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［Japanese Journal of Water Treatment Biology Vol.53 No.2 57－67 2017］
Polyhydroxybutyrate Accumulation Ability and Relevant Microbial Populations in Acetate-fed Mixed Microbial Cultures Enriched from Activated Sludge under Different Temperature Conditions
DAISUKE INOUE1,2,3*, YUTA SUZUKI2, JOTA MOROHOSHI1, and KAZUNARI SEI1,2
1Department of Health Science, School of Allied Health Sciences, Kitasato University /1－15－1 Kitasato, Sagamihara-Minami, Kanagawa 252－0373, Japan 2Environment and Medical Sciences Course, Graduate School of Medical Sciences, Kitasato University /1－15－1 Kitasato Sagamihara-Minami, Kanagawa 252－0373, Japan 3Division of Sustainable Energy and Environmental Engineering, Osaka University /2－1 Yamadaoka, Suita, Osaka 565－0871, Japan
Abstract This study investigated the influence of enrichment temperature on the accumulation ability of polyhydroxybutyrate (PHB) and the composition of PHB－accumulating microorganisms (PHBAMOs) in mixed microbial cultures obtained from activated sludge. PHBAMOs were enriched using the feast-famine method in sequencing batch reactors that were fed with acetate as the sole external carbon source and controlled at 20℃ or 28℃. Batch PHB accumulation experiments under nitrogen- and phosphorus-deficient conditions revealed that the maximum content and yield of PHB in the two enrichment cultures were twice as high as the seed sludge. The results indicated that the PHB accumulation ability of the sludge can be greatly enhanced by enrichment using the feast-famine method, irrespective of temperature. Through clone library analysis targeting the phaC genes, PHBAMOs belonging to Rhodocyclales, specifically Thauera, were found to be notably increased after enrichment, although Burkholderiales were dominant in the seed sludge. The increase of PHBAMOs belonging to Rhodocyclales was greater in the enrichment culture obtained at 20℃, which had a slightly stronger PHB accumulation ability, as compared with that at 28℃. These results suggest that Rhodocyclales may be the key members enhancing PHB accumulation abilities of the enrichment cultures.
Key words: Activated sludge, Feast-famine method, phaC gene, Polyhydroxybutyrate (PHB), PHB－accumulating microorganisms
INTRODUCTION resources and energy from waste sludge. However, although there are many beneficial Activated sludge processes, which are uses of inorganic components, the recycling of employed worldwide in wastewater treatment, organic components is still quite limited－ produce a large amount of waste sludge. around 23% in the fiscal year 2006 in Japan1). Sludge management is a crucial issue because Increasing the beneficial use of organic its treatment is costly and consumes much components in waste sludge is a significant energy, and landfill sites for disposal are challenge in sustainable wastewater treat- limited. A possible solution is producing ment. ＊ Corresponding author Tel: +81－6－6879－7673, Fax: +81－6－6879－7675 E－mail: [email protected] 58 Japanese J. Wat. Treat. Biol. Vol.53 No.2
Polyhydroxyalkanoates (PHAs) are linear effective in enhancing the capability of polyesters synthesized by a variety of bacteria various sludge to accumulate PHA, including as intracellular carbon and energy reserves. PHB7),9). Some recent studies that employed PHAs are completely biodegradable, biocom- the feast-famine method succeeded in patible and thermoplastic2),3), and are regarded constructing very effective PHB－accumulating as environmentally compatible bioplastic mixed microbial cultures that were capable of materials that can be a promising alternative accumulating up to 90% of PHB from to conventional petroleum-based plastics. In acetate10) or lactate11). contrast, because PHAs are easily biodegraded To optimize the enrichment process using to fatty acids under anaerobic conditions, the feast-famine method, the infl uence of PHA accumulation in sludge can enhance operational parameters on the PHB accu- methane production by anaerobic digestion4)－6). mulation ability of the resultant enrichment Therefore, the transformation of organics to cultures has been examined. However, PHAs can improve the value of waste sludge contrasting results have been reported on the as a bioresource. A concept of enhanced infl uence of enrichment temperature: for an resource and energy recovery from sludge via enrichment temperature range of 15 to PHA accumulation is depicted