount of N2 gas evolved increased with time (data not shown). Anaerobic denitrifying growth with PHB powder resulted in a similar cell yield but the growth rate was lower than that with 3HB (data not shown). This was the case in denitrifying growth with PHBV powders, as it took 60^72 h to remove nitrate completely and yield a cell mass of 0.4^0.5 mg dry wt ml31 (Fig. 3). However, denitri¢cation was possible with a much smaller amount of PHBV than of 3HB. The amounts of PHBV required for the complete removal of 0.28 mg N-NO3 3 were 0.87 mg for PHBV5, 0.80 mg for PHBV8, and 1.0 mg for PHBV12. These kinetic data indicate that the co-HV content of PHBV had minor e¡ects on the denitri¢cation rate and that approximately 0.7 g of PHBV is required to reduce 1 g of NO3 3 to N2 . As shown in Fig. 3, the denitri¢cation rate was slightly higher with PHBV12 than with PHBV8. This might not result from the di¡erence in the co-HV content but from a small di¡erence in particle size between the two. When nitrate was added again to the cultures with PHBV noted above, it was sharply removed at a denitri¢cation rate of 31 31 19 mg N-NO3 g dry wt of cells. 3 removed h 4. Discussion Although the biodegradation of PHB and PHBV in the environment is a well-known phenomenon, the available information on anaerobic PHB degradation by denitrifying bacteria is still limited [4^6]. In this study, a new denitrifying bacterium capable of anaerobic degradation of PHB and PHBV was isolated from activated sludge and characterized. The results of this study may not only give
Fig. 3. Anaerobic denitrifying growth of strain NA10B with di¡erent grades of PHBV powders as the sole carbon and energy source. Closed circles, results with PHBV8; open circles, results with PHBV12. a: Growth as shown by dry weight of cells. b: Change in PHBV concentration. c: Change in nitrate concentration. The plotted data indicate the average values with standard deviations for ¢ve di¡erent determinations.
a new insight into our understanding of the biodegradability of the bioplastic in wastewater environments but also provide basic information useful for exploiting new biotechnology using PHB-degrading denitri¢ers. 16S rDNA sequence comparisons have shown that the new isolate has 99.9% similarity to strain LW1, which was reported to be a 1-chloro-4-nitrobenzene-degrading bacterium but described as not being able to degrade or denitrify PHB [7]. The present study has shown that strain DSM 13225 ( = strain LW1) cannot degrade PHB but grows under denitrifying conditions with simple organic compounds. Our concurrent study has shown that the DNA^DNA hybridization level between strains NA10B and DSM 13225 is ca. 50% (unpublished data). These data indicate that, although our isolate and strain DSM 13225 may share many phenotypic characteristics, they are distinguishable from each other at the species level. The phylogenetic analysis on the basis of 16S rDNA sequences has indicated that the new isolate forms a tight cluster together with strain LW1 within the family Comamona-
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daceae. This cluster is separated from all generic groups of this family. Recently, PHB-degrading, denitrifying strains identi¢ed as Acidovorax sp. have been reported [6], but it is di¤cult, at this time, to compare these strains with ours due to the lack of 16S rDNA sequence information on the former strains. In view of the collective data so far obtained, it seems more appropriate to classify our isolate together with strain LW1 into a new genus than to assign it to a previously known genus of Comamonadaceae. We will report a formal taxonomic proposal for this elsewhere. As reported in this study, strain NA10B showed similar denitri¢cation rates with di¡erent grades of the PHBV powders which had the same particle size but di¡ered in the co-HV content (5^12%). This observation suggests that the co-HV content variance has minor e¡ects on the e¤ciency of denitri¢cation by this bacterium, provided that these grades of PHBV powders have a similar size. Our ¢nding that such a high nitrate removal rate as 19 mg N31 31 NO3 g dry wt of cells was achieved ac3 removed h tually using PHBV provides a basis for the possibility of the development of a new nitrogen removal system using the bioplastic as the electron donor and solid matrix. The use of the bioplastic in a denitri¢cation system for nitrogen removal from wastewater may have some advantages over the conventional system, e.g., constant supply of reducing power and immobilization of bacterial cells on the solid matrix. A better understanding of the kinetics, biochemistry, and genetics of PHB degradation and denitri¢cation by strain NA10B should be of much help to reach this goal. Acknowledgements We are grateful to Japan Monsanto Co., Tokyo, for providing us with various grades of PHBV and the bioplastic. We also thank M. Yamamoto for his technical assistance. References [1] Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450^472. [2] Hankermeyer, C.R. and Tjeerdema, R.S. (1999) Polyhydroxybutyrate: plastic made and degraded by microorganisms. Rev. Environ. Contam. Toxicol. 159, 1^24. [3] Abou-Zeid, D., Muller, R. and Deckwer, W.J. (2001) Degradation of natural and synthetic polyesters under anaerobic conditions. J. Biotechnol. 86, 113^126.
