Effect of process parameters on the production and drying of Leuconostoc mesenteroides cultures

Leuconostoc mesenteroides BLAC was grown on MRS broth or on a carrot juice medium, and the effects of sugar concentration, type of pH control, aeration and fermentor size on viable counts were examined. The effect on viability of the type of centrifuge used to concentrate the bacterial culture was also examined. When the MRS broth had the traditional 110 mM glucose, pH control did not increase the final population. However, using a zone pH control mode, increasing the glucose content of MRS both from 110 to 220 mM almost doubled the population. In MRS broth, the amount of acetic acid produced was the same for all treatments, and was proportional to the amount of citrate consumed. There was a significantly lower cell yield in the carrot juice medium when the pH was not regulated. In the carrot juice medium, pH had a more pronounced effect on the final population level than did aeration, even though the quantity of viable cells was greater when the culture was aerated. In MRS broth, glucose was completely consumed during fermentation, but this was not the case in carrot juice medium. Aeration resulted in increased acetic acid content of the fermented medium. Viable counts were not affected by scaling the volume of the fermentation from 2 to 15 l ,or by the type of centrifuge used to concentrate the cells. Cells were concentrated by a factor of 10, but in both centrifuge types, viable counts showed only an eightfold average increase. However, freeze-dried powders obtained from the continuous pilot-plant-centrifuged cultures had, on the average, 33% lower populations than those obtained from the laboratory unit. Journal of Industrial Microbiology & Biotechnology (2002) 28, 291–296 DOI: 10.1038/sj/jim/7000245 Received 09 July 2001/ Accepted in revised form 25 January 2002     

An investigation of the seasonal pattern of mannitol content in deciduous and evergreen species of the oleaceae growing in northern Sicily

In several species of the Oleaceae, mannitol, already present at considerable levels, accumulates in response to stress. This family comprises both deciduous and evergreen species, and we investigated the role of mannitol in deciduous malacophyll and evergreen sclerophyll species growing under the same conditions in the field. The relationship between mannitol content and changes in rainfall or temperature was also studied. The mannitol content of leaves of Fraxinus ornus L., F. angustifolia Vahl., Olea europaea L. and Phillyrea media L. was determined by gas chromatography. Leaf samples were collected once a month for 1 year. In the two ash species, the seasonal pattern of mannitol content appeared the same: a gradual increase in spring, peaking in summer, followed by a gradual decrease. The mannitol content was similar in both species, ranging between 260 and 720 micromol g(-1) d. wt. The seasonal pattern of mannitol content in Olea and Phillyrea was similar for both species, but unlike that of Fraxinus did not show a summer peak. Rainfall was negatively correlated with the seasonal increase of mannitol content in ash. Mannitol content increased gradually during drought, reaching a maximum value at the end of the dry season. Temperature did not have a direct influence on mannitol content. In Olea and Phillyrea, variations in mannitol content were poorly correlated with rainfall or temperature, indicating that mannitol does not have a primary role in the response of these species to the hot, dry summer conditions.     

Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos.     

Industrial media and fermentation processes for improved growth and protease production by Tetrahymena thermophila BIII

Tetrahymena thermophila was cultivated on industrial by-product media. The composition of the best medium (with milk proteins) was optimised by a central composite design for growth and protease secretion. The optimal combination [1.07% (w/v) of yeast extract and 0.99% (w/v) of skimmed milk] improved biomass production by 46%. In a fermentation strategy, the pH must be regulated to produce no cell damage, lengthening the stationary phase and resulting in a more abundant protease production. To increase cell concentration and protease secretion, a continuous culture with cell recycling by microfiltration was successfully tested on ciliated protozoa. Journal of Industrial Microbiology & Biotechnology (2000) 24, 285–290. Received 28 July 1999/ Accepted in revised form 20 January 2000     

Studies on fermentative production of rifamycin using Amycolatopsis mediterranei

The fermentative production of rifamycin by Amycolatopsis mediterranei (MTCC17) has been studied. Both qualitative and quantitative aspects of the fermentation were determined by optimizing cultural conditions and medium design to improve the production of rifamycin. A pH value of 7.0, a temperature of 26°C, an aeration rate of 250rev/min for a 50ml volume, a level of inoculum of 10% grown aeration for 48h and a fermentation period of 11days were found to be optimum. Among the nitrogen sources, and culture conditions, peanut meal and aeration were found to be critical for rifamycin production respectively. The above mentioned exercise increased the yields of rifamycin from 350mg/l to 2000mg/l.     

Culture of Staphylococcus xylosus in fish processing by‐product‐based media for lipase production

Aims: The objective of this study was to demonstrate that fish‐processing by‐products could be used as sole raw material to sustain the growth of Staphylococcus xylosus for lipase production. Methods and Results: Bacterial growth was tested on supernatants generated by boiling (100°C for 20 min) of tuna, sardine, cuttlefish and shrimp by‐products from fish processing industries. Among all samples tested, only supernatants generated from shrimp and cuttlefish by‐products sustained the growth of S. xylosus. Shrimp‐based medium gave the highest growth (A₆₀₀ = 22) after 22 h of culture and exhibited the maximum lipase activity (28 U ml^?1). This effect may be explained by better availability of nutrients, especially, in shrimp by‐products. Standard medium (SM) amendments to sardine and tuna by‐product‐based media stimulated the growth of S. xylosus and the highest A₆₀₀ values were obtained with 75% SM. Lipase activity, however, remained below 4 U ml^?1 for both sardine and tuna by‐product‐based media. Conclusions: Fish by‐products could be used for the production of highly valuable enzymes. Significance and Impact of the Study: The use of fish by‐products in producing S. xylosus‐growth media can reduce environmental problems associated with waste disposal and, simultaneously, lower the cost of biomass and enzyme production.     

Enhancement of optical coherence microscopy in turbid media by an optical parametric amplifier

We report the enhancement in imaging performance of a spectral‐domain optical coherence microscope (OCM) in turbid media by incorporating an optical parametric amplifier (OPA). The OPA provides a high level of optical gain to the sample arm, thereby improving the signal‐to‐noise ratio of the OCM by a factor of up to 15 dB. A unique nonlinear confocal gate is automatically formed in the OPA, which enables selective amplification of singly scattered (ballistic) photons against the multiply‐scattered light background. Simultaneous enhancement in both imaging depth and spatial resolution in imaging microstructures in highly light‐scattering media are demonstrated with the combined OPA‐OCM setup.

Typical OCM inteferograms (left) and images (right) without and with OPA.     

Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells

Advantages of using internally developed chemically‐defined (CD) media for cell culture‐based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in‐house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large‐scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large‐scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb‐expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1163–1171, 2015     

Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches.