Overproduction of bacterial cellulose from Acetobacter xylinum BPR2001 using food industries wastes.

In this study, a cost-effective complex culture media containing molasses and corn steep liquor (CSL) was developed for the high production of bacterial cellulose (BC) by investigating the effect of four effective factors on BC production at three levels using Taguchi and combined methods. The predicted and actual values of BC production in optimal conditions by Taguchi and combined methods were 8.41 and 14.52 g/L, respectively. These results showed that the combined method was more suitable for predicting the optimal conditions in the optimization of BC production, the cost of developed culture medium was around 94% cost of HS medium preparation, molasses was the most effective factor in both experimental design methods, and initial pH adjustment had little impact on BC production. Then, the effect of inoculation conditions containing three factors of inoculation age, ethanol addition time, and agitation rate on the increase of BC production at three levels was investigated using the response surface methodology with the Box-Behnken design algorithm. Under the optimal conditions including inoculum age of 3 days, ethanol addition time of 10 days, and stirring speed of 100 rpm, the predicted and experimental results of BC production were 21.61 and 20.21 g/L, respectively. This is among the highest ever reported for BC production, which was achieved with a more cost-effective culture medium containing molasses and CSL.

Economical Optimization of Industrial Medium Culture for Bacterial Cellulose Production.

The competitiveness of bacterial cellulose (BC) production with plant cellulose can be achieved by production on cost-effective media. It was found that the bacterial cell number ratio of BC to culture medium increases over time so that from the fourth day, the entrapped cell number in the cellulose network exceeds the suspended cells. Optimization based on 23-full factorial showed that inoculum development at 50 rpm and the main culture process under static conditions significantly increases BC production. A cost-effective culture medium containing molasses (ML) and corn steep liquor (CSL) was developed based on the same C/N ratio to HS medium, with 7.24 g/l cellulose at C/N ratio 12.6 is competitive with maximum production 8.7 g/L in HS medium. The BC production cost was reduced about 94% using the proposed cheap and locally available medium containing ML and CSL, while BC mechanical properties increased by about 50%.

High cell density culture of recombinant E. coli in the miniaturized bubble columns.

Miniaturized bubble columns (MBCs) can provide mass transfer characteristics similar to stirred tank bioreactors. In this study, a new application was developed for MBCs to investigate the effect of feeding strategy and medium type on the fed-batch culture of recombinant E. coli. The results showed that the exponential feeding strategy and defined M9 medium were more suitable to achieve the high cell density culture (HCDC). The maximum obtained cell concentration in exponential feeding strategy in the defined medium without induction, was at OD600 of 169, while glucose concentration was maintained under 2 g/L. To the best of our knowledge, this cell concentration cannot be achieved in lab or pilot scale bubble columns. At the end of the process, adverse effect of the metabolic burden due to induction and mass transfer limitations decreased the obtained final cell concentration to OD600 of 116. Finally, a comparison of the results for fed-batch culture in the stirred tank bioreactor with those of the MBCs showed that their lower cell concentrations were due to the hydrodynamics limitations of MBCs. Yet, it was found that the MBCs are efficient tools in development of feeding strategies and evaluation of medium components for HCDC of recombinant E. coli.

Hydrodynamics and mass transfer in miniaturized bubble column bioreactors.

Miniaturized bubble columns (MBCs) have different hydrodynamics in comparison with the larger ones, but there is a lack of scientific data on MBCs. Hence, in this study, the effect of gas hold-up, flow regimes, bubble size distribution on volumetric oxygen mass transfer coefficient at different pore size spargers and gas flow rates in MBCs in the presence and absence of microorganisms were investigated. It was found that flow regime transition occurred around low gas flow rates of 1.18 and 0.85 cm/s for small (16-40 µm) and large (40-100 µm) pore size spargers, respectively. Gas hold-up and KLa in MBC with small size sparger were higher than those with larger one, with an increasing effect in the presence of microorganisms. A comparison revealed that the wall effect on the flow regime and gas hold-up in MBCs was greater than bench-scale bubble columns. The KLa values significantly increased up to tenfold using small pore size sparger. In the MBC and stirred tank bioreactors, the maximum obtained cell concentrations were OD600 of 41.5 and 43.0, respectively. Furthermore, it was shown that in MBCs, higher KLa and lower turbulency could be achieved at the end of bubbly flow regime.

Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli.

BACKGROUND: The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. METHODS: An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h⁻¹ at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. RESULTS: Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l⁻¹ and 32.1 g.l⁻¹. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g⁻¹ DCW and 1.83 g.l⁻¹.h⁻¹, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen and PD-Grastim showed that all proteins have a similar secondary structure. CONCLUSION: Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity.