Metabolic Behavior and Enzymatic Aspects of Denitrifying EBPR Sludge in a Continuous-Flow Anaerobic–Anoxic System

The metabolic aspects of enhanced biological phosphorus removal (EBPR) were investigated for the first time in a continuous-flow anaerobic–anoxic plant fed with acetate, propionate, or substrates which are involved in the tricarboxylic acid and/or glyoxylate cycle, i.e., fumarate, malate, or oxaloacetate, as the sole carbon source. Although the polyphosphate-accumulating organisms (PAOs) population remained stable with any carbon source examined, no typical EBPR metabolism was observed during fumarate, malate, or oxaloacetate utilization. Specific enzymatic activities related to EBPR were determined in activated sludge homogenates and directly correlated with the nutrient metabolic rates. The experimental results indicated the direct involvement of alkaline phosphatase, pyrophosphatase, and exopolyphosphatase in the denitrifying EBPR process. Metabolic aspects of glyoxylate cycle enzymes are discussed with regard to the biomass anaerobic and anoxic activity. Process performance was highly influenced by the kind of substrate utilized, indicating that specific metabolic pathways should be followed to favor efficient EBPR.     

Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems

The present study focuses on predicting the concentration of intracellular storage polymers in enhanced biological phosphorus removal (EBPR) systems. For that purpose, quantitative image analysis techniques were developed for determining the intracellular concentrations of PHA (PHB and PHV) with Nile blue and glycogen with aniline blue staining. Partial least squares (PLS) were used to predict the standard analytical values of these polymers by the proposed methodology. Identification of the aerobic and anaerobic stages proved to be crucial for improving the assessment of PHA, PHB and PHV intracellular concentrations. Current Nile blue based methodology can be seen as a feasible starting point for further enhancement. Glycogen detection based on the developed aniline blue staining methodology combined with the image analysis data proved to be a promising technique, toward the elimination of the need for analytical off-line measurements.     

The influence of process parameters on the characteristics of polyhydroxyalkanoates produced by mixed cultures

The characterization of polyhydroxyalkanoates (PHA) produced by mixed cultures is fundamental for foreseeing the possible final applications of the polymer. In this study PHA produced under aerobic dynamic feeding (ADF) conditions are characterized. The PHA produced shows a stable average molecular weight ([symbol: see text]) in the range (1.0-3.0) x 10(6), along three years of reactor operation. Attempts to improve the amount of PHA produced did not introduce significant variations on the values [symbol: see text]. Along this period, the polydispersity indices (PDI) were between 1.3 and 2.2. The use of different carbon sources allowed the tailoring of polymer composition: homopolymers of poly(3-hydroxybutyrate), P(3HB), were obtained with acetate and butyrate, whereas a mixture of acetate and propionate, and propionate and valerate, gave terpolymers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 2-methyl-3-hydroxyvalerate (2M3HV). All of these PHA had [symbol: see text] between 2.0 x 10(6) and 3.0 x 10(6). Thermal characterization of the produced polymers showed values of glass transition temperature, melting temperature, melting enthalpy, and crystallinity slightly lower than those obtained for PHA from pure cultures. The introduction of a purification step during the polymer extraction process allowed the elimination of possible contaminants but did not significantly improve the polymer quality.     

Production of Polyhydroxyalkanoates (PHAs) by Vibrio alginolyticus Strains Isolated from Salt Fields

Vibrio alginolyticus is a halophilic organism usually found in marine environments. It has attracted attention as an opportunistic pathogen of aquatic animals and humans, but there are very few reports on polyhydroxyalkanoate (PHA) production using V. alginolyticus as the host. In this study, two V. alginolyticus strains, LHF01 and LHF02, isolated from water samples collected from salt fields were found to produce poly(3-hydroxybutyrate) (PHB) from a variety of sugars and organic acids. Glycerol was the best carbon source and yielded the highest PHB titer in both strains. Further optimization of the NaCl concentration and culture temperature improved the PHB titer from 1.87 to 5.08 g/L in V. alginolyticus LHF01. In addition, the use of propionate as a secondary carbon source resulted in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). V. alginolyticus LHF01 may be a promising host for PHA production using cheap waste glycerol from biodiesel refining.     

