Cell Mass Concentration Research Articles

KINETICS OF PHENOL AND BENZOATE BIODEGRADATION IN STATIC CULTIVATION SYSTEM BY Burkholderia cepacia G4.

Kinetics of phenol and benzoate degradation by Burkholderia cepacia G4 in classical batch mode of static culture were investigated over a wide range of initial substrate concentrations applied as single substrates. Due to better adaptation to changing conditions, faster degradation of both phenol and benzoate was observed when higher concentrations were used. The results show decrease of the biomass yield coefficient, YX/S, from 0.83 to 0.31 g g-1 when the initial phenol concentration was increased from 0.54 to 0.73 g L-1, supporting the well known inhibitory effect of phenol. On the other hand, linear increasing of the yield coefficient was observed with increasing benzoate concentration. During all experiments, maximum specific substrate consumption rates, rSmax, were reached to 0.27 g g-1h-1 for phenol and to 0.40 g g-1h-1 when benzoate was the sole source of carbon in the bioreactor. In the present study, minor inhibitory effect of benzoate was observed during their investigated concentration range. To explain their degradation modelling, experimental data of both phenol and benzoate biodegradation were fitted using various kinetic models. The results demonstrated that the Yano and Koga equation gave the best fit, compared with the other models. It means that biodegradation of phenol and benzoate can be described by the same kinetic model. Based on the kinetic data, all experiments were not sensitive to change in the saturation constant, KS; therefore, KS-value was fixed at 0.042 and 0.068 g L-1 for phenol and benzoate, respectively. After that, rSmax and the inhibition constant, Ki, parameters were re-fitted in all experiments. In general, the kinetic parameters of both phenol and benzoate degradation were influenced by the initial substrate and bacterial cell mass concentrations, in addition to adaptation with changing in the culture conditions.

Asymmetric Reduction of Acetophenone to α-Phenylethanol by Candida valida under Compressed CO2

The asymmetric reduction of acetophenone to optically active α-phenylethanol catalyzed by cells of Candida valida CICC1444 under compressed CO2 without agitation was investigated. The reaction by the unimmobilized whole cells under compressed CO2 was feasible, and the R-enantiomer of α-phenylethanol was excessive. The yield of α-phenylethanol was greatly influenced by operating parameters such as concentration of acetophenone, cell mass, temperature, and pressure. Compared with the reaction at ambient pressure without agitation, the conversion of acetophenone and yield of α-phenylethanol under compressed CO2 were increased by 20% and 40%, respectively, and the enantiomeric excess of (R)-α-phenylethanol was increased by 5%–10%.

Bioreactor design for enhanced carrier-assisted surfactin production with Bacillus subtilis

Bacillus subtilis ATCC 21332 was used to produce a lipopeptide-type biosurfactant (namely, surfactin) in an innovative bioreactor tailored to solve the problems of severe foaming arising from production of the biosurfactant. To cope with the rapid foam generation, a conventional jar fermentor was integrated with a foam collector, a cell recycler, and a surfactin precipitation unit. Meanwhile, solid carriers (e.g., activated carbon) were added into the fermentation broth to increase cell mass concentration and surfactin yield. The proposed bioreactor allowed stable and efficient surfactin fermentation under intensive foaming conditions without the need of adding antifoam agents. The effect of oxygen transfer rate and mass transfer efficiency on surfactin production was also explored by employing various combinations of aeration and agitation rates. The best combination was 1.5 vvm and 300 rpm, giving an excellent maximum production rate, overall production rate, surfactin concentration, and surfactin yield of 190 mg L −1 h −1, 106 mg L −1 h −1, 6.45 g L −1, and 161 mg surfactin (g glucose) −1, respectively. It was also found that the oxygen volumetric mass transfer coefficient ( K L a) was highly correlated with the performance of surfactin production. The highest surfactin productivity was achieved when the fermentation was carried out at a K L a value of 0.0132 s −1.

On-line estimation of cell growth from agitation speed in DO-stat culture of a filamentous microorganism,Agaricus blazei

A simple, but effective on-line method for estimating the mycelial cell mass concentration from agitation speed data, a most readily-available process variable, has been developed for DO-stat cultures ofAgaricus blazei. The dynamic change of dissolved oxygen concentration (DOC) in the initial transient period and the change in yield were considered in the development of the estimation algorithm or estimator. Parameters in the estimation algorithm were calculated from the agitation speed data at 20% of DOC. The proposed estimator could accurately predict the cell mass concentration regardless of DOC levels in the tested range of 10–40%, showing a good extrapolation capability.

