Microbes Environ. Vol. 22, No. 1, 20–31, 2007 http://wwwsoc.nii.ac.jp/jsme2/

Activity and Community Composition of Denitrifying in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-Using Solid-phase Denitrification Processes

SHAMS TABREZ KHAN1, YOKO HORIBA1, NAOTO TAKAHASHI1 and AKIRA HIRAISHI1*

1 Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441–8580, Japan

(Received September 4, 2006—Accepted November 13, 2006)

Two laboratory-scale solid-phase denitrification (SPD) reactors, designated reactors A and B, for nitrogen removal were constructed by acclimating activated sludge with pellets and flakes of poly(3-hydoxybutyrate-co-3- hydroxyvalerate) (PHBV) as the sole added substrate under denitrifying conditions, respectively. The average − −1 −1 denitrification rate in both reactors was 60 mg NO3 -N g (dry wt) h under steady-state conditions, whereas −1 −1 washed sludge taken from the reactors showed an average denitrification rate of 20 mg NO3-N g (dry wt) h with fresh PHBV as the sole substrate. The difference in the denitrification rate between the two might be due to the bioavailability of intermediate metabolites as the substrate for denitrification, because acetate and 3-hydroxy- butyrate were detected in the reactors. Most of the predominant denitrifiers isolated quantitatively by the plate- counting method using non-selective agar medium were unable to degrade PHBV and were identified as mem- bers of genera of the class by studying 16S rRNA gene sequence information. nirS and nosZ gene clone library-based analyses of the microbial community from SPD reactor A showed that most of the nirS and nosZ clones proved to be derived from members of the family and other phylogenetic groups of the Betaproteobacteria. These results suggest that the efficiency of denitrification in the PHBV-SPD process is affected by the availability of intermediate metabolites as possible reducing-power sources as well as of the solid substrate, and that particular species of the Betaproteobacteria play the primary role in denitrification in this process.

Key words: Solid-phase denitrification, poly(3-hydroxybutyrate), nirS gene, nosZ gene, microbial community

Biological denitrification is a series of distinct bioener- supply of reducing power and ease in handling and opera- getic reactions in which nitrate is reduced to dinitrogen gas tion. Promising solid substrates for denitrification are poly- through nitrite, nitric oxide and nitrous oxide46). This bio- hydroxyalkanoates (PHAs) and some other biodegradable chemical process is important not only as a key step in the aliphatic polyesters. To date, SPD processes using poly(3- nitrogen cycle in nature but also as a means of nutrient hydroxybutyrate) (PHB)31) and its copolymer, poly(3- removal in engineered wastewater treatment processes. Bio- hydroxybutyrate-co-hydroxyvalarate) (PHBV)6,26,30), have logical denitrification processes using biodegradable solid been most intensively studied. The application of poly(ε- polymers, termed the solid-phase denitrification (SPD) pro- caprolactone) (PCL) to SPD processes has also been cess, have received attention as promising methods for reported4–6,22). removing nitrogen from water and wastewater17). The SPD Functional and structural analyses of the microbial com- process has some advantages over conventional nitrogen munities involved in SPD processes are necessary to pro- removal systems using soluble substrates, e.g., a constant vide a basis for their practical application to nitrogen removal. In this connection, several strains of PHB- and * Corresponding author. E-mail address: [email protected]; Tel.: PHBV-degrading denitrifying bacteria have been isolated +81–532–44–6913; Fax: +81–532–44–4929. and characterized from activated sludge and PHA-utilizing Denitrifying Microbial Communities with PHBV 21

