A fed-batch fermentation process for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production by Yangia sp. ND199 using molasses as substrate

A locally isolated halophilic bacterium, Yangia sp. ND199 was able to use molasses as substrate for copolymers poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production. Cell dry weight (CDW) of 6.37 g/l, P(3HB-co-3HV) content of 43.1 wt% and P(3HB-co-3HV) concentration of 2.75 g/l were obtained by Yangia sp. after 60 h of cultivation in flask. In a batch cultivation mode in a fermentor, the CDW was increased to 9.1 g/l but P(3HB-co-3HV) content was decreased to 37 wt%. Fed-batch fermentation with two different nutrient feeding strategies was used. High CDW of 54.8 g/l was obtained after 54 h of cultivation but P(3HB-co-3HV) content was still low (39.8 wt%). Two-step fed-batch fermentation with two different nutrient feeding strategies was then designed. High CDW of 50 g/l and P(3HB-co-3HV) content of 52.9 wt% were obtained after 54 h of cultivation. The two-step fed-batch process designed here for the production of P(3HB-co-3HV) by Yangia sp. ND199 can be developed and used for further studies

BIOCONVERSION OF GLYCEROL TO POLYHYDROXYALKANOATE BY HALOPHILIC BACTERIA ISOLATION FROM MANGROVE SOIL SAMPLES

Polyhydroxyalkanoate (PHA) is a group of biodegradable polymer, synthesize by many microorganisms. PHA provide a good fully degradable alternative to petrochemical plastics. One of the major bottlenecks in the commercial application of PHAs is their high price as compared to the conventional petroleum-based plastic materials. . A potential source for low-cost PHA production is to utilize glycerol (byproduct of biodiesel production process) as carbon source. In this study, we isolated seventeen bacteria strains, which were able to synthesize PHA from glycerol. Among them, three of the strains named VK75, VK98 and VK129 (fast growth and high PHA accumulation) were chosen for further study. They are moderately halophilic and neutrophilic bacteria, able to metabolize different nitrogen sources. Highest PHA content of 61% was obtained by strain VK75 after 30 h of cultivation in flask using KNO3 as nitrogen source. Maximum CDW of 6 g/l was obtained by strain VK75 after 30 h of cultivation in bioreactor. Futher studies are being doing to optimize condition for both cell growth and PHA accumulation by three selected strains

DIFFERENT FRUCTOSE FEEDING STRATEGIES FOR POLY(3-HYDROXYBUTYRATE) PRODUCTION BY Yangia sp. ND199

Yangia sp. ND199 is a halophilic bacterium isolated from mangrove soil sample. This strain was able to produce polyhydroxyalkanoate (PHA) from different carbon sources. Only homopolymer poly(3-hydroxybutyrate) (PHB) was synthesized when fructose was used as carbon source. The bacterium can accumulate high PHB content during exponential phase. Maximum cell dry weight (CDW) of 7.8 g/l and PHB content of 49 wt% were obtained after 27 h of cultivation in batch fermentation. High CDW and PHB content were achieved by using fed-batch fermentation with different fructose feeding strategies. The highest CDW of 78.5 g/l, PHB content of 67.5 wt%, and PHB productivity of 1 g/l/h were obtained by using two-stage fed-batch fermentation, is among the highest reported so far for PHB production by halophilic bacteria.       

RECOVERY OF POLY(3-HYDROXYBUTYRATE) FROM Yangia sp. ND199 BY SIMPLE DIGESTION WITH SODIUM HYPOCHLORITE

Polyhydroxyalkanoate (PHA) is a biodegradable polymer synthesized intracellularly by many microorganisms. After extraction from the cells, they possess the common features of non-toxic, biocompatible, biodegradable and recyclable. These features render them highly competitive with polypropylene or other petroleum-derived plastics, especially in medicine. The isolation and purification of PHA from bacterial cells are the key step of PHA production process. There are several methods have been used for OHA purification, these include solvent extraction, enzymatic hydrolysis, and chemical digestion. In this study we used chemical digestion method for purification of PHB from Yangia sp. ND199. Among various chemical tested (NaOH, HCl, KOH, NaOCl, and SDS), NaOCl was found as an efficient chemical for PHB recovery and PHB purity from Yangia sp. ND199. The optimal purity and recovery conditions were a ratio of 1:1 (v/v, solution containing 100 g/l bacterial cells and solution containing 6% NaOCl), a 30 oC temperature and a 1 h treatment time. Under such conditions, a purity of 99% and a recovery of 94% were obtained. This method is sumple and can be developed and used for industrial scale

