Analysis of polyhydroxyalkanoate (PHA) synthase gene in activated sludge that produces PHA containing 3-hydroxy-2-methylvalerate

The identification of phaC which encodes PHA synthase, that is involved in the formation of polyhydroxyalkanoate (PHA) containing 3-hydroxy-2-methylvalerate (3H2MV), was attempted. As of now, PHA containing 3H2MV has been reported to be produced only by mixed microbial cultures in activated sludge, but no pure bacterial cultures. A laboratory-scale activated sludge process was operated for 67 days. During the operation of the activated sludge process, its capacity to produce PHA containing 3H2MV, and the diversity of the partial phaC genes in the activated sludge microorganisms were monitored periodically. Analysis of the partial phaC genes was conducted by PCR followed by cloning and DNA sequencing, or by PCR followed by terminal-restriction fragment length polymorphism (T-RFLP). The cloning-sequencing of the 263 clones gave 11 distinct genetic groups (GGs). All of the genetic groups had similarities to known phaC higher than 48%, and one of them had similarity as high as 96% to that of Alcaligenes sp. The behavior of each of the genetic groups during the operation of the activated sludge process was monitored by the T-RFLP method. The restriction enzyme AccII, with the help of MboI, enabled the monitoring of each of the genetic groups. One of the genetic groups was found to have a strong correlation with the capability of the activated sludge to produce PHA containing 3H2MV, and its DNA sequence together with its amino acid sequence are reported.     

Fine-scale population structure of Accumulibacter phosphatis in enhanced biological phosphorus removal sludge

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting G1PAO, G2PAO, and G3PAO groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non- Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (G1NPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the G4PAO group of Accumulibacter phosphatis, which suggests that G1NPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.     

Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater

Production of polyhydroxyalkanoates (PHAs) in activated sludge treating wastewater represents an economical and environmental promising alternative to pure culture fermentations. A process for production of PHA from a paper mill wastewater was examined at laboratory scale. The three stage process examined consisted of acidogenic fermentation to convert wastewater organic matter to volatile fatty acids (VFAs), an activated sludge system operating under feast/famine conditions to enrich for PHA producing organisms and accumulation of PHA in batch experiments. After fermentation of the wastewater, 74% of the soluble COD was present as VFA (acetate, propionate, butyrate and valerate) and the resulting PHA after batch accumulation consisted of 31-47 mol% hydroxybutyrate and 53-69 mol% hydroxyvalerate. The maximum PHA content achieved was 48% of the sludge dry weight and the three stage process exhibited a potential to produce 0.11 kg of PHA per kg of influent COD treated.     

剩余活性污泥完全资源化利用微生物集成技术

剩余活性污泥完全资源化利用微生物集成技术包括: 使用土著PHA合成菌回注法驯化并发酵活性污泥, 生产生物降解材料聚羟基脂肪酸酯(PHA); 采用土著嗜酸性氧化亚铁硫杆菌和氧化硫硫杆菌进行生物淋滤, 去除重金属; 以解磷菌和解钾菌为菌种, 进行固态发酵, 生产生物菌肥。结果表明, 500 L中试PHA占挥发性悬浮固体的20%以上; 重金属含量达到国家排放要求; 生物菌肥中活菌数大于1×108 个/g以上。实现了剩余活性污泥的近零排放。     

染料脱色菌的分子分类和有关基因与其脱色特性的关系

从印染废水处理系统中分离到12株具有不同脱色特性的细菌,比较分析这12株细菌的质粒携带情况、分子分类地位和黄素还原酶基因(fre)的拷贝数与其脱色特性之间的关系。结果发现,所分离到的这12株菌分别归属于希瓦氏菌属、克雷伯氏菌属、假单胞菌属、气单胞菌属和苍白杆菌属。具有较广谱脱色能力的细菌在系统进化树上基本聚为一类,分别归属于希瓦氏菌属和气单胞菌属,其质粒携带率也较低。fre基因在不同菌属中的基因型存在很大的差异,仅仅采用一套引物无法对不同菌属中的fre基因进行有效的扩增。     

Inhibition of acetate and propionate assimilation by itaconate via propionyl-CoA carboxylase in isocitrate lyase-negative purple bacterium Rhodospirillum rubrum

