Low Agitation Research Articles

Relationship between morphology and citric acid production in submerged Aspergillus niger fermentations

Morphology and citric acid production of an industrial strain of Aspergillus niger (R3) were investigated in 5 l fermentations using different inoculum types and agitation conditions. The fermentations were inoculated using dispersed and pelleted forms, and the effect on subsequent morphology and citric acid production in the 5 l fermenter were studied. The morphology was characterised in depth by automatic image analysis. Using on-line gas analysis by a mass spectrometer, and glucose, citric acid and oxalic acid analysis by an HPLC, some important metabolic parameters have been determined and compared for three citric acid fermentations. A relatively high yield of citric acid (0.74 g/g glucose) was obtained from a fermentation inoculated with dispersed mycelia. Two pelleted fermentations arose from pelleted inocula; one was conducted with low agitation in the early stages and the other with high agitation. With low agitation (300 rpm) the pellets of initial mean equivalent diameter 1.4 mm grew larger with fermentation time, until the agitation was increased at 96 h, to a very high level of 800 rpm when the pellets fragmented rapidly from 3.2 to 1.6 mm diameter. Both the citric acid yield (0.49 g/g glucose) and cell growth from this fermentation were very low. The glucose uptake rate, oxygen uptake rate (OUR), and carbon dioxide production rate (CPR) were also low compared to the non-pelleted fermentation although all showed a sharp rise after the fragmentation of the pellets. The high agitation speed of the second pelleted fermentation (500–800 rpm) retarded continued pellet size increases. The mean equivalent diameter at inoculation was 1.4 mm and this rose to only 1.5 mm towards the end of the fermentation (168 h). Both the citric acid yield (0.58 g/g glucose) and cell growth were higher than those obtained from the fermentation with large pellets but were still lower than in the dispersed morphology case. These results support previous qualitative ideas that initial inoculum morphology and fermentation conditions have significant effects on the subsequent culture morphology and productivity of the citric acid fermentation. In the present work however, these effects have been quantified, particularly using image analysis to characterise morphology and using HPLC to measure concentrations of important broth components. This information will be valuable for optimisation and better control of the citric acid fermentation.

Vancomycin production is enhanced in chemostat culture with biomass-recycle.

Production of the glycopeptide antibiotic vancomycin by Amycolatopsis orientalis ATCC 19795 was examined in phosphate-limited chemostat cultures with biomass-recycle, employing an oscillating membrane separator, at a constant dilution rate (D= 0. 14 h-1). Experiments made under low agitation conditions (600 rpm) showed that the biomass concentration could be increased 3.9-fold with vancomycin production kinetics very similar to that of chemostat culture without biomass-recycle. The specific production rate (qvancomycin) was maximal when the biomass-recycle ratio (R) was 0.13 (D= 0.087 h-1). When the dissolved oxygen tension dropped below 20% (air saturation), the biomass and vancomycin concentrations decreased and an unidentified red metabolite was released into the culture medium. Using increased agitation (850 rpm), used to maintain the dissolved oxygen tension above 20% air saturation, maximum increases in biomass concentration (7.9-fold) and vancomcyin production 1.6-fold (0.6 mg/g dry weight/h) were obtained when R was 0.44 (D= 0.056 h -1) compared to chemostat culture without biomass-recycle. Moreover, at this latter recycle ratio the volumetric vancomycin production rate was 14.7 mg/L/h (a 7-fold increase compared to chemostat culture without biomass-recycle). These observations encourage further research on biomass-recycling as a means of optimising the production of antibiotics.