in Fig. 1. In 30/35℃, Johnson et al.12) and Jiang et al.13) this study, polyhydroxybutyrate (PHB), a reported the enhancement of PHB accu- polymer of 3－hydroxybutyrate, was targeted mulation abilities with increasing tem- as the representative PHA. perature, while Krishna and van Loosdrecht14) Although it has been known that PHB can reported the opposite trend and Pittmann be accumulated in activated sludge, the PHB and Steinmetz15) observed the strongest accumulation abilities of activated sludge ability at 20℃. In addition, although the in wastewater treatment plants (WWTPs) PHBAMOs compositions in the enrichment are generally low, possibly due to the cultures would affect their PHB accumulation coexistence of PHB－accumulating micro- abilities, the changes in PHBAMOs organisms (PHBAMOs) and non－ composition by enrichment at different tem- PHBAMOs7),8). Therefore, selective enrichment peratures have rarely been studied. of PHBAMOs with eminent PHB accumulation Therefore, this study aimed to elucidate ability is crucial to enhancing the PHB the infl uence of enrichment temperature on accumulation ability of the sludge. The feast- the PHB accumulation ability and PHBAMOs famine method, in which the sludge is composition in the resultant mixed microbial subjected to successive periods of external cultures. PHBAMOs were enriched in carbon source accessibility (feast) and acetate-fed sequencing batch reactors (SBRs) unavailability (famine), has been proven operated at 20℃ and 28℃. The enrichment
Fig. 1 Conceptual diagram of enhanced energy and material recovery from organic components in waste activated sludge through PHA accumulation. Infl uence of Temperature on Enrichment of PHBAMOs 59 processes in both SBRs were monitored PHBAMOs from activated sludge sample was periodically. PHB accumulation abilities conducted with two SBRs operated at 28±1℃ before and after enrichment were evaluated (SBR－A) and at 20±1℃ (SBR－B). The reactor by single batch PHB accumulation apparatus is shown in Fig. 2. An SBR experiments under nitrogen- and phosphorus- consisted of a polymethylpentene beaker with defi cient conditions. The composition of the a 2 L working volume, equipped with a PHBAMOs before and after enrichment was stirrer (SM－101; AS ONE, Osaka, Japan) analyzed by the clone library method and an air pump (W600; Japan Pet Design, targeting phaC genes. Tokyo, Japan). The SBRs were controlled at aforementioned temperatures using a water MATERIALS AND METHODS bath equipped with a thermostat (ET300X; Sludge sample Activated sludge sample Kotobuki-Kogei, Nara, Japan) and heater was collected from municipal wastewater (SH80W; Kotobuki-Kogei). The airfl ow to the treatment plant in Tokyo, Japan, on October SBRs was maintained at 1.0 L/min. The pH 1, 2013. This plant employs a conventional of both SBRs was not controlled. To activated sludge process. Water temperature continuously measure dissolved oxygen (DO) and pH in the aeration tank at sampling concentration and pH, portable DO and pH were 22.6℃ and 6.6, respectively. The mixed meters were installed in the SBRs. liquor suspended solid (MLSS) concentration Both SBRs were operated with a sludge of the activated sludge sample was 1603 retention time of 5 d, hydraulic retention mg/L. The sample was transported on ice to time of 1 d, and cycle duration of 12 h. An the laboratory, harvested by centrifugation SBR cycle consisted of a start phase (10 (1500 × g, room temperature, 10 min), washed min), an infl uent phase (10 min) in which 1 twice with nitrogen- and phosphorus-free L of fresh medium was supplied, a reaction basal salt medium (BSM－NP: MgSO4•7H 2O phase (675 min), a biomass withdrawal phase 68.4 mg/L, KCl 26.8 mg/L, trace element (3 min) in which 200 mL of mixed reactor solution16) 1.5 mL/L, pH 7.2). The washing liquor were withdrawn, a settling phase (15 step was conducted by resuspension to the min), and an effl uent withdrawal phase (7 aforementioned medium and centrifugation. min) in which 800 mL of reactor supernatant
Finally, the sludge sample was suspended in were withdrawn. Basal salt medium (NH4Cl the same medium to make an MLSS 362 mg/L (94.5 mg－N/L), KH2PO4 338 mg/L concentration of 3000 mg/L to be used for (77 mg－P/L), MgSO4•7H 2O 137 mg/L, KCl PHBAMOs enrichment. 53.5 mg/L, allylthiourea 10 mg/L, trace Enrichment of PHBAMOs Enrichment of element solution 3 mL/L, pH 7.2) supple-
