Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers

Azotobacter vinelandii is a gram-negative soil bacterium that produces two biopolymers of biotechnological interest, alginate and poly(3-hydroxybutyrate), and it has been widely studied because of its capability to fix nitrogen even in the presence of oxygen. This bacterium is characterized by its high respiration rates, which are almost 10-fold higher than those of Escherichia coli and are a disadvantage for fermentation processes. On the other hand, several works have demonstrated that adequate control of the oxygen supply in A. vinelandii cultivations determines the yields and physicochemical characteristics of alginate and poly(3-hydroxybutyrate). Here, we summarize a review of the characteristics of A. vinelandii related to its respiration systems, as well as some of the most important findings on the oxygen consumption rates as a function of the cultivation parameters and biopolymer production.

Bioconversion of sugarcane molasses to poly(3-hydroxybutyrate-co-3- hydroxyvalerate) by endophytic Bacillus cereus RCL 02

Sugarcane molasses, the by-product of sugar industry are rich in nutrients, growth factors and minerals and areefficiently utilized by the microorganisms for growth and production of primary as well as secondary metabolitesof commercial importance. In this communication, we report the utilization of sugarcane molasses as the solesource of carbon for the production of copolymers of polyhydroxyalkanoates (PHAs) with improved materialproperties. The endophytic bacterium Bacillus cereus RCL 02 (MCC 3436) produced 7.8 g/L of PHA whengrown in MS medium with 4% (w/v) sugarcane molasses under batch cultivation. Proton nuclear magneticresonance (1H NMR) analysis revealed that the copolymer so produced contain 12.4 mol% 3-hydroxyvalerate(3HV) along with 87.6 mol% 3-hydroxybutyrate (3HB). The copolyester, P(3HB-co-12.4 mol%-3HV)has been isolated and purified following standard solvent extraction method and partially characterized byFourier-transform infrared (FTIR), X-ray diffraction (XRD), thermogravimetric (TG) and differential scanningcalorimetric (DSC) analysis. The material and thermal properties of the copolyester so produced indicated itspotential for industrial application.

Effect of reducing the activity of respiratory chain on biosynthesis of poly(3-hydroxybutyrate-co-lactate) in Escherichia coli / 生物工程学报

Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] belongs to the polyhydroxyalkanoates (PHA) family and possesses promising properties including biocompatibility and biodegradability. In this study, we directly synthesized P(3HB-co-LA) with glucose by introducing the β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha, the engineered propionate CoA transferase from Clostridium propionicum and the engineered polyhydroxyalkanoate synthase from Pseudomonas fluorescens strain 2P24 into Escherichia coli. The polymer content was 83.9% (W/W), and the molar percentage of lactate reached 1.6%. On this basis, in order to accumulate lactate, we reduced the activity of respiratory chain by deleting the ubiX gene, which is involved in the synthesis of coenzyme Q8. Moreover, we removed the dld gene to avoid the conversion of lactate to pyruvate during the fermentation. With these manipulations, the molar percentage of lactate in the polymer was improved to 14.1%, with an 81.7% (W/W) of polymer content. The test results indicated that the strategy of reducing the activity of respiratory chain effectively increased the lactate units in the polymer, and it contributed a new approach to change the content of monomer components in the polymer.

Evaluation of poly(3-hydroxybutyrate) production by microbiota endogenous to oleaginous plants

