Operation of an SBR with activated sludge for PHA production
To increase the PHA production rate, bacteria that are capable of accumulating PHA must be enriched in the activated sludge. In this study, a SBR inoculated with the activated sludge was operated under ‘feast or famine’ conditions in which activated sludge was submitted to consecutive periods of external substrate accessibility and unavailability (Serafim et al., 2004). Under these conditions, PHA-accumulating bacteria were enriched, thereby becoming dominant in the complex microbial community. Since Nile red does not affect the growth of bacteria and the Nile red-PHA conjugates produce a strong orange fluorescence, the viable-colony staining method was used in this study to detect colonies of PHA-accumulating bacteria. The numbers of PHA-producing bacteria were 5.2 × 106 and 2.4 × 108 CFU g−1 activated sludge, respectively, at days 5 and 10. At day 10, a large number of colonies grown on the Nile red plate emitted bright orange fluorescence (Fig. S2), which indicated that the PHA-producing bacteria were effectively enriched in the activated sludge.
During domestication, the samples were withdrawn from the SBR for the batch experiments. In the initial 5 days, the amounts of PHA produced by activated sludge were low in the batch experiments, which only accounted for 3–16% of CDW. In contrast, the amounts of PHA significantly increased in the latter half of the operation of the reactor, accounting for 54% of CDW on the 10th day.
T-RFLP profiles of activated sludge bacterial community in an SBR
During domestication, it is useful to monitor the changes of activated sludge community over time. For this purpose, T-RFLP was applied for analyzing community 16S rRNA gene. The community T-RFLP profiles at different time points are shown in Fig. S3. On the 1th, 3th, 5th, 7th and 9th day the total numbers of T-RFs were 24, 17, 18, 11 and 13 T-RFs, respectively (Fig. 1). Seven T-RFs – 78, 86, 93, 99, 202, 367 and 558 bp – were common in all samples. Among them, two T-RFs, 99 and 558 bp, showed a higher relative abundance than the other T-RFs and a regular change in the relative abundance was also observed for the two T-RFs. Five unique T-RFs – 108, 192, 214, 226 and 277 bp – were found on the 1st day. A T-RF of 75 bp was found on the 5th day, and a T-RF of 563 bp on the 7th day.
Figure 1. T-RFLP profiles of bacterial 16S rRNA genes amplified from total DNAs extracted from activated sludge samples. Histograms show the results after cleavage with HhaI. The relative abundance of T-RFs is given as percentage of total peak height. Numbers indicate the lengths of the T-RFs for fragments with a relative abundance of more than 1%.
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In this study, the T-RFLP profiles at different domestication stages were different from each other, indicating that the structure of the bacterial community changed significantly during domestication. Moreover, the types of bacterial species (i.e. the phylogenetic diversity) decreased with the prolonged domestication, as judged by the reduction in the number of T-RFs. These results from T-RFLP suggest that some specific microbes such as PHA-accumulating bacteria may be enriched in activated sludge by domestication.
DGGE profiles of activated sludge bacterial community in an SBR
DGGE analysis of amplified 16S rRNA gene permits investigation of the spatial and temporal changes of the population in the environment, and it allows identification of the predominant species in a community. To better define the composition of the enriched mixed cultures, bacterial communities were analyzed by PCR–DGGE. The 16S rRNA V6–V8 variable regions were PCR-amplified from community DNA and further analyzed by DGGE. Bands 1–10 on the DGGE gel (Fig. 2) were sequenced. The closest matches for the resulting sequences were determined by a blast search at GenBank (Table 1). The sequence extracted from band 2 showed the highest identity with the genus Thauera. Previous studies have shown that the genus Thauera was the dominant microbial population reasonably responsible for PHASCL accumulation in activated sludge enriched by periodic feeding with a mixture of organic acids (Dionisi et al., 2005, 2006). Two DGGE bands (bands 7 and 8) appeared at days 8, 9 and 10 (Fig. 2). The sequences extracted from the two bands showed the highest similarity with the genera Pseudomonas and Acinetobacter, respectively (Table 1). It is well known that many species from the genera Pseudomonas and Acinetobacter are typical PHA producers. In particular, many Pseudomonas species possess the Class II PHA synthases and can produce a wide variety of PHAMCL (Solaiman et al., 2000; Zhang et al., 2001). Two specific genera, Dechloromonas and Nitrosomonas, which significantly correlate with the functions and performance of wastewater treatment, were also identified by DGGE analysis (bands 9 and 10). Several differential bands (bands 3, 4 and 5) in DGGE profiles were not classified into any genera.