Seasonal variation of paralytic and amnesic shellfish toxicities in bivalves and microalgae in Haiphong area, Vietnam

Monitoring survey was conducted to know the distribution and seasonal variation of PSP and ASP toxicities in bivalves and the abundance of toxic microalgae in Haiphong area, Vietnam. Sampling was carried out at the stations in Cat Ba and Do Son every two weeks from February 2002 to March 2004. Low levels of PSP and DA were detected in shellfish samples from both stations, showing seasonal and yearly variation. Toxicity of plankton samples also showed fluctuation, though the clear correlation could not be observed with the shellfish toxicities. HPLC or LC-MSMS analysis provided clear evidence of shellfish contamination with these toxins and indicated the existence of causative microalgae for these toxicities in this area. Several species of Alexandrium such as A. minutum, A. tamiyavanichii, A. ostenfeldii, A. tamarense were found, though the abundance of them was very low. On the other hand, massive bloom of Pseudo-nitzschia occurred in January at both stations. However, these blooms did not always cause the increase of DA level both in plankton and shellfish samples

Seasonal variation of paralytic and amnesic shellfish toxicities in bivalves and microalgae in Haiphong area, Vietnam

Monitoring survey was conducted to know the distribution and seasonal variation of PSP and ASP toxicities in bivalves and the abundance of toxic microalgae in Haiphong area, Vietnam. Sampling was carried out at the stations in Cat Ba and Do Son every two weeks from February 2002 to March 2004. Low levels of PSP and DA were detected in shellfish samples from both stations, showing seasonal and yearly variation. Toxicity of plankton samples also showed fluctuation, though the clear correlation could not be observed with the shellfish toxicities. HPLC or LC-MSMS analysis provided clear evidence of shellfish contamination with these toxins and indicated the existence of causative microalgae for these toxicities in this area. Several species of Alexandrium such as A. minutum, A. tamiyavanichii, A. ostenfeldii, A. tamarense were found, though the abundance of them was very low. On the other hand, massive bloom of Pseudo-nitzschia occurred in January at both stations. However, these blooms did not always cause the increase of DA level both in plankton and shellfish samples

Biogeochemical Impacts of a Black Carbon Wet Deposition Event in Halong Bay, Vietnam

Black carbon (BC) is emitted to the atmosphere during biomass, biofuel, and fossil fuel combustion, and leaves the atmosphere via dry or wet deposition on land and on the ocean. On a global scale, wet deposition accounts for about 80% of the total atmospheric BC inputs to the ocean. The input of BC particles to the ocean can enrich surface waters with carbon and associated elements, and owing to high porosity and surface-active properties, BC can alter biogeochemical cycles by sorbing dissolved compounds and promoting aggregation. The rain-mediated input of BC to the ocean and its consequences on nutrient concentrations and particle dynamics were studied in Halong Bay, Vietnam, during a 24-h cycle impacted by short and heavy rainfall events. This study suggests that once introduced in the surface ocean via wet deposition, BC sorbs dissolved organic matter (DOM) and stimulates aggregation processes. The observed wet deposition events were characterized by sudden and pulsed inputs of BC particles that created a thin layer of sinking surface-active aggregates, acting as a net-like scavenger for DOM, nutrients (especially phosphate), and small particles. In addition, the wet deposition events coincided with an enrichment of nutrients in the surface microlayer, with an excess input of nitrogen relative to phosphorus leading to an increase of the molar N:P ratio from 24:1 to 37:1. In the underlying water, the molar N:P ratio also increased (i.e., from 39:1 to 64:1), and this can be attributed to the preferential scavenging of dissolved P-compounds on sinking BC-aggregates

Pichia pastoris versus Saccharomyces cerevisiae:a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. RESULTS: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. CONCLUSION: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production