SPD processes3,24–26,30,38). A representative of PHB-degrad- the other, with PHBV flakes (reactor B). At the beginning, ing denitrifiers is Diaphorobacter nitroreducens25). Our pre- mineral base RM2 (pH 7.0)18), 20 g of PHBV and 20 mM vious study on a 16S rRNA gene clone library constructed KNO3 were added to the reactors. During the first 4 weeks, from a PHBV-utilizing SPD reactor has shown that mem- half of the supernatant in the reactor was exchanged with bers of the family Comamonadaceae, a major phylogenetic fresh mineral medium containing 40 mM KNO3 every 3–4 group of the Betaproteobacteria, predominated in this days of operation. During the subsequent period, the reac- process26). However, the question of what kinds of microor- tors were loaded daily with the same medium by exchang- ganisms are actually involved in denitrification in PHBV- ing half of the supernatant and also supplemented with 5 g SPD processes has remained unanswered. each of PHBV every week. The concentration of microbial Although 16S rRNA genes are powerful molecular mark- sludge was adjusted to ca. 700 mg dry wt L−1 every week. ers for studying the entire prokaryotic community structure During the acclimation, the reactors were gently stirred at in an environment, they can provide no direct information 70 rpm with a magnetic stirrer; under these conditions, the about the physiologic and functional nature of the commu- dissolved oxygen (DO) tension in the core of the reactors nity. In the present study, therefore, nirS and nosZ genes, was less than 0.5 mg L−1, indicating that the reactors were encoding cytochrome cd1 nitrite reductase and nitrous oxide continuously operated under semi-anaerobic conditions. reductase, respectively45), were used as molecular markers to assess the community composition of denitrifying bacte- Measurement of denitrifying activity ria in the PHBV-SPD process. These denitrifying-enzyme The rate of denitrification in the reactors was measured genes as well as the Cu-containing nitrite reductase gene, by monitoring the change in the concentration of nitrate and nirK, have been used for the molecular characterization of nitrite in each batch cycle, for which ion chromatography denitrifying microbial communities in different environ- was used as described previously24,26). Also, the denitrifica- ments including wastewater treatment systems2,13,32,44,45). In tion rate was measured separately using washed sludge and addition, the predominant culturable denitrifying bacteria fresh PHBV powders as the sole substrate. For this, sludge from PHBV-SPD reactors were isolated, phylogenetically samples from the reactors at the end of each batch cycle identified, and studied in terms of their substrate specificity. were collected by centrifugation, washed twice with 50 mM Relationships between the activity and community structure phosphate buffer (pH 7.0), and concentrated in a small vol- of denitrifiers in the PHBV-SPD process are discussed. ume of this buffer. An aliquot of the concentrated sludge suspension was introduced into rubber-plugged test tubes Materials and Methods (30-ml capacity) containing 20 mM KNO3, 0.5 g of PHBV powder, and 25 ml of RM2 mineral medium (pH 7.0) from Biodegradable plastics which (NH4)2SO4 was eliminated. The tubes were sparged PHBV pellets and powder containing 5% poly(3-hydroxy- with argon to establish anoxic conditions and incubated on a valerate) (PHV) were obtained from Japan Monsanto Co., reciprocal shaker at 25°C for 24–48 h. Then, the concentra- Tokyo, Japan. PHBV sheets (5% PHV) were kindly pro- tions of nitrate, nitrite, nitrous oxide, and N2 gas were moni- vided by the Mikawa Textile Research Institute, Aichi Pre- tored by ion chromatography and gas chromatography as fectural Government, Gamagori, Japan. The PHBV sheets described previously24,26). Preliminary experiments showed were cut into small flakes (5 mm×5 mm). All PHBV pellets that the amounts of nitrite and nitrous oxide produced as and flakes were washed with ethanol and then pure water intermediates both in the reactors and in the test tubes were prior to use. negligible in almost all cases. Therefore, the nitrate removal rate was considered as the denitrification rate in this study. Construction of PHBV-acclimated reactors Activated sludge samples were collected from the main Measurement of organic acids aerobic treatment tank of a sewage treatment plant in Toyo- Samples were taken from the reactors at appropriate hashi, Japan, and used as the seed for constructing denitrifi- intervals and centrifuged at 12,600×g for 10 min. The cation reactors. Two glass flasks (2 L capacity) containing resultant supernatant samples were filtered through a mem- 1,000 ml each of culture medium was inoculated with the brane filter (pore size, 0.2 µm) and stored at −20°C until sludge and semi-anaerobically incubated at 25°C for more analyzed. Lower fatty acids and 3-hydroxybutyrate (3HB) than 2 months in the presence of nitrate added. One of the were measured by ion-pair HPLC as described previously33). reactors was acclimated with PHBV pellets (reactor A) and 22 KHAN et al.