Determination of cell mass and polymyxin using multi-wavelength fluorescence

Multi-wavelength fluorescence was applied for on-line monitoring of cell mass and the antibiotic polymyxin B in Bacillus polymyxa cultivations. By varying the phosphate and nitrogen content of the medium different polymyxin–cell mass ratios could be obtained. Using this strategy, it was possible to investigate if multi-wavelength fluorescence is able to give independent prediction of the two parameters. Partial least square (PLS) regression was applied to establish mathematical relationships between off-line determined cell mass and polymyxin concentrations and on-line collected fluorescence data. For polymyxin one universal PLS model, with a correlation of 0.95 and a root mean square error of cross validation (RMSECV) of 35 mg l −1, could be constructed. However, correlation between fluorescence and cell mass dry weight could not be established including data from all three types of cultivations. For data from each type of cultivation, separate models with high correlation and low RMSECV values were built. A large variation in cellular composition as a result of the different levels of nitrogen and phosphorus in the cultivations was the probable reason to the necessity of building three models. The results of the present investigation indicate that production of polymyxin is concomitantly regulated by phosphate and nitrogen as the highest polymyxin yield on cell mass, 0.17 ± 0.01 g g −1, was reached in the cultivations where both nitrogen and phosphate concentrations were kept low.

A comparison between batch and fed-batch simultaneous saccharification and fermentation of steam pretreated spruce

In order to improve the process economy it is important to use as high dry matter content as possible in simultaneous saccharification and fermentation (SSF). However, too high dry matter content often gives rise to severe inhibition of the yeast metabolism, due to the increased levels of toxic compounds. The aim of the present work was to increase the fibrous content in SSF of steam pretreated spruce to 10% by adapting the yeast to the inhibitory substrate and by using a fed-batch process. Both batch and fed-batch approaches were evaluated. The fed-batch experiments were started with a batch fermentation containing 6% dry matter. Fibrous slurry from the pretreatment was then added four times during the first 24 h giving a final dry matter content corresponding to 10%. The yeast used in the fermentation was produced aerobically on the hemicellulose hydrolysate obtained from the pretreatment. SSF batch and fed-batch experiments with a cell mass concentration of 2, 3 and 5 g/L were carried out. When adapted yeast was used, the available hexoses were completely converted within 72 h and the final ethanol concentrations reached 40–44 g/L. No major differences in performance between batch and fed-batch were seen, but the ethanol productivity during the first 24 h was higher in the fed-batch SSF experiments, particularly during the experiments with a cell mass concentration of 2 and 3 g/L.

Poly(β-hydroxybutyrate) production by a moderate halophile,Halomonas boliviensisLC1 using starch hydrolysate as substrate

The objective of the present work was to enable the use of starch hydrolysate, generated by the action of a recombinant maltooligosaccharide forming amylase from Bacillus halodurans LBK 34, as the carbon source for the production of poly-beta-hydroxybutyrate (PHB) by Halomonas boliviensis LC1. In this work, different amounts of the alpha-amylase (Amy 34) were utilized for starch hydrolysis, resulting in the production of mixtures of maltooligosaccharides (G1-G6) at varying ratios. The highest PHB accumulation (56 wt%) by H. boliviensis cultivated in shake flasks (with agitation at 160 rev min(-1)) was obtained when 6.4 U ml(-1) of the amylase was used for starch hydrolysis. When H. boliviensis cells were grown in a fermentor with no oxygen limitation the accumulation of PHB was decreased to 35 wt%. Although some improvements in PHB accumulation and cell mass concentration were reached by the addition of peptone and phosphate, respectively, major enhancements were attained when oxygen limitation was induced in the fermentor. Halomonas boliviensis uses preferentially maltose for PHB formation from starch hydrolysate. It is also able to hydrolyse higher sugars if no other simpler carbon source is available but with a significantly lower polymer yield. Furthermore, H. boliviensis is able to adjust its metabolism to oxygen limitation, most probably by directing the excess NAD(P)H to PHB accumulation. There have been no reports related to PHB production amongst the members of the genus Halomonas. The use of a maltooligosaccharide forming alpha-amylase, which is active at a temperature and pH close to that required for growth of H. boliviensis, and the versatility of this bacterium in the selection of the carbon source may provide an attractive alternative for the utilization of starch-derived raw materials.

