High density cultivation of plant cells in a new aeration-agitation type fermentor,maxblend fermentor®

The applicability of a new aeration-agitation type fermentor with a grid-paddle type impeller and a spiral-sparger, Maxblend Fermentor® (MBF) for high density cultivation of plant cells, was investigated. The MBF showed a high capacity for oxygen supply and extremely low hydrodynamic stress in aeration and mixing compared with a conventional fermentor (CF). When Oryza sativa cells were cultivated at a k_La of 20 h⁻¹, a high cell density cultivation of about 30 g dry cell weight per liter was accomplished in both fermentors and there were few differences in culture performance between the two. On the contrary, considerable differences were observed when Catharanthus roseus cells, which seemed to be sensitive to physical stress, were cultivated at a k_La of 20 h⁻¹ in both fermentors. The MBF exhibited excellent cell growth characteristics, achieving about 19 g dry cell weight per liter, because of its superior oxygen supply and low hydrodynamic stress in aeration and mixing in highly viscous cultures containing high density cells. In CF only about 9.5 g dry cell weight per liter was achieved because of its high hydrodynamic stress.     

Studies on the production of Scytalidium acidophilum biomass

Summary The conversion of nutrient-supplemented and non-supplemented acid-extracts from peat to biomass of the acid-resistant fungus Scytalidium acidophilum is described. Yeast extract and several inorganic salts were tested as nutrient supplement to the peat extract in shake-flask experiments and in an agitated and aerated fermentor. The best results were obtained in the fermentor, with 0.3% yeast extract, which produced 6.2 g dry biomass/1 with a yield of 41% and an efficiency of 30%, at 200 rpm and 1.75 vvm in 8 days. Although higher agitation speeds appeared detrimental to the mycelial physiology, it was detected that the dissolved oxygen concentration at 200 rpm could limit the growth of S. acidophilum if higher biomass concentrations are obtained. The potential for utilizing the residual fermentation medium as a plant liquid fertilizer is reported.     

The Role of Ethylene in the Inhibition of Rooting under Low Oxygen Tensions

A 60-fold increase in ethylene content was observed in stem cuttings of chrysanthemum (Chrysanthemum × morifolium Ramat.) held in aero-hydroponics under anoxic conditions during the 8 to 12 days necessary for adventitious root formation. Ethylene, 1-aminocyclopropane-1-carboxylic acid, and 10-(malonylamino) cyclopropane-1-carboxylic acid contents were highest in the immersed portion of the cuttings, but there was substantial ethylene produced by the anoxic, misted portions of the cutting above the liquid. Application of ethylene (10 microliters per liter) to chrysanthemum cuttings stimulated root development in cuttings held in high dissolved oxygen concentrations (8.0 milligrams per liter). Since the application of ethylene did not inhibit rooting in cuttings held at low dissolved oxygen concentrations (2.0 milligrams per liter), the inhibition of rooting under low oxygen concentrations is not mediated by the observed increase in endogenous ethylene content.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Production of Bacterial Cells from Methane

A mixed methane-oxidizing bacterial culture capable of stable and predictable growth in continuous culture was isolated. The culture consisted of two types of gram-negative nonsporulating rods resembling pseudomonads. The culture grew well at 45 C on an inorganic medium without asepsis. Specific metal requirements for Ca²⁺, Cu²⁺, MoO₄²⁻, Zn²⁺, Mn²⁺, Mg²⁺, and Fe³⁺ (or Fe²⁺) were shown. The cells grown in continuous culture contained 11.7 to 12.1% total nitrogen. From an animal nutrition standpoint, the distribution of amino acids was satisfactory. The continuous fermentation was operated over a range of steady-state dilution rates from 0.085 to 0.301 hr⁻¹. The maximum specific growth rate for the culture, μ_max, was 0.303 hr⁻¹ (doubling time 2.29 hr). The average yield for all fermentations analyzed was 0.616 g (dry weight of cells per g of methane used and 0.215 g (dry weight) of cells per g of oxygen used. The yields on both methane and oxygen were higher for the oxygen-limited than for the methane-limited fermentations. The maximum productivity attained in the fermentor was 2.39 g (dry weight) of cells per hr per liter at a dilution rate of 0.187 hr⁻¹ and a cell concentration of 12.8 g (dry weight) of cells per liter. The limit on maximum cell productivity was determined only by the mass transfer rate of oxygen in the fermentor. The simultaneous volumetric mass-transfer coefficients (k_La in hr⁻¹) for oxygen and methane were determined. The results appear to indicate an oxygen to methane mass-transfer coefficient ratio of approximately 1.4.     