samples noted above was plated and incubated anaerobi- Stoichiometric analyses cally at 25°C using the AnaeroPak system (Mitsubishi Gas The overall mixture of microbial sludge and PHBV pel- Chemicals, Niigata, Japan). Inoculated plates were incu- lets remaining in the reactors were collected at the end of bated for 2 to 4 weeks before the final reading of CFUs. operations. The sludge and PHBV were separated manually Few if any detectable colonies appeared on the 4 agar media from each other using tweezers, washed twice with pure without nitrate added, thereby confirming that colonies water, and subjected to a dry weight analysis as described recovered on the test media with nitrate were denitrifiers. 24) previously to estimate the growth yield coefficient Yx/s. For counting CFUs of PHBV-degrading denitrifiers, colo- The substrate consumed/oxidant consumed (S/O) ratio was nies exhibiting a cleared halo formation were taken as being calculated based on the amounts of PHBV and nitrate con- positive. Colonies recovered on the TSN plates were ran- sumed. The theoretical relationship between Yx/s and the S/O domly selected and purified by repeatedly streaking on the ratio was based on information from Hiraishi and Khan17) same agar medium under anaerobic conditions. Since all (see also chemical reaction formula [1] and [2] given below). isolates thus obtained were aerobic chemoorganotrophic denitrifying bacteria, they were maintained aerobically on Direct cell counting PBY agar slants. Ten milliliters of sludge suspension from the reactors at the end of batch cycles was sonicated on ice for 90 sec with 16S rRNA gene amplification and sequencing 2-sec intermittent bursts (20 kHz; output power, 50 W) and Crude cell lysates as the DNA source were prepared for diluted with filter-sterilized phosphate-buffered saline (PBS). PCR use as described previously19). 16S rRNA gene frag- Aliquots (10–50 µl) of these diluted samples were taken and ments that corresponded to positions 8 to 1543 in Escheri- used for direct cell counting. Total bacterial counts were chia coli 16S rRNA8) were PCR-amplified from the cell measured by epifluorescence microscopy with ethidium lysate with a set of bacterial universal primers, fD1 and bromide (EtBr) or SYBR Green II (Molecular Probes, Inc., rP142), as described previously19). PCR products were puri- Eugene, USA) staining as described previously15,43). Detec- fied by the polyethylene glycol precipitation method16), tion of viable cells using a LIVE/DEAD BacLight Bacte- sequenced with a SequiTherm Long Read cycle sequencing rial Viability kit (Molecular Probes) was performed accord- kit (Epicentre Technologies, Madison, USA), and analyzed ing to the manufacturer’s instructions and as described with an Amersham-Pharmacia ALFexpress DNA sequencer. previously43). Stained specimens were observed under an Olympus BX-50 epifluorescence microscope equipped with Quinone profiling a Flovel FD-120 M digital CCD camera (Flovel Co., Tokyo, Quinones from sludge samples and the isolates were Japan). The number of stained cells was counted using the extracted with a chloroform-methanol mixture and partially image analysis program WINROOF (Flovel): 10–15 fields purified by column chromatography. Quinone components per sample and a total of 1,000–2,000 cells per sample. were separated and identified by reverse-phase HPLC and photodiode array and mass spectrometric detection with Enumeration, isolation and characterization of denitrifiers external ubiquinone (Q-n) and menaquinone (MK-n) stan- Sludge and mixed liquor samples taken from the reactor dards. Detailed information on the analytical procedures has were diluted with PBS as described above, and appropriate been given previously14,15). dilutions were used for the enumeration of viable bacteria. Plate counts of aerobic heterotrophic bacteria were obtained DNA extraction and purification by the smear-plating method using 1/10-diluted PBY12) agar Bulk DNA was extracted from sludge samples as des- medium or 1/10-diluted tryptic soy agar medium. Denitrify- cribed previously26). The crude DNA extracted was further ing bacteria were enumerated by the pour-plating method purified by a standard method including deproteinization using agar media containing mineral base RM2, 20 mM with chloroform-isoamylalcohol and RNase treatment29). 