High Cell Mass Concentration Research Articles

Economical lipid production from crude glycerol using Rhodosporidiobolus fluvialis DMKU-RK253 in a two-stage cultivation under non-sterile conditions

To enhance the microbial lipid production through Rhodosporidiobolus fluvialis DMKU-RK253, a two-stage cultivation process with a focus on cost reduction was developed. Low-cost raw materials, which are biodiesel-derived crude glycerol and monosodium glutamate were utilized to produce microbial lipids. A two-stage cultivation process was performed under non-sterile cultivation, and a response surface methodology was used to determine the effects of the interaction among factors in the first and second stages of cultivation while using shaking flask cultivation. In the first stage of cultivation, cells were cultivated in a nitrogen-rich medium for cell mass production. The optimal crude glycerol (30 g/L) and monosodium glutamate (5 g/L) concentrations, and the inoculum age (96 h), were found to improve cell mass production. In the second stage, a non-sterile crude glycerol solution was used to enhance lipid production without any aseptic techniques. An optimal crude glycerol concentration of 57 g/L with an initial pH of 7 and an initial cell concentration of optical density at 600 nm of 20 resulted in a high lipid production of 27.81 ± 1.86 g/L and maximum cell mass of 39.14 ± 0.08 g/L. These high lipid and cell mass concentrations consequent to achieve a high lipid content of 71.42% of the dry cell mass, a lipid yield of 0.34 g/g glycerol and a cell mass yield of 0.48 g/g glycerol. The low-cost raw materials were estimated to be US$0.80/kg for microbial lipid production. Therefore, a potential condition for the cultivation process of yeast that produces a high amount of inexpensive lipids has been demonstrated.

Assessing safety of Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 using in vitro approaches and an in vivo murine model

Lactobacilli have a long history of safe use in human nutrition, however, inclusion of any new strain, despite its safe usage evidence, warrants proper analysis of its safety and toxicity under the purview of existing regulations. In the present investigation, Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 were evaluated for their safety and toxicity using both in vitro and in vivo approaches. The in vitro assays included mucin degradation, hemolytic activity, biogenic amine production and platelet aggregation assay. The safety was also assessed using acute, subacute and subchronic assays, bacterial translocation studies, intravenous and intravenous administration and genotoxicity assay in murine model. The outcome of this toxicological safety assessment indicated that both the test strains lacked any harmful metabolic activity or any genotoxic effects. Furthermore, the results of oral toxicity studies in mice revealed that short term administration of high cell mass concentration of 1012 cfu/animal as well as long term feeding of the probiotic strains did not alter any hematological, general health parameters or cause any organ specific disorder. Based upon these scientific assessments and supported by long history of safe use, both MTCC 5690 and MTCC 5689 may be considered safe for human consumption.

Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds.

Assessment of pectinase production by Aspergillus oryzae in growth-limiting liquid medium under limited and non-limited oxygen supply

The cultivation of Aspergillii in liquid media is often hindered by the intense consumption of oxygen by the fungi. In this work, pectinase production by Aspergillus oryzae IPT-301 was assessed in cultures grown with limiting and non-limiting oxygen supplies to evaluate the effects of pH and the concentration of dissolved oxygen on the process. Different strategies – such as the use of a growth-limiting medium and the delayed addition of inducer (citrus pectin) to the bioreactor – were employed to provide an effective oxygen supply to the microorganisms and to avoid excessively high impeller speeds that could damage the fungal hyphae. In a limited oxygen environment, it was observed that the maintenance of a constant pH value of 4.0 throughout the cultivation resulted in high cell mass concentration but no enzyme activity. However, when the pH was kept at 4.0 only during the period of intense cell growth followed by sustained control at pH 2.7, significant enzyme activity was detected (43UmL−1). It was observed that this low pH favors both the production of enzyme and their stability. Under a non-limiting dissolved oxygen concentration, over 30% of saturation, the delayed pH control approach resulted in the maximum pectinase activity of 120UmL−1.

Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO2 using a two-stage cultivation system