The role of dissolved oxygen and function of agitation in hyaluronic acid fermentation

The role of dissolved oxygen (DO) and function of agitation in hyaluronic acid fermentation by Streptococcus zooepidemicus ATCC 39920 were explored. With controlled DO levels, it was found that the present strain grew as well under both aerobic and anaerobic conditions. Dissolved oxygen plays a role as a stimulant in the HA synthesis; the intrinsic factor affecting the efficiency of HA synthesis is DO level; and there existed a critical DO level of 5% air saturation for the HA synthesis. On the other hand, agitation functions to mix the broth, to enhance oxygen absorption, but not to release HA capsule. In addition, vigorous agitation would lengthen the operation time. It therefore suggests that the relevant criteria for scaling up the fermentor are to maintain DO level above the critical value, and to provide a mild agitation for homogeneity in the fermentor.     

Development of a new commercial-scale airlift fermentor for rapid growth of yeast

To grow yeast rapidly, it is necessary to supply sufficient oxygen to the yeast and to effectively remove the heat of the fermentation. We succeeded in developing a commercial-scale fermentor for growing a food yeast (Candida utilis) to produce RNA rapidly. This fermentor is an internal-loop airlift type with vertical heat transfer tubes between inner and the outer columns. The volume of the fermentor is 145 m³ (working volume 75 m³). The oxygen transfer rate (OTR) was 9.9 kg-O₂/m³/h using a superficial gas velocity of 30 cm/s based on the outer column. Much of the heat of fermentation and the energy resulting from aeration could be removed effectively by the heat transfer tubes. This unique airlift fermentor was driven at a dilution rate of 0.43 h⁻¹ for about 70 d, with the yeast concentration being maintained at 22.8 kg-dry cell/m³. The yeast production rate was 9.79 kg-dry cell/m³/h. Compared with a traditional stirred-type fermentor, two Vogelbush-type fermentors and another airlift fermentor, our fermentor was far superior with respect to OTR and yeast productivity.     

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.     

Selective fengycin production in a modified rotating discs bioreactor

Production of lipopeptides fengycin and surfactin in rotating discs bioreactor was studied. The effects of rotation velocity and the addition of agitators between the discs on volumetric oxygen transfer coefficient k _L a were firstly studied in model media. Then the production of lipopeptides was also studied at different agitation conditions in the modified bioreactor (with agitators). The effect of agitation on dissolved oxygen, on submerged and immobilized biomass, on lipopeptide concentrations and yields and on the selectivity of the bioreaction was elucidated and discussed. The proposed modified rotating discs bioreactor allowed to obtain high fengycin concentrations (up to 787 mg L^?1), but also better selectivity of the bioreaction towards fengycin (up to 88 %) and better yields of fengycin per glucose (up to 62.9 mg g^?1), lipopeptides per glucose (up to 71.5 mg g^?1), fengycin per biomass (up to 309 mg g^?1) and lipopeptides per biomass (up to 396 mg g^?1) than those reported in the literature. Highest fengycin production and selectivity were obtained at agitation velocity of 30 min^?1. The proposed non-foaming fermentation process could contribute to the scale-up of lipopeptide fermentors and promote the industrial production of fengycin. The proposed bioreactor and bioprocess could be very useful also for the production of other molecules using bioprocesses requiring bubbleless oxygen supply.     