21) −1 KNO3, vitamin solution PV1 (1 ml L ) and 1.8% agar as The DNA solution thus obtained was diluted in TE buffer the basal medium. The agar media were supplemented with (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) as needed and either 0.2% tryptic soy broth, 10 mM acetate, 10 mM 3HB used for PCR experiments. or 2% PHBV powder (w/v) as the sole carbon and energy source: designated TSN, ACN, 3HBN and PHBN agar Amplification and sequencing of denitrifying enzyme genes media, respectively. For plating, 1 ml of each of the diluted Fragments of nitrite reductase genes, nirK and nirS, from Denitrifying Microbial Communities with PHBV 23 the bulk DNA purified from sludge samples were PCR- which were isolated from SPD reactor A in this study, were amplified with the oligonucleotide primer pairs nirK1F subjected to the nosZ gene analysis. This study is the first to (GG[A/C]ATGGT[G/T]CC[C/G]TGGCA)-nirK5R (GCC- determine the nosZ gene sequences of these 4 denitrifying TCGATCAG[A/G]TT[A/G]TGG)7) and nirS2F’ (5'-GAAT- species. A[C/T]CACCC[C/G]GAGCCG-3')-nirS6R’ (5'-A[C/G][A/ G]CGTTGAACTT[A/G]CCGGT-3'), respectively. The pri- Sequence comparisons and phylogenetic analyses mer pair nirS2F’-nirS6R’ was a modification of the nirS2F- All sequence data were compiled with the GENETYX- nisS6R pair described by Braker et al.7). The expected sizes MAC program (Software Development Co., Osaka, Japan). of PCR-amplified nirK and nirS fragments were 0.51 and 16S rRNA gene sequence data were compared with those 0.80 kb, respectively. For PCR amplification of nosZ frag- retrieved from Ribosomal Database Project II9). Nucleotide ments, we first used previously described primers36), but this sequences and translated amino acid sequences of nirS and attempt gave unsatisfactory results. In this study, therefore, nosZ clones were analyzed using the BLAST homology we designed a new pair of PCR primers, nosZ2f (5'-GT- search system1,37). The multiple alignment of sequences and GCCGAAGAACCC[G/C]CACGG-3' and nosZ3r (5'-T[C/ calculation of the nucleotide substitution rate with Kimura’s G]GCCGGAGATGTCGATCA-3'), based on nosZ gene two parameter model27) were performed using the CLUSTAL sequences retrieved from databases (see Fig. 4). The se- W program40). Distance matrix trees were constructed by the quences of nosZ2f and nosZ3r correspond to positions 1006 neighbor-joining method34), and the topology of the trees to 1025 and 1466 to 1484 in the nosZ gene of Ralstonia eu- was evaluated by bootstrapping with 1,000 resamplings11). tropha strain H16 (database accession number AY305378), and the expected size of PCR-amplified nosZ fragments was Nucleotide sequence accession numbers 0.48 kb. All thermocycling reactions were performed by the The nucleotide sequences determined in this study have touchdown PCR method10) using an rTaq DNA polymerase been deposited with the DDBJ under the accession numbers kit (Takara, Otsu, Japan), one of the primer sets and a Takara AB277845 to AB277852 for 16S rRNA genes, AB278071 Thermal Cycler. The first half of the PCR procedure includ- to AB278090 for nirS genes, and AB278091 to AB278115 ed an initial preheating step of 2 min at 94°C and 20 cycles for nosZ genes. of a touch-down procedure consisting of denaturation for 1 min at 94°C, annealing for 1 min at a temperature decreas- Results ing from 60 to 51°C with 1°C decremental steps of 2 cycles each and extension for 1 min at 72°C. Following this, addi- Denitrifying activity of PHBV-using reactors tional 20 cycles of the thermocycling reaction with anneal- PHBV-containing reactors A and B were operated at 3- to ing at 50°C was performed. The final step was followed by 4-day batch intervals during 30 days of start-up and with a extension at 72°C for 5 min. PCR products were separated 24-h batch cycle thereafter. Both the reactors exhibited sim- by agarose gel electrophoresis, cut from the gel, and then ilar denitrifying activity during the overall period of opera- purified using a GENECLEAN Spin kit (Bio 101, Vista, tion. As an example, changes in denitrifying activity in USA). Purified PCR fragments were subcloned using a pT- reactor A are shown in Fig. 1. Nitrate remained in detect- Blue Perfectly Blunt cloning kit (Novagen, Madison, USA). able amounts at the end of each batch cycle during the first Transformation of Escherichia coli competent cells was 2 weeks of operation but was not detected thereafter (Fig. carried out according to a standard manual of molecular 1a). Also, little nitrite was detected after 2 weeks of opera- cloning35). Plasmid DNA was isolated and purified by using tion. During the first 40 days of operation, the denitrifi- the Wizard Plus Minipreps DNA Purification System cation rate exhibited by the reactor in each batch cycle (Promega Inc., Madison, USA) according to the manufac- increased markedly. After that, the denitrification rate turer’s instructions. Nucleotide sequences of nirS and nosZ exhibited by the reactor became constant at around 60 mg − −1 −1 gene clones were determined as described for 16S rRNA NO3 -N g (dry wt) h (Fig. 1b). To estimate the actual gene sequences. For comparison, nosZ genes from the pre- denitrification rate with PHBV more accurately, sludge viously described denitrifying bacteria Brachymonas deni- samples were taken from the reactors at the end of each trificans strain AS15220) and Diaphorobacter nitroreducens batch cycle, washed and separately tested for denitrification strain NA10BT 24,25) were PCR-amplified, subcloned and with fresh PHBV as the sole substrate. The denitrification sequenced as described above. In addition, Aquitalea sp. rate with the fresh PHBV also increased linearly during the − strain PGP-1 and sp. strain TSL-h, both of first 30 days of operation and reached around 20 mg NO3 - 24 KHAN et al.