The technical feasibility of Cupriavidus necator DSM 545 for sustainable autotrophic polyhydroxybutyrate (PHB) production from CO2 using a two-stage cultivation system was evaluated. In this cultivation method, cell mass growth occurred under heterotrophic conditions using two different organic substrates, namely glucose and waste glycerol. In both cases, PHB biosynthesis was triggered by applying nitrogen and oxygen limitation at three different cell mass concentrations under autotrophic conditions using a gas mixture of H2, O2 and CO2. To ensure that the test conditions were relevant for later industrial application, O2 concentration was kept below the safety value during autotrophic PHB production. PHB production from CO2 on waste-glycerol grown cell mass resulted in a PHB concentration of 28g/L, which is the highest reported value in literature for PHB synthesized from CO2 at an O2 concentration below the lower explosion limit of 5vol%. The fermentation performance decreased when nutrient limitation was delayed at higher cell mass concentrations. Furthermore, it was shown that PHB production from CO2 at high cell mass concentration is metabolically feasible, but under the tested conditions the mass transfer of O2 was limiting PHB accumulation. Characterization of the produced polymers showed that the organic carbon source affected the properties of PHB and that the cultivation method developed in this study provided PHB with properties similar to a commercial PHB and PHB typically found in literature. It can be concluded that heterotrophic–autotrophic production of PHB by C. necator is a promising cultivation method to reduce the overall production cost of PHB. In order to compete with the current heterotrophic cultivation system, the oxygen transfer rate must however be enhanced to achieve a higher PHB productivity.

Shortening the Light Path Length of Photobioreactors to Elevate the Cell Mass Concentration of Cyanobacterium Synechococcus sp. Strain PCC6301

To explore high-cell density cultivation of photolithotrophs in the production of high-value compounds, a model cyanobacterium Synechococcus sp. strain PCC6301 was grown in rectangular photobioreactors with light path lengths of 0.1, 0.3, 0.5, 1.4 and 5 cm. A mathematical model to correlate cell growth with medium concentration, light transmittance and light path length was formulated. Logarithmic and linear growth phase were prolonged, and linear growth rate was elevated by shortening the light path length and by avoiding monohydrogen phosphate ion depletion. With sufficient monohydrogen phosphate ions, a high cell mass concentration of 24.5 mg·mL–1 was accomplished using a 0.1 cm light path length photobioreactor.

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.

Engineering aspects of the production of sugar alcohols with the osmophilic yeast Moniliellatomentosa var pollinis. Part I. Batch and fed-batch operation in stirred tank

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 cultivation. Rates of growth, substrate uptake and production, yields and yield coefficients as well as OUR, CPR and RQ were evaluated with different medium composition and operating conditions in a 30 l stirred tank by batch and fed-batch operation. At high substrate concentrations in batch operation high (dry) cell mass concentrations (65 g l −1), high erythritol concentrations (90 g l −1) erythritol yield (36%) and yield coefficients (0.35 g g −1) as well as polyol concentrations (150 g l −1) polyol yield (62%) and yield coefficients (0.65 g g −1) were obtained under optimal conditions. With fed-batch operation better results were obtained: high (dry) cell mass concentrations (up to 116 g l −1), high erythritol concentrations (up to 170 g l −1), polyol concentrations (up to 188 g l −1) were obtained under optimal conditions. Under oxygen limitation ethanol was formed, and under nitrogen limitation foam formation impaired the production process. By suitable aeration and a fed-batch process using a mixture of glucose and nitrogen source for feeding, ethanol and foam formation could be reduced and eliminated, respectively.

Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels.

Ralstonia eutropha grows on and produces polyhydroxyalkanoates (PHAs) from fermentation acids. Acetic acid, one major organic acid from acidogenesis of organic wastes, has an inhibitory effect on the bacterium at slightly alkaline pH (6 g HAc/L at pH 8). The tolerance of R. eutropha to acetate, however, was increased significantly up to 15 g/L at the slightly alkaline pH level with high cell mass concentration. A metabolic cell model with five fluxes is proposed to depict the detoxification mechanism including mass transfer and acetyl-CoA formation of acetic acid and the formation of three final metabolic products, polyhydroxybutyrate (PHB), active biomass, and CO(2). The fluxes were measured under different conditions such as cell mass concentration, acetic acid concentration, and medium composition. The experimental results indicate that the acetate detoxification by high cell mass concentration is attributed to the increased fluxes at high extracellular acetate concentrations. The fluxes could be doubled to reduce and hence detoxify the accumulated intracellular acetate anions.

Kinetic analysis on formation of poly(3-hydroxybutyrate) from acetic acid by Ralstonia eutropha under chemically defined conditions.

Batch cultures of Ralstonia eutropha in chemically defined media with acetic acid (HAc) as the sole carbon source were conducted to investigate acetate utilization, formation of poly(3-hydroxybutyrate) (PHB) and growth of active biomass (ABM) under different carbon to nitrogen (C/N) weight ratios. The specific acetate utilization rate based on ABM approached 0.16 g/g ABM h(-1), which was not affected very much by the extracellular HAc concentration from 1 to 5 g/l, but was affected by the C/N weight ratio. A low C/N ratio or high nitrogen supply sped up the specific acetate utilization rate to produce more ABM and less PHB. A high HAc concentration (>6 g/l), however, depressed acetate utilization as well as the ABM growth and PHB formation. A high cell mass concentration enhanced the tolerance of R. eutropha to the toxicity of HAc at pH 7 to 8.5. The viscosity-average molecular size of PHB generally increased first and then declined in batch cultures. Larger PHB molecules and less PHB per ABM were produced at a low C/N ratio with enough nutrient nitrogen than those under a high C/N ratio with less nutrient nitrogen available.