Model for Aerobic Growth of Bacillus Amyloliquefaciens in Processed Soy Sauce under Various Conditions of Temperature, Initial Dry Cell Mass and Ethanol Concentration

Construction of a predictive model for hygenic management of liquid foods was attempted by investigating the effects and interactions of storage conditions on the aerobic growth kinetics of Bacillus amyloliquefaciens in a soup product. The Gompertz curves were generated from the experimental data obtained under three temperatures (298 to 310K) in combination with four levels of inoculation (5.32×10-3 to 5.32×10-6g·L-1) and five levels of ethanol concentration (2.7 to 4.7g·L-1). The microbial growth kinetic analysis disclosed that ethanol was effective for prolonging lag phase duration of the growth in a processed soy sauce. The Gompertz function in combination with nonlinear equations generated by stepwise regression analysis was suitable for predicting the growth of organisms. The lag phase duration was about 18h longer than the quality guarantee period of a soup product containing a perceptible amount of contaminant organisms.

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. However, fermentation using a microbubble dispersion (MBD) can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved oxygen mass-transfer of the microbubbles used for the sparging. Conventional air-sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, 750 rpm. These experiments were compared to MBD-sparged fermentation at 350 and 500 rpm agitation rates. After 97 h of air-sparged fermentation, cell masses were 27.9, 48.9, and 138.2 g/l at agitation rates of 350, 500, and 750 rpm, respectively. For MBD-sparged fermentation, cell mass concentrations after 97 h were 129.9 and 137.7 g/l at 350 and 500 rpm, respectively. Protein concentrations were 36.3 and 67.8 mg/l for air-sparged at 350 and 500 rpm, while 267.3 and 301.2 mg/l for MBD-sparged experiments at 350 and 500 rpm, respectively. The volumetric oxygen mass-transfer coefficient, k L a for the MBD system at 500 rpm was 268.6 h −1 compared to 76.1 h −1 for the conventional sparger at 500 rpm. There was significant improvement in oxygen transfer to the microorganism relative to the conventional sparger.

Biokinetic evaluation and modeling of continuous thiocyanate biodegradation by Klebsiella sp.

Biokinetics for autotrophic degradation of thiocyanate using batch culture of Klebsiella sp. were evaluated both analytically and numerically. A sequential approach with an analytical method followed by a numerical approximation was used to evaluate and to ensure the accuracy of the parameter estimation. The nonlinear least-squares method with a 95% confidence interval was employed. The growth conditions were maintained at pH 7 and 38 degrees C for all experiments. With an automated incubation and turbidity reader, a total of 16 different initial thiocyanate concentrations, ranging from 10 to 300 mg L(-1), were used to develop a kinetic expression of specific growth rate as a function of substrate concentration. The biodegradation of thiocyanate with Klebsiella sp. followed a substrate inhibition pattern. Three identical automated bioreactors with working volumes of 1.5 L, equipped with sterilizable sampling ports, were also used for the numerical approximation of the biokinetic parameters in batch mode. A fourth order Runge-Kutta method was used to approximate the substrate inhibition kinetics of the Klebsiella sp. utilizing thiocyanate. Although the kinetic coefficients estimated by analytical and numerical methods were not statistically different at a 0.05 alpha level, model responses of numerical approximation generated a better prediction of changes in thiocyanate and cell mass concentrations. The hypothetical maximum growth rate, micro m, half saturation coefficient, Ks, microbial yield coefficient, Y, cell mass decay rate coefficient, kd, and substrate inhibition coefficient, Ksi, were evaluated as being 0.62 +/- 0.05 d(-1), 85 +/- 8 mg SCN- L(-1), 0.076 +/- 0.011 mg cell mass (mg SCN)(-1), 0.03 +/- 0.002 d(-1), and 131 +/- 22 mg SCN- L(-1), respectively. The calculated maximal substrate concentration, Sm, and apparent maximum specific growth rate, micro'm, were 105.5 +/- 8.7 mg SCN- L(-1) and 0.24 +/- 0.01 d(-1), respectively. Using these estimated parameters, the theoretical performance of the continuous operation was also illustrated, which depicts the residual thiocyanate and Klebsiella sp. concentrations in the non-steady and steady states at different hydraulic retention times (HRTs). Assuming the influent concentration of 250 mg SCN- L(-1), the expected treatment efficiency ranged from 94.9% to 69.4% between 20 and 5 days HRT, respectively. Klebsiella sp. was expected to be washed out at 4.8 days HRT, thus resulting in no treatment of thiocyanate.