Fig. 2 Schematic of sequencing batch reactors (SBRs) used to enrich PHBAMOs. Thermostat was set at 28℃ and 20℃ in SBR－A and SBR－B, respectively. 60 Japanese J. Wat. Treat. Biol. Vol.53 No.2
mented with CH3COONa•3H 2O 3.68 g/L (649 Japan) and with an InertCap WAX－HT mg－C/L) as the sole carbon source was used capillary column (30 m × 0.25 mm ID, 0.25 as the influent. At the beginning of the µm df; GL Science, Tokyo, Japan). PHB reactor operation, 1 L of seed sludge prepared content was expressed as the weight above was inoculated into each SBR to percentage of PHB of the total sludge biomass prepare an initial MLSS concentration of (100 × g－PHB/g－MLSS). PHB yield was 1500 mg/L. determined by dividing the accumulated PHB During SBR operation, DO concentration by the consumed acetate (C－mol PHB/C－mol and pH were measured at 5 min intervals. acetate). DO was measured with a HQ30d portable Molecular methods Microbial DNA was meter (HACH, Loveland, CO, USA) or a extracted from seed sludge and enrichment ProODO portable meter (YSI, Yellow Springs, cultures with an ISOIL for Beads Beating kit OH, USA). pH was measured with a DM－32P (Nippon Gene, Tokyo, Japan), and further portable meter (DKK－TOA, Tokyo, Japan). At purified with a MagExtractor－PCR&Gel designated cycles, MLSS concentration, Clean up kit (Toyobo, Tokyo, Japan). acetate concentration in the liquid phase, and Clone library analysis targeting the phaC PHB content in the sludge pellet were genes, which encode the class I and II PHA determined. synthase, was employed for analyzing the Batch PHB accumulation experiment PHB composition of PHBAMOs. PCR amplification accumulation abilities of seed sludge and of phaC genes was conducted using the CF1 enrichment cultures after stabilization in two and CR4 primer set as described previously19). SBRs were evaluated by single batch The PCR products were purified with a experiments under nitrogen- and phosphorus- NucleoSpin Extract II kit (Macherey－Nagel, deficient conditions. Seed sludge prepared as Düren, Germany), and then purified and described above and enrichment cultures concentrated by ethanol precipitation. were harvested by centrifugation (1500 × g, Purified PCR products were ligated into room temperature, 10 min), washed twice plasmid pTAC－1 and cloned into competent with BSM－NP and inoculated to an MLSS Escherichia coli DH5α using a DynaExpress concentration of 1000 mg/L in a 500－mL TA PCR Cloning Kit (pTAC－1) with Jet Erlenmeyer flask containing 300 mL of BSM－ Competent Cell (BioDynamics Laboratory, NP supplemented with 1000 mg－C/L of Tokyo, Japan). The cloned plasmids were sodium acetate as the sole carbon source. extracted from E. coli DH5α cultures with a The flasks were incubated at 28℃ on a FastGene Plasmid Mini kit (Nippon Genetics, rotary shaker at 120 rpm for 24 h. Acetate Tokyo, Japan). Then, the insertion regions of and MLSS concentrations and PHB content the plasmids were sequenced on an Applied were measured at 0, 6, 12, and 24 h. Biosystems 3730XL DNA analyzer (Applied Analytical methods MLSS concentrations Biosystems, Foster City, CA, USA) at of sludge samples were measured according Macrogen Japan (Tokyo, Japan). The to standard methods17). Acetate concentration nucleotide sequences of all cloned plasmids in was determined by a DR890 colorimeter each sample were aligned and compared (HACH) with prepacked volatile acids reagent using CLUSTALW 20), and the clones with (method 8196). ≥97% identity were classified into operational PHB content was determined by gas taxonomic units (OTUs). The nucleotide chromatography-mass spectrometry (GC/MS) sequence of the representative clone from analysis of methyl－3－hydroxybutyrate, which individual OTU was compared with those in is a derivative of PHB through the the NCBI database using the BLAST search methanolytic decomposition according to the program (http://www.ncbi.nlm.nih.gov/blast/). method described previously8),18). Benzoic acid According to the search results, taxonomic was used as the internal standard. GC/MS positions of PHBAMOs possessing the analysis was performed using Shimadzu targeted phaC genes were determined with GC－2010 Plus equipped with Shimadzu thresholds of ≥70% coverage and ≥70% GCMS－QP2010 Ultra (Shimadzu, Kyoto, sequence identity because the phylogeny of Influence of Temperature on Enrichment of PHBAMOs 61 the phaC genes closely reflects the 16S rRNA that the feast and famine conditions were gene phylogeny19). Based on the alignment maintained in both SBRs