Table 1. Stoicheometric estimation of microbial biomass, PHBV and nitrate in the reactor

Measured value for Parameter Reactor A Reactor B Total PHBV added (g) 45.0 45.0 PHBV consumed (g) 20.1 20.6 − Total NO3 -N added and removed (g) 7.28 7.28 Excess biomass produced (g dry wt) 8.7 9.1 −1 Yx/s (g g ) 0.43 0.44 S/O ratio (g g−1) 0.624 0.640

tion are shown in Table 1. Based on the amounts of PHBV consumed and excess biomass produced, the growth yield coefficient for the reactors was estimated to be 0.43–44. These growth yields are slightly lower than those recorded previously for a PHB-using denitrifying reactor31) and a pure culture of Diaphorobacter nitroreducens with PHB as the substrate24). The denitrification reactions with PHB as + the substrate and with or without NH4 as the nitrogen source are given by the following formula17):

− 5[C4H6O2]+18NO3 − Fig. 1. Changes in nitrate removal activity of SPD reactor A during → 9N2+18HCO3 +2CO2+6H2O(1) the acclimation with PHBV under denitrifying conditions. Figure − − − + 1a shows changes in the concentrations of NO3 -N and NO2 -N in 10[C4H6O2]+14NO3 +6NH4 − − the reactor at each batch cycle: diamonds, concentration of NO3 - → 7N2+10CO2+6[C5H7O2N]+12H2O+18OH (2) N at the start of each batch cycle; open squares, concentration of − NO3 -N at a 24 h-interval of sampling; open triangles, concentra- − Based on the biochemical reactions (1) and (2), it can be tion of NO2 -N at a 24 h-interval of sampling. Figure 1b shows changes in the nitrate removal rate as estimated the reactor (dia- predicted that a Yx/s value of 0.43–44 corresponds to a S/O 17) monds) and the washed sludge with fresh PHBV (squares). ratio of 0.58–0.59 . However, the experimental values of the S/O ratio obtained (0.624–0.640) were considerably higher than the predicted values. This suggests that PHBV N g−1 (dry wt) h−1 under steady-state conditions (Fig. 1b). as the substrate was consumed not only for denitrification HPLC experiments revealed that significant amounts of but also for other biochemical processes, e.g., aerobic respi- acetate and 3HB were constantly produced in both the reac- ration. tors under steady-state conditions. The concentrations of acetate and 3HB detected in the supernatant at the end of Quinone profiling of microbial communities operation were 2.9–4.8 and 1.9–2.4 mmol L−1, respectively. Sludge samples were collected from reactors A and B Thus, the marked difference in the denitrification rate under steady-state conditions (on days 48, 56 and 66) for between the reactor itself and the washed sludge with fresh quinone profiling. In both the reactors, ubiquinone Q-8 PHBV was possibly due to the bioavailability of intermedi- accounted for 83–85% of the total quinone content. The ate metabolites, e.g., organic acids, as a usable substrate for remaining fractions were composed mainly of Q-9 (3–5%) denitrification. In fact, the washed sludge taken from the and Q-10 (7–9%). Menaquinones constituted only minor reactors at the end of operation exhibited 2.0–2.5 fold proportions of the total content. In light of the available higher denitrification rates with acetate than with PHBV information on microbial quinone systems14), it was evident (data not shown). that Q-8-containing proteobacterial species, especially those Stoichiometric data on PHBV, nitrate, and microbial bio- of the Betaproteobacteria, constituted the overwhelming mass in reactors A and B during the overall period of opera- majority of the microbial communities in the reactors. The Denitrifying Microbial Communities with PHBV 25

highest countable plates of TSN agar used for the enumera- tion and subjected to the standard purification procedure. Thus, a total of 50 strains were isolated from reactors A and B and further examined as described below.