On-line cell mass monitoring of Saccharomyces cerevisiae cultivations by multi-wavelength fluorescence

The catalyst in bioprocesses, i.e. the cell mass, is one of the most challenging and important variables to monitor in bioprocesses. In the present study, cell mass in cultivations with Saccharomyces cerevisiae was monitored on-line with a non-invasive in situ placed sensor measuring multi-wavelength culture fluorescence. The excitation wavelength ranged from 270 to 550 nm with 20 nm steps and the emission wavelength range was from 310 to 590 nm also with 20 nm steps. The obtained spectra were analysed chemometrically with the multi-way technique, parallel factor analysis (PARAFAC), resulting in a decomposition of the multivariate fluorescent landscape, whereby underlying spectra of the individual intrinsic fluorophors present in the cell mass were estimated. Furthermore, gravimetrically determined cell mass concentration was used together with the fluorescence spectra for calibration and validation of multivariate partial least squares (PLS) regression models. Both two- and three-way models were calculated, the models behaved similarly giving root mean square error of prediction (RMSEPs) of 0.20 and 0.19 g l −1, respectively, when test set validation was used. Based on this work, it is evident that on-line monitoring of culture fluorescence can be used for estimation of the cell mass concentration during cultivations.

On the Nonlinear Dynamic State Reconstruction Problem for Chemical/Biochemical Reaction Systems in the Presence of Model Uncertainty

A new approach to the unmeasurable state reconstruction problem for nonlinear chemical reaction systems in the presence of model uncertainty is proposed. In particular, a new robust nonlinear state estimation method is developed that explicitly uses all the available useful information associated with: (i) a dynamic model inevitably characterized by uncertainty, and (ii) a set of sensor measurements in order to accurately reconstruct other key quantities/variables that cannot be measured on-line due to physical and/or technical limitations. The problem of interest is conveniently formulated and addressed within the context of singular partial differential equations (PDE) theory, leading to a nonlinear state estimator that possesses a state-dependent gain computed through the solution of a system of first-order singular PDEs. A set of necessary and sufficient conditions is presented that ensure the existence and uniqueness of a locally analytic solution to the aforementioned system of singular PDEs, and a series solution method that can be easily implemented via a MAPLE code is developed. Under these conditions, the convergence of the estimation error or the mismatch between the actual unmeasurable states and their estimates is analyzed and characterized in the presence of model uncertainty. Finally, the performance of the proposed nonlinear etimator and its convergence properties are evaluated in an illustrative biochemical reaction system that exhibits nonlinear behavior coupled with parametric uncertainty, and the estimation objective is to accurately reconstruct the unmeasurable substrate concentration using the available cell mass concentration measurements and the model of the system under consideration.

High-Efficiency Production of Bioplastics from Biodegradable Organic Solids

Microbial polyhydroxyalkanoates (PHAs) have been extensively studied as environmentally friendly biodegradable thermoplastics. The major obstacle to wide acceptance of PHAs is their high price, mainly attributed to the costs of raw materials and polymer recovery. A large amount of organic solids are discarded from food production and consumption and may be used as carbonaceous raw materials for production of PHAs. A novel technology was investigated at bench-top scale to produce PHAs from food scraps. The harvested cell mass had a high PHA content (72.6% of dry cell mass), the same as obtained from pure glucose and organic acids. The organic solid was first digested in an acidogenic reactor in which about 60% solid was converted to fermentative products, including short-chain fatty acids. The four major acids were acetic, propionic, butyric, and lactic acids at concentrations of 6, 2, 27, and 33 g/L, respectively. The acids were transported through a membrane barrier via molecular diffusion to an airlift bioreactor, where the acids were utilized by an enriched culture of Ralstonia eutropha for PHA synthesis. Purification of fermentative acids was not performed in this molecular diffusion–based integration of acidogenesis and polymerization. By using a dialysis membrane as the barrier, the dry cell mass concentration and PHA content reached 22.7 g/L and 72.6%, respectively. The PHA was a copolymer of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 2.8 mole % of hydroxyvalerate.

