Anand Sundararajan

Flotation characteristics of cyanobacterium Anabaena flos‐aquae for gas vesicle production

Cyanobacterium Anabaena flos‐aquae was cultivated in photobioreactors for production of intracellular gas vesicles (GVs), as potential oxygen microcarriers. Natural flotation of the buoyant culture was investigated as a potential means of cell harvesting, because filtration and centrifugation tended to destroy the vesicles. Best flotation was found with actively growing culture and when conducted in the dark. The flotation‐related cell properties, including the specific GV content, vesicle‐collapsed filament density, and intracellular carbohydrate content, were measured to understand the phenomena. During the batch culture, the specific GV content remained relatively constant at 370 μL/(g dry cells) but the filament density (ranging 1.02 to 1.08 g/cm3) showed a decrease‐then‐increase profile. The increase began when the growth slowed down because of the reduced light availability at high cell concentrations. The dark flotation was studied with both actively growing (μ ≈ 0.2 day−1) and stationary‐phase cultures. The specific GV content of the stationary‐phase culture remained relatively constant while that of the growing culture increased slightly. The intracellular carbohydrate content of the growing culture decreased much faster and more significantly, from 57 to 10 mg/(g dry cells) in ⩽ 8 h. The filament density also decreased, apparently parallel to the profiles of carbohydrate content.

The effects of cells on oxygen transfer in bioreactors

Cells may affect oxygen transfer rates by three mechanisms: respiration of cells accumulated at the gas/liquid interface, physical presence of cells as solid particles, and modification of the medium by cells. These effects were studied experimentally in bubble-aerated bioreactors using bakers yeast at different cell concentrations, agitation speeds, aeration rates, and specific oxygen uptake rates. The overall effect of cells was to enhance oxygen transfer rates. The physical presence of cells as solid particles was found to retard oxygen transfer, presumably due to the lower oxygen permeability in the cell layer accumulated near the bubble surfaces. Cell respiration and medium modification, on the other hand, enhanced oxygen transfer rates. The retardation by nonrespiring cells and the enhancement due to cell respiration were found stronger at higher agitation speeds and lower aeration rates employed. This was attributed to the higher interfacial cell accumulation associated with the smaller bubbles produced under these conditions in the systems studied.

Evaluation of oxygen permeability of gas vesicles from cyanobacterium Anabaena flos-aquae.

The enhancement of oxygen permeability in aqueous medium by addition of cyanobacterial gas vesicles (GVs) has been examined. The GVs were isolated from cultures of Anabaena flos-aquae that had been cultivated in photobioreactors and harvested by dark flotation. Prior to the permeability experiments, the collected GVs were treated with glutaraldehyde for improved stability. Measurements of oxygen permeability were made with a polarographic oxygen electrode in suspensions of various GV volume fractions (0-2.1%). The experimental results were compared with the values predicted theoretically (Frickes equation) assuming different permeability through the GVs (PmGV), ranging from 0 to 8.30 x 10(-4) mol m-1 atm-1 s-1. The former corresponded to impermeable vesicles, the latter to air at 22 degrees C as if there were no vesicle wall. The best-fit value of PmGV was 9.9 x 10(-7) mol m-1 atm-1 s-1, ca. 36-fold higher than that in water. GVs were therefore very permeable to oxygen. However, the value was much lower than that predicted for air, implying the existence of wall resistance.

The effects of cells on oxygen transfer in bioreactors: physical presence of cells as solid particles

Abstract Oxygen transfer into broths of non-respiring live bakers yeast was studied at different cell concentrations, agitation speeds and aeration rates. Under all the studied conditions, the physical presence of cells as solid particles was found to reduce the oxygen transfer rate. The effect of physical blockage by the less permeable cells accumulated near the bubble surface was dominant in the studied systems, while the hydrodynamic enhancement by the “snowball” effect (G.F. Andrews, J.P. Fonta, E. Marrotta and P. Stroeve, Chem. Eng. J., 29 (1980) B39–B46, B47–B55) was insignificant. The reduction in volumetric oxygen transfer coefficient k L a increased with cell concentration, but with decreasing marginal effect. Higher agitation speeds and the lower aeration rate used in the study (1 vs. 2 vvm), which produced smaller bubbles with higher cell capture efficiencies, were also found to promote the blocking effect of non-respiring cells.

