In situ based surface-enhanced Raman spectroscopy (SERS) for the fast and reproducible identification of PHB producers in cyanobacterial cultures.

The production of polyhydroxybutyrate (PHB) by autotrophic fermentation of cyanobacteria has received increasing interest in the light of carbon emission reducing process strategies. Biotechnological approaches are in development to optimize the yield of PHB, including adapted cultivation media, characterized by a limitation of key nutrients: cyanobacteria accumulate PHB as energy storage molecules under limited growth conditions. Since there is an increasing demand for fast, simple and reliable analytics, we report the establishment of surface enhanced Raman spectroscopy (SERS) as a suitable monitoring tool for up scaled PHB production processes. Both, pure Ag-colloids mixed with bacterial culture, and in situ prepared colloids (Ag-Synechocystis), generated on the cell surface directly, were successfully applied and evaluated for this purpose. SERS measurements with in situ prepared Ag-colloids improved the reproducibility of Raman signals from 54.8% to 93.9%. The measurement time could be reduced significantly, completing our secondary goal. The quality of classically and in situ prepared Ag-colloids was monitored by zeta potential measurements and scanning electron microscopy (SEM) respectively. For data interpretation and statistical model-building an in house written code in the open source software RStudio was implemented. It was applied for the differentiation of PHB producers at the cellular level, revealing heterogeneities within sample groups regarding the PHB amount accumulated. The results obtained using the statistical model were validated as well and were complementary to the reference HPLC analysis. Therefore, a fast and reliable identification in situ SERS tool for the selection of the most promising cyanobacterial PHB production was established.

Novel unexpected functions of PHA granules.

Polyhydroxyalkanoates (PHA), polyesters accumulated by numerous prokaryotes in the form of intracellular granules, have been for decades considered being predominantly storage molecules. However, numerous recent discoveries revealed and emphasized their complex biological role for microbial cells. Most of all, it was repeatedly reported and confirmed that the presence of PHA granules in prokaryotic cells enhances stress resistance and robustness of microbes against various environmental stress factors such as high or low temperature, freezing, oxidative, and osmotic pressure. It seems that protective mechanisms of PHA granules are associated with their extraordinary architecture and biophysical properties as well as with the complex and deeply interconnected nature of PHA metabolism. Therefore, this review aims at describing novel and unexpected properties of PHA granules with respect to their contribution to stress tolerance of various prokaryotes including common mesophilic heterotrophic bacteria, but also extremophiles or photo-autotrophic cyanobacteria. KEY POINTS: • PHA granules present in bacterial cells reveal unique properties and functions. • PHA enhances stress robustness of bacterial cells.

Comparative assessment of environmental impacts of 1st generation (corn feedstock) and 3rd generation (carbon dioxide feedstock) PHA production pathways using life cycle assessment.

Polyhydroxyalkanoates (PHA) are bio-based and biodegradable alternatives to conventional plastic types and have the potential to reduce the environmental impacts along the life cycle. In comparison to already established production routes for PHA (heterotrophic production) based on renewable feedstock like glucose (first generation feedstock), novel production routes, such as the photoautotrophic production of PHA based on CO2 as feedstock (third generation feedstock) could offer new perspectives with regard to the reduction in the environmental impacts. To quantify the environmental impacts of PHA produced via photoautotrophic and heterotrophic production pathways, life cycle assessment (LCA) methodology based on ISO 14040/44 was applied, thus conducting a first of its kind comparative study for PHA based on third generation feedstock. The results show that the photoautotrophic production of PHA has advantages in comparison to heterotrophic PHA based on glucose originating from corn as feedstock in all the assessed environmental impact categories, thus showing the environmental potential of novel production routes for bioplastics. Additionally, the results of the LCA show that the chloroform-based extraction method, commonly used in the downstream processes of both the technologies, has a significant contribution of environmental impacts in the production of PHA. Therefore, the reduction of chloroform loss during the extraction process can reduce its environmental impact. Our results indicate that PHA production from CO2 using the photoautotrophic production route is a promising technology with regard to the environmental impacts when compared to the heterotrophic production based on glucose feedstock.

PHB Producing Cyanobacteria Found in the Neighborhood-Their Isolation, Purification and Performance Testing.

Cyanobacteria are a large group of prokaryotic microalgae that are able to grow photo-autotrophically by utilizing sunlight and by assimilating carbon dioxide to build new biomass. One of the most interesting among many cyanobacteria cell components is the storage biopolymer polyhydroxybutyrate (PHB), a member of the group of polyhydroxyalkanoates (PHA). Cyanobacteria occur in almost all habitats, ranging from freshwater to saltwater, freely drifting or adhered to solid surfaces or growing in the porewater of soil, they appear in meltwater of glaciers as well as in hot springs and can handle even high salinities and nutrient imbalances. The broad range of habitat conditions makes them interesting for biotechnological production in facilities located in such climate zones with the expectation of using the best adapted organisms in low-tech bioreactors instead of using "universal" strains, which require high technical effort to adapt the production conditions to the organism's need. These were the prerequisites for why and how we searched for locally adapted cyanobacteria in different habitats. Our manuscript provides insight to the sites we sampled, how we isolated and enriched, identified (morphology, 16S rDNA), tested (growth, PHB accumulation) and purified (physical and biochemical purification methods) promising PHB-producing cyanobacteria that can be used as robust production strains. Finally, we provide a guideline about how we managed to find potential production strains and prepared others for basic metabolism studies.