operated in this results of sequences of all representative study. clones in this study and of the reference The feast/famine ratio (F/F ratio) has been sequences in the database, a phylogenetic used as an indicator to judge the progress of tree was produced using the njplotWIN95 the enrichment of PHBAMOs and the software21). stabilization of PHBAMO cultures. Based on Nucleotide sequence accession numbers the relationship between temporal changes of The partial sequences of the phaC genes acetate (substrate) and DO as described determined in this study were deposited in above, DO concentration can be used to the DDBJ/EMBL/GenBank databases under determine the F/F ratio. That is, in this the accession numbers LC190841 to study, the feast period in a cycle was defined LC190898. to be from the beginning of the cycle to the time when DO concentration increased to 5 RESULTS AND DISCUSSION mg/L or higher, and the F/F ratio of each Confirmation of the establishment of feast and cycle was calculated as the percentage of famine conditions To understand the fates feast period to a total cycle length (12 h). of PHB accumulation during a cycle, temporal Our results also suggested that maximal variations of PHB contents and concentrations PHB content in an enrichment cycle should of acetate, DO, and MLSS were investigated be determined when DO concentration in some cycles. Fig. 3 shows their temporal increases again. variations at the 6th cycle in SBR－A as a Enrichment of PHBAMOs Fig. 4 shows typical result. DO decreased to 0 mg/L after the variations of F/F ratio, MLSS concen adding the influent within the initial period tration and PHB content during enrichment of a cycle, then started increasing when in two SBRs. The MLSS concentration shown acetate was consumed almost completely and in the figure was the one of the sludge fluctuated between 6 and 7 mg/L. PHB collected during the biomass withdrawal content of sludge was increased and reached phase, while PHB content was determined at the maximal value around when acetate was the end of the feast phase in a cycle. In both consumed almost completely and DO started SBRs, pH increased from neutral to weakly increasing, then decreased gradually to the basic during the 1st cycle, and thereafter end of the cycle. Similar temporal trends of fluctuated between 8.5 and 9.2 throughout substrate and DO concentrations and PHB content in a cycle have also been observed in 11),22) 100 1800 previous studies . Thus, it was confirmed 90 (A) 1600 )
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st / 30 0123456789 10 11 PHB 20 400 Fe a Time (h) 10 200 0 0 Fig. 3 Typical profile of acetate concentration (closed 0 10 20 30 40 50 60 70 Cycle number square), DO concentration (open triangle) and PHB content in sludge (closed circle) during a Fig. 4 Variations over time of feast/famine ratio (closed cycle of enrichment with the feast-famine triangle), MLSS concentration (open square), method. An example at the 6th cycle in SBR－A and PHB content (closed circle) during the is shown. In this figure, 0 h indicate the onset of enrichment process in SBR－A (A) and SBR－B reaction phase. (B). 62 Japanese J. Wat. Treat. Biol. Vol.53 No.2 the remaining cycles (data not shown). The mechanism of instability and the method for F/F ratio was 51% and 91% at the 1st cycle improvement of enrichment stability. in SBR－A and SBR－B, respectively, after PHB accumulation ability of enrichment cultures which it decreased cycle by cycle in both To evaluate and compare the PHB accu SBRs. Lower F/F ratios in SBR－A than in mulation abilities before and after enrichment SBR－B by the 12th cycle suggested faster of PHBAMOs, single batch PHB accumulation acetate consumption in SBR－A than SBR－B experiments were conducted for 24 h. Seed during the initial cycles. From the 13th cycle sludge and enrichment cultures of SBR－A onward, the F/F ratios in both SBRs stabilized and SBR－B after the 65th cycle were used at around 15% excluding transient increases. and the experiments were fed with high MLSS concentrations in both SBRs were acetate loading under the nitrogen- and nearly 1600 mg/L at the 1st cycle. Thereafter, phosphorus-deficiency. Seed sludge consumed in SBR－A, MLSS concentration declined to around 60% of the acetate by 6 h, but no around 700 mg/L during the initial 20 cycles, further acetate consumption occurred until then fluctuated between 630 and 1275 mg/L, 24 h (Fig. 5). PHB content in seed sludge