Use of cyanobacterial gas vesicles as oxygen carriers in cell culture

The gas vesicles isolated from the cells of filamentous cyanobacterium Anabaena flos-aquae were treated and sterilized with glutaraldehyde and then evaluated for their effectiveness as gas carriers in cell culture. Anchorage-dependent Vero cells were grown in a packed bed of microcarrier beads under the perfusion of Dulbecco’s Modified Eagle’s Medium with 1% serum. The culture medium supplemented with 1.8% (v/v) gas vesicles was found to support a 30% higher maximum glucose utilization rate than the same medium without gas vesicles. The gas vesicle suspension was confirmed to have no apparent effects on cell metabolism in T-flask cultures. The study results indicated that the gas vesicles, with high oxygen carrying capacity, can be used to increase the oxygen supply in cell culture systems.

Glutaraldehyde treatment of proteinaceous gas vesicles from cyanobacterium Anabaena flos-aquae.

As potential gas microcarriers, gas vesicles (GVs) were isolated from cultures of the filamentous cyanobacterium Anabaena flos‐aquae and treated with glutaraldehyde. The effects of glutaraldehyde treatment on the stability of GVs, against elevated temperatures (40−121 °C) and protein‐stripping agents such as urea and sodium dodecyl sulfate (SDS), were then examined with the pressure collapse curves generated using pressure nephelometry. The treatment was very beneficial to GVs against the exposure to SDS and urea; however, it did not make the evolution‐optimized vesicle structure stronger or more temperature‐resistant. In the presence of these protein‐stripping agents, the treated vesicles had higher median (50%) collapse pressures (by ≥1 atm) than the untreated ones, at both room temperature and 40 °C. This increase has been presumably attributed to the cross‐linking of the large GvpC protein to the ribbed GvpA shell, thereby resisting the stripping of GvpC that provides the primary mechanical strength to the vesicle wall. The glutaraldehyde treatment also restored the strength of GVs weakened by a 5‐week storage in a refrigerator and, therefore, is expected to improve the stability of GVs for long‐term storage. GVs could not be autoclaved. If necessary for the intended applications, glutaraldehyde treatment may also serve to chemically sterilize the vesicles, with the glutaraldehyde subsequently removed by dialysis.

MODEL SIMULATION OF WATER-IN-OIL XANTHAN FERMENTATION

ABSTRACT The W/O xanthan fermentation is simulated by integrating the microbial kinetic behaviors and the multiple-phase process characteristics. Model 1 assumes uniform redistribution of cells, substrates and product by frequent droplet breakup and coalescence. Model 2 simulates the system of viscous aqueous phase with minimal droplet breakup and component redistribution. The real fermentation should proceed within the bounds set by the two models. Effects of various parameters are evaluated. The aqueous-phase xanthan concentration (Xn) and volumetric productivity (QP) achieved at 200 h are used as the indicators. Xn and QP increase with nitrogen-source concentration (SNO) initially but plateau (Model 1) or decrease slightly (Model 2) at high SNO. Xn (at 200 h) decreases with increasing aqueous-phase volume fraction (f). QP, however, increases with f reflecting its basing on the total dispersion volume. Increasing agitation and aeration result in higher Xn and QP. The higher agitation enhances the G/O in...

LIQUID-FILM TIME CONSTANT ASSESSMENT FOR kLa MEASUREMENTS IN RESPIRING FERMENTATION BROTHS

Abstract To determine the liquid-film time constant (τF) of the dissolved oxygen electrode for kLa measurements with the dynamic method in respiring fermentation broths, an approach using the steady-state readings of the electrode in the broths containing respiring and non-respiring cells, respectively, has been established. The results showed that τF in microbial suspensions with high volumetric oxygen uptake rates could be very large under the conditions of low aeration and agitation, indicating the slower response of the oxygen electrode caused by the more difficult penetration of oxygen molecules through the thicker films with active cell respiration. The significance and adequacy of the τF corrections for kLa measurements were evaluated experimentally.