Cell Biomass Yield Research Articles

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l −1) and rhamnolipids yield (4.1 g l −1) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate ( μ) of 0.272 h −1, specific product yield ( q p) of 0.318 g g −1 h and volumetric productivity ( P V) of 0.024 g l −1 h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l −1 and surface tension as 31.2 mN m −1.

Fluid mixing in shaken bioreactors: Implications for scale-up predictions from microlitre-scale microbial and mammalian cell cultures

Pressures on pharmaceutical companies to speed bioprocess development have led to significant interest in small scale, parallel experimentation. A particular focus is cell cultivation and the optimisation of protein synthesis because of the number of biological and engineering variables involved. In this work, we briefly review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view to defining criteria for the scale-up of results obtained in shaken microwell systems to conventional laboratory scale. Scale-up approaches are illustrated for two different cell cultures. The first involves an automated microscale process ( 1000 μ l ) for the aerobic fermentation of E. coli JM107:pQR706 overexpressing transketolase (TK) which is subsequently used for asymmetric carbon–carbon bond formation. The kinetics of both the fermentation and bioconversion stages are first quantified as a function of fermentation medium composition (LB or LB-glycerol) and shaking frequency with oxygen transfer rates being identified as rate limiting in certain cases. Successful scale-up of the microwell process (in terms of maximum cell growth rate, biomass yield and specific TK activity) to a 1.4 l scale mechanically stirred bioreactor is then demonstrated based on experiments performed at constant k L a values. The second process investigated involved antibody production in suspension cultures of VPM8 hybridoma cells. Initial results suggest that experiments performed at constant mean energy dissipation rates provide a satisfactory basis for scale translation from shaken microwells ( 800 μ l ) to conical flasks (100 ml) and are indicative of results obtained in a mechanically stirred bioreactor (3.5 l). Overall this work provides an initial insight into the engineering characterisation of shaken bioreactors and how key parameters may be used to define suitable scale-up criteria for different cell cultures.

Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae

The effects of amino acids on glutathione (GSH) production by Saccharomyces cerevisiae T65 were investigated in this paper. Cysteine was the most important amino acids, which increased intracellular GSH content greatly but inhibited cell growth at the same time. The suitable amino acids addition strategy was two-step addition: in the first step, cysteine was added after two hours culture to 2 mM and then, the three amino acids (glutamic acid, glycine, and serine) were added after seven hours culture. The optimum concentration of those three key amino acids (10 mM glutamic acid, 10 mM glycine, and 10 mM serine) was obtained by orthogonal matrix method. With the optimum amino acids addition strategy a 1.63% intracellular GSH content was obtained in shake flask culture. Intracellular GSH content was 55.2% higher than the experiments without three amino acids addition. The cell biomass and GSH yield were 9.4 g/L and 153.2 mg/L, respectively. Using this amino acids addition strategy in the fed-batch culture of S. cerevisiae T65, GSH content, the biomass, and GSH yield reached 1.41%, 133 g/L, and 1875 mg/L, respectively, after 44 hours fermentation. GSH yield was about 2.67 times as that of amino acids free.

Simultaneous and comparative assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and enhanced polyhydroxyalkanoate (PHA) production using optimization studies

Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K 2HPO 4 (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K 2HPO 4/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K 2HPO 4 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K 2HPO 4 1.0 g/l and inoculum 150.58 ml/l.