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[4] Biedermann, J., Owen, A.J., Schloe, K.T., Gassner, F. and Sussmuth, R. (1997) Interaction between poly-3-hydroxybutyrate-co-3-hydroxyvalerate and a denitrifying Pseudomonas strain. Can. J. Microbiol. 43, 561^568. [5] Biedermann, J., Staniszeweski, M., Wais, S. and Sussmuth, R. (1992) Poly-L-hydroxybutyrate/L-hydroxyvalerate-copolymers as a substrate and a matrix for microorganisms in denitri¢cation of drinking water. FEMS Microbiol. Rev. 103, 473^474. [6] Schloe, K., Gillis, M., Hoste, B., Pot, B., Vancanney, M., Mergaert, J., Swings, J., Biedermann, J. and Sussmuth, R. (2000) Polyphasic characterization of poly-3-hydroxubutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains. Syst. Appl. Microbiol. 23, 364^372. [7] Katsivela, E., Wray, V., Pieper, D.H. and Wittich, R.M. (1999) Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium sp., strain LW1. Appl. Environ. Microbiol. 65, 1405^1412. [8] Kasuya, K., Inoue, Y., Tanaka, T., Akehata, T., Iwata, T., Fukui, T. and Doi, Y. (1997) Biochemical and molecular chacterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM 1609, isolated from freshwater. Appl. Environ. Microbiol. 63, 4844^ 4852. [9] Hiraishi, A. and Komagata, K. (1989) E¡ects of the growth medium composition on menaquinone homolog formation in Micrococcus luteus. J. Gen. Appl. Microbiol. 35, 311^318. [10] Hiraishi, A. and Kitamura, H. (1984) Distribution of phototrophic purple nonsulfur bacteria in activated sludge systems and other aquatic environments. Bull. Jpn. Sci. Soc. Fish. 50, 1929^1937. [11] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265^275. [12] Brosius, J., Palmer, J.L., Kennedy, J.P. and Noller, H.F. (1978) Complete nucleotide sequence of a 16S rRNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75, 4801^4805. [13] Hiraishi, A., Shin, Y.K., Ueda, Y. and Sugiyama, J. (1994) Automated sequencing of PCR-ampli¢ed 16S rDNA on `hydrolink' gels. J. Microbiol. Methods 19, 145^154. [14] Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991) 16S ribosomal DNA ampli¢cation for phylogenetic study. J. Bacteriol. 173, 697^703. [15] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. [16] Saitou, N. and Nei, M. (1987) The neighbor-joining method : a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406^425. [17] Felsentein, J. (1985) Con¢dence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783^791. [18] Adachi, J. and Hasegawa, M. (1996) Computer Science Monographs, No. 28. MOLPHY Version 2.3. Programs for Molecular Phylogenetics Based on Maximum Likelihood. Institute of Statistical Mathematics, Tokyo. [19] Mahne, I. and Tiedje, J.M. (1995) Criteria and methodology for identifying respiratory denitri¢ers. Appl. Environ. Microbiol. 61, 1110^1115.