Phenotypic and phylogenetic characterization of isolates Results of phylogenetic and phenotypic characterization of the isolates are summarized in Table 2. The isolates were categorized into 12 groups (groups 1 to 12) at the species or a similar taxonomic level, all of which were assigned to the phylum by 16S rRNA gene sequencing and quinone profiling. Most of the isolates (90%) were assigned to genera of the Betaproteobacteria, especially those of the family Comamonadaceae (i.e., Acidovorax, Brachymonas, Comamonas, Diaphorobacter and Simplicispira). The major established genera to which more than 15% of the Fig. 2. Total and viable bacterial population densities in SPD reac- tors A (closed histograms) and B (open histograms) under steady- isolates were assigned were Brachymonas, Comamonas and state conditions. TC, direct total count with EtBr or SYBR Green Diaphorobacter. The Brachymonas and Diaphorobacter staining; TVC, direct viable (LIVE/DEAD) count; PC, plate isolates, designated groups 5 and 9, respectively, were affil- count of aerobic heterotrophic bacteria; TSN, denitrifying bacte- iated with previously known species, B. denitrificans and D. rial count on TSN agar; ASN, denitrifying bacterial count on nitroreducens, respectively. The Comamonas isolates were ASN agar; 3HB, denitrifying bacterial count on 3HB agar; PHBN, denitrifying bacterial count on PHBN agar. classified into two groups, one of which (group 7) was most closely related to a previously described uncultured bacte- rium, clone OS1L-1626), and one of which (group 8) was results of quinone profiling are in agreement with those of a most similar to a denitrifying bacterium, Comamonas sp. previous study on a PHBV-acclimating denitrification strain PD-1422). The group-7 Comamonas isolates was also process26). closely associated with Comamonas denitrificans (strain 123T, accession no. AF233877) at a 99% similarity level, Enumeration and isolation of denitrifiers whereas the group-8 isolates showed less than 97% similar- Population densities of denitrifying bacteria as CFUs as ity to any established species of the genus Comamonas. In well as direct total and viable counts were measured in reac- addition, the isolates belonging to the recently described tors A and B under steady-state conditions (Fig. 2). No genus Aquitalea28) (group 4), whose closest relative was an marked differences were noted in these bacterial counts uncultured bacterium, clone OS1L-2426), accounted for 16% between the two reactors. Among the media used for enu- of all the isolates. meration, TSN agar medium yielded the highest counts of The uncultured bacteria OS1L-16 and OS1L-24 noted denitrifiers, in the order of 107 to 108 CFU ml−1. The TSN above as well as many other Comamonas-related clones count accounted for 7.5% of the direct total count, 10% of were detected previously as the major uncultured clones the total viable (LIVE/DEAD) count and 83% of the plate from a PHBV-SPD process26). Therefore, the PHBV-SPD count of aerobic heterotrophic bacteria on average. Compa- reactors reported previously26) and in this study might share rable or slightly lower counts were obtained with ACN agar a similar bacterial community. medium, accounting for 7.2% of the total count and 80% of All of the test isolates were aerobic chemoorganotrophic the aerobic plate count. The average denitrifying bacterial bacteria that grew well on ACN and 3HB agar media under numbers obtained with 3HBN and PHBN agar media anaerobic denitrifying conditions, thereby confirming them accounted for 1.7 and 0.34% of the direct total counts, to be acetate- and 3HB-utilizing denitrifiers. In all isolates, respectively. These data suggest that most of the viable het- the formation of nitrogen gas from nitrate was confirmed by erotrophic bacteria in the reactors detected as CFUs were gas chromatography (not shown). With the exception of the acetate-utilizing denitrifiers, whereas PHB-degrading deni- group-9 Diaphorobacter isolates and the group-8 Comamo- trifiers constituted rather minor populations. nas isolates, no degradation of PHBV by the denitrifying Several single colonies were chosen at random from the isolates was observed (Table 2). 26 KHAN et al.

Table 2. Phylogenetic affiliations and phenotypic characterization of denitrifying bacteria isolated from reactor A

a Class and Isolate group 16S rRNA gene sequence comparison Major Utilization of genus assigned (and no. of isolates) Closest relative (and accession number) Similarity (%)quinone 3HB PHB Brevundimonas 1 (1) Brevundimonas sp. strain LMG 19834 (AJ30077) 99 Q-10 NTb − Paracoccus 2 (1) P. pantotrophus strain ATCC 35512T (Y16933) 100 Q-10 NT − Betaproteobacteria Acidovorax 3 (5) Acidovorax sp. strain PD-10 (AB195159) 99–100 Q-8 + − Aquitalea 4 (9) Uncultured bacterium OS1L-24 (AB076875) 99–100 Q-8 + − A. magnusonii TRO-001DR8T (DQ018117) 99 Brachymonas 5 (8) B. denitrificans strain AS-P1T (D14320) 99–100 Q-8+RQ-8 + − Castellaniella 6 (1) C. defragrans strain PD-19 (AB195161) 100 Q-8 + − Comamonas 7 (7) Uncultured bacterium OS1L-16 (AB076869) 99 Q-8 + − C. denitrificans 123T (AF233877) 99 8 (4) Comamonas sp. PD-14 (AB195167) 99–100 Q-8 + + Diaphorobacter 9 (8) D. nitroreducens strain NA10BT (AB064317) 99–100 Q-8 + + Simplicispira 10 (4) Simplicispira sp. strain R-23033 (AM236310) 99–100 Q-8 + − Gammaproteobacteia Klebsiella 11 (1) Klebsiella sp. strain zmmo (U31075) 99 Q-8+MK-8c NT − Pseudomonas 12 (1) Uncultured Pseudomonas sp. OS1L-17 (AB076873) 100 Q-9 NT − a Results of testing under denitrifying conditions. b NT, not tested. c A trace amount of demethylmenaquinone-8 was present in addition.