except for a temporary drop to less than 500 increased from 2.7% to 31% within 12 h, mg/L at the 55th cycle (Fig. 4A). MLSS then reached 34% at 24 h. The PHB concentration in SBR－B fluctuated between accumulation ability of the seed sludge was 1240 to 1630 mg/L throughout of the slightly higher than those of activated sludge enrichment process, except for a temporary collected from various wastewater treatment drop to nearly 1000 mg/L at the 29th and processes in municipal WWTPs reported 40th cycles (Fig. 4B). These results suggested previously8). higher stability in SBR－B than in SBR－A in Enrichment cultures from SBR－A and the case of MLSS concentration. PHB SBR－B consumed acetate completely within contents were 7.9% at the 1st cycle in both 12 h, with a concomitant notable increase of SBRs. Then, PHB contents in SBR－A and PHB contents from 13% to 58% and from SBR－B, respectively, increased until the 34th 7.4% to 62%, respectively (Fig. 5). After that, and 40th cycles, with the maximal PHB PHB contents in both enrichment cultures contents of 66% and 63% (Fig. 4). However, declined slightly until 24 h. The maximum at the 55th cycle, PHB contents dropped to PHB contents in enrichment cultures were 44% and 24% in SBR－A and SBR－B, nearly twice as high as that in seed sludge. respectively. Low acetate concentrations at 0 h in the It has been reported that a low F/F ratio, experiments for enrichment cultures could be consisting of short feast and long famine periods, is needed to improve the PHA 10),23) 24) 70 1200 accumulation ability . Dionisi et al. ) L /
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observed in SBR－A than in SBR－B until the A ℃ 0 0 40th cycle. Thus, it was indicated that 28 0 6 12 18 24 may be a preferable temperature for early Time (h) enrichment of PHBAMOs. In contrast, the reason for the decrease of PHB contents Fig. 5 Temporal variations of PHB content (closed during the latter period of enrichment in symbols) and acetate concentration (open symbols) during single batch PHA accumulation both SBRs was obscure, and therefore further experiments using seed sludge (triangle), study should be conducted to elucidate the SBR－A (circle), and SBR－B (square). Influence of Temperature on Enrichment of PHBAMOs 63 caused by parts of acetate being very rapidly SBR－A operated at 28℃, despite single batch taken by the cultures just after these were PHA accumulation experiments being per inoculated (possibly within several minutes formed at 28℃. The trend of the temperature from the inoculation of cultures to the effect observed in this study is similar to collection of 0 h samples), which reflected low those reported in Krishna and van F/F ratios described above. In addition, the Loosdrecht14) and Pittmann and Steinmetz15), decline of PHB content from 12 h to 24 h and opposite to those in Johnson et al.12) and would be attributable to the degradation of Jiang et al.13). PHB by PHBAMOs for cell growth and Composition of PHAAMOs Clone library maintenance. analysis of phaC genes was performed using Based on the experimental results, the seed sludge and enrichment cultures of PHB yields by seed sludge and enrichment SBR－A and SBR－B after the 55th cycle. The cultures were determined. When data from 0 results of clone library analysis are h to 12 h were applied, the PHB yields for summarized in Table 1. A phylogenetic tree seed sludge and enrichment cultures in of representative clones for each OTUs is SBR－A and SBR－B were estimated to be shown in Fig. 6. A total of 58 clones were 0.23, 0.70 and 0.88 C－mol PHB/C－mol retrieved from seed sludge and two acetate, respectively. As mentioned above, the enrichment cultures. The sequences of all low acetate concentrations at 0 h for SBR－A clones had the highest similarity to those of and SBR－B seemed to be due to the rapid phaC genes registered in the GenBank, most acetate consumption after the acetate supply. of which were possessed by members in β－ If the initial concentrations in the PHA Proteobacteria, more specifically the orders accumulation experiments for enrichment Burkholderiales (the genera Acidovorax, cultures are the same as that for seed sludge, Alcaligenes, Alicycliphilus, Hydrogenophaga, the PHB yields for SBR－A and SBR－B Rubrivivax, and Variovorax) and Rhodocyclales enrichment cultures are calculated to be 0.43 (the genus Thauera). In seed sludge, almost and 0.52 C－mol PHB/C－mol acetate, respec all clones (85%) were