The PCR cloning strategy for type II polyhydroxyalkanoate (PHA) biosynthesis genes established previously for Pseudomonas was successfully applied to Burkholderia caryophylli strain AS 1.2741. The whole pha locus containing PHA synthase genes phaC1, phaC2 and PHA depolymerase gene phaZ was cloned. The complete open reading frames of phaC1(Bc), phaC2(Bc) and phaZ(Bc) were identified. Sequence analyses of the phaC1(Bc), phaZ(Bc) and phaC2(Bc) showed more than 77.7%, 73.7% and 68.5% identities compared with the corresponding pha loci of the known Pseudomonas strains, respectively. The functional expression of the phaC1(Bc) or phaC2(Bc) in Escherichia coli strain KM32B (fadB deleted mutant) showed the abilities of PHA production by the estimated PHA synthase genes. Over 1% PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) was detected from cells of recombinant E. coli KM32B (pHXM11) harboring phaC1(Bc), grown on octanoate. At the same time over 3% of PHA consisting of 3HO and 3HD was produced from cells of recombinant E. coli KM32B (pHXM21) harboring phaC2(BC), grown on decanoate. Results showed the PCR cloning strategy developed previously can be applied to non-Pseudomonas strains such as Burkholderia in this case. This result also provided evidence for the presumption that the Burkholderia strain possesses not only polyhydroxybutyrate synthase genes, but also synthase for medium-chain-length polyhydroxyalkanoates consisting of 3HHx, 3HO and 3HD.     

Polyhydroxyalkanoate (PHA) accumulation in sulfate-reducing bacteria and identification of a class III PHA synthase (PhaEC) in Desulfococcus multivorans

Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE(Dm) and phaC(Dm) genes. PhaC(Dm) and PhaE(Dm) were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaC(Dm) alone (pBBRMCS-2::phaC(Dm)) and of phaE(Dm)C(Dm) (pBBRMCS-2::phaE(Dm)C(Dm)) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB(-)4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaE(Dm)C(Dm) small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaC(Dm) and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.     

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

Engineering of chimeric class II polyhydroxyalkanoate synthases

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.     

The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.     

Batch production of polyhydroxyalkanoate by low-polyphosphate-content activated sludge at varying pH

Polyhydroxyalkanoate (PHA), a biodegradable plastic, can be produced from excess activated sludge by utilizing intracellular glycogen and polyphosphate as energy sources under growth-limiting conditions. Activated sludge of 2%, 6%, and 8% polyphosphate with similar glycogen content of 33% was investigated for batch PHA production by varying the pH values from 6 to 8. Acetate applied at 1000 mg COD/L was almost exhausted within 80 min of anaerobic stage. The remaining glycogen in the sludge was higher at a lower pH because of less energy used for acetate uptake. Highest PHA content of 51% was obtained from sludge with an 8% polyphosphate content at pH 8. PHA production occurred rapidly within the first 20 min, with a productivity rate of 2.19 g PHA/L-h. The results in this study indicate that PHA production by using activated sludge is a promising alternative to a typical pure culture approach.     

Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation

To reduce the production cost of polyhydroxyalkanoates (PHA) and disposal amount of excess sludge simultaneously, the feasibility of using fermentative volatile fatty acids (VFAs) as carbon sources to synthesize PHA by activated sludge was examined. At pH 11.0, 60 degrees C and fermentative time of 7d, the VFAs yield was 258.65 mgTOC/gVSS. To restrain cell growth during PHA production, the released phosphorus and residual ammonium in the fermentative VFAs was recovered by the formation of struvite precipitation. Acetic acid was the predominant composition of the fermentative VFAs. PHA accumulation in excess sludge occurred feeding by fermentative VFAs with aerobic dynamic feeding process. The maximum PHA content accounted for 56.5% of the dry cell. It can be concluded from this study that the VFAs generated from excess sludge fermentation were a suitable carbon source for PHA production by activated sludge.     

Engineered Aeromonas hydrophila for enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers composition

Aeromonas hydrophila 4AK4 produces poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 3-hydroxybutyrate (3HB) and about 15 mol% 3-hydroxyhexanoate (3HHx) from dodecanoate. To study the factors affecting the monomer composition and PHBHHx content, genes encoding phasin (phaP), PHA synthase (phaC) and (R)-specific enoyl-CoA hydratase (phaJ) from Aeromonas punctata (formerly named Aeromonas caviae) were introduced individually or jointly into A. hydrophila 4AK4. The phaC gene increased 3HHx fraction more significantly than phaP, while phaJ had little effect. Expression of phaC alone increased the 3HHx fraction from 14 to 22 mol%. When phaC was co-expressed with phaP and phaJ, the 3HHx fraction increased from 14 to 34 mol%. Expression of phaP or phaC alone or with another gene enhanced PHBHHx content up to 64%, cell dry weight (CDW) as much as 4.4 gL(-1) and PHBHHx concentration to 2.7 gL(-1) after 48 h in shake flask culture. The results suggest that a higher PHA synthase activity could lead to a higher 3HHx fraction and PHBHHx content. Co-expression of phaJ with phaC or phaP would favor PHA accumulation, although over-expression of phaJ did not affect PHA synthesis much. In addition, inhibition of beta-oxidation by acrylate in A. hydrophila 4AK4 enhanced PHBHHx content. However, no monomers longer than 3HHx were detected. The results show that genetic modification of A. hydrophila 4AK4 enhanced PHBHHx production and altered monomer composition of the polymer.     

