Kefir yeast technology: scale-up in SCP production using milk whey

A three-step process to scale-up kefir biomass production at a semi-industrial scale employing whey is reported. Aerobic fermentations were initially performed at laboratory scales, in 1.5- and 4-L bioreactors, yielding 79 g/L final kefir biomass (0.89 g/g of lactose utilized), in 7 h of fermentation time. The use of whey as carbon source even in solid cultures led to the formation of a granular biomass. These results encouraged scale-up at a semi-industrial-scale pilot plant employing 100- and 3,000-L bioreactors, leading to the development of a process for granular kefir biomass production. The results validated the laboratory-scale experiments and the avoidance of centrifugal separators due to granular biomass formation. Pilot-plant operations showed kefir to be highly resistant to contamination under actual industrial conditions and no serious problems in handling of raw materials and equipment were observed. Economic analysis showed a 20% higher cost of the market price of products, with added value of up to 15.9 x 10(9) within the European Union.     

Transient impairment of the adaptive response to fasting in FXR-deficient mice

The farnesoid X receptor (FXR) has been suggested to play a role in gluconeogenesis. To determine whether FXR modulates the response to fasting in vivo, FXR-deficient (FXR^−/−) and wild-type mice were submitted to fasting for 48 h. Our results demonstrate that FXR modulates the kinetics of alterations of glucose homeostasis during fasting, with FXR^−/− mice displaying an early, accelerated hypoglycaemia response. Basal hepatic glucose production rate was lower in FXR^−/− mice, together with a decrease in hepatic glycogen content. Moreover, hepatic PEPCK gene expression was transiently lower in FXR^−/−mice after 6 h of fasting and was decreased in FXR^−/−hepatocytes. FXR therefore plays an unexpected role in the control of fuel availability upon fasting.     

Automated expression and solubility screening of His-tagged proteins in 96-well format

A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand of postgenomics, and it is now generally accepted that the long-term solution to this problem will be based on automation, on industrial standard-formatted experiments, and on downsizing samples and consumables. In agreement with this consensus, we have set up a fully automated method based on a dot-blot technology and using 96-well format consumables for assessing by immunodetection the amount of total and soluble recombinant histidine (His)-tagged proteins expressed in Escherichia coli. The method starts with the harvest of expression cells and ends with the display of solubility/expression results in milligrams of recombinant protein per liter of culture using a three-color code to assist analysis. The program autonomously processes 160 independent cultures at a time.     

Human red cell glycolysis in high altitude chronic hypoxia

We have found that glycolysis in human red blood cells under the hypoxic conditions found at high altitudes is connected with changes in enzyme activities and levels of various metabolic intermediates. The sensitivity of the four kinases to hypoxia results in 1) glycolytic hyperactivity leading to a higher intracellular energy state, and 2) accumulation of 2–3 DPG, whose role in the adaptation of red blood cell respiration to high altitude has been shown by previous research. PEP, 3PG, and G6P appear to be the main regulating intermediates in glycolysis in this system. The reason for the very large increase in G1-6DP is still not clear.     

Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor

The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina ( < 44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity.     

Stimulating in-soil rhamnolipid production in a bioslurry reactor by limiting nitrogen

A soil with aged contamination from lubricating oil (LO) and polychlorinated biphenyls (PCBs) was treated in a bioslurry reactor to investigate in-soil biosurfactant production by Pseudomonas aeruginosa, the most abundant indigenous, culturable, hydrocarbon-degrading microorganism. After 2 days of growth on LO, a depletion of nitrogen stimulated the production and accumulation of rhamnolipids to levels roughly 20 times the critical micelle concentration. Surface tensions and concentrations of monorhamnolipid and dirhamnolipid, PCBs, and total petroleum hydrocarbons (TPH) were measured in a slurry filtrate. Soil-bound PCBs and TPH were also quantified. Rhamnolipid production was observed within 1 to 2 days after nitrogen depletion in each of the 10 batches tested. By day 6, total rhamnolipid concentrations increased from below detection to average values over 1,000 mg/L, which caused over 98% of soil-bound PCBs and over 99% of TPH to be emulsified and recovered in the filtrate. After 70 days, rhamnolipid concentrations were only reduced by 15%, because of nitrogen-limited rates of rhamnolipid biodegradation. The results show that in-soil biosurfactant production can be stimulated in a controlled way with nutrient limitation and can be used to achieve soil washing.     

Bioprocess design and economic analysis for the commercial production of environmentally friendly bioinsecticides from Bacillus thuringiensis HD-1 kurstaki

A production process for B. thuringiensis (Bt) bioinsecticides was designed in detail, including alternative batch, low-density fed-batch (LDFB), and high-density fed-batch (HDFB) fermentation configurations. Capital and operating costs, as well as profitability based on simple rate of return, were performed using a purpose-written FORTRAN program, explicitly analyzing production of a water-based flowable product used in forestry applications.The total capital cost was 18 million dollars (Canadian dollars) for a stand-alone plant with base-scale capacity of 3 x 10(7) billion international units (BIU)/year. Raw material costs amounted to 1.5 million dollars yearly, of which approximately half was for formulation ingredients. Per-unit production cost rose sharply for scales of less than 1 x 10(7) BIU/year, but was little affected by scale above 3 x 10(7) BIU/year. Product cost was much lower at all scales for a LDFB as opposed to batch fermentation process, but HDFB gave relatively little additional cost benefit. Profitability analysis performed by co-varying scale and selling price showed that break-even occurred at a price of 0.45 dollars/BIU for a batch process at base scale, while with LDFB fermentation the same production volume sold at 0.35 dollars/BIU gave a 12% rate of return. Since the assumed base scale would represent 8-15% of current world Bt bioinsecticide production, based on value or volume, it was concluded that profitability would require some or all of the following elements: targeting higher-value markets such as disease vector control, in addition to forestry; a potentially lower plant capacity (although at least 1 x 10(7) BIU/year;) and coproduction of other large-volume microbial products to absorb capacity and match bioinsecticide output to market demand.     

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.