Effects of sludge retention time, carbon and initial biomass concentrations on selection process: From activated sludge to polyhydroxyalkanoate accumulating cultures

Four sequence batch reactors (SBRs) fed by fermented sugar cane wastewater were continuously operated under the aerobic dynamic feeding (ADF) mode with different configurations of sludge retention time (SRT), carbon and initial biomass concentrations to enrich polyhydroxyalkanoate (PHA) accumulating mixed microbial cultures (MMCs) from municipal activated sludge. The stability of SBRs was investigated besides the enrichment performance. The microbial community structures of the enriched MMCs were analyzed using terminal restriction fragment length polymorphism (T-RFLP). The optimum operating conditions for the enrichment process were: SRT of 5 days, carbon concentration of 2.52 g COD/L and initial biomass concentration of 3.65 g/L. The best enrichment performance in terms of both operating stability and PHA storage ability of enriched cultures (with the maximum PHA content and PHA storage yield (Y_PHA/S) of 61.26% and 0.68 mg COD/mg COD, respectively) was achieved under this condition. Effects of the SRT, carbon concentration and initial biomass concentration on the PHA accumulating MMCs selection process were discussed respectively. A new model including the segmentation of the enrichment process and the effects of SRT on each phase was proposed.     

不同比例乙酸和丙酸对活性污泥微生物合成聚羟基脂肪酸酯的影响

聚羟基脂肪酸酯（PHA）是一种生物可降解塑料,利用活性污泥合成PHA具有节约成本、操作方便的优点。研究选择污泥发酵液挥发性脂肪酸（VFAs）中比重相对较高的乙酸和丙酸作为碳源自配营养液,深入研究不同质量浓度比对PHA合成的影响。结果表明,乙酸为主要碳源时更有利于PHA的合成,最大合成量12.3%出现在乙酸作为唯一碳源的条件下。此外,PHA合成量随着底物中有机酸比重的增加而增加,充分展现了利用挥发性脂肪酸大量合成PHA的良好前景。     

Synthesis of Short-chain-length/Medium-chain-length Polyhydroxyalkanoate (PHA) Copolymers in Peroxisome of the Transgenic Arabidopsis Thaliana Harboring the PHA Synthase Gene from Pseudomonas sp. 61-3

In this paper, the photosynthetic production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers is reported. The wild-type and highly active doubly mutated PHA synthase 1 (S325T/Q481K, abbreviated ST/QK) genes from Pseudomonas sp. 61-3 were introduced into Arabidopsis thaliana. Peroxisome targeting signal 1 (PTS1) was used to target PHA synthases into the peroxisome to synthesize PHA from the intermediates of the β-oxidation pathway. The transgenic Arabidopsis produced PHA copolymers consisting of 40–57 mol% 3-hydroxybutyrate, 21–49 mol% 3-hydroxyvalerate, 8–18 mol% 3-hydroxyhexanoate, and 2–8 mol% 3-hydroxyoctanoate. The maximum PHA contents were 220μ g/g cell dry weight (cdw) in leaves, and 36μ g/g cdw in stems, respectively. The expression of the ST/QK mutated PHA synthase in leaves gene did not lead to significant difference in PHA content and monomer composition of PHAs, compared to the wild-type PHA synthase gene, suggesting that the supply of monomers may be a rate-determining step of PHA biosynthesis in the peroxisome. However, in stems, there were significant differences dependent on whether the wild-type or ST/QK mutated PHA synthase was expressed. These results suggest that tissue-specific monomer availability is important in determining the final mol% composition of PHA copolymers produced by the peroxisome in plants.     

Reconsideration on the effect of nitrogen on mixed culture polyhydroxyalkanoate production toward high organic loading enrichment history