Table 1. Sequence similarities of excised DGGE bands shown in Fig. 2
|Band no.||Closest relative (accession no.)||Similarity (%)||Phylum (or class)|
|Band 1||Uncultured Rhodocyclaceae bacterium clone 408 (FM207908)||419/431 (97)|| Betaproteobacteria |
|Band 2||Thauera sp. R-24450 (AM231040)||421/430 (98)|| Betaproteobacteria |
|Band 3||Uncultured bacterium clone AS-26 (HQ609686)||416/428 (97)||Unclassified|
|Band 4||Uncultured bacterium clone Er-MS-1 (EU542425)||425/430 (99)||Unclassified|
|Band 5||Uncultured bacterium clone 244ds10 (AY212692)||426/431 (99)||Unclassified|
|Band 6||Uncultured Saprospiraceae bacterium clone Epr10 (EU177733)||416/428 (97)|| Bacteroidetes |
|Band 7||Pseudomonas sp. (EU770402.1)||427/431 (99)|| Gammaproteobacteria |
|Band 8||Acinetobacter sp. (FJ660569)||418/429 (97)|| Gammaproteobacteria |
|Band 9||Dechloromonas sp. RCB (AY032610)||422/430 (98)|| Betaproteobacteria |
|Band 10||Nitrosomonas sp. Nm86 (AY123798)||418/429 (97)|| Betaproteobacteria |
Figure 2. DGGE analysis of the V6–V8 variable regions of the bacterial 16S rRNA genes amplified from the activated sludge community DNA. Band numbers correspond to the sequences retrieved in Table 1. Lanes (from left to right) are the 1–10 day samples.
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Phylogenetic analysis of PHA synthase genes in activated sludge
In general, these PHA synthase genes possess high G+C contents. DNA templates with a high G+C content usually hamper PCR amplification; the reagents formamide, glycerol, DMSO and betaine are often used as PCR additives to improve the PCR amplification of GC-rich DNA sequences (Dieffenbach & Dveksler, 1995). In this study, DMSO was added to the PCR reaction mixture. PCR with the CF1-CR4 primer set gave products with sizes around 500 bp. The sizes of the PCR products were as expected (Sheu et al., 2000). The amplicons from the day 9 sample were cloned and sequenced. In total, 80 phaC genes were obtained. A phylogenetic tree of the phaC genes was constructed based on their nucleotide sequences (Fig. 3). Clones with similarities higher than 97% were classified as the same operational taxonomic units (OTU). As shown in Fig. 3, of the 80 phaC genes found, 62 were classified into 13 OTUs (OTU 1–13). Of the 13 OTUs defined after the phylogenetic analysis, 12 were closest to the Class I PHA synthase, and the remaining one (OUT 12) was affiliated to the Class II PHA synthase. Our results suggested that Class I PHA synthase is the dominant type of PHA synthase in activated sludge, which is consistent with previous reports on the investigation of the diversity of the phaC genes in activated sludge (Michinaka et al., 2007; Ciesielski et al., 2008). Moreover, the two main types of PHA produced by activated sludge are PHB and PHBV (Salehizadeh & Van Loosdrecht, 2004; Serafim et al., 2008), which indirectly provides evidence for the predominance of Class I PHA synthase in activated sludge.
Figure 3. Phylogenetic tree of the PHA synthase genes obtained from the constructed phaC gene clone library. Thirteen OTU were found in the tree, and are highlighted in boxes. The tree was constructed based on the Kimura 2 parameter model and the neighbor-joining algorithm using the mega 4.0 package. Bootstrap values from 1000 replicates are indicated at the nodes of branches. The bar represents 0.1 substitution per nucleotide position.
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In a review summarized by Rehm (2003), 59 PHA synthases from various bacteria were extensively compared, showing that these enzymes exhibit strong similarities. Six conserved blocks, which include a putative lipase box that is the catalytic active site of PHA synthases, were identified in the amino acid sequences of the PHA synthases. After the multiple alignments of the amino acid sequences, of the total of 80 PHA synthases obtained, 72 were found to have a putative lipase box (GXCXG; data not shown). Among 80 PHA synthases obtained, 76 were closest to the Class I PHA synthase, and the remaining four were closer to the Class II PHA synthase (Table S1). Class I PHA synthase is represented by that of Ralstonia eutropha and utilizes hydroxyalkanoate unit comprising 3–5 carbon atoms, whereas Class II PHA synthase is represented by that of P. aeruginosa and utilizes a hydroxyalkanoate unit comprising 6–14 carbon atoms (Lee, 1996a b).
Production of PHAMCL containing 3HDD monomer by activated sludge
Since the first finding of PHB in 1926, more than 100 different monomer units have been identified as constituents of PHA in > 300 different microorganisms (Lee, 1996a b). Many studies have been conducted to evaluate the potential for PHA production by activated sludge using various external carbon substrates (Salehizadeh & Van Loosdrecht, 2004; Serafim et al., 2008). However, most studies focus on the synthesis of PHASCL by activated sludge, such as PHB, and its copolymer with 3HV (PHBV).