closely related to phaC tively, nearly double compared with that for genes possessed by members of seed sludge. Burkholderiales, and the other two clones The results obtained here clearly showed from seed sludge were highly different from that the PHB accumulation ability was the other clones (similarity: 41% to 55%) and largely enhanced by the enrichment of closely related to those of uncultured bacteria PHBAMOs in light of the substrate in sludge samples in an SBR operated under consumption, the maximum PHB content and the anaerobic-aerobic condition18). In SBR－A PHB yield. This indicated that PHBAMOs enrichment culture, most of the isolated with a strong PHB accumulation ability can clones (76%) were also closely related to be selectively enriched by the feast-famine phaC genes of Burkholderiales, but 5 clones method, irrespective of the enrichment tem (20%) had the highest similarities to those of perature at 20℃ and at 28℃. It should be Rhodocyclales, specifically the genus Thauera. noted that the enrichment culture in SBR－B In SBR－B enrichment culture, 30% of the operated at 20℃ exhibited better PHB obtained clones were closely related to phaC accumulation performance (higher maximum genes of Burkholderiales, and those of PHB content and PHB yield) than that in Rhodocyclales accounted for 65% of the total
Table 1 Summary of clone library analysis of phaC genes retrieved from seed sludge and enrichment cultures in SBR-A and SBR-B Total clone The order of PHBAMO possessing the phaC gene Sample number Burkholderiales Rhodocyclales Unknown Seed sludge 13 11 0 2 SBR-A 25 19 5 1 SBR-B 20 6 13 1 64 Japanese J. Wat. Treat. Biol. Vol.53 No.2
Fig. 6 Phylogenetic tree constructed based on nucleotide sequences of phaC genes obtained from three clone libraries with reference sequences. Clone numbers for each OTU are indicated in brackets after the clone name. Numbers adjacent to the branches indicate the bootstrap values based on 1000 replicates. Bar indicates 0.05 Substitutions per sequence position. clones. those studies. In addition, Carvalho et al.30) The results of clone library analysis reported that the sum of the abundance of suggested that members of Burkholderiales Azoarcus and Thauera in a mixed microbial were dominant in activated sludge in culture enriched on fermented molasses by municipal WWTPs, but that the enrichment the feast-famine method was correlated with by the feast-famine method using acetate as the maximum content of PHA (PHB + the sole external substrate led to increase polyhydroxyvalerate) observed in the fed- the relative abundance of Rhodocyclales. The batch experiments with multiple pulses of occurrence and predominance of members of fermented molasses. Between the two Rhodocyclales such as the genera Azoarcus enrichment cultures in this study, SBR－B and Thauera in mixed microbial cultures had higher relative abundance of with enhanced PHA accumulation abilities Rhodocyclales PHBAMOs than SBR－A (Table that were enriched by the feast-famine 1), and exhibited slightly stronger PHB method with various substrates has been accumulation ability than SBR－A in single reported previously11),25)－30). However, changes batch experiments (Fig. 5). Therefore, it was in PHBAMO compositions before and after indicated that PHBAMOs belonging to enrichment have not been investigated in Rhodocyclales, which were minor in activated Influence of Temperature on Enrichment of PHBAMOs 65 sludge in municipal WWTPs, would be the REFERENCES key to enhancing PHB accumulation ability 1 ） Ministry of the Environment, Japan: Solid in the enrichment cultures. However, waste management and recycling tech although it has been known that some nology of Japan–Toward a sustainable members of Rhodocyclales are capable of society.
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under different operating conditions using 30） Carvalho, G., Oehmen, A., Albuquerque, M. cell-sorting RT－PCR approach. Appl. G. E., and Reis, M. A. M.: The relationship Microbiol. Biotechnol., 78, 351－360 (2008) between mixed microbial culture compo 29） Liu, Z., Wang, Y., He, N., Huang, J., Zhu, K., sition and PHA production performance Shao, W., Wang, H., Yuan, W., and Li, Q.: from fermented molasses. N. Biotechnol., Optimization of polyhydroxybutyrate 31, 257－263 (2014) (PHB) production by excess activated sludge and microbial community analysis. (Submitted 2017. 1. 23) J. Hazard. Mater., 185, 8－16 (2011) (Accepted 2017. 5. 1)