Aims@#Microbiota endogenous to oleaginous plants have attracted special attention in recent years for their biotechnological potentials and applications including the production of biodegradable biopolyester poly(3- hydroxybutyrate) [P(3HB)] as an alternative to thermoplastics. The present study is aimed to screen the endophytic bacteria of selected oleaginous plants such as Arachis hypogaea L., Brassica napus L., Brassica nigra L., Helianthus annuus L., Ricinus communis L. and Sesamum indicum L. for the production of P(3HB). @*Methodology and results@#Bacteria endogenous to the oleaginous plants were isolated from surface sterilized healthy tissues following sterilization with 70% ethanol and 0.5% sodium hypochlorite and screened for P(3HB) production in mineral salts medium. Nile blue A staining method was used for detection of intracellular P(3HB), while the accumulated biopolyester was quantified spectrophotometrically following chemical conversion to chrotonic acid by treating with sulfuric acid. Five potent P(3HB) accumulating isolates have been selected and identified as Cellulosimicrobium cellulans AHS 01 (KX458038), Beijerinckia fluminensis AHR 02 (KX458039), Exiguobacterium acetylicum BNL 103 (KX458037), Bacillus toyonensis BNS 102 (KX458036) and Bacillus cereus RCL 02 (KX458035) based on morphological, physio-biochemical and 16S rDNA sequence analysis. These endogenously growing bacterial isolates accumulated intracellular biopolyester accounting 43-62% of their cell dry weight (CDW) when grown in mineral salts medium supplemented with yeast extract. Intracellular accumulation of P(3HB) by these isolates have also been confirmed by FTIR spectral analysis of lyophilized cell mass and 1HNMR spectra of the extracted polymer. @*Conclusion, significance and impact of study@#These findings, first of its kind point to exploration of endogenous bacterial communities of oil-producing plants as a potential bioresource for production of P(3HB) bioplastics in a sustainable manner.

Hypoxic three-dimensional culture microenvironment promotes proliferation of bone marrow mesenchymal stem cells through HIF-1α signaling pathway / 中国胸心血管外科临床杂志

@#Objective To investigate the effects of hypoxic three-dimensional culture microenvironment on the proliferation of bone marrow mesenchymal stem cells and its mechanism. Methods P5 generation mouse bone marrow mesenchymal stem cells and P (3HB-co-4HB) were co-cultured under normoxic three-dimensional (20%) and hypoxic three-dimensional microenvironment (4%) respectively. After 24 hours, the proliferation of the two groups was determined by CCK-8 method. The expression of HIF-1α gene was detected by real-time quantitative PCR after 12 hours. Western blotting was used to detect the expression of HIF-1α protein after 24 hours. Results After 24 hours, the CCK-8 method showed that the OD value of the hypoxia group was significantly higher than that of the normoxia group (0.455±0.027 vs. 0.352±0.090, n=12, P<0.05). After 12 hours of hypoxic culture, the expression level of HIF-1α mRNA in the hypoxia group was significantly higher than that in the normoxia group (P<0.05). Compared with the normoxia group (0.47± 0.05), the relative expression level of HIF-1α protein in the hypoxia group (0.63±0.06) significantly increased in the Western blotting after 24 hours (n=3, P<0.05). Conclusion The hypoxic three-dimensional microenvironment can promote the proliferation of bone marrow mesenchymal stem cells, which may be related to the activation of HIF-1α signaling pathway.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

Isolation and identification of bacteria and fungi growing spontaneously on polyhydroxyalkanoate pellets recovered by a new biological process

Aims: The study was carried out to isolate and identify the spontaneously growing populations of bacteria and fungi on the surface of biologically recovered polyhydroxyalkanoate (PHA) copolymer, poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) [P(3HB-co-3HHx)]. Methodology and results: Large-scale PHA biosynthesis was carried out using 300 L fermenter and a biological methodology developed in our laboratory was utilized for PHA recovery. Using standard microbiological and molecular biology techniques the naturally growing microbial populations on the surface of biologically recovered PHA were identified. Scanning electron microscopy (SEM) analysis showed that the identified bacterial (Bacillus cereus and Burkholderia cepacia) and fungal isolates (Aspergillus niger, Byssochlamys nivea, Penicillium citrinum and Penicillium griseofulvum) were able to grow on and degrade the P(3HB-co-3HHx) copolymer. Conclusion, significance and impact of study: This is the first report on biologically recovered PHA pellet addressing the occurrence of microorganisms that grew spontaneously on it during storage under laboratory conditions. Fungi appeared to be dominant over bacteria in their ability to colonize the biologically recovered PHA.

Screening and Production of Biodegradable Polyester Poly(3-hydroxybutyrate) by Bacteria Endophytic to Brassica nigra L.