Effect of nitrogen on mixed culture PHA production was reconsidered. Enrichment history of PHA accumulating culture was discussed. Higher PHA content and biomass growth were achieved in presence of nitrogen. Enrichment strategy toward higher PHA accumulation was investigated. Microbial community succession in PHA accumulation phase was investigated. In most of the operating strategies for mixed microbial cultures polyhydroxyalkanoate (PHA) production, moderate organic loads and low nitrogen concentrations are used, however, the real waste streams contain variable concentrations of carbon and nitrogen. To evaluate the effect of enrichment history on PHA producer and production the various carbon and nitrogen levels were utilized during the accumulation phase. Different operating strategies were applied in three sequencing batch reactors (SBRs) subjected to aerobic dynamic feeding. The maximum PHA production of the enriched cultures under nutrient excess, limitation and starvation (Cmol/Nmol ratio of 8, 40 and ∞, respectively) was evaluated in batch assays. A higher PHA content and biomass growth were achieved in the nutrients presence in comparison to the nutrient starvation condition. The cultures from the SBR treated under short sludge retention time, high organic loading rate, short cycle length (SBR#3) and nutrient excess reached the maximum PHA content (54.9%) and biomass increase (38.9%). Under nutrient limitation, the negative biomass growth was observed under nutrient starvation because of the sampling loss. The succession of microbial communities in SBRs and batch assays was analyzed using terminal restriction fragment length polymorphism. The SBR#3 had the best overall PHA production performance considering its high PHA content and productivity in all nutrient content, it indicates that nitrogen has a substantial impact on PHA yield especially when high organic loading rate enrichment history is involved.     

A Processing,Characterization and Marine Biodegradation Study of Melt-Extruded Polyhydroxyalkanoate (PHA) Films

A series of polyhydroxyalkanoates (PHA), all containing 1% nucleating agent but varying in structure, were melt-processed into films through single screw extrusion techniques. This series consisted of three polyhydroxybutyrate (PHB) and three polyhydroxybutyrate-valerate (PHBV) resins with varying valerate content. Processing parameters of temperature in the barrel (165–173 °C) and chill rolls (60 °C) were optimized to obtain cast films. The gel-permeation chromatography (GPC) results showed a loss of 8–19% of the polymer’s initial molecular weight due to extrusion processing. Modulated differential scanning calorimetry (MDSC) displayed glass transition temperatures of the films ranging from −4.6 to 6.7 °C depending on the amount of crystallinity in the film. DSC data were also used to calculate the percent crystallinity of each sample and slightly higher crystallinity was observed in the PHBV series of samples. X-ray diffraction patterns did not vary significantly for any of the samples and crystallinity was confirmed with X-ray data. Dynamic mechanical analysis (DMA) verified the glass transition trends for the films from DSC while loss modulus (E′) reported at 20 °C showed that the PHBV (3,950–3,600 MPa) had the higher E′ values than the PHB (3,500–2,698 MPa) samples. The Young’s modulus values of the PHB and PHBV samples ranged from 700 to 900 MPa and 900 to 1,500 MPa, respectively. Polarized light microscopy images revealed gel particles in the films processed through single-screw extrusion, which may have caused diminished Young’s modulus and tensile strength of these films. The PHBV film samples exhibited the greatest barrier properties to oxygen and water vapor when compared to the PHB film samples. The average oxygen transmission rate (OTR) and water vapor transmission rate (WVTR) for the PHBV samples was 247 (cc-mil/m²-day) and 118 (g-mil/m²-day), respectively; while the average OTR and WVTR for the PHB samples was 350 (cc-mil/m²-day) and 178 (g-mil/m²-day), respectively. Biodegradation data of the films in the marine environment demonstrated that all PHA film samples achieved a minimum of 70% mineralization in 40 days when run in accordance with ASTM 6691. For static and dynamic incubation experiments in seawater, microbial action resulting in weight loss as a function of time showed all samples to be highly biodegradable and correlated with the ASTM 6691 biodegradation data.     

Cell-recycle fed-batch production of a highly unsaturated polyhydroxyalkanoate from 1,3-butanediol byPseudomonas sp. A33

The recently isolatedPseudomonas sp. A33 was investigated for the production of a highly unsaturated polyhydroxyalkanoate (PHA) containing various alkyl and alkenyl pendent groups from 1,3-butanediol in a cell-recycle fed-batch production mode. The monomer composition and degree of unsaturation in PHA were dependent on the environmental conditions. The production temperature markedly influenced the content, composition, and degree of unsaturation of PHA. As the production temperature decreased from 30 to 10°C, the degree of unsaturation and content of PHA were increased, while the mole percentage of 3-hydroxybutyrate (3HB) was decreased. These temperature effects on the composition of PHA imply that the production can be used as a control variable for the biosynthesis of a highly unsaturated PHA and for the specific regulation of the composition of PHA. The biosynthetic pathway for a highly unsaturated PHA which is based on de novo fatty acid biosynthetic pathway is proposed. For the enhanced production of this functional PHA, a high cell density was achieved by cell-recycle continuous culture at 30°C, and then a large amount of PHA was accumulated at 15°C by fed-batch addition of the feeding solution containing excess 1,3-butanediol. The structures of monomer constituents of polymer were confirmed by gas chromatography—mass spectrometric analysis of trimethylsiyl derivatives of 3-hydroxyalkanoic acids methyl esters.     