Stability of beta-carotene in spray dried preparation of Rhodotorula glutinis mutant 32.

To obtain beta-carotene-rich dry cell preparation from mutant 32 of Rhodotorula glutinis and determination of its pigment stability. The mutant 32 of R. glutinis was grown in a 14 l stirred tank fermenter. Cell mass was concentrated 10-fold by cross-flow microfiltration and then spray dried. Butylated hydroxy toluene (BHT) and d-tocopherol were used as protecting agents. A two-level, three-variable, factorial optimization was performed to achieve moisture-free, non-viable and beta-carotene-rich feed additive. The beta-carotene and cell mass in stirred tank fermenter were found to be 54 +/- 5 mg l-1 and 12.8 +/- 2 g l-1, respectively. In the presence of BHT, 97 +/- 3% (w/w) beta-carotene was recovered for all the inlet temperatures studied. The best beta-carotene and yeast powder recoveries were obtained at 160 degrees C, 11.6% (w/v) cell mass concentration and 1 g l-1 BHT. The pigments inside dried yeast powder were stable in dark and cold condition for at least 10 weeks. The purified beta-carotene got almost totally denatured, under similar conditions of storage, within 76 h. Spray dried and stable preparation of beta-carotene-rich yeast, R. glutinis can provide alternative source of beta-carotene for use in animal nutrition.

Poly(3-hydroxy butyrate) synthesis from a mutant strain Azotobacter vinelandii utilizing glucose in a batch reactor

The synthesis of the biopolymer poly(3-hydroxy butyrate) P(3HB) was studied in a batch reactor using a mutant strain of Azotobacter vinelandii utilizing glucose. The objective of the present study was to optimize the cost of production by using glucose as substrate. In order to achieve this, a mutant strain was developed using a UV-mutagenic technique which is capable of utilizing glucose as substrate and giving a better yield. The effect of temperature, pH, glucose concentration and inoculum concentration on the production of P(3HB) using the optimized media was studied. The optimum temperature, pH, glucose concentration and inoculum concentration for growth and P(3HB) synthesis were found to be 30 °C, 7.5, 2.4% (w/v), and 0.63 g/l, respectively. The experiments were carried out to establish the kinetics of P(3HB) production by measuring cell mass, P(3HB) and glucose concentrations as a function of time in the batch reactor under optimum conditions. Growth kinetic models such as Monod, logistic and modified logistic models were used to predict the experimental data. The respective growth models incorporated in the Leudeking–Piret model and modified Leudeking–Piret models were used to predict P(3HB) formation rates and glucose consumption rates.

Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium

Multi-analogue resistant mutants of Corynebacterium lilium were developed using UV and N-methyl- N′-nitro-nitrosoguanidine (NTG) mutagenesis in order to overproduce methionine. Although the methionine yield at each stage of mutation by UV and NTG mutagenesis were comparable, it was observed that the NTG mutants had higher cell growth and glucose utilization rates. From the parent NTG mutant C. lilium E 4 resistant to ethionine, the highest methionine producing strain C. lilium M-128 resistant to 3.5 mg ml −1 of ethionine, 3.0 mg ml −1 of norleucine, 3.8 mg ml −1 of methionine sulphoxide and 3.8 mg ml −1 of methionine methyl sulphoniumchloride was derived. When C. lilium M-128 was grown under optimised conditions, it produced 2.3 g l −1 of methionine in a 15 l batch reactor. The maximum cell mass concentration obtained was 17 g l −1 dry cell weight resulting in a cell yield coefficient of 0.34 g g −1. The dissolved oxygen concentration was controlled at 40% saturation by cascade control of the airflow rate and agitation speed.

Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation: development of an enzyme-linked immunosorbent assay-based method with application to Clostridium thermocellum batch cultures.

