Degradation of high concentrations of azithromycin when present in a high organic content wastewater by using a continuously fed laboratory-scale UASB bioreactor.

As the presence of emergent contaminants in wastewater, such as antibiotics, has become a threat for public health, the evaluation of strategies to treat them has been gaining importance. A critical example of this situation can be found in wastewaters coming from the pharmaceutical industry, where high concentrations of antibiotics are sometimes accompanied by high organic contents. Even the agroindustry can be affected by a similar problem when cattle infections are treated with antibiotics and part of the antibiotic-contaminated milk has to be wasted. With these situations in mind, in the present study we evaluated a progressive acclimation strategy for a granular sludge in a UASB reactor treating a high organic-content synthetic wastewater contaminated with azithromycin. In parallel, we tested a previously reported low-cost method for azithromycin determination by spectrophotometry, obtaining results comparable with liquid chromatography coupled to mass spectrometry. Although azithromycin has been reported as recalcitrant and resistant to biological degradation, the antibiotic was removed with efficiencies over 50% for wastewater with 10 mg L-1 of azithromycin and a COD of more than 4000 mgO2 L-1. Furthermore, efficiencies over 40% were achieved for wastewater with higher azithromycin concentrations (80 mg L-1) and a COD of 20,000 mgO2 L-1. A careful acclimation strategy permitted the partial removal of azithromycin from wastewater when treating concentrations comparable and higher than what would be expected for domestic and hospital wastewaters, even when its chemical oxygen demand is considerably higher than the average maximum of around 1000 mgO2 L-1.

Novel external-loop-airlift milliliter scale bioreactors for cell growth studies: Low cost design, CFD analysis and experimental characterization.

Many researchers have limited access to fully equipped laboratory-scale batch bioreactors and chemostats due to their relatively high cost. This becomes particularly prohibitive when multiple replicas of the same experiment are required, but not enough bioreactors are available to operate simultaneously. Additionally, experiments using shaken flasks are common but show significant limitations in terms of maintaining homogeneous conditions in liquid cultures or installing instrumentation for monitoring. Here, we proposed to tackle this significant hurdle by providing a route to make available the manufacture of low-cost, milliliter-scale bioreactors. This approach seems plausible for enabling proof-of-concept experiments before moving to a larger scale without significant investments. The conceptually designed systems were based on external-loop bioreactors due to their flexibility, simplicity, and ease of assembling and testing. Designs were initially evaluated in silico with the aid of COMSOL Multiphysics. The successfully evaluated systems were then constructed via additive manufacturing and assembled for hydrodynamics testing via tracer methods. This was enabled by a newly home-made optical absorbance sensor (OAS) for in-line and real-time measurements. Both the in silico and experimental results indicated close to ideal mixing conditions and low shear stress. Cell growth curves were prepared by culturing Escherichia coli and following its cell density in real-time. Our cell growth rate and maximum cell density were similar to those previously obtained in closely related systems. Therefore, the proposed bioreactors are an affordable alternative for batch and continuous cell growth studies rapidly and inexpensively.

Formulation and Characterization of Gelatin-Based Hydrogels for the Encapsulation of Kluyveromyces lactis-Applications in Packed-Bed Reactors and Probiotics Delivery in Humans.

One of the main issues when orally administering microorganism-based probiotics is the significant loss of bioactivity as they pass through the gastrointestinal (GI) tract. To overcome these issues, here, we propose to encapsulate the probiotic yeast Kluyveromyces lactis on chemically crosslinked gelatin hydrogels as a means to protect the bioactive agents in different environments. Hydrogels were prepared by the chemical crosslinking of gelatin, which is commercially available and inexpensive. This is crucial to ensure scalability and cost-effectiveness. To explore changes in key physicochemical parameters and their impact on cell viability, we varied the concentration of the crosslinking agent (glutaraldehyde) and the gelatin. The synthesized hydrogels were characterized in terms of morphological, physical-chemical, mechanical, thermal and rheological properties. This comprehensive characterization allowed us to identify critical parameters to facilitate encapsulation and enhance cell survival. Mainly due to pore size in the range of 5-10 µm, sufficient rigidity (breaking forces of about 1 N), low brittleness and structural stability under swelling and relatively high shear conditions, we selected hydrogels with a high concentration of gelatin (7.5% (w/v)) and concentrations of the crosslinking agent of 3.0% and 5.0% (w/w) for cell encapsulation. Yeasts were encapsulated with an efficiency of about 10% and subsequently tested in bioreactor operation and GI tract simulated media, thereby leading to cell viability levels that approached 95% and 50%, respectively. After testing, the hydrogels' firmness was only reduced to half of the initial value and maintained resistance to shear even under extreme pH conditions. The mechanisms underlying the observed mechanical response will require further investigation. These encouraging results, added to the superior structural stability after the treatments, indicate that the proposed encapsulates are suitable to overcome most of the major issues of oral administration of probiotics and open the possibility to explore additional biotech applications further.

An engineered eukaryotic protein glycosylation pathway in Escherichia coli.

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.