The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy

Previous studies dealing with bacterial identification by means of Raman spectroscopy have demonstrated that micro-Raman is a suitable technique for single-cell microbial identification. Raman spectra yield fingerprint-like information about all chemical components within one cell, and combined with multivariate methods, differentiation down to species or even strain level is possible. Many microorganisms may accumulate high amounts of polyhydroxyalkanoates (PHA) as carbon and energy storage materials within the cell and the Raman bands of PHA might impede the identification and differentiation of cells. To date, the identification by means of Raman spectroscopy have never been tested on bacteria which had accumulated PHA. Therefore, the aim of this study is to investigate the effect of intracellular polymer accumulation on the bacterial identification rate. Combining fluorescence imaging and Raman spectroscopy, we identified polyhydroxybutyrate (PHB) as a storage polymer accumulating in the investigated cells. The amount of energy storage material present within the cells was dependent on the physiological status of the microorganisms and strongly influenced the identification results. Bacteria in the stationary phase formed granules of crystalline PHB, which obstructed the Raman spectroscopic identification of bacterial species. The Raman spectra of bacteria in the exponential phase were dominated by signals from the storage material. However, the bands from proteins, lipids, and nucleic acids were not completely obscured by signals from PHB. Cells growing under either oxic or anoxic conditions could also be differentiated, suggesting that changes in Raman spectra can be interpreted as an indicator of different metabolic pathways. Although the presence of PHB induced severe changes in the Raman spectra, our results suggest that Raman spectroscopy can be successfully used for identification as long as the bacteria are not in the stationary phase.     

Polyhydroxyalkanoate granules quantification in mixed microbial cultures using image analysis: Sudan Black B versus Nile Blue A staining

Polyhydroxyalkanoates (PHA) can be produced and intracellularly accumulated as inclusions by mixed microbial cultures (MMC) for bioplastic production and in enhanced biological phosphorus removal (EBPR) systems. Classical methods for PHA quantification use a digestion step prior to chromatography analysis, rendering them labor intensive and time-consuming. The present work investigates the use of two quantitative image analysis (QIA) procedures specifically developed for PHA inclusions identification and quantification. MMC obtained from an EBPR system were visualized by bright-field and fluorescence microscopy for PHA inclusions detection, upon Sudan Black B (SBB) and Nile Blue A (NBA) staining, respectively. The captured color images were processed by QIA techniques and the image analysis data were further treated using multivariate statistical analysis. Partial least squares (PLS) regression coefficients of 0.90 and 0.86 were obtained between QIA parameters and PHA concentrations using SBB and NBA, respectively. It was found that both staining procedures might be seen as alternative methodologies to classical PHA determination.     

Effect of Cycle Time on Polyhydroxybutyrate (PHB) Production in Aerobic Mixed Cultures

The aim of this study was to investigate the effect of cycle time on polyhydroxybutyrate (PHB) production under aerobic dynamic feeding system. The acetate-fed feast and famine sequencing batch reactor was used to enrich PHB accumulating microorganism. Sequencing batch reactor (SBR) was operated in four different cycle times (12, 8, 4, and 2 h) fed with a synthetic wastewater. The system performance was determined by monitoring total dissolved organic carbon, dissolved oxygen, oxidation-reduction potential, and PHB concentration. In this study, under steady-state conditions, the feast period of the SBR was found to allow the PHB storage while a certain part of stored PHB was used for continued growth in famine period. The percentage PHB storages by aerobic microorganism were at 16, 18, 42, and 55 % for the 12, 8, 4, and 2-h cycle times, respectively. The PHB storage was increased as the length of the cycle time was decreased, and the ratio of the feast compared to the total cycle length was increased from around 13 to 33 % for the12 and 2-h cycle times, respectively.     

聚羧基脂肪酸酯细菌合成的生长环境依赖性

聚羟基脂肪酸酯（Ｐｏｌｙｈｙｄｒｏｘｙａｌｋａｎｏａｔｅ,ＰＨＡ）是一类由许多细菌合成的、结构多变的能量和碳源的储藏物质,为了得到能合成新型ＰＨＡ的菌种,或得到能在便宜简单碳源上合成ＰＨＡ的菌种,以我们实验室开发的傅立叶红外（ＦＴ－ＩＲ）细胞无损检测技术和常规气相色谱（ＧＣ）法对全国各地的采集的不同样品中分离的菌种进行了筛选,往往在不同的地理环境中,筛选出的菌株所合成的ＰＨＡ的单体组成不同,有以含四个或五个碳原子的短链单体ＰＨＡ（Ｓｈｏｒｔ－ｃｈａｉｎ－ｌｅｎｇｔｈＰＨＡ,ｓｃｌＰＨＡ）为主,有的以含六个到十六个碳原子的中长链单体ＰＨＡ（Ｍｅｄｉｕｍ－ｃｈａｉｎ－ｌｅｎｇｔｈＰＨＡ,ｍｃｌ ＰＨＡ）为主,在我们以六种底物为碳源培养的３７１株形态不一的菌株中,有４０％的菌可以合成ＰＨＡ,而其中许多可以同时合成ＰＨ     