Biological treatment of paper pulping effluents by using a fungal reactor

Pulp and paper plants are amongst the most polluter industries in this country and elsewhere. Most of the organic halides (AOX) and colour from pulp bleaching units are discharged to the receiving basins without being fully treated. A fungus, which is able to affect over 50% AOX and colour removals from soft-wood bleachery effluents within two days contact time, have been isolated in this laboratory. Optimum condition for dechlorination by this fungus in batch tests was determined as pH 5.5 and 25 °C. The low agitation speeds required by the fungus indicated its tendency towards immobilisation on a solid substrate. Glass wool was chosen as a suitable immobilising matrix to be used in the continuous experiments. An up-flow column was packed with glass wool and operated successfully for over one and half years with AOX removals around 70% in 7-8 hours contact time. Fungal dehalogenation required very low supplemental carbon and no DO. The fungal reactor was also effective in dechlorinating polychlorinated aromatics, e.g. PCP, though dehalogenation ability decreased considerably with the chlorinated aliphatics. High PCP concentrations presumably toxified the fungus, even at short exposures, thereby irreversibly damaging the column reactor.

Parameters affecting the synthesis of geranyl butyrate by esterase 30,000 from Mucor miehei

AbstractThe factors affecting the synthesis of geranyl butyrate by esterase 30,000 of Mucor miehei were studied in a solvent‐free system. The effects of substrate molar ratio, temperature, agitation speed, and initial addition of water were investigated. The equimolar ratio was most interesting for ester production in batch. There were no diffusion limitations, and the reaction could be realized at low agitation. The catalytic activity of the enzyme was irreversibly deactivated at 60°C, and the initial addition of water decreased the rate of conversion after 75 h of reaction. The enzyme activity increased with increased linear chainlength of the acid and was also affected by the alcohol structure. Esterase 30,000 gave the highest conversion of butyric acid with hexanol and terpenic alcohols (citronellol, nerol) and the lowest with the secondary alcohol (2‐hexanol). Finally, five other industrial enzymatic preparations were investigated for their ability to synthesize geranyl butyrate and to hydrolyze olive oil. We observed, for the lipase from Rhizopus javanicua, that there is no relationship between hydrolytic and synthetic activities; this example shows that the hydrolytic lipase activity data cannot predict the capability of lipases in esterification reactions.

Parameters affecting the synthesis of geranyl butyrate by esterase 30,000 from Mucor miehei

AbstractThe factors affecting the synthesis of geranyl butyrate by esterase 30,000 of Mucor miehei were studied in a solvent‐free system. The effects of substrate molar ratio, temperature, agitation speed, and initial addition of water were investigated. The equimolar ratio was most interesting for ester production in batch. There were no diffusion limitations, and the reaction could be realized at low agitation. The catalytic activity of the enzyme was irreversibly deactivated at 60°C, and the initial addition of water decreased the rate of conversion after 75 h of reaction. The enzyme activity increased with increased linear chainlength of the acid and was also affected by the alcohol structure. Esterase 30,000 gave the highest conversion of butyric acid with hexanol and terpenic alcohols (citronellol, nerol) and the lowest with the secondary alcohol (2‐hexanol). Finally, five other industrial enzymatic preparations were investigated for their ability to synthesize geranyl butyrate and to hydrolyze olive oil. We observed, for the lipase from Rhizopus javanicua, that there is no relationship between hydrolytic and synthetic activities; this example shows that the hydrolytic lipase activity data cannot predict the capability of lipases in esterification reactions.

Environmental correlates of resident agitation in Alzheimer's disease special care units.

To determine the point prevalence of agitated behaviors in a representative sample of Alzheimer's disease Special Care Units, and to determine the extent to which agitation is associated with aspects of the treatment environment. A cross-sectional study in which nonparticipant observers recorded 3723 observations of resident behaviors in 53 Alzheimer's disease Special Care Units. Observational data were gathered on the physical environment and staff treatment in these settings, and resident characteristics were extracted from a data base developed in the study states by the Health Care Financing Agency. Analyses studied the association between aspects of the staff and physical environment and resident agitation levels, controlling for resident cognitive and functional status. Special Care Units in nursing homes in Kansas, Maine, Mississippi, and South Dakota. All residents and staff of the participating units. Eight specific agitated behaviors and two indexes of resident agitation were measured by direct observation by research assistants on three to four data collection walk-throughs in each of the study facilities. The most common agitated behaviors noted were repetitive mannerisms (4.5% of resident observations) and non-loud verbal excess (3.8%). Wandering, which frequently reflects agitation, was noted in 6.5% of resident observations. The proportion of residents exhibiting an agitated behavior varied from none in some units to 38% in one unit. Independent correlates of low unit agitation levels included favorable scores on measures of the physical environment and of staff treatment activities, low rates of physical restraint use, a high proportion of residents in bed during the day, small unit size, low levels of resident functional dependency, and fewer numbers of comorbid conditions. While the prevalence of agitation tends to increase as Alzheimer's disease progresses, modifiable treatment factors appear to have a strong influence on the prevalence of agitation. Both physical design and staff treatment appear to influence agitation rates, as do some measures consistent with a low stimulus approach to Alzheimer's care.

