Improving Bioprocess Conditions for the Production of Prodigiosin Using a Marine Serratia rubidaea Strain.

The enormous potential attributed to prodigiosin regarding its applicability as a natural pigment and pharmaceutical agent justifies the development of sound bioprocesses for its production. Using a Serratia rubidaea strain isolated from a shallow-water hydrothermal vent, optimization of the growth medium composition was carried out. After medium development, the bacterium temperature, light and oxygen needs were studied, as was growth inhibition by product concentration. The implemented changes led to a 13-fold increase in prodigiosin production in a shake flask, reaching 19.7 mg/L. The conditions allowing the highest bacterial cell growth and prodigiosin production were also tested with another marine strain: S. marcescens isolated from a tide rock pool was able to produce 15.8 mg/L of prodigiosin. The bioprocess with S. rubidaea was scaled up from 0.1 L shake flasks to 2 L bioreactors using the maintenance of the oxygen mass transfer coefficient (kLa) as the scale-up criterion. The implemented parameters in the bioreactor led to an 8-fold increase in product per biomass yield and to a final concentration of 293.1 mg/L of prodigiosin in 24 h.

Isolation and Characterization of a Serratia rubidaea from a Shallow Water Hydrothermal Vent.

Microbial life present in the marine environment has to be able to adapt to rapidly changing and often extreme conditions. This makes these organisms a putative source of commercially interesting compounds since adaptation provides different biochemical routes from those found in their terrestrial counterparts. In this work, the goal was the identification of a marine bacterium isolated from a sample taken at a shallow water hydrothermal vent and of its red product. Genomic, lipidomic, and biochemical approaches were used simultaneously, and the bacterium was identified as Serratia rubidaea. A high-throughput screening strategy was used to assess the best physico-chemical conditions permitting both cell growth and production of the red product. The fatty acid composition of the microbial cells was studied to assess adaptation at the lipid level under stressful conditions, whilst several state-of-the-art techniques, such as DSC, FTIR, NMR, and Ultra-High Resolution Qq-Time-of-Flight mass spectrometry, were used to characterize the structure of the pigment. We hypothesize that the pigment, which could be produced by the cells up to 62 °C, is prodigiosin linked to an aliphatic compound that acts as an anchor to keep it close to the cells in the marine environment.

Optimization of Multiparameters for Increased Yields of Cytochrome B5 in Bioreactors.

The production of recombinant proteins is gaining increasing importance as the market requests high quality proteins for several applications. However, several process parameters affect both the growth of cells and product yields. This study uses high throughput systems and statistical methods to assess the influence of fermentation conditions in lab-scale bioreactors. Using this methodology, it was possible to find the best conditions to produce cytochrome b5 with recombinant cells of Escherichia coli. Using partial least squares, the height-to-diameter ratio of the bioreactor, aeration rate, and PID controller parameters were found to contribute significantly to the final biomass and cytochrome concentrations. Hence, we could use this information to fine-tune the process parameters, which increased cytochrome production and yield several-fold. Using aeration of 1 vvm, a bioreactor with a height-to-ratio of 2.4 and tuned PID parameters, a production of 72.72 mg/L of cytochrome b5 in the culture media, and a maximum of product to biomass yield of 24.97 mg/g could be achieved.

Cultivation-based strategies to find efficient marine biocatalysts.

Marine bacteria have evolved to survive in the marine environment by using unique physiological, biochemical and metabolic features and the ability to produce enzymes and compounds which may have commercial value. The Azores archipelago presents several ecosystems with strong volcanic activity where bacteria thrive under e.g. high temperatures. In this study, samples collected in the island of São Miguel were screened for biocatalysts possessing e.g. lipase, esterase, amylase, and inulinase activities. After isolation of several hundred bacterial strains, high throughput screening methods allowed the fast identification of biocatalysts. The first cultivation tests were performed on 24-wells microtiter plates with online oxygen monitoring and bacteria able to grow within 24 h were selected for further process development. Bacteria able to produce the desired enzymes were selected for the first round of tests. Four Bacillus strains presented high inulinase activity. The next step in process development was the determination of key parameters for enzyme activity such as temperature, pH, salinity and substrate concentration. The highest inulinase activity, 2.2 gsugars /gprotein h, was attained when the supernatant of a culture of a Bacillus subtilis strain was used in a magnetically stirred bioreactor. This study demonstrates how bacterial strains from marine environments may be used successfully in biotechnological processes.