聚羟基脂肪酸酯细菌合成的生长环境依赖性

聚羟基脂肪酸酯 (Polyhydroxyalkanoate,PHA)是一类由许多细菌合成的、结构多变的能量和碳源的储藏物质 .为了得到能合成新型PHA的菌种 ,或得到能在便宜简单碳源上合成PHA的菌种 ,以我们实验室开发的傅立叶红外 (FT IR)细胞无损检测技术和常规气相色谱 (GC)法对全国各地的采集的不同样品中分离的菌种进行了筛选 .往往在不同的地理环境中 ,筛选出的菌株所合成的PHA的单体组成不同 ,有的以含四个或五个碳原子的短链单体PHA(Short chain lengthPHA ,sclPHA)为主 ,有的以含六个到十六个碳原子的中长链单体PHA(Medium chain lengthPHA ,mclPHA)为主 .在我们以六种底物为碳源培养的 371株形态不一的菌株中 ,有 40 %的菌可以合成PHA ,而其中许多可以同时合成PHB与中长链PHA共混的聚合物 .本研究为进行PHA研究的高分子同行提供了寻找能合成PHA的微生物菌种的依据     

Synthesis of polyhydroxyalkanoate (PHA) from excess activated sludge under various oxidation-reduction potentials (ORP) by using acetate and propionate as carbon sources

Accumulation of poly hydroxyalkanoate (PHA) from excess activated sludge (EAS) was monitored and controlled via the oxidation-reduction potential (ORP) adjusting process. The ORP was adjusted and controlled by only regulating the gas-flow rate pumped into the cultural broth in which sodium acetate (C2) and propionate (C3) were used as carbon sources. Productivity of PHA and the PHA compositions at various C2 to C3 ratios were also investigated. When ORP was maintained at +30 mV, 35% (w/w) of PHA of cell dry weight obtained when C2 was used as sole carbon source. The PHA copolymer, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), accumulated by EAS with different 3-hydroxyvalarate (3HV) molar fractions ranged from 8% to 78.0% when C2 and C3 was used as sole carbon source, By using ORP to monitor and control the fermentation process instead DO meter, the ORP system provided more precise control to the PHA accumulation process from EAS under low dissolved oxygen (DO) concentrations. Adjusting the C2 to C3 ratios in the media could control the composition such as the 3HV/3HB ratios of the PHBV. Furthermore, it might be an effective way to adjust the 3HV molar fractions in PHBV by controlling the DO concentration via the ORP monitoring system. The 3HV molar fractions in the PHBV declined with increasing ORP from −30 mV to +100 mV by adjusting the gas-flow rate (i.e. the DO concentration). It is concluded that the DO plays a very important role in the synthesis of 3HV subunits in PHBV co-polymer from the EAS. Therefore, a hypothetic metabolic model for PHA synthesis from EAS was proposed to try to explain the results in this study.     

甲烷氧化细菌生物催化合成聚-β-羟基丁酸酯的分子量调控

在甲烷氧化细菌Methylosinus trichosporium IMV3011细胞内生物催化合成聚-β-羟基丁酸酯(PHB)的过程中,对影响聚合物分子量的各种因素进行了研究.发现碳源、培养基组分NH4+,NO3-,HPO24-,Mg2+,某些导向PHB合成的关键中间产物以及PHB的提取方法均会对PHB的分子量产生影响.同时,通过对胞内PHB合成酶系中关键作用酶的活性变化进行研究,发现β-酮硫解酶催化着控制进入PHB循环入口的关键反应,而PHB分子量的变化则主要取决于PHB合成酶和PHB降解酶的协同作用.     

Isolation of PHA–Producing Bacteria from Date Syrup Waste

Since the major problem associated with the industrial production of Polyhydroxyalkanoates (PHAs) is their high production cost, this study was carried out using date syrup as the major carbon source to decrease the production cost and also help to supply other nutrient requirements. To isolate PHA–producing bacteria for this purpose, microorganisms were isolated from the syrup waste of a local date factory. These purified colonies were screened for intracellular granules by staining with Sudan Black. The positive-staining strains were grown for production of PHAs in 5% date syrup as carbon source supplemented with mineral salt medium. The culture was incubated at 30 °C with shaking at 140 rpm for 60 h. Among positively stained bacteria, the best PHA producers were selected on the basis of cell growth, cell dry weight, PHA content and the monomer composition of PHA. One of them could utilize date syrup for growth and produce the homopolymer of Polyhydroxybutyrate (PHB) with a cell density of about 5.1 g/L and maximum concentration of PHB equal to 3.6 g/L which is 71% of cell dry weight. Another one produces copolymer of Poly (hydroxybutyrate-hydroxyvalerate) in date syrup media with a maximum concentration of 2.2 g/L containing 10 wt % valerate in shake flask cultivation.     