Mechanistic analysis of xanthan gum production in a stirred tank

The overall effect of agitation on xanthan gum production by Xanthomonas campestris ATCC13951 in a stirred vessel was mechanistically analyzed considering local variation of the specific production rate due to variation of shear stress in the vessel. The whole liquid volume in a fermentor was roughly divided into three regions; the micromixing region around the impeller with high shear stress, the macromixing region dominated by a circulating flow and the stagnant region. The value of the shear rate was first ascertained by experiments in order to obtain a picture of shear rate variation in a radial direction from the impeller, and the equivalence between the volumes of the high shear stress region and micromixing region was confirmed. The shear stress obtained using a correlation between the shear rate at the impeller tip and Reynolds number of Wichterle et al. was used as a representative of the shear stress in the micromixing region, and the shear stress estimated by use of an empirical correlation between the average shear rate in a fermentor and agitation speed derived by Metzner et al. was adopted as a representative of the shear stress in the macromixing region. The information about the circulation time distribution was also used to take into account oxygen deficiency during circulation of liquid elements in the macromixing region, considering that oxygen from the gas phase was supplied mainly in the high shear region. The calculated values of xanthan gum concentrations which were obtained by the proposed simulation method agreed well with the experimental data in the time course of xanthan gum production at various agitation speeds. Experimental results of the relationship between the overall specific production rate and ND (N, agitation speed, and D, impeller diameter) was also verified by the proposed method.     

Studies on the production of bacterial rennet in a pilot plant fermentor

An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K-26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K-26 using 5% wheat bran medium in a 13 liter fermentor.     

Degradation of n-Hexadecane and Its Metabolites by Pseudomonas aeruginosa under Microaerobic and Anaerobic Denitrifying Conditions

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O₂-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O₂-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO₂⁻-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO₂⁻-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO₃⁻-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N₂). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.     

Effect of glucose concentration on the rate of fructose consumption in native strains isolated from the fermentation of Agave duranguensis

Studies on hexose consumption by Saccharomyces cerevisiae show that glucose is consumed faster than fructose when both are present (9:1 fructose to glucose) in the medium during the fermentation of Agave. The objective of this work was to select strains of S. cerevisiae that consume fructose equal to or faster than glucose at high fructose concentrations by analyzing the influence of different glucose concentrations on the fructose consumption rate. The optimal growth conditions were determined by a kinetics assay using high performance liquid chromatography (HPLC) using 50?g of glucose and 50?g of fructose per liter of synthetic medium containing peptone and yeast extract. Using the same substrate concentrations, strain ITD-00185 was shown to have a higher reaction rate for fructose over glucose. At 75?g of fructose and 25?g of glucose per liter, strain ITD-00185 had a productivity of 1.02 gL^?1?h^?1 after 40?h and a fructose rate constant of 0.071?h^?1. It was observed that glucose concentration positively influences fructose consumption when present in a 3:1 ratio of fructose to glucose. Therefore, adapted strains at high fructose concentrations could be used as an alternative to traditional fermentation processes.     

Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-¹⁴C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH₃ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-¹⁴C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of ¹⁴CO₂ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.     

Mixing state of xanthan gum solution in an aerated and agitated fermentor—Effects of impeller size on volumes of mixing regions and circulation time distribution

The effects of the impeller diameter and width on the volumes of the micromixing and macromixing regions, and on the circulation time distribution were investigated at various agitation speeds to formulate the relationships of them in emperical equations. A fermentor was a 10-l capacity, which was equipped with a turbine impeller with six flat balades and aerated at 1 vvm. It was found that the volumes of the micromixing and macromixing regions depended on the tip speed of the impeller, ND, and the discharging performance of the impeller, ND²W, respectively, in the xabthan gum solution with concentrations of 0.9, 1.8, 2.7, and 3.9%. Empirical equations were derived to estimate the volume of each mixing region from the impeller diameter, D, impeller width, W, agitation speed, N, and consistency coefficient of the xanthan gum solution. On the other hand, the circulation time distribution could be estimated empirically from only the impeller diameter and agitation speed, regardless of variation in the impeller width and consistency coefficient of the xanthan gum solution tested.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Role of agitation conditions in nitrification