PHAMCL are considered to be much more suitable biomaterials for various applications based on their physical properties (Chen & Wu, 2005). Some Pseudomonas sp. strains, such as Pseudomonas oleovorans, Pseudomonas putida and Pseudomonas resinovorans, which are the most common PHAMCL-producing species, can accumulate PHAMCL from fatty acids (Ouyang et al., 2007). PHAMCL consisting of mainly 3-hydroxydodecanoate (3HDD) monomer showed improved thermal and mechanical properties over the conventional PHAMCL, which usually have low Tm and weak tensile strength (Liu et al., 2011). In this study, the production of PHAMCL containing 3HDD monomer by activated sludge was demonstrated for the first time. The PHA copolymer produced by activated sludge at day 10 was found to be composed of three types of monomers: 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 3-hydroxydodecanoate (3HDD), by comparing their mass spectrogram with that of the standard in GC–MS analysis (Fig. 4). The molar percentages of 3HB, 3HV and 3HDD monomer were 62.41%, 34.17% and 3.42%, respectively, as determined by the relative abundance of each monomer in GC analysis. Previously, open mixed cultures enriched in glycogen-accumulating organisms with fermented sugar cane molasses were shown to produce PHAMCL containing 3-hydroxyhexanoate (3HHx) monomer (Bengtsson et al., 2010). Recently, the PHAMCL produced by activated sludge enriched by periodic feeding with nonanoic acid contained 3-hydroxynonanoate (3HN) and 3-hydroxyheptanoate (3HHp) monomers (Lee et al., 2011). So far, 3HDD is the longest carbon chain of monomer (C12) that has been found in the PHA produced by activated sludge.
Figure 4. GC–MS analysis of PHAMCL produced by activated sludge. (a) Mass spectrometry data of 3-hydroxybutyrate (3HB). (b) Mass spectrometry data of 3-hydroxyvalerate (3HV). (c) Mass spectrometry data of 3-hydroxydodecanoate (3HDD).
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The Class I PHA synthases listed in Table S1 were responsible for the catalysis of the polymerization of 3HB and 3HV monomers. One of the four Class II PHA synthases (clone 18) listed in Table S1 was shown to be associated with the ability of the activated sludge to produce PHAMCL containing 3HDD monomer. The PHA synthase from clone 18 showed the highest similarity (99%) with the PHA synthase 1 (PhaC 1) from Pseudomonas mendocina NK-01. The PHAMCL produced by NK-01 mainly consists of 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) monomers (13). The other three Class II PHA synthases from clones 47, 55 and 80 showed 97–99% identity with those PHA synthases that had substrate preferences for hydroxyalkanoate monomers of 6–8 carbon atoms (Table S1). These results suggest that the PHA synthase from clone 18 may correlate with the polymerization of 3HDD monomer in the synthesis of PHAMCL containing 3HDD. In the phylogenetic analysis of the phaC genes, clone 18 was affiliated with OTU 12 (Fig. 3), and it is suggested that OTU 12 most probably corresponds to the formation of PHAMCL containing 3HDD.
The production of PHA containing 3HDD by activated sludge was more active in the last 3 days, and was much less in the first 7 days. The compositions of PHA produced in the batch experiments were determined by gas chromatograph analysis. The PHA produced by activated sludge consisted of 3HB and 3HV monomer in the initial 5 days, and no 3HDD monomer was detected. However, the 3HDD monomer was detected with a molar percentage of 0.12% and 0.38%, respectively, at days 6 and 7. Interestingly, the molar percentage of 3HDD monomer significantly increased in the last 3 days, and it reached 3.42% on the 10th day.
This observation most probably is due to the changes in the population of the PHA-producing bacteria. Our speculation was supported by the evidence below. A band (band 7 in DGGE profiles) appeared at days 8, 9 and 10, and the sequence extracted from band 7 showed the highest identity with the genus Pseudomonas. Many species from the genus Pseudomonas, such as P. putida KT2442, are well-known PHAMCL producers and can produce PHAMCL containing 3HDD monomer (Ouyang et al., 2007; Liu et al., 2011). The results from DGGE suggest that the Pseudomonas strain identified from band 7 may play a key role in the production of PHAMCL containing 3HDD.
In this study, the monomer compositions of PHAMCL were shown to be related to the types of PHA-producing bacteria and PHA synthase genes. Importantly, PHAMCL containing 3HDD monomer was produced by activated sludge. The present study provides valuable information for the directed synthesis of PHAMCL with different monomer compositions.