碳源对SBR单级好氧工艺中微生物摄磷能力的影响及其机理研究

以两种典型基质：葡萄糖（R1）和乙酸钠（R2）作为单一碳源,考察了SBR单级好氧工艺在R1和R2中的除磷效果,并通过比较各自微生物体内储能物质的变化,探讨了SBR单级好氧工艺以不同基质作为碳源时影响其微生物摄磷能力的根本原因．结果表明：R1和R2中均观察到明显的超量摄磷现象,但在运行条件完全相同的情况下,两系统中微生物摄磷能力以及体内储能物质有很大的差别．稳定运行后,R1和R2中单位混合液挥发性悬浮固体（MLVSS）的总磷（TP）去除量分别为6．7～7．4、2．7～3．2mg·g^-1．R1中微生物体内储能物质多β羟基烷酸盐（PHA）含量没有明显的变化,另一储能物质糖原质在好氧段外碳源（葡萄糖）存在时有明显的积累现象（糖原质的最大积累量为3．21mmol-C·g^-1）,在好氧段外碳源消耗完后呈下降趋势,并在好氧结束时下降到好氧前水平;R2中PHA与糖原质在好氧段外碳源（乙酸钠）存在时均有明显的积累现象（外碳源消耗完时PHA与糖原质积累量分别为2．1,0．55mmol—C·g^-1）,PHA在好氧段外碳源消耗完后呈下降趋势,并在好氧结束时也几平下降到好氧前水平,而糖原质却在外碳源消耗完后继续积累,并在好氧2h左右时达到最大值（糖原质最大积累量为0．88mmol—C·g^-1）,此后下降明显,在好氧结束时也几乎恢复到原始水平．在整个好氧过程中,R1中内碳源（PHA与糖原质）的积累／转化量大于R2系统．在闲置期内,R1与R2中PHA与糖原质均没有明显变化,但却观察到明显的释磷现象,且R1释磷量多于R2系统．此研究显示,由于R1与R2中进水碳源不同,在其反应系统内碳源的好氧代谢途径有所差别,使各自微生物在好氧段体内储能物质的类型与积累／转化量也有所不同,各自微生物在好氧摄磷时得到的可利用的能量也不同,因而各自微生物的摄磷能力亦有所差别．     

On the preparation of methyl and ethyl (r)-(−)-3-hydroxy-valerate by depolymerization of a mixed phb/phv biopolymer

The microbial polyester containing 70–80% 3-hydroxy-butanoate and 20–30% 3-hydroxy-pentanoate (PHB/PHV, 2) is depolymerized to give monomeric esters of (R)-configuration. These are separated by fractional distillation. (R)-3-Hydroxy-pentanoate (4) is thus made readily available as enantiomerically pure starting mortal for synthesis.     

Purification and Properties of an Extracellular Polyhydroxybutyrate Depolymerase from Pseudomonas mendocina DSWY0601

An extracellular polyhydroxybutyrate(PHB) depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was approximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20―50 ℃ and sensitive to pH value within a pH range of 8.0―9.5. PHB depolymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate(P3/4HB) and poly-3-hydroxybutyrate-co-3- hydroxyvalerate(PHBV) but did not degrade poly(lactic acid)(PLA), poly(butylene succinate)(PBS) or poly- (caprolactone)(PCL). PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride(PMSF), H₂O₂ and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer(3HB) by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis of phaZ_pm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA(dPHA_SCL, PHA=polyhydroxyalkanoate) depolymerase containing catalytic domain, linker and substrate-binding domain.     

Degradation of medium-chain-length polyhydroxyalkanoates in tropical forest and mangrove soils