Triphase catalysis: a new rotating disk contactor for measuring mass transfer coefficients

Triphase catalysis (TPC) is a practically attractive way of carrying out phase transfer catalyzed reactions. The analysis of triphase catalytic systems requires a knowledge of mass transfer coefficients, but no correlations for estimating these are available. Therefore, a new rotating disk contactor (RDC) for measuring the external mass transfer coefficients of TPC systems has been designed and operated. Details of the design and construction of the contactor are discussed. In order to test the apparatus, experiments were carried out on a test system: synthesis of benzyl acetate from benzyl chloride and sodium acetate with polymer supported tributylmethylammonium chloride as the immobilized phase transfer catalyst. This reaction was found to be mass transfer controlled at low agitation speeds, and it was therefore possible to determine the external mass transfer coefficient as a function of the bulk agitation speed. The values ranged from 1.4×10 -3 to 2.0×10 -3 cm/min.

Decreasing extracellular pH increases CD13 receptor surface content and alters the metabolism of HL60 cells cultured in stirred tank bioreactors.

Flow cytometry and Northern blotting were used to examine the effects of extracellular pH on CD13 receptor surface content and mRNA levels of HL60 (human promyelocytic leukemia) cells. Decreasing culture pH (7.4, 7.2, and 7.0) increased the CD13 receptor surface content of HL60 cells cultured at low agitation intensity (80 rpm) in 2-L bioreactors. Unlike our earlier findings on the effects of increasing agitation rates and serum concentrations, changes in CD13 receptor content in response to decreasing culture pH did not correlate with changes in CD13 mRNA levels. Decreasing culture pH also decreased the average HL60 cell size. HL60 cells cultured at pH 7.0 and 7.2 exhibited glucose consumption and lactate production rates that were approximately 30-40% and 20-30% lower, respectively, than values of cells cultured at pH 7.4.

Improved oxygen transfer in cultures of Nocardia lactamdurans maintains the cephamycin biosynthetic proteins for prolonged time and enhances the conversion of deacetylcephalosporin into cephamycin C

Batch cultures of Nocardia lactamdurans MA4213 in stirred tank fermentors (400 rpm) became oxygen limited after 45 h of incubation. When the stirrer speed was increased at 36 h from 400 to 600 rpm there was a 5-fold increase in the dissolved oxygen (DO) in the broth what resulted in a 2-fold stimulation of cephamycin biosynthesis. Immunoblotting studies using antibodies against the α-aminoadipyl-cysteinyl-valine synthetase and the protein P7 (cephem-7-hydroxylase) component of the methoxylation system showed that cultures with increased DO concentrations maintained high levels of these proteins for an extended period of time. Under these conditions there was an enhanced conversion of cephamycin intermediates into cephamycin C. Allophane and alumina exerted a strong stimulatory effect on cephamycin biosynthesis that correlated with high DO levels in allophane- or alumina-supplemented cultures at low agitation speeds. Allophane or alumina supplementation of N. lactamdurans cultures resulted in a delayed decay of the cephem-7-hydroxylase protein in the cells favoring conversion of the cephamycin intermediates into cephamycin C.