PHA production by activated sludge.

The production of polyhydroxyalkanoate by anaerobic-aerobic activated sludge was reviewed concentrating on the biochemical mechanisms and on the trials to increase polyhydroxyalkanoate (PHA) content in activated sludge. The anaerobic aerobic activated sludge system selects microorganisms with the capabilities to couple glycolysis, polyphosphate degradation, and PHA accumulation for anaerobic substrate uptake. Some of the PHA-related metabolisms observed there have not been seen in pure cultures so far. Such metabolisms are the formation of PHA containing 3-hydroxy-2-methylvalerate, and '3-hydroxyvalerate fermentation' in which glucose or glycogen is converted to 3-hydroxyvalerate-rich PHA while yielding energy. The PHA content of activated sludge can be increased up to 62% by applying a microaerophilic-aerobic activated sludge process. PHA production by activated sludge is worth investigation.     

分子检测技术对活性污泥中氨氧化细菌的比较研究

采用PCR扩增、随机克隆测序等技术,分析处理含高浓度氨氮的废水处理系统不同驯化时期的4个活性污泥样品,对样品中氨氧化细菌（AOB）的种类和氨单加氧酶（AMO）的活性进行分析比较,并在国内首次采用PCR变性梯度凝胶电泳（DGGE）相结合的技术对样品中总的细菌类群的差异进行研究。结果表明所检测到的氨氧化细菌优势菌群均属于变形细菌的β亚类,与Nitrosomonas sp.具有较高的相似性。活性污泥驯化成熟后,废水处理系统中AMO的活性有明显提高,活性污泥中的细菌类群更加集中,优势菌群相对稳定,系统对废水的处理效率也相应提高。结果表明采用分子检测技术有利于更全面地了解AOB的类群和功能,进而改善废水处理系统的处理效果。     

Occurrence of ammonia-oxidizing Archaea in activated sludges of a laboratory scale reactor and two wastewater treatment plants

Aims:  Characterization of the ammonia-oxidizing archaea (AOA) community in activated sludge from a nitrogen removal bioreactor and wastewater treatment plants (WWTPs).
Methods and Results:  Three primer sets specific for ammonia mono-oxygenase α -subunit ( amoA ) were used to construct clone libraries for activated sludge sample from a nitrogen removal bioreactor. One primer set resulted in strong nonspecific PCR products. The other two clone libraries retrieved both shared and unique AOA amoA sequences. One primer set was chosen to study the AOA communities of activated sludge samples from Shatin and Stanley WWTPs. In total, 18 AOA amoA sequences were recovered and compared to the previous reported sequences. A phylogenetic analysis indicated that sequences found in this study fell into three clusters.
Conclusions:  Different primers resulted in varied AOA communities from the same sample. The AOA found from Hong Kong WWTPs were closely similar to those from sediment and soil, but distinct from those from activated sludge in other places. A comparison of clone libraries between Shatin WWTP and bioreactor indicated the AOA community significantly shifted only after 30-day enrichment.
Significance and Impact of the Study:  This study confirmed the occurrence of AOA in a laboratory scale nitrogen removal bioreactor and Hong Kong WWTPs treating saline or freshwater wastewater. AOA communities found in this study were significantly different from those found in other places. To retrieve diverse AOA communities from environmental samples, a combination of different primers for the amoA gene is needed.     

PHB synthase from Streptomyces aureofaciens NRRL 2209

An approximately 4.9 kb Sau3A I genomic DNA fragment from the Streptomyces aureofaciens NRRL 2209 aiding in the biosynthesis of PHB in recombinant Escherichia coli has been sequenced and analysed for phaC gene. The putative phaC(Sa) gene of 2 kb is 79.1% GC rich and encodes a 63.5 kDa protein. It expressed under its own promoter and significant PHA synthase activity was detected in the recombinant E. coli. This is the first putative PHA synthase gene reported from a Streptomyces sp. with serine as the active nucleophile in the conserved lipase box. The phaC(Sa) was found in close proximity to a regulatory gene, which apparently regulated the phaC expression.     

Expression of PHA polymerase genes of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.