A methodology was developed to determine the mass concentrations of cellulase and cells applicable to studies of microbial cellulose utilization in systems for which a substantial fraction of cellulase is cell-associated. Antibodies raised against a 14-amino acid synthetic peptide with sequence taken from the cohesin domain of the scaffoldin protein of Clostridium thermocellum ATCC 27405 were used to develop an indirect ELISA protocol. Six cellulase calibration standards were prepared using affinity digestion (Morag, E.; Bayer, E. A.; Lamed, R. Enzyme Microb. Technol. 1992, 14, 289-292.). These included supernatant and pellet samples from an Avicelgrown culture with fractional cellulose conversion (X) = 0.98, as well as supernatant, pellet, cell-associated, and cellulose-associated samples from an Avicel-grown culture with X = 0.8. All six standards displayed a very similar absorbance versus concentration relationship when subjected to ELISA, essentially identical SDS-PAGE banding patterns, and similar cellulase specific activity in relation to both other purified cellulase preparations and crude samples. Coefficients of variation for cellulase concentration measurements were 5.2% for supernatant samples and 5.9% for pellet samples. The ELISA method was applied to batch cultures of C. thermocellum grown on Avicel. Cell concentration was calculated from the pellet protein concentration and the cell protein fraction of a cellobiose-grown control. Two alternative methods appeared to overpredict the cell concentration and were not capable of quantifying cells as distinct from cellulase. Cellulase protein production by Avicel-grown batch cultures represented approximately 20% of cell mass exclusive of cellulase. It is concluded that the reported protocols establish a reasonable methodological basis for quantitative determination of the mass concentration of cellulase protein produced by C. thermocellum and for calculation of cell mass concentration as distinct from cellulase concentration.

Increasing ethanol productivity during xylose fermentation by cell recycling of recombinant Saccharomyces cerevisiae.

The influence of cell recycling of xylose-fermenting Saccharomyces cerevisiae TMB3001 was investigated during continuous cultivation on a xylose-glucose mixture. By using cell recycling at the dilution rate ( D) of 0.05 h(-1), the cell-mass concentration could be increased from 2.2 g l(-1) to 22 g l(-1). Consequently, the volumetric ethanol productivity increased ten-fold, from 0.5 g l(-1) h(-1) to 5.35 g l(-1) h(-1). By increasing the biomass concentration, the xylose consumption rate increased from 0.75 g xylose l(-1) h(-1) without recycling to 1.9 g l(-1) h(-1) with recycling. The specific ethanol productivity was in the range of 0.23-0.26 g g(-1) h(-1) with or without cell recycling, showing that an increased cell-mass concentration did not influence the efficiency of the yeast.

Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3).

Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-beta- D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l(-1)), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.

Engineering aspects of the production of sugar alcohols with the osmophilic yeast Moniliella tomentosa var pollinis: Part 2. Batch and fed-batch operation in bubble column and airlift tower loop if reactors

Moniliella tomentosa var pollinis was cultivated on complex medium consisting of glucose as carbon source, urea, (NH 4) 2SO 4, corn steep liquor and yeast extract as nitrogen sources, respectively. The pH and the concentrations of (dry) cell mass, glucose, erythritol, ribitol, glycerol, disaccharides, trisaccharides, polysaccharides, amino acids in medium, oxygen and carbon dioxide in off-gas were monitored during the cultivation. Rates of growth, substrate uptake and production, yields and yield coefficients as well as OUR, CO 2 production rate and respiratory coefficient were evaluated with different medium composition and operating conditions with two mutants in 80 l bubble column and 80 l airlift tower loop reactors in batch and fed-batch operation. Foam height, gas hold up, bubble velocity, and specific gas/liquid interfacial area were measured in situ. The proteins in medium were investigated with SDS-PAGE. At high concentrations (up to 500 g l −1) high (dry) cell mass concentrations (up to 65 g l −1), high erythritol concentrations (up to 175 g l −1) erythritol yield (43%) and yield coefficients (0.58 g g −1) as well as polyol concentrations (250 g l −1) polyol yield (60%) and yield coefficients (0.70 g g −1) were obtained under optimal conditions. With a mutant somewhat better results were obtained. Under oxygen limitation ethanol was formed, and under nitrogen limitation strong foam formation impaired the production process. By suitable aeration and fed-batch process using mixture of glucose and nitrogen source for feeding, ethanol and foam formation could be reduced and eliminated, respectively. The strong effect of antifoam agent in the medium on the fluid dynamic of the two-phase system and the dissolved oxygen concentration was demonstrated.