Effect of Flow Rate Pattern on Glucose‐6‐phosphate Dehydrogenase Synthesis in Fed‐Batch Culture of Recombinant Saccharomycescerevisiae

A strain of genetically modified Saccharomyces cerevisiae (S. cerevisiae) W303 181 was used to improve glucose-6-phosphate dehydrogenase (G6PDH) production in aerobic culture. Fed-batch cultures were carried out in a 5 L fermentor at variable values of the parameter K, namely, 0.2, 0.3, 0.5, 0.7, and 0.8 h(-)(1). The highest G6PDH production (1164 U/L) and specific activity (517 U/g(cell)) were obtained using the following conditions: glucose, 5.0 g/L; adenine, 8 microg/mL; histidine, 8 microg/mL; tryptophan, 8 microg/mL; temperature, 30 degrees C; inoculum, 1.28 g/L; pH, 5.7; agitation, 400 rpm; aeration, 2.2 vvm; and K, 0.2 h(-)(1). The exponential feeding pattern increased cell density (2.14 g/L), enzyme productivity (149.27), and biomass yield (0.18 g(glu)/g(cell)( )(mass)). The level of G6PDH in the genetically modified S. cerevisiae was approximately 4.1-fold higher than that found in a commercial strain.

Development of suspension culture of Podophyllum hexandrum for production of podophyllotoxin

A suspension culture of Podophyllum hexandrum was established. As the cultures grew, reduction in cell viability, biomass and product yield were associated with browning of culture medium, clumping of cells and drop in medium pH. Supplementation of the medium with both polyvinylpyrrollidone (PVP) and pectinase eliminated these problems. PVP at 10 g l−1 was optimum for both growth of and product formation in P. hexandrum suspension cultures.

Inhibitory effect of mineral ion accumulation on high density growth of the hyperthermophilic archaeon Sulfolobus solfataricus

A fed-batch operation for high density cultivation of Sulfolobus solfataricus (DSM 1617) in a bench-top fermentor using a feed medium composed of glucose and yeast extract was investigated. The highest maximal cell density obtained in controlled fed-batch cultures was 21.7 g/ l. Although higher yeast extract concentrations in the medium favored greater cell biomass yield, cell growth ceased with low cell densities. It was observed that large amounts of inorganic ions, such as sulfate, ammonium, potassium and phosphate ions, were accumulated in the culture broth at higher yeast extract concentrations. This was due to either the addition of the titrant or feeding of yeast extract during cultivation. Fed-batch cultures with additional mineral salts in the feed medium showed much lower cell biomass, indicating that accumulation of inorganic ions has a significant inhibitory effect on the growth of S. solfataricus. Inhibition of cell growth by the presence of mineral ions was further confirmed by the batch culture experiments. Some plausible mechanisms which can account for the growth inhibition at higher mineral ion concentrations have been suggested.

Effect of additives and fed-batch culture strategies on the production of glutathione by recombinant Escherichia coli

The effect of additives including yeast extract, precursor amino acids and ATP on the production of glutathione (GSH) by recombinant E. coli was investigated. The addition of precursor amino acids (glutamate, cysteine and glycine) and ATP improved intracellular GSH accumulation. When 2.0 g/l ATP and 9 mmol/l precursor amino acids were added at the beginning and after 12 h of fermentation, respectively, cell concentration and intracellular GSH content was 24% and 1.4 times higher than that of culture without additions. Growth inhibition was observed while the initial glucose concentration was above 20 g/l. Based on the analysis of the batch culture process, different fed-batch cultures in jar fermenters were carried out in which three kind of simple feeding strategies were applied. An exponential feeding strategy increased cell density, productivity and biomass yield. Using this strategy, cell density and total GSH quantity in broth reached 80 g(DCW)/l and 880 mg/l at 25 h without pure oxygen, respectively.

Variation in polar-group content in lipids of cowpea (Vigna unguiculata) Cell cultures as a mechanism of haloadaptation

Callus and cell suspension cultures of cowpea (Vigna unguiculata) were induced with 2,4-dichlorophenoxyacetic acid and grown at different NaCl concentrations. The cell biomass yield and its total lipid content decreased with increasing salinity. However, while the hexose content in lipids was higher, the amount of lipid phosphorus was significantly lower in both agar and cell suspension cultures. Ion-transport rates with artificial membranes prepared with different lipid fractions showed that lipids from cells grown in a saline medium were less permeable to Na+ and to Cl- than those grown in a non-saline medium. Also the permeability of membranes prepared with glycolipids was lower than those prepared with phospholipids and whole lipids. Apparently, the increase of hexose/phosphorus ratio in membrane lipids is induced in response to the halo-adaptation process.