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Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge Shams Tabrez Khan, Akira Hiraishi * Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441-8580, Japan Received 29 July 2001; received in revised form 22 October 2001; accepted 22 October 2001 First published online 13 November 2001
Abstract A new denitrifying chemoorganotrophic bacterium capable of aerobic and anaerobic degradation of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) was isolated from activated sludge. A phylogenetic analysis based on 16S rDNA sequences indicated that the new isolate is a member of the L subclass of the Proteobacteria and represents a distinct line of descent within the family Comamonadaceae. During denitrifying growth with 3-hydroxybutyrate, PHB, or PHBV as the sole carbon source, the isolate reduced nitrate to N2 without appreciable accumulation of nitrite and nitrous oxide as intermediate products. Kinetic analyses of the denitrification with different grades of PHBV indicated that approximately 0.7 g of PHBV was required to reduce 1 g of NO3 3 . A high 31 31 denitrification rate (19 mg N-NO3 removed h g dry wt of cells) was found with PHBV as the electron donor. ß 2001 Federation of 3 European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Poly(3-hydroxybutyrate); Poly(3-hydroxybutyrate-co-hydroxyvalerate); Denitrifying bacterium; Nitrogen removal; Activated sludge
1. Introduction Poly(3-hydroxybutyrate) (PHB), which is intracellular storage material produced by a large variety of bacteria, comprises a class of biodegradable polymers that o¡er an environmentally sustainable alternative for the conventional plastics (for reviews, [1,2]). While aerobic degradation by microorganisms of external PHB and the bioplastic made of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) has been studied extensively, much less information is available on their biodegradation under anaerobic conditions [3]. One of the major concerns in this respect is anaerobic degradation of PHB and PHBV under denitrifying conditions. Although some investigators have reported on denitrifying bacteria that are capable of anaerobic degradation of these polymers [4^6], the available information on the phylogeny and taxonomy of such bacteria is limited. An interesting aspect of PHB-degrading, denitrifying bacteria is involved with possible high denitri-
* Corresponding author. Tel. : +81 (532) 44-6913; Fax: +81 (532) 44-6929. E-mail address : [email protected] (A. Hiraishi).
¢cation with PHB and PHBV as the electron donor. Here we report the phenotypic and phylogenetic characteristics of a new PHB-degrading, denitrifying bacterium isolated from activated sludge. We also report the kinetics of denitri¢cation with concomitant degradation of PHBV by this bacterium. 2. Materials and methods 2.1. Chemicals used Di¡erent grades of PHB and PHBV powders and pellets, all of which were generous gifts from Japan Monsanto Co. (Tokyo, Japan), were used. The powder types of PHBV designated PHBV5, PHBV8, and PHBV12 had an average particle size (in diameter) of 50, 100, and 60 Wm and a co-hydroxyvalerate (HV) content of 5, 8, and 12%, respectively. The PHBV pellets had an average diameter of 2 mm (see Fig. 1) and a co-HV content of 8%. 2.2. Sludge sample and bacterial strains Activated sludge was collected from a sewage treatment
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 8 7 - 6
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plant in Osaka Prefecture, Japan, and used for isolation of PHB-degrading denitri¢ers immediately upon return to the laboratory. A new strain (designated strain NA10B) isolated from this sludge was studied. Strain NA10B has been deposited with the Japan Collection of Microorganisms under accession number JCM 11421. Acidovorax sp. DSM 13225 ( = strain LW1) and Comamonas testosteroni JCM 10170, which have been reported to be a chlorobenzene-degrading bacterium [7] and a PHB degrader [8], respectively, were used as reference organisms. 2.3. Growth media and cultivation A peptone-containing complex medium designated PBY [9] and PBYN medium (PBY+0.2% KNO3 ) were used for preculture. For main culture, mineral base RM2 (pH 7.0) [10] was used as the basal medium. For isolation and cultivation of PHB-degrading denitri¢ers, the basal medium was supplemented with 2 g of PHB powder, 2 g of KNO3 , and 0.1 g of yeast extract (Difco Laboratories) per liter (pH 7.0). This medium, designated PHBN medium, was modi¢ed by replacing PHB with an equal weight of PHBV, 3-hydroxybutyrate (3HB), and other simple organic compounds in some experiments. All media contained 1.8% agar when used as solid media. Cultivation with liquid medium was carried out in 20-ml screw-capped test tubes or 100^500-ml bottles. For anaerobic growth, the