Bacterial polyhydroxyalkanoates (PHAs) are perceived to be a suitable alternative to petrochemical plastics because they have similar material properties, are environmentally degradable, and are produced from renewable resources. In this study, the in situ degradation of medium-chain-length PHA (PHA_MCL) films in tropical forest and mangrove soils was assessed. The PHA_MCL was produced by Pseudomonas putida PGA1 using saponified palm kernel oil (SPKO) as the carbon source. After 112 d of burial, there was 16.7% reduction in gross weight of the films buried in acidic forest soil (FS), 3.0% in the ones buried in alkaline forest soil by the side of a stream (FSst) and 4.5% in those buried in mangrove soil (MS). There was a slight decrease in molecular weight for the films buried in FS but not for the films buried in FSst and in MS. However, no changes were observed for the melting temperature, glass transition temperature, monomer compositions, structure, and functional group analyses of the films from any of the burial sites during the test period. This means that the integral properties of the films were maintained during that period and degradation was by surface erosion. Scanning electron microscopy of the films from the three sites revealed holes on the film surfaces which could be attributed to attack by microorganisms and bigger organisms such as detritivores. For comparison purposes, films of polyhydroxybutyrate (PHB), a short-chain-length PHA, and polyethylene (PE) were buried together with the PHA_MCL films in all three sites. The PHB films disintegrated completely in MS and lost 73.5% of their initial weight in FSst, but only 4.6% in FS suggesting that water movement played a major role in breaking up the brittle PHB films. The PE films did not register any weight loss in any of the test sites.     

作物秸秆降解中稀有机酸的萃取回收

作物秸秆有机降解的研究中,有机酸的回收利用是一个非常重要的环节,本研究采用低沸点的乙酸乙酯和高沸点的磷酸三丁酯（TBP）作萃取剂,对作物秸秆有机酸（乙酸,丙酸）降解中不同浓度的稀有机酸溶液（汽提液和洗涤液）用液－液淬取法回收,结果表明：磷酸三丁酯的萃取能力大于乙酸乙酯,同一萃取剂对丙酸的萃取效果比对乙酸好。     

Safer solvents for reactive organometallic reagents

This paper describes the use of poly(α -olefin)s (PAOs) as safer alternatives to cyclohexane, hexanes, and heptane as solvents for alkyllithium reagents. While PAOs like any alkane are flammable, PAOs do not readily catch on fire because they contain 20 or more carbon atoms, a low volatility, and have a high flash point vis-à-vis alkanes like hexane. Also unlike conventional alkanes, PAOs can be quantitatively separated from polar organic solvents and polar organic products either by a simple gravity separation or by an extraction after a reaction. Any leaching of the PAO solvent into a polar phase during such a separation can be minimized by addition of small amounts of water to the polar phase. However, while these PAO solvents have some physical differences from conventional low molecular weight volatile alkanes, they otherwise behave like alkanes and alkyllithium reagents in these PAO solvents can used in their conventional reactions in these PAO solvents.     

Effect and mechanism of carbon sources on phosphorus uptake by microorganisms in sequencing batch reactors with the single-stage oxic process

To investigate the chief reason for phosphorus uptake by microorganisms affected by substrates in sequencing batch reactors with the single-stage oxic process, two typical substrates, glucose (R1) and acetate (R2) were used as the sole carbon source, and the performances of phosphorus removal and the changes of intracellular storage were compared. The experimental results showed that the phenomenon of excess phosphorus uptake was observed in two reactors, but bacteria’s capability to take in phosphorus and its intracellular storage were obviously different under the same operational condition. After steady-state operation, total phosphorus (TP) removed per MLVSS in R1 and R2 was 6.7–7.4 and 2.7–3.2 mg/g, respectively. The energy storage of poly-β-hydroxyalkanoates (PHA) was nearly constant in R1 during the whole period, and another aerobic storage of glycogen was accumulated (the max accumulation of glycogen was 3.21 mmol-C/g) when external substrate was consumed, and then was decreased to the initial level. However in R2, PHA and glycogen were both accumulated (2.1 and 0.55 mmol-C/g, respectively) when external substrate was consumed, but they showed different changes after the period of external consumption. Compared to rapid decrease of PHA to the initial level, glycogen continued accumulating to the peak (0.88 mmol-C/g) in 2 h of aeration before decreasing. During the aeration, the accumulations/transformations of internal carbon sources in R1 were higher than those in R2. In addition, obvious TP releases were both observed in R1 and R2 other than PHA and glycogen during the long-term idle period; moreover, the release content of phosphorus in R1 was also higher than that in R2. The researches indicated that different aerobic metabolism of substrate occurred in R1 and R2 due to the different carbon sources in influent, resulting in different types and contents of aerobic storage accumulated/translated in bacteria of R1 and R2. As a result, ATP content provided for phosphorus uptake was different in R1 and R2, and the capability to take up phosphorus was also different from each other.     

环己酮与丙烯酸甲酯及(S)-3-(2′-氧环己基)-丙酸与醇的酯化反应

在(R)TTCA·K催化下由环己酮直接与丙烯酸甲酯进行Michael加成反应得到了(S)-3-(2′-氧环己基)-丙酸甲酯, [α]20D-4.14(41.5% e.e.).