from the 4 isolates. As shown in Fig. 3, the phylogenetic Community analysis based denitrifying-enzyme gene clones tree based on the nirS gene sequence showed that the nirS PCR amplification of the denitrifying-enzyme genes was clones from the reactor were classified into three major performed for the microbial community in SPD reactor A, clusters designated I, II and III. The nirS clones of clusters I the previously described denitrifiers Brachymonas denitrifi- and II accounted for 63 and 22% of all the clones detected cans strain AS152 and Diaphorobacter nitroreducens strain and formed a major cluster together with the nirS genes of NA10BT and the 2 isolates in this study, Comamonas sp. Comamonadaceae genera, including Alicylophilus, Aci- strain TSL-h (group 8) and Aquitalea sp. strain PGP-1 dovorax and Comamonas, with nucleotide sequence simi- (group 4). PCR with primer pairs nirS2F7-nirS6R’ and larities of more than 80%. In particular, the cluster I nirS nosZ2f-nosZ3r resulted in the successful amplification of clones grouped with the uncultured nirS clone R2-s02 nirS and nosZ gene fragments of expected sizes from the (accession no. AB118885) as their nearest neighbor, which SPD reactor and all of the test organisms, whereas the was detected previously from a wastewater treatment amplification with the primer pair nirK1F-nirK5R failed to system44). The nucleotide sequence similarity between the produce nirK gene fragments from any of the test organisms cluster I nirS clones and the R2-s02 clone ranged from 89 to (data not shown). Also, nirK-targeted PCR with the reactor- 94%, and the deduced amino acid sequence similarity community DNA gave a weak signal of the product (not ranged from 91 to 97%. The remaining nirS clones (cluster shown). These results suggest that microorganisms contain- III) were most closely associated to the nirS sequence of the ing cytochrome cd1 nitrite reductase rather than Cu-type betaproteobacterial species tolulyticus. nitrite reductase were the major constituents of the denitri- The phylogenetic tree based on the nosZ gene sequence fying microbial community in the PHBV-SPD process. gave a similar topology to that based on the nirS sequence Based on the aforementioned results, we constructed nirS (Fig. 4). The nosZ clones detected were separated into four and nosZ gene clone libraries from the microbial commu- major clusters designated I to IV. The majority (68%) of the nity in SPD reactor A and determined the sequences of 32 clones fell into cluster I and grouped with the nosZ genes of nirS clones and 34 nosZ clones as well as of the nosZ gene the betaproteobacterial species Azoarcus sp., Burkholderia Denitrifying Microbial Communities with PHBV 27

Fig. 3. Neighbor-joining phylogenetic tree of nirS gene clones from the microbial community of SPD reactor A and those from denitrifying strains. Colwellia psychrerythraea strain 34H nirS (CP000083) was used as the outgroup to root the tree. Scale=5% substitution (Knuc). The nodes supported by more than 80% of bootstrap confidence are shown by solid circles.

malei and Rhodoferax ferrireducens and group-4 isolate Discussion PGP-1 as their closest relative; the similarity to the PGP-1 nosZ gene was 77–78% in nucleotide sequence and 75–77% The two PHBV-SPD reactors studied herein exhibited − −1 in translated amino acid sequence. Thus, unlike the case of high rates of denitrification (ca. 60 mg NO3 -N g [dry wt] the nirS clones noted above, nosZ genes showing more than h−1) under steady-state conditions. These rates are much 80% similarity to the major clones detected were not found higher than those recorded for activated sludge with various in the databases. The cluster II nosZ clones accounted for soluble substrates13). However, the SPD reactor-derived 21% of the total and were associated with the nosZ genes of washed sludge showed approximately one-third lower deni- Acidovorax sp. strain JS42, Diaphorobacter nitroreducens trification rates with fresh PHBV as the substrate than did strain NA10BT and the group-8 Comamonas sp. strain TSL- the reactors themselves. This difference between the two is h. The nosZ clones of clusters III and IV grouped with the possibly due to the difference in the bioavailability of solu- genes of some species of the Alphaproteobacteria and the ble intermediate metabolites that could serve as electron , respectively. donors for denitrification. Similar results have been obtained with a PCL-utilizing SPD process22). Results of organic acid analyses suggest that, in the PHBV-SPD pro- 28 KHAN et al.