Effect of impeller geometry on gas-liquid mass transfer coefficients in filamentous suspensions

Volumetric gas-liquid mass transfer coefficients were measured in suspensions of cellulose fibers with concentrations ranging from 0 to 20 g/L. The mass transfer coefficients were measured using the dynamic method. Results are presented for three different combinations of impellers at a variety of gassing rates and agitation speeds. Rheological properties of the cellulose fibers were also measured using the impeller viscometer method. Tests were conducted in a 20 L stirred-tank fermentor and in 65 L tank with a height to diameter ratio of 3:1. Power consumption was measured in both vessels. At low agitation rates, two Rushton turbines gave 20% better performance than the Rushton and hydrofoil combination and 40% better performance than the Rushton and propeller combination for oxygen transfer. At higher agitation rates, the Rushton and hydrofoil combination gave 14 and 25% better performance for oxygen transfer than two Rushton turbines and the Rushton and hydrofoil combination, respectively.

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO2 manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO2 levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO2 at a low level during the initial phase of the fermentation. However, both the timing of the pO2 decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates.

Neuropsychiatric aspects of progressive supranuclear palsy.

Administering the Neuropsychiatric Inventory (NPI), we examined the behavioral symptoms of 22 patients with progressive supranuclear palsy (PSP), 50 patients with Alzheimer's disease, and 40 controls. PSP patients exhibited apathy (91%), disinhibition (36%), dysphoria (18%) and anxiety (18%), but rarely (< 9%) irritability, abnormal motor behaviors, or agitation. Apathy in PSP was significantly associated with executive dysfunction. The presence of high apathy and low agitation and anxiety scale scores correctly identified the PSP patients 85% of the time. Evaluating the behavioral abnormalities of patients with neurodegenerative disorders will aid diagnosis and facilitate management.

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K( L ) a and of mixing (via pH measurements) in bioreactors up to 8 m(3) at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO(2) has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.

Characterization of non-newtonian behavior during mixing of bacterial cellulose in a bioreactor

The mixing properties of bacterial cellulose (BC) in an aerated and agitated vessel are described. To characterize the mixing of BC culture broth, which can affect the productivity of BC, non-Newtonian behavior during mixing of a 1% BC suspension was studied using an image processor capable of detecting decoloration of a pH indicator and was compared with that of a 2% carboxy methyl cellulose (CMC) solution. CMC solution was mixed homogeneously within the measured range of agitation speed, but the BC suspension was not mixed homogeneously at agitation speeds lower than 15 rps because mixing was delayed in some areas of the vessel. A possible reason for the inhomogeneity of the BC suspension at low agitation speeds is the non-Newtonian behavior which increases viscosity at low shear rates.

Efficient production of acetic acid from glucose in a mixed culture of Zymomonas mobilis and Acetobacter sp.

Efficient production of acetic acid from glucose was investigated in a mixed culture of Zymomonas mobilis ATCC10988 and Acetobacter sp. TC-1. The culture was initially incubated under low aeration and low agitation conditions to allow ethanol fermentation. A shift from ethanol fermentation to acetic acid fermentation was achieved by enhancing the aerobic conditions when the glucose, whose concentration was monitored using an on-line glucose sensor, dropped below a certain level. Ethanol production decreased when a higher aeration rate and agitation speed were used during the early stage of the cultivation. Acetic acid production was also markedly dependent on agitation and aeration during the early stage. The acetic acid yield from glucose was 0.64 g/g (95.5% of the theoretical value) under the optimum conditions.

LIQUID-FILM TIME CONSTANT ASSESSMENT FOR kLa MEASUREMENTS IN RESPIRING FERMENTATION BROTHS

Abstract To determine the liquid-film time constant (τF) of the dissolved oxygen electrode for kLa measurements with the dynamic method in respiring fermentation broths, an approach using the steady-state readings of the electrode in the broths containing respiring and non-respiring cells, respectively, has been established. The results showed that τF in microbial suspensions with high volumetric oxygen uptake rates could be very large under the conditions of low aeration and agitation, indicating the slower response of the oxygen electrode caused by the more difficult penetration of oxygen molecules through the thicker films with active cell respiration. The significance and adequacy of the τF corrections for kLa measurements were evaluated experimentally.

