Increased carbohydrate production from carbon dioxide in randomly mutated cells of cyanobacterial strain Synechocystis sp. PCC 6714: Bioprocess understanding and evaluation of productivities.

Recently, several mutants of Synechocystis sp. PCC 6714 were obtained showing superior PHB content and productivities. Here, the most promising mutant named MT_a24 is compared in detail with the wild-type in controlled photobioreactors. In order to provide an easily scalable and alternative approach to the normally done two-step process -comprising of growth phase and limitation phase- a one-step cultivation was optimized. The multivariate experimental design approach was used for the optimization of the one-step, self-limiting media. During one-step cultivation of MT_a24 with optimized media 30 ±â€¯4% (DCW) corresponding to 1.16 g L-1 PHB was obtained. Using pulse experiments it was demonstrated that phosphate is the key driver of glycogen synthesis in Synechocystis sp. PCC 6714 and it can be used to boost glycogen productivity. The maximum glycogen content acquired was 2.6 g L-1 (76.2% DCW) for mutant MT_a24 using phosphate feeding and carbon dioxide as carbon source.

Bioprocess Engineering Aspects of Sustainable Polyhydroxyalkanoate Production in Cyanobacteria.

Polyhydroxyalkanoates (PHAs) are a group of biopolymers produced in various microorganisms as carbon and energy reserve when the main nutrient, necessary for growth, is limited. PHAs are attractive substitutes for conventional petrochemical plastics, as they possess similar material properties, along with biocompatibility and complete biodegradability. The use of PHAs is restricted, mainly due to the high production costs associated with the carbon source used for bacterial fermentation. Cyanobacteria can accumulate PHAs under photoautotrophic growth conditions using CO2 and sunlight. However, the productivity of photoautotrophic PHA production from cyanobacteria is much lower than in the case of heterotrophic bacteria. Great effort has been focused to reduce the cost of PHA production, mainly by the development of optimized strains and more efficient cultivation and recovery processes. Minimization of the PHA production cost can only be achieved by considering the design and a complete analysis of the whole process. With the aim on commercializing PHA, this review will discuss the advances and the challenges associated with the upstream processing of cyanobacterial PHA production, in order to help the design of the most efficient method on the industrial scale.

Increased poly-ß-hydroxybutyrate production from carbon dioxide in randomly mutated cells of cyanobacterial strain Synechocystis sp. PCC 6714: Mutant generation and characterization.

Photosynthetic Poly-ß-hydroxybutyrate (PHB) productivity in cyanobacteria needs to be increased to make cyanobacterial derived bioplastics economically feasible and competitive with petroleum-based plastics. In this study, high PHB yielding mutants of Synechocystis sp. PCC 6714 have been generated by random mutagenesis, using UV light as a mutagen. The selection of strains was based on PHB content induced by nitrogen and phosphorus starvation. The fast growing mutant MT_a24 exhibited more than 2.5-fold higher PHB productivity than that of the wild-type, attaining values of 37 ±â€¯4% dry cell weight PHB. The MT_a24 was characterized for phenotypes, CO2 uptake rate and gene expression levels using quantitative PCR. Genome sequencing showed that UV mutagenesis treatment resulted in a point mutation in the ABC-transport complex, phosphate-specific transport system integral membrane protein A (PstA). The MT_a24 shows potential for industrial production of PHB and also for carbon capture from the atmosphere or point sources.

Prediction of filamentous process performance attributes by CSL quality assessment using mid-infrared spectroscopy and chemometrics.

Every biopharmaceutical production process aims for control strategies to achieve process robustness in order to ensure consistent product quality. Process variability can origin from process parameters, the biological nature as well as from high lot-to-lot variability of raw materials. In filamentous processes raw materials with very complex matrices, such as corn steep liquor (CSL), are used, which are especially challenging to characterize. In this study, CSL was characterized in detail for its ingredients presenting an overall composition of its matrix of 50 analyzed components (19 amino acids, 5 organic acids, 8 reducing sugars, 7 water-soluble vitamins and 11 trace elements/minerals) in order to facilitate analytical reduction to fingerprinting methods FT-MIR was evaluated as fast and non-destructive spectroscopic fingerprinting method for adequate assessment of CSL quality. Feasibility of this method was shown by the correlation of certain bands in the spectra to substance groups present in CSL, such as the Amide I and II band and amino acids, respectively. Additionally, applicability of FT-MIR could be shown for classification of different CSL lots differing in provider and corn quality as well as for predictability of process performance attributes. The latter was demonstrated on a fed-batch filamentous fungi process for the production of antibiotics. By multivariate data analysis, it could be shown that CSL quality assessment via FT-MIR can be used for the prediction of maximal biomass generated in the process, with a correlation coefficient R2 of 0.964, as well as for the prediction of an unwanted impurity. The combination of a fast and easy method for CSL quality assessment and correlations of this quality with process performance attributes may facilitate the establishment of a risk-based acceptance criteria for raw material quality release of CSL. As CSL is a frequent used raw material, we believe that this method will also be useful for other processes and that CSL quality assessment is of high relevance in academia and industry.

Photosynthetic poly-ß-hydroxybutyrate accumulation in unicellular cyanobacterium Synechocystis sp. PCC 6714.

Poly-ß-hydroxybutyrate (PHB) production from CO2 has the potential to reduce the production cost of this biodegradable polyesters, and also to make the material more sustainable compared to utilization of sugar feedstocks. In this study the unicellular cyanobacterium, Synechocystis sp. PCC 6714 has been identified as an unexplored potential organism for production of PHB. Synechocystis sp. PCC 6714 was studied under various cultivation conditions and nutritional limitations. Combined effects of nitrogen and phosphorus deficiency led to highest PHB accumulation under photoautotrophic conditions. Multivariate experimental design and quantitative bioprocess development methodologies were used to identify the key cultivation parameters for PHB accumulation. Biomass growth and PHB accumulation were studied under controlled defined conditions in a lab-scale photobioreactor. Specific growth rates were fourfold higher in photobioreactor experiments when cultivation conditions were controlled. After 14 days of cultivation in nitrogen and phosphorus, limited media intracellular PHB levels reached up to 16.4% from CO2. The highest volumetric production rate of PHB was 59 ± 6 mg L-1 day-1. Scanning electron microscopy of isolated PHB granules of Synechocystis sp. PCC 6714 cultivated under nitrogen and phosphorus limitations showed an average diameter of 0.7 µm. The results of this study might contribute towards a better understanding of photoautotrophic PHB production from cyanobacteria.