Design of an efficient fermentation process for the production of Metarhizium acridum blastospores

Using a sequential approach, we described efficient blastospore production in a stirred tank bioreactor (3?L capacity). We used the response surface methodology to optimise the media ingredients and fermentation parameters to obtain the maximum production of blastospores by a locally collected isolate of Metarhizium acridum (Ascomycota: Hypocreales). The results showed that a liquid culture medium supplemented with monopotassium phosphate (15.17?g/L), corn steep liquor (69.25?g/L), and casamino acids (80.68?g/L) in a stirred tank bioreactor under operating conditions constant at 635?rpm, a temperature of 26°C, and pH 3.3 produced 1.25?×?10⁸?blastospores (bls)/ml, with 93% viability after 120?h of fermentation. This bioreactor yield compares favourably with the yields obtained by shake flask production and confirms the suitability of the media and production parameters for the potential scale-up fermentation production of M. acridum.     

A method for the quantification of bacterial protein in the presence of Jarosite

A variation on Peterson's modification of the Lowry method for microbial protein determination was developed in which 10% (w/v) oxalic acid was used to remove jarosite. This allowed the quantification of Thiobacillus ferrooxidans entrapped in solid jarosite or in aqueous suspensions containing jarosite. The quantity of protein measured was not affected by the amount of jarosite in the culture, the concentration of oxalic acid, or the time of exposure (up to 72 h) of the sample to oxalic acid. An application of this method was demonstrated in the quantification of biomass immobilized in jarosite on the surface of polystyrene beads in an inverse fluidized bed bioreactor used for the rapid microbial oxidation of ferrous iron.     

Xylitol production from high xylose concentration: evaluation of the fermentation in bioreactor under different stirring rates

AIMS: To investigate the production of xylitol by the yeast Candida guilliermondii FTI 20037, in a bioreactor, from rice straw hemicellulosic hydrolysate with a high xylose concentration. METHODS AND RESULTS: Batch fermentation was carried out with rice straw hemicellulosic hydrolysate containing about 85 g xylose l(-1), in a stirred-tank bioreactor at 30 degrees C, under aeration of 1.3 vvm (volume of air per volume of medium per min) and different stirring rates (200, 300 and 500 rev min(-1)). The bioconversion of xylose into xylitol by the yeast depended on the stirring rate, the maximum xylitol yield (YP/S = 0.84 g g(-1)) being achieved at 300 rev min-1, with no need to pretreat the hydrolysate for purification. CONCLUSIONS: To determine the most adequate oxygen transfer rate is fundamental to improving the xylose-to-xylitol bioconversion by C. guilliermondii. SIGNIFICANCE AND IMPACT OF THE STUDY: For the microbial production of xylitol to be economically viable, the initial concentration of xylose in the lignocellulosic hydrolysate should be as high as possible, as with high substrate concentrations it is possible to increase the final product concentration. Nevertheless, there are few reports on the use of high xylose concentrations. Considering a process in bioreactor, from rice straw hemicellulosic hydrolysate, this is an innovator work.     

植物生物反应器生产药物蛋白的前景

转基因植物作为生物反应器生产重要的药用蛋白,如抗体、血液替代品和疫苗,为健康保健和科学研究提供充足的生物药物,满足人们日益增长的需要。本文综述了这一领域的研究进展以及商业化前景。     

Serum‐free scaled up expansion and differentiation of murine embryonic stem cells to osteoblasts in suspension bioreactors

The use of embryonic stem cell (ESC) derived cells has emerged as a potential alternative treatment for a number of degenerative diseases, including musculoskeletal diseases. Conventional ESC culturing methods use fetal bovine serum (FBS) as a major supplemental component of culture media, which is undesirable for clinical applications. These cultures are usually performed in small‐scale static vessels (gelatin‐coated dishes), which limit the number of cells that can be generated. It is essential to develop effective, reproducible protocols for efficient scalable production of ESC‐derived cells. Here we present serum‐free bioreactor protocols for (1) expansion and (2) differentiation of embryonic stem cells to osteoblasts. Cultivation of mESCs in serum‐free media, supplemented with 15% knockout serum replacement (KSR) resulted in a 27.1‐ and 48.6‐fold expansion in static culture and suspension respectively by day 5 of culture. Further induction to osteoblasts with a differentiation cocktail was verified by up‐regulation of osterix and osteocalcin. Mineralization was also enhanced, as indicated by an increase in the calcium deposition by osteogenic cells by day 28. These results will serve as the basis for developing protocols with human ESCs as a new treatment alternative for musculoskeletal diseases. Biotechnol. Bioeng. 2010;106: 829–840. © 2010 Wiley Periodicals, Inc.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzyme-binding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3 degrees C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed.     

Removal and destruction of high concentrations of gaseous toluene in a two-phase partitioning bioreactor by Alcaligenes xylosoxidans

A two-phase bioreactor consisting of hexadecane dispersed in an aqueous, cell-containing medium (organic fraction = 0.33) was used to trap toluene vapours from an air stream. The affinity for toluene by the solvent resulted in high efficiency of removal and transfer to the aqueous phase based on equilibrium transfer. The system was readily able to handle a loading capacity of 748 mg l^–1 h^–1 at a toluene degradation efficiency of greater than 98%.     

Stereoselective reduction of 2-substituted cyclohexanones by Saccharomyces cerevisiae

A comparative study of two modifications of enzymic reduction of ethyl N-{2-{4-[(2-oxo-cyclohexyl)methyl]phe- noxy}ethyl} carbamate (1), an insect juvenile hormone bioanalog, was performed using Saccharomyces cerevisiae in two bioreactors of different size, 250-ml shake-flask and 1-l fermenter. The two major products of this reduction were obtained in 45–49% (w/w) yields but with > 99% enantiomeric purity. Their absolute configurations were assigned as ethyl (1S,2S)-N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate (2a) and ethyl (1R,2S)-N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate (3a).     

Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor

Conversion of paper sludge to ethanol was investigated with the objective of operating under conditions approaching those expected of an industrial process. Major components of the bleached Kraft sludge studied were glucan (62 wt.%, dry basis), xylan (11.5%), and minerals (17%). Complete recovery of glucose during compositional analysis required two acid hydrolysis treatments rather than one. To avoid the difficulty of mixing unreacted paper sludge, a semicontinuous solids-fed laboratory bioreactor system was developed. The system featured feeding at 12-h intervals, a residence time of 4 days, and cellulase loading of 15 to 20 FPU/g cellulose. Sludge was converted to ethanol using simultaneous saccharification and fermentation (SSF) featuring a -glucosidase-supplemented commercial cellulase preparation and glucose fermentation by Saccharomyces cerevisiea. SSF was carried out for a period of 4 months in a first-generation system, resulting in an average ethanol concentration of 35 g/L. However, steady state was not achieved and operational difficulties were encountered. These difficulties were avoided in a retrofitted design that was operated for two 1-month runs, achieving steady state with good material balance closure. Run 1 with the retrofitted reactor produced 50 g/L ethanol at a cellulose conversion of 74%. Run 2 produced 42 g/L ethanol at a conversion of 92%. For run 2, the ethanol yield was 0.466 g ethanol/g glucose equivalent fermented and >94% of the xylan fed to the reactor was solubilized to a mixture of xylan oligomers and xylose.