test tubes or bottles were completely ¢lled with the same medium. Anaerobic denitrifying growth was also performed in rubber-plugged test tubes (30 ml capacity) containing 25 ml of the medium, in which anoxic conditions were obtained by sparging argon. Because the optimal temperature for growth of our new isolate was 28^30³C, incubation was at 28³C in all cases. 2.4. Measurement of growth Cell growth was monitored spectrophotometrically by measuring the optical density at 660 nm. For strain NA10B, the cell suspension giving an OD660 of 1.0 corresponded to 0.59 mg dry wt of cells per ml. When growth media contained PHB or PHBV, growth was monitored by measuring cell protein because of the interference of the insoluble particles in measuring the optical density. Protein was extracted and measured colorimetrically by Lowry's method [11]. On the basis of the relationship between dry weight of cells and cell protein concurrently determined, 1 mg of cell protein corresponded to 1.7 mg dry wt of cells. 2.5. 16S rDNA sequencing and phylogenetic analysis 16S rDNA fragments (positions 8^1543 of Escherichia coli 16S rRNA [12]) from the crude cell lysate [13] were ampli¢ed by PCR with a pair set of universal primers [14], sequenced directly with SequiTherm Long Read sequencing kit (Epicentre Technologies, Madison, WI, USA), and analyzed with a Pharmacia DNA sequencer. Sequence data were complied with the GENETYX-MAC program (Software Developing Co., Tokyo, Japan) and compared with those available from the DDBJ/EMBL/GenBank databases. The CLUSTAL W program [15] was used for multiple sequence alignments and construction of neighbor-joining [16] phylogenetic trees. The topology of trees was evaluated by bootstrapping with 1000 resamplings [17]. The maximum likelihood inference of phylogeny was carried out using MOLPHY version 2.3 [18]. Alignment positions with gaps were excluded from all calculations. The 16S rDNA sequences determined in this study have been deposited under DDBJ accession numbers AB064317 and AB64318. 2.6. Other analytical techniques.
Fig. 1. PHB degradation by strain NA10B during anaerobic denitrifying growth. a: Growth on PHBN agar. See the clear zone around colonies of strain NA10 (left) in contrast to no growth of the PHB-degrading non-denitrifying bacterium Comamonas testosteroni JCM 10170 used as the reference (right). b: PHBV pellets from the liquid culture before (left) and after 96 h of incubation (right).
The concentration of 3HB in liquid media was monitored by the spectrophotometric method using a L-HBA reagent kit (Sigma, St. Louis, MO, USA). An aliquot of 3HB culture was sampled and centrifuged to remove cells and large debris. The resultant supernatant was ¢ltered through a Millipore Millex ¢lter and subjected to the 3HB dehydrogenase assay in the presence of NAD and hydrazine monohydrate. The increasing concentration of NADH was measured at 340 nm. The amount of PHBV
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degraded in liquid media was estimated by direct measurement of dry weight of them. An aliquot of culture was sampled in a dry pre-weighed centrifuge tube and centrifuged at 15 000Ug for 10 min. The pellet in the tube was washed twice with pure water and dried at 110³C for 24 h. The amount of PHBV was determined by subtracting the dry weight of cells present (measured as noted above) from the total weight of the pellet. The concentrations of nitrate and nitrite were simultaneously determined with a Hitachi L-7110 ion chromatograph equipped with a Hitachi #2740 column in a column oven at 40³C and monitored with a Hitachi L-7470 conductivity detector. Samples were run with 2.3 mM phthalic acid and 2.5 mM Tris as the mobile phase at a £ow rate of 1.5 ml min31 . Dinitrogen gas and nitrous oxide produced during denitri¢cation were measured by gas chromatography as described previously [19]. 3. Results 3.1. Isolation For the enrichment of PHB-degrading denitri¢ers, the sludge sample was diluted serially with 50 mM phosphate bu¡er (pH 7.0) and a 100-Wl aliquot of each dilution was added to 20-ml screw-capped test tubes containing 10 ml of PHBN medium and a Durham tube which were then completely ¢lled with the same medium. After 3^7 days of incubation, most of the PHBN tubes inoculated with the sludge exhibited signi¢cant growth with gas production. These positive cultures were spread onto PHBN agar plates and then incubated anaerobically for 2 weeks using the AnaeroPack system (Mitsubishi Gas Chemicals, Japan). Several positive colonies showing zones of clearance were picked up and subjected to the standard puri¢cation procedure. During this procedure, however, most strains isolated were found to exhibit only weak denitrifying growth in PHBN medium. As a result of the isolation, we selected one strain that well grew anaerobically in PHBN medium and designated it strain NA10B.
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denitrifying growth of strain NA10B with PHBV pellets as the sole carbon source resulted in reducing the average weight of the pellets to 33% after 96 h of incubation (Fig. 1b). The new isolate was able to grow on a wide variety of simple organic compounds as well as PHB and PHBV as the sole carbon and energy source under both aerobic conditions and anaerobic denitrifying conditions. However, the isolate was unable to grow with sugars as the carbon source under any growth conditions. Nearly complete sequences of the 16S rRNA gene of strains NA10B and JCM 10170 were determined and compared with those available from the DNA databases. Results of homology searches indicated that the 16S rDNA sequence of the new isolate was most similar to the sequence of an unidenti¢ed bacterium, strain LW1 [7] (99.9% similarity). A neighbor-joining phylogenetic tree based on 16S rDNA sequences showed that strain NA10B was a member of the L subclass of the Proteobacteria and formed a tight distinct cluster together with strain LW1 within the family Comamonadaceae (Fig. 2). Although this cluster grouped with Aquaspirillum sinosum as its nearest phylogenetic neighbor, the bootstrap value for this node was low. A maximum likelihood analysis gave a similar topography of the tree showing that strains
3.2. Phenotypic and phylogenetic analyses Strain NA10B was a Gram-negative, motile, rodshaped, chemoorganotrophic bacterium that had a strictly respiratory type of metabolism with oxygen or nitrate as the terminal electron acceptor. Aerobic growth on 3HB and PBY agar media was relatively fast, and 36 h incubation was enough to get visible colonies. In the case of anaerobic growth on PHBN agar plates, however, 48 h was required to produce visible colonies, and the clear zone of PHB degradation started appearing after 48^60 h of incubation (Fig. 1a). Under this condition, the PHB-degrading bacterium C. testosteroni JCM 10170 used as a reference formed no visible colonies. Anaerobic
Fig. 2. Neighbor-joining distance matrix tree showing the phylogenetic position of strain NA10B based on 16S rDNA sequences. The 16S rDNA sequence of Paucimonas lemoignei was used as an outgroup to root the tree. Bootstrap values are given at branching points of interest. Scale = 10% nucleotide substitution (Knuc ).
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NA10B and LW1 formed a distinct line of descent within the family Comamonadaceae (data not shown). Because of the high phylogenetic similarity between our isolate and strain LW1, strain DSM 13225 ( = strain LW1) was tested for PHB degradation and denitri¢cation. This bacterium did not degrade PHB but grew anaerobically under denitrifying conditions with simple organic compounds as the electron donor and carbon sources. 3.3. Denitri¢cation with 3HB, PHB, and PHBV Anaerobic denitrifying growth of strain NA10B was studied with 3HB, PHB, and di¡erent grades of PHBV as the electron donor and carbon sources. As 3HB is the monomer of PHB and is completely soluble in water, it was most readily utilized by the isolate. Under anoxic denitrifying growth conditions with 3HB, strain NA10B exhibited a doubling time of 3.5 h and a maximum cell yield of 0.59 mg dry wt ml31 , and removed nitrate in the 31 medium (0.28 mg N-NO3 3 ml ) completely within 24 h of incubation with concomitant consumption of 1.3 mg 3HB ml31 . During this denitrifying growth, nitrite and nitrous oxide as intermediate products were not detected at any growth stage, and the amount of N2 gas evolved increased with time (data not shown). Anaerobic denitrifying growth with PHB powder resulted in a similar cell yield but the growth rate was lower than that with 3HB (data not shown). This was the case in denitrifying growth with PHBV powders, as it took 60^72 h to remove nitrate completely and yield a cell mass of 0.4^0.5 mg dry wt ml31 (Fig. 3). However, denitri¢cation was possible with a much smaller amount of PHBV than of 3HB. The amounts of PHBV required for the complete removal of 0.28 mg N-NO3 3 were 0.87 mg for PHBV5, 0.80 mg for PHBV8, and 1.0 mg for PHBV12. These kinetic data indicate that the co-HV content of PHBV had minor e¡ects on the denitri¢cation rate and that approximately 0.7 g of PHBV is required to reduce 1 g of NO3 3 to N2 . As shown in Fig. 3, the denitri¢cation rate was slightly higher with PHBV12 than with PHBV8. This might not result from the di¡erence in the co-HV content but from a small di¡erence in particle size between the two. When nitrate was added again to the cultures with PHBV noted above, it was sharply removed at a denitri¢cation rate of 31 31 19 mg N-NO3 g dry wt of cells. 3 removed h 4. Discussion Although the biodegradation of PHB and PHBV in the environment is a well-known phenomenon, the available information on anaerobic PHB degradation by denitrifying bacteria is still limited [4^6]. In this study, a new denitrifying bacterium capable of anaerobic degradation of PHB and PHBV was isolated from activated sludge and characterized. The results of this study may not only give
Fig. 3. Anaerobic denitrifying growth of strain NA10B with di¡erent grades of PHBV powders as the sole carbon and energy source. Closed circles, results with PHBV8; open circles, results with PHBV12. a: Growth as shown by dry weight of cells. b: Change in PHBV concentration. c: Change in nitrate concentration. The plotted data indicate the average values with standard deviations for ¢ve di¡erent determinations.
a new insight into our understanding of the biodegradability of the bioplastic in wastewater environments but also provide basic information useful for exploiting new biotechnology using PHB-degrading denitri¢ers. 16S rDNA sequence comparisons have shown that the new isolate has 99.9% similarity to strain LW1, which was reported to be a 1-chloro-4-nitrobenzene-degrading bacterium but described as not being able to degrade or denitrify PHB [7]. The present study has shown that strain DSM 13225 ( = strain LW1) cannot degrade PHB but grows under denitrifying conditions with simple organic compounds. Our concurrent study has shown that the DNA^DNA hybridization level between strains NA10B and DSM 13225 is ca. 50% (unpublished data). These data indicate that, although our isolate and strain DSM 13225 may share many phenotypic characteristics, they are distinguishable from each other at the species level. The phylogenetic analysis on the basis of 16S rDNA sequences has indicated that the new isolate forms a tight cluster together with strain LW1 within the family Comamona-
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daceae. This cluster is separated from all generic groups of this family. Recently, PHB-degrading, denitrifying strains identi¢ed as Acidovorax sp. have been reported [6], but it is di¤cult, at this time, to compare these strains with ours due to the lack of 16S rDNA sequence information on the former strains. In view of the collective data so far obtained, it seems more appropriate to classify our isolate together with strain LW1 into a new genus than to assign it to a previously known genus of Comamonadaceae. We will report a formal taxonomic proposal for this elsewhere. As reported in this study, strain NA10B showed similar denitri¢cation rates with di¡erent grades of the PHBV powders which had the same particle size but di¡ered in the co-HV content (5^12%). This observation suggests that the co-HV content variance has minor e¡ects on the e¤ciency of denitri¢cation by this bacterium, provided that these grades of PHBV powders have a similar size. Our ¢nding that such a high nitrate removal rate as 19 mg N31 31 NO3 g dry wt of cells was achieved ac3 removed h tually using PHBV provides a basis for the possibility of the development of a new nitrogen removal system using the bioplastic as the electron donor and solid matrix. The use of the bioplastic in a denitri¢cation system for nitrogen removal from wastewater may have some advantages over the conventional system, e.g., constant supply of reducing power and immobilization of bacterial cells on the solid matrix. A better understanding of the kinetics, biochemistry, and genetics of PHB degradation and denitri¢cation by strain NA10B should be of much help to reach this goal. Acknowledgements We are grateful to Japan Monsanto Co., Tokyo, for providing us with various grades of PHBV and the bioplastic. We also thank M. Yamamoto for his technical assistance. References [1] Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450^472. [2] Hankermeyer, C.R. and Tjeerdema, R.S. (1999) Polyhydroxybutyrate: plastic made and degraded by microorganisms. Rev. Environ. Contam. Toxicol. 159, 1^24. [3] Abou-Zeid, D., Muller, R. and Deckwer, W.J. (2001) Degradation of natural and synthetic polyesters under anaerobic conditions. J. Biotechnol. 86, 113^126.