Aims: Poly(3-hydroxybutyrate) [P(3HB)], the microbially produced biodegradable thermoplastics has find wide range of applications in recent years. Development of low cost production strategies utilizing novel organisms is a crucial challenge. Present study is aimed to isolate and screen bacterial endophytes of Brassica nigra L. for the production of P(3HB). Place and Duration of Study: The experiments were performed in the Microbiology Laboratory, Department of Botany, University of Calcutta, Kolkata during 2012-2014. Methodology: Culturable bacterial endophytes were isolated from surface sterilized healthy tissues of B. nigra L. and screened for P(3HB) production in mineral salts medium. The chloroform extracted dried polymer was treated with H2SO4 and quantified spectrophotometrically at 235 nm. Results: About 78% of the bacterial endophytes recovered from surface sterilized B. nigra L. tissues showed different degrees of P(3HB) accumulation. Isolates (9) showing P(3HB) accumulation exceeding 10% of the cell dry weight (CDW) were characterized and tentatively identified as members of Bacillus, Pseudomonas, Xanthomonas, Alcaligenes and Acetobacter. The most potent isolate, BNL 06 identified as Bacillus pumilus BNL 06 (GenBank Accession No. KP202723), accumulated P(3HB) accounting 18% of CDW with an yield of 0.55 g/l. Finally the nature of the polymer was further confirmed by FTIR analysis. Conclusion: Exploration of the endophytic bacterial diversity of B. nigra L. have clearly revealed the potential of Bacillus pumilus BNL 06 for P(3HB) production as an alternative source of thermoplastics.

Mixed Cultures Fermentation for the Production of Poly- ß-hydroxybutyrate

Poly-ß-hydroxybutyrate (PHB) is a biodegradable intracellular microbial product produced by many bacteria and it is comparable to some of the petrochemical derived thermoplastics such as polypropylene. One of the main barriers for the commercial exploitation is the high cost of the substrate for the production of biopolymer. The utilization of mixed microbial cultures facilitates the use of complex substrates thereby reducing the cost of PHB production. In the present study, mixed culture systems were evaluated for PHB production. Bacillus firmus NII 0830 was used for the production of PHB since it accumulates a large amount of PHB and a second organism Lactobacillus delbrueckii NII 0925 was used to provide lactic acid. FTIR and 1H NMR analyses revealed that the PHB extracted from pure culture and mixed culture showed exact match to that of standard PHB. Biodegradation studies of the PHB blends showed 87% degradation. It was also found that a consortium of organisms degraded the films faster than a single organism.

Preparation and cytocompatibility of novel porous calcium metaphosphate composite membranes / 中国组织工程研究

BACKGROUND:Porous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes prepared previously is too thick and uneven in holes. OBJECTIVE:To prepare the thin even porous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membrane, and to evaluate the cytocompatibility and differentiation capacity. METHODS:Porous and nonporous, thin and even poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes were prepared by phase separation method. Its thickness and weight loss rate were determined. Human bone marrow mesenchymal stem cel s were cocultured with porous and nonporous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes for 7 days. Ultrastructure of composite membranes was observed under the scanning electron microscopy. Surface markers of the bone marrow mesenchymal stem cel s on the composite membranes were analyzed using flow cytometry. RESULTS AND CONCLUSION:The thickness of the porous and nonporous composite membranes was (0.041 ± 0.005) mm and (0.058±0.004) mm. Weight loss rates of porous and nonporous composite membranes were respectively 19.93%and 7.64%at 24 hours. Calcium metaphosphate particles were evenly distributed in porous and nonporous composite membrane. Cel s spread entirely, showing spindle shape. Calcium metaphosphate particles were evenly distributed in porous composite membrane. Pore in porous composite membranes was also uniformly distributed, and pore size was about 2-8μm. Cel s spread entirely, showing polygonal shape with multiple tentacles. The tentacles of some cel s entered into the scaffold. CD105, CD90, CD44, CD29 and CD73 expression was detected in porous and nonporous composite membranes. There was no significant difference in cel-positive rate. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes prepared in this study has good biocompatibility and could not promote cel differentiation.