Fig. 4. Neighbor-joining phylogenetic tree of nosZ gene clones from microbial community of SPD reactor A and those from denitrifying strains. Colwellia psychrerythraea strain 34H nosZ (CP000083) was used as the outgroup to root the tree. Scale=5% substitution (Knuc). The nodes supported by more than 80% of bootstrap confidence are shown by solid circles. The strains analyzed in this study are asterisked.

cess, acetate and 3HB as intermediate metabolites become our finding that the experimentally obtained value of the available as good substrates for denitrification by non- S/O ratio was higher than the theoretically predicted value. PHBV-degrading microorganisms. In fact, the plate counts In the biodegradation of PHBV, extracellular PHB depoly- of denitrifying bacteria with acetate or 3HB as the carbon merase is the key enzyme, catalyzing the hydrolysis of the and energy source were much higher than those with polymer to its monomer 3HB23,41). Also, extracellular 3HB- PHBV. In view of this, together with the fact that the major- oligomer hydrolases may be involved in 3HB production39). ity of the denitrifying strains isolated were unable to While it is known that 3HB is converted to acetoacetate by degrade PHBV, it is likely that the predominant denitrifiers 3HB dehydrogenase within the cell, there remains the ques- in the PHBV-SPD process denitrify with intermediate tion of how acetate is produced in the PHBV-SPD process. metabolites (e.g., acetate and 3HB) as electron donors con- Further studies on the metabolism of PHBV and the produc- stantly produced by co-existing microorganisms. tion of intermediate metabolites in the SPD microbial com- Since the SPD reactors were operated under semi-anaero- munity is necessary to understand how non-PHB-degrading bic conditions, PHBV was possibly degraded not only by denitrifying bacteria are provided with soluble substrates anaerobically growing, denitrifying bacteria but also by aer- for denitrification. obically growing bacteria. This assumption is supported by Based on the results of 16S rRNA gene sequencing and Denitrifying Microbial Communities with PHBV 29 quinone profiling, most of the predominant denitrifying iso- to conclude that members of the Betaproteobacteria, espe- lates recovered quantitatively by the plate-counting method cially those of the family Comamonadaceae, not only con- were assigned to genera of the Betaproteobacteria, particu- stitute the major population of the microbial community but larly those of the family Comamonadaceae. Although the also actually play the primary role in denitrification in the culturable population of denitrifiers represented a small pro- PHBV-SPD process. The majority of the predominant deni- portion of the total population (7.5%), this culture-dependent trifying bacteria may be non-PHBV degraders and denitrify information is relatively consistent with the results of quinone with soluble metabolites of PHBV produced by co-exiting profiling of the whole community and our previous finding bacteria. In further studies, denitrifying-enzyme gene expres- that most of the uncultured 16S rRNA gene clones in a sion profiling and the proteomic and metabolomic analyses PHBV-SPD process belonged to the Comamonadaceae26). of the SPD microbial community should clarify the relation- The close phylogenetic similarities between the Aquitalea ships between the ecology of active denitrifying bacteria (group 4) and Comamonas (group 7) isolates obtained in and nitrogen removal efficiency in the PHBV-SPD process. this study and the uncultured clones detected previously26) suggest that PHBV-SPD processes share a common micro- Acknowledgements bial community regardless of the origin of seed sludge. In general, nirS gene phylogenetic trees have a similar The help of the staff of the sewage treatment plants which topology to 16S rRNA gene phylogenetic trees7). In the provided sludge samples is gratefully acknowledged. Spe- present study, consistent with the results of the 16S rRNA cial thanks are directed to the Mikawa Textile Research gene-based phylogenetic analysis of the isolates, most of the Center, Aichi Industrial Technology Institute, Gamagori, nirS genes from the SPD microbial community (i.e., those and Japan Monsant Co., Tokyo, for generously providing of clusters I and II) proved to be derived from members of PHBV. This work was supported in part by a grant-in-aid the Comamonadaceae. This finding is also consistent with for scientific research (No. 17310046) from the Ministry of the results of our previous 16S rRNA gene-based analysis Education, Culture, Sports, Science and Technology, Japan. of a PHBV-SPD microbial community26). The cluster I nirS This work was also performed as a part of the 21st Century clones, comprising the largest group of nirS clones detected, COE Program “Ecological Engineering and Homeostatic was most closely related to the uncultured nirS clone R2- Human Activities” founded by the Ministry of Education, s02, which was found previously in an activated sludge Sports, Culture, Science and Technology, Japan. system44). This means that the predominant species of deni- trifying bacteria present in the PHBV-SPD process are not specific to this process but are common in wastewater treat- References ment systems. 1) Altschul, S.F., T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, As in the case of the nirS gene clone library, most of the W. Miller and D.J. Lipman. 1997. Gapped BLAST and PSI- nosZ clones formed a tight major cluster (cluster I) associ- BLAST: a new generation of protein database search programs. 25 ated with some members of the Betaproteobacteria. 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