The objective of this study was to determine the role of agitation conditions in the oxidation of nitrite ions by Nitrobacter. Batch reaction kinetic experiments were conducted in baffled stirred tanks. The range of agitation conditions studied was 6200 ? 95700 ergs/cm³ sec. This power input corresponds to 3.2 ? 45.6 hp/ 1000 gal, or a “hem Scale” of 3 ? 9. After a lag phase, the reaction kinetics were found to be zero order with respect to nitrite over a concentration range of 590 to 10 mg/liter nitrite nitrogen (NO₂^?-N). The zero-order rate constants were found to significantly decrease with increasing impeller power input per volume of liquid (P / V).     

Optimization of Banana Juice Fermentation for the Production of Microbial Oil

Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D), a lipid-accumulating yeast, was grown in banana juice. The optimum conditions for biomass production in shake flasks were 30°C growth temperature, efficient aeration, a juice concentration of 25%, and preliminary heat treatment at less than sterilization conditions. Under controlled conditions in a fermentor, 20% banana juice was optimum. High concentrations of yeast extract (0.3%) increased biomass production by 40% but decreased oil production by 30%. A lower yeast extract concentration (0.05%) increased biomass production by 2% and oil production by 25%. The best growth and oil production were observed when asparagine (1.4 g/liter) and mineral salts were added to the banana juice. The addition of minerals seemed to improve the utilization of carbon. Growth inhibition was observed when the fermentor was aerated with pure oxygen, even when additional nutrients were present. A fed-batch process permitted the juice concentration to be increased from 15 to 82%; biomass accumulation was three times higher than in batch fermentations. However, the cellular lipid content was only 30% of dry weight, and chemical oxygen demand reduction was slow and inefficient.     

An energy cost minimizing strategy for fermentation dissolved oxygen control

A cost-minimizing mathematical model for on-line control of dissolved oxygen using agitation speed and aeration rate was developed. In pilot scale monensin fermentation using Streptomyces cinnamonensis, this algortihm provided stable control of dissolved oxygen at 40%, reducing energy usage 27.8%. The agitation and aeration profiles provided by the algorithm respresent the pathway of least energy cost for control at the desired dissolved oxygen level. Other observed advantages of bivariable control were reduction of foaming, evaporation, and gas holdup. Reduced maintenance of compressors and agitator motors could also be expected due to decreased load. Monensin productivity equivalent to fermentation with constant agitation and aeration was not obtained, however, with potency reduced 14.8% with the dissolved oxygen control strategy.List of Symbols A m² cross sectional area of fermentor - A ₁, A ₂, A ₃, A ₄ constants of polynomial fit to Calderbank's equations - BP N/m² gauge back pressure - C _ag $/W/s cost of electrical power - C _Q $/m³ cost of compressed air - CE mol/m³/s carbon dioxide evolution rate - D m impeller diameter - DO, DO _meas, DO _sp % dissolved oxyen saturation at any time, measured, and setpoint respectively - h m height of liquid in fermentor - H N/m²/mmol Henry's constant for oxygen in water - H _av average gas holdup in fermentor - k _L a, k _L a _meas, k _L k _sp s^–1 oxygen mass transfer coefficient at any time, measured, and setpoint respectively - N, N _sp s^–1 agitation speed at any time and setpoint respectively - N _a, N _{a, sp} aeration number at any time and setpoint respectively - N _i total number of impellers - N _p impeller power number - N _s number of impellers into which air is directly sparged - OU, OU _meas mol/m³/s Oxygen uptake rate at any time and measured respectively - P W ungassed agitation power - P _g, P _g,meas, P _g,sp W gassed agitation power at any time, measured, and set point respectively - Q, Q _meas, Q _sp m³/s aeration rate at any time, measured, and setpoint respectively - T K fermentation temperature - u _g m/s linear gas velocity - V m³ fermentation liquid volume - mole fraction of oxygen in fermentation off-gas - calculation constant - motor efficiency - $/s sum of agitation and aeration costs - kg/m³ liquid density