Waste cooking oil as substrate for biosynthesis of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate): Turning waste into a value-added product

Aims: Improper disposal of domestic wastes, such as waste cooking oil (WCO), contributes to the deterioration of the environment and may lead to health problems. In this study, we evaluated the potential of plant-based WCO as a carbon source for the commercial biosynthesis of the bio-plastics, poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). The consumption of WCO for this purpose would mitigate their pollution of the environment at the same time. Methodology and Results: WCO collected from several cafeterias in USM was tested as the carbon source for polyhydroxyalkanoates (PHA) production. A selection of suitable nitrogen source was first conducted in order to obtain an acceptable number of dry cell weight (DCW) and PHA content. Urea was found to be a suitable nitrogen source for the two bacterial strains used in our study, Cupriavidus necator H16 and its transformed mutant, C. necator PHB¯4 harboring the PHA synthase gene of Aeromonas caviae (PHB¯4/pBBREE32d13). With WCO as the sole carbon source, C. necator H16 yielded a relatively good dry cell weight (DCW=25.4 g/L), with 71 wt% poly(3-hydroxybutyrate) P(3HB) content. In comparison, the DCW obtained with fresh cooking oil (FCO) was 24.8 g/L. The production of poly(3 hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from WCO by the transformant C. necator PHB¯4 was comparable, yielding a DCW of 22.3 g/L and P(3HB-co-3HHx) content of 85 wt%. Lipase activities for both bacterial strains reached a maximum after 72 h of cultivation when time profile was conducted. Conclusion, significance and impact of study: The use of WCO as a carbon source in the biosynthesis of the bioplastic, PHA, turns a polluting domestic waste into a value-added biodegradable product. This renewable source material can thus be exploited for the low cost production of PHA.

Experimental study on biocompatibility between canine bone marrow stem cells and copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate / 中国病理生理杂志

AIM:To evaluate the biocompatibility between copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) and bone marrow stem cells (BMSCs). METHODS: Canine BMSCs were isolated and cultured. The cells in passage 3-4 were seeded onto the PHBV films and three-dimensional foams. The seeded cells were observed under inverted microscope for morphology and cell attachment onto the PHBV films at 1, 2 or 3 weeks after seeding. With 4% paraformaldehyde formalin and staining, the protein content in seeded cells was determined by bicinchoninic acid assay (BCA). The content of DNA was quantified using the Hoechst 33258 assay. RESULTS: Observation under inverted microscope showed that the PHBV fabric was fairly thickness, lucency is weak. Unser contrast phase microscope, PHBV fabric was uneasy to be observed. Most cells attached onto the PHBV films 2 h after seeding, and extended well and acquired a spindle fibrecyte-like morphology 3 d later. Moreover, on the three-dimensional foams, the seeded cells lay in micropores and grew tri-dimensionally. The conjunction of cells appeared about 1 week, and extended at 3 weeks, with a large amount of extracellular matrix around cells. The content of DNA and protein has no significant difference with control group. CONCLUSION: As a kind of tissue engineering material for BMSCs seeding, PHBV has an excellent biocompatibility.

Studies on Synthesis of Poly (3-Hydroxybutyrate-co-3-Hydroxyhexanoate) by a Sinorhizobium fredii Strain / 微生物学通报

The potential of a Sinorhizobium fredii strain producing a copolymer polyhydroxyalkanoate(PHA)from glucose and sodium decanoate substrates was studied in this paper.Using orthogonal design in a flask-shaker culture system,the culture medium,some culture conditions and vital regulation conditions for polymer synthesis were optimized.These optimized results were applied into further studies in two-stage fed-batch with a 10L fermentor.The whole culture process consisted of two stages,that is,the cell growth and the copolymer production.The first stage was for the cell growing to a desired biomass and the second was for the copolymer synthesis.For producing PHA polymers,the selected 8 mM sodium decanoate was added into the broth by adopting a two-step adding method for avoiding of foaming when the biomass had approached 28.5g/L dry cell.The maximum P(HB-HH)production could be 17.55 g/L with a monomer ratio of 79.4%(W/W)3-HB and 20.6%(W/W)3-HH.The molecule constitute of the copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)[P(HB-HH)] and its molecular weight(MW)is 1.4?105D.The results demonstrated that the employed S.fredii strain could be a potential candidate for industrial production of the copolymer.The fermentation parameters acquired in the experimental system offered some valuable references for studying large-scale production of the copolymer.