Holdup measurements in a Scheibel column by means of an ultrasonic technique

AbstractAn ultrasonic technique developed by Bonnet and Tavlarides for dispersed phase holdup determination in liquid–liquid contactors has been applied in a 0·10 m diameter Scheibel extraction column. The column consists of three alternate mixing and packing sections of 0·03 and 0·055 m in height, respectively. The packings were built with polypropylene mesh with 96% voidage. The liquid–liquid systems were toluene (dispersed)/water (continuous) and MIBK (dispersed)/water (continuous); the Rushton‐type impellers were operated at 400, 500 and 600 rpm, and with four dispersed and continuous phase flow rates. In one of the mixing chambers two different holdup measuring devices were installed: two ultrasonic transducers and one controlled dispersion sampler. The values of dispersed phase holdup obtained by both methods were compared using statistical methods. It was found that at low agitation and for high interfacial tension, conditions for which the dispersion is not completely uniform, the difference was significant, whereas at high agitation and low interfacial tension the values obtained through both methods were statistically equal. This work demonstrates the applicability of the ultrasonic technique for holdup measurements to Scheibel columns, in which the only technique used so far was sampling. This ultrasonic technique allows us to solve the axial monitoring and control problems of these columns.

Influence of a rotating sieve on pumping and mixing performances of an internal loop reactor

Internal loop reactors equiped with an axial impeller are often used in biotechnology but their mixing characteristics under low agitation conditions have not been studied extensively. Mixing performances of a pilot scale (15 L) loop reactor equipped with an independently driven rotating sieve and an axial impeller have been studied by a pH pulse technique. Smaller mixing times and lower recirculation times were obtained for the same impeller rotation speed when fluid was recirculated upward in the draft tube. The influence of sieve rotation speed on mixing performances has been studied in a second step. A negligible effect was obtained on recirculation time, while lower mixing times were obtained, especially under low agitation conditions. Data analysis shows that classical correlations enable a correct description of experimental results; sieve rotation is shown to induce a better pumping as well as overall mixing efficiency.

Enhanced biodegradation of phenanthrene in a biphasic culture system

Degradation of phenanthrene byPseudomonas aeruginosa AK1 was examined in (i) an aqueous mineral salts medium to which phenanthrene particles of varying size (i.e. diameter) were added, and (ii) an aqueous/organic biphasic culture system consisting of mineral salts medium supplemented with 2,2,4,4,6,8,8-heptamethylnonane (HMN) as the phenanthrene-carrying organic phase. In both systems, the rate of phenanthrene biodegradation could be significantly enhanced by manipulations leading to improved phenanthrene mass transfer into the aqueous phase. With crystalline phenanthrene, the rate of biodegradation was found to be directly correlated to the particle surface area, whereas in the biphasic system the rate of biodegradation of the dissolved phenanthrene was mainly governed by the HMN/water interface area. In the latter system, exponential growth with a doubling time t d of 6–8 hours has been achieved under conditions of intensive agitation of the medium indicating that phenanthrene degradation by strain AK1 is limited mainly by physicochemical parameters. Addition of selected surfactants to the culture medium was found to accelerate phenanthrene degradation by strain AK1 only under conditions of low agitation (in the presence of HMN) and after pretreatment of phenanthrene crystals by ultrasonication (in the absence of HMN). Evidence is presented that the stimulating effect of the surfactants was primarily due to improved dispersion of phenanthrene particle agglomerates (in the aqueous mineral salts medium supplemented with phenanthrene crystals) or of the phenanthrene-carrying lipophilic solvent drops (in the aqueous/organic biphasic culture system) whereas the solubilizing activity towards phenanthrene was neglectible. Under conditions of intensive mixing of the culture medium (i.e. if a high particle surface area or HMN/water interface area, respectively, is provided), the addition of surfactants did not enhance phenanthrene biodegradation.

A simple staining procedure for the characterisation of Basidiomycetes pellets by image analysis

A simple staining procedure, associated with image analysis, was used to discriminate two zones (the filamentous zones and the compacted cores) in aggregates of fungal biomass according to their accessibility for an agent of the medium. The application of the method to batch fermentations of Schizophyllum commune showed a higher proportion of loosely entangled particles (clumps) and thinner filamentous outer zones obtained at a high agitation speed (500 rpm) compared to low agitation speeds (120–200 rpm).