Continuous growth ofCandida utilis under periodic change of growth-limiting substrate

A periodic change of limitation by glucose and ammonia was effected during continuous cultivation of Candida utilis. Values of feed parameters providing periodic nitrogen limitation were established. Cell biomass yield, macromolecular composition and parameters of individual cells (cell mass, budding percentage and cell-wall polysaccharide per surface square unit) were examined. The cyclic regime was found to result in culture synchrony. Parameters obtained on the basis of cell counts displayed a high sensitivity to changes in limitation where as the remaining parameters were less sensible.

Monitoring Growth of Microorganisms using the Methods of Mass-Energy Balance and Utilization of Computer on Line with Fermenter in Real Time Processing (in Russian)

The effective means of microbial culture monitoring is the measurement of low-inertial parameters (respiration rate, rates of supply of alkali for pH maintenance and the limiting substrate) and utilization of computer on line with fermenter for recalculation of these rates into the instant values of mass and energy cell yields, specific rates of cell growth and substrate and oxygen consumption, using the method of mass-energy balance. In this paper, the equations of mass-energy balance are presented both in general form and in the form of numerical algorythms for computer programming. The installation for automation of microbial cultivation experiment is described. Experimental data are presented which indicate the effectiveness of the method of indirect measurement of cell biomass yield and specific rates of physiological processes.

Growth of the thermotolerant yeast, Candida valida, on ethanol: Dependences of maximal growth rate and cell biomass yield on temperature

Growth of Candida valida on ethanol in pH-auxostat and chemostat has been studied. Maximal growth rate, μm, and cell biomass yield, Ys, display the Arrhenius dependence on temperature within the ranges 18°–30° C and 30°–36° C and an abrupt fall above 36° C. The temprature dependence of both parameters has breaks at 30° C and 36° C. Activation energies have been measured for both μm and Ys. The reason for a weaker effect of temperature on Ys than on μm is discussed.

The effect of temperature on the kinetics of ethanol production by strains of Zymomonas mobilis

Two strains of Z. mobilis were evaluated for temperature sensitivity between 25°C and 40°C. At higher temperatures the cell viability, biomass yield, ethanol yield and final ethanol concentration decreased, and there was evidence of increased ethanol inhibition. However the kinetic parameters μ, qs and qp were largely unaffected by temperature over this range.

Microbial utilization of Pinus radiata bark.

A screening program using suspensions of ground bark in mineral salts media, or extracts prepared from ground bark by treating with hot water, sulfuric acid, ammonium hydroxide, or sodium hydroxide, yielded more than 200 pure cultures of fungi, yeasts, and bacteria. Only 38 of these have good growth on liquid bark media. All were filamentous fungi, although many bacteria and yeasts were among the cultures that failed to give appreciable growth. Species of Penicillium, Scopulariopsis, Aspergillus, Trichoderma, Cladosporium, and Fusarium were among the most actively growing cultures. Cell biomass yields, as measured by cell nitrogen determination, were too low for economic production of single cell protein.

Microbial Utilization of Pinus radiata Bark

A screening program using suspensions of ground bark in mineral salts media, or extracts prepared from ground bark by treating with hot water, sulfuric acid, ammonium hydroxide, or sodium hydroxide, yielded more than 200 pure cultures of fungi, yeasts, and bacteria. Only 38 of these have good growth on liquid bark media. All were filamentous fungi, although many bacteria and yeasts were among the cultures that failed to give appreciable growth. Species of Penicillium, Scopulariopsis, Aspergillus, Trichoderma, Cladosporium, and Fusarium were among the most actively growing cultures. Cell biomass yields, as measured by cell nitrogen determination, were too low for economic production of single cell protein.