The pH‐auxostat as a tool for studying microbial dynamics in continuous fermentation

If a microorganism has a growth coupled production or consumption of acid or alkali, it is possible to use the pH-auxostat as a means of control in continuous fermentation. In using the pH-auxostat, it is possible to separate the inlet substrate flow in two different streams. These will both be pH controlled, with one main flow, consisting of nutrients and a second minor but concentrated flow, of acid or alkali. Hereby, it is possible to vary the difference in pH between the fermentor and the inlet medium. This pH difference is proportional to the steady-state cell mass concentration.(1,2) It is shown that by separating the inlet flow in two different streams and cultivating without any substrate limitation, the maximum growth rate may be obtained while the cell mass concentration will be controlled. This will also give the possibility to reach high cell mass concentrations at micro(max) without the risk of wash-out. A modified expression, based on hydrogen, of the steady-state bio-mass concentration, X, is developed as X = Y(X/H x [FHin/(FHin +FMin)] x (CHin -CHFerm) where Y(X/H) is the yield coefficient of cell mass per acid produced. The indexes Hin and Min refer to the inflows of alkali and medium, respectively; C(Hin) is the inlet concentration of hydrogen ions. The boundary condition for the cell mass shows that S(in) > X/Y(X/S), where S(in) is the medium substrate concentration and Y(X/S) is the yield of biomass per consumed substrate. It is shown that when the cell mass concentration exceeds this value, the flow stops. The applicability of the pH-auxostat method is then verified from different experiments. It is hereby used to detect a deviation from the maximal growth rate showing effects on the microbial physiology. With Escherichia coli used as the model organism, the effect on the growth rate of temperature and high concentration of ammonia were investigated.

Economic significance of increasing cell mass concentration for metabolite production -A theoretical study-

The economic significance of increasing cell mass to high concentrations for metabolite production has been discussed theoretically. First, the direct cost of the metabolite production has been formulated as the sum of the raw material cost, the running cost, and the direct labor cost. By definition, the raw material cost is related to the metabolite yield from the nutrient consumed and the running cost is related to the productivity (or space-time yield) of the metabolite formed. Two characteristics of fed-batch culture at high cell mass concentration, i.e. the increase in the total volume of the culture broth and a definite volumetric occupation of wet cells in the total culture broth, have been taken into mathematical consideration. 1. (1) When the metabolite accumulates intracellularly, the direct production cost is minimized by obtaining the greatest amount of cells from a fixed amount of the nutrient consumed in the shortest operating time at which the cells have the highest content of the metabolite under consideration. In the two stage cultivation method (the early cell growth phase followed by the metabolite accumulation phase), an increase in cell mass concentration reduces the raw material cost only a little, but it does reduce the running cost. 2. (2) When the metabolite accumulates extracellularly in the culture supernatant, the raw material cost rises with increasing yield of the cell mass which is formed from a given amount of the raw material. The raw material cost cannot be lowered unless the metabolite yield is raised. The running cost tends to be reduced by increases in the cell mass concentration. In the two-stage cultivation method of extracellular metabolite production, the raw material cost increases with increasing cell mass concentration unless the metabolite yield at the second phase is predominant, while there is usually a particular cell mass concentration at which the running cost is minimized.

Automatic supplementation of minerals in fed‐batch culture to high cell mass concentration

Automatic constant-value control of mineral ions was attempted in semibatch culture of high cell mass concentration (more than 150 g dry cell/L) with ethanol and ammonia feeds. Equations were derived from the mass balance principle to calculate the required concentration of each mineral ion in the mineral feed solution, taking into account both the decrease in the volume of the culture supernatant as a proportion of the whole culture broth and the increase in the volume of the whole culture broth during the cultivation. The mineral solution was supplied automatically, linked either with ethanol feed or ammonia water feed. The actual concentrations of mineral ions could be kept within small variations. To adjust the supplementation in accordance with the culture change from oxygen sufficiency (early growth phase) to oxygen deficiency (later growth phase), the concentration of each mineral ion was altered stepwise when the dissolved oxygen concentration fell to zero. The mineral supplementation gave better results coupled with ethanol feed than with ammonia feed. The mineral ions studied were K(+), Mg(2+), Na(+), Fe(2+), Zn(2+), Ca(2+), Co(2+), Cu(2+), Mn(2+), NH(+) (4), PO(4) (3-) and SO(4) (2-).