Process Development for Benzyl Alcohol Production by Whole-Cell Biocatalysis in Stirred and Packed Bed Reactors.

The ocean is an excellent source for new biocatalysts due to the tremendous genetic diversity of marine microorganisms, and it may contribute to the development of sustainable industrial processes. A marine bacterium was isolated and selected for the conversion of benzaldehyde to benzyl alcohol, which is an important chemical employed as a precursor for producing esters for cosmetics and other industries. Enzymatic production routes are of interest for sustainable processes. To overcome benzaldehyde low water solubility, DMSO was used as a biocompatible cosolvent up to a concentration of 10% (v/v). A two-phase system with n-hexane, n-heptane, or n-hexadecane as organic phase allowed at least a 44% higher relative conversion of benzaldehyde than the aqueous system, and allowed higher initial substrate concentrations. Cell performance decreased with increasing product concentration but immobilization of cells in alginate improved four-fold the robustness of the biocatalyst: free and immobilized cells were inhibited at concentrations of benzyl alcohol of 5 and 20 mM, respectively. Scaling up to a 100 mL stirred reactor, using a fed-batch approach, enabled a 1.5-fold increase in benzyl alcohol productivity when compared with batch mode. However, product accumulation in the reactor hindered the conversion. The use of a continuous flow reactor packed with immobilized cells enabled a 9.5-fold increase in productivity when compared with the fed-batch stirred reactor system.

Cultivating marine bacteria under laboratory conditions: Overcoming the "unculturable" dogma.

Underexplored seawater environments may contain biological resources with potential for new biotechnological applications. Metagenomic techniques revolutionized the study of bacterial communities but culture dependent methods will still be important to help the biodiscovery of new products and enzymes from marine bacteria. In this context, we promoted the growth of bacteria from a marine rock pond by culture dependent techniques and compared the results with culture independent methods. The total number of bacteria and diversity were studied in different agar plate media during 6 weeks. Agar plate counting was of the same order of magnitude of direct microscopy counts. The highest efficiency of cultivation was 45% attained in marine agar medium. Molecular analysis revealed 10 different phyla of which only four were isolated by the culture dependent method. On the other hand, four taxonomic orders were detected by cultivation but not by the molecular technique. These include bacteria from the phyla Bacillota and Actinomycetota. Our study shows that it is possible to grow more than the traditionally considered 1% of bacteria from a seawater sample using standard agar plate techniques and laboratorial conditions. The results also demonstrate the importance of culture methods to grow bacteria not detected by molecular approaches for future biotechnological applications.

Marine Bioprospecting, Biocatalysis and Process Development.

Oceans possess tremendous diversity in microbial life. The enzymatic machinery that marine bacteria present is the result of extensive evolution to assist cell survival under the harsh and continuously changing conditions found in the marine environment. Several bacterial cells and enzymes are already used at an industrial scale, but novel biocatalysts are still needed for sustainable industrial applications, with benefits for both public health and the environment. Metagenomic techniques have enabled the discovery of novel biocatalysts, biosynthetic pathways, and microbial identification without their cultivation. However, a key stage for application of novel biocatalysts is the need for rapid evaluation of the feasibility of the bioprocess. Cultivation of not-yet-cultured bacteria is challenging and requires new methodologies to enable growth of the bacteria present in collected environmental samples, but, once a bacterium is isolated, its enzyme activities are easily measured. High-throughput screening techniques have also been used successfully, and innovative in vitro screening platforms to rapidly identify relevant enzymatic activities continue to improve. Small-scale approaches and process integration could improve the study and development of new bioprocesses to produce commercially interesting products. In this work, the latest studies related to (i) the growth of marine bacteria under laboratorial conditions, (ii) screening techniques for bioprospecting, and (iii) bioprocess development using microreactors and miniaturized systems are reviewed and discussed.

Phenotypic Adaptations Help Rhodococcus erythropolis Cells during the Degradation of Paraffin Wax.

During crude oil extraction, the reduction in temperature and pressure results in the precipitation of paraffin wax that contains 20-40 carbon chain hydrocarbons. The paraffin wax may accumulate inside production tubes, pipelines, and processing facilities, and also in tankers during petroleum transportation. There are few bacterial strains that are able to degrade solid substrates. In the present study, the biodegradation of paraffin is evaluated using Rhodococcus erythropolis cells. This bacterium is able to grow using paraffin wax from an oil refinery plant as the sole carbon source. The cells grow as a thick biofilm over the solid substrate, make scale-like structures that increase the area of the initially smooth surface of paraffin, produce biosurfactants, and become more negatively charged than ethanol- or glucose-grown cells. When paraffin wax is supplied as microparticles, to increase the cell-substrate contact area and to simulate paraffin precipitation, the cells also adjust the composition of the fatty acids of the phospholipids of the cellular membrane to decrease its fluidity and paraffin biodegradation increases considerably. The study suggests that the phenotypic adaptation of R. erythropolis cells may be used to degrade paraffin wax under real conditions.

Determining transaminase activity in bacterial libraries by time-lapse imaging.

Transaminase activity was determined by time-lapse imaging using a colourimetric reaction and image analysis. A correlation between the benzaldehyde conversion and relative luminance was determined, allowing the identification of the most promising biocatalysts, the determination of kinetic parameters, and the assessment of the effect of the substrate concentration on activity.

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.

Rhodococcus erythropolis cells adapt their fatty acid composition during biofilm formation on metallic and non-metallic surfaces.

Several parameters are involved in bacterial adhesion and biofilm formation including surface type, medium composition and cellular surface hydrophobicty. When the cells are placed inside tubes, parameters such as oxygen availability should also influence cell adhesion. To understand which cellular lipids are involved in the molecular events of biofilm formation in Rhodococcus erythropolis, cell adhesion was promoted on different metallic and non-metallic surfaces immersed in culture media. These cells were able to modulate the fatty acid composition of the cell membrane in response to both the surface to which they adhered and the growth medium used. To assess the response of the cells to both surfaces and operational conditions, biofilms were also promoted inside a reactor built with five different types of tubes and with medium recirculation. The biofilm biomass could be directly related not to the hydrophobicity of the tubes used but to the oxygen permeability of the tubes. Besides this, cell age influenced the adhesion of the R. erythropolis cells to the tubes. Principal component analysis showed that the lipid composition of the cells could separate cells attached to metallic from those on non-metallic surfaces in the plane formed by PC1 and PC2, and influence biofilm biomass.