Enhancement of simultaneous nitrogen and phosphorus removal using intermittent aeration mechanism

Biological nutrient removal grows into complicated scenario due to the microbial consortium shift and kinetic competition between phosphorus (P)-accumulating and nitrogen (N)-removing microorganisms. In this study, three sequential batch reactors with constant operational conditions except aeration patterns at 6 h cycle periods were tested. Intermittent aeration was applied to develop a robust nutrient removal system aimed to achieve high energy saving and removal efficiency. The results showed higher correspondence of P-uptake, polymeric substance synthesis and glycogen degradation in intermittent-aeration with longer interval periods compared to continuous-aeration. Increasing the intermittent-aeration duration from 25 to 50 min, resulted in higher process performance where the system exhibited approximately 30% higher nutrient removal. This study indicated that nutrient removal strongly depends on reaction phase configuration representing the importance of aeration pattern. The microbial community examined the variation in abundance of bacterial groups in suspended sludge, where the 50 min intermittent aeration, favored the growth of P-accumulating organisms and nitrogen removal microbial groups, indicating the complications related to nutrient removal systems. Successful intermittently aerated process with high capability of simple implementation to conventional systems by elemental retrofitting, is applicable for upgrading wastewater treatment plants. With aeration as a major operational cost, this process is a promising approach to potentially remove nutrients in high competence, in distinction to optimizing cost-efficacy of the system.     

A spectrophotometric method for detection of enzymatic degradation of thin polymer films

An assay method has been developed for monitoring the enzymatic degradation of thin films of translucent polymers. The method was based on the observation that when a solution-cast film of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was exposed to a solution of a depolymerase fromPseudomonas lemoignei, the surface of the film roughened and the film became visibly turbid. This increase in turbidity could be measured spectrophotometrically and was reproducible during the initial stage of degradation. Turbidity correlated very closely with film weight loss early in the degradation but reached a maximum value before extensive degradation had taken place. For a given set of films, this correlation was independent of the concentration of the enzyme used, although it did vary with the mode of enzyme exposure. The turbidity was associated with the exposure of crystalline domains due to the removal of amorphous material from the film surface. The increase in crystallinity at the surface was verified by attenuated total reflectance infrared spectroscopy (ATRIR). In conjunction with SEM, weight loss, and ATRIR, the film turbidity assay provided much semiquantitative insight into the mechanism of the enzymatic degradation reaction. This assay was used to study the enzymatic degradation of films of PHBV solution blended with cellulose acetate esters (CAE). The presence of only 25% of CAE of degree of substitution 2.9 severely hampered the enzymatic degradability of PHBV, a result which is consistent with the environmental degradation of these same samples exposed to activated sludge.     

Degradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Some Soil Aspergillus spp.

Systematic screening of 45 soil fungi for degradation polyhydroxyalkanoic acids (PHAs) has led to the selection of 6 potent Aspergillus isolates belonging to A. flavus, A. oryzae, A. parasiticus, and A. racemosus. Degradation of PHAs as determined by tube assay method revealed that these Aspergillus spp. were more efficient in degrading poly(3-hydroxybutyrate) [P(3HB)] compared to copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB-co-16% 3HV). Moreover, the extent of degradation in mineral base medium was much better than those in complex organic medium. For all the Aspergillus spp. tested, maximum degradation was recorded at a temperature of 37°C with significant inhibition of growth. The optimum pH range for degradation was 6.5–7.0 with degradation being maximum at pH 6.8. The extent of polymer degradation increased with increase in substrate concentration, the optimum concentration for most of the cultures being 0.4% and 0.2% (w/v) for P(3HB) and P(3HB-co-16%3HV) respectively. Supplementation of the degradation medium with additional carbon sources exerted significant inhibitory effect on both P(3HB) and P(3HB-co-16%3HV) degradation.