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[4] Biedermann, J., Owen, A.J., Schloe, K.T., Gassner, F. and Sussmuth, R. (1997) Interaction between poly-3-hydroxybutyrate-co-3-hydroxyvalerate and a denitrifying Pseudomonas strain. Can. J. Microbiol. 43, 561^568. [5] Biedermann, J., Staniszeweski, M., Wais, S. and Sussmuth, R. (1992) Poly-L-hydroxybutyrate/L-hydroxyvalerate-copolymers as a substrate and a matrix for microorganisms in denitri¢cation of drinking water. FEMS Microbiol. Rev. 103, 473^474. [6] Schloe, K., Gillis, M., Hoste, B., Pot, B., Vancanney, M., Mergaert, J., Swings, J., Biedermann, J. and Sussmuth, R. (2000) Polyphasic characterization of poly-3-hydroxubutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains. Syst. Appl. Microbiol. 23, 364^372. [7] Katsivela, E., Wray, V., Pieper, D.H. and Wittich, R.M. (1999) Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium sp., strain LW1. Appl. Environ. Microbiol. 65, 1405^1412. [8] Kasuya, K., Inoue, Y., Tanaka, T., Akehata, T., Iwata, T., Fukui, T. and Doi, Y. (1997) Biochemical and molecular chacterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM 1609, isolated from freshwater. Appl. Environ. Microbiol. 63, 4844^ 4852. [9] Hiraishi, A. and Komagata, K. (1989) E¡ects of the growth medium composition on menaquinone homolog formation in Micrococcus luteus. J. Gen. Appl. Microbiol. 35, 311^318. [10] Hiraishi, A. and Kitamura, H. (1984) Distribution of phototrophic purple nonsulfur bacteria in activated sludge systems and other aquatic environments. Bull. Jpn. Sci. Soc. Fish. 50, 1929^1937. [11] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265^275. [12] Brosius, J., Palmer, J.L., Kennedy, J.P. and Noller, H.F. (1978) Complete nucleotide sequence of a 16S rRNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75, 4801^4805. [13] Hiraishi, A., Shin, Y.K., Ueda, Y. and Sugiyama, J. (1994) Automated sequencing of PCR-ampli¢ed 16S rDNA on `hydrolink' gels. J. Microbiol. Methods 19, 145^154. [14] Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991) 16S ribosomal DNA ampli¢cation for phylogenetic study. J. Bacteriol. 173, 697^703. [15] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. [16] Saitou, N. and Nei, M. (1987) The neighbor-joining method : a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406^425. [17] Felsentein, J. (1985) Con¢dence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783^791. [18] Adachi, J. and Hasegawa, M. (1996) Computer Science Monographs, No. 28. MOLPHY Version 2.3. Programs for Molecular Phylogenetics Based on Maximum Likelihood. Institute of Statistical Mathematics, Tokyo. [19] Mahne, I. and Tiedje, J.M. (1995) Criteria and methodology for identifying respiratory denitri¢ers. Appl. Environ. Microbiol. 61, 1110^1115.
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