Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

Role of hemoglobin in improving biodegradation of aromatic contaminants under hypoxic conditions

Genetic engineering of bacteria using the Vitreoscilla (bacterial) hemoglobin gene has been used to enhance bioremediation of several compounds which are models for, or are themselves, toxic chemicals which may contaminate soil and water. Initial experiments, done mostly in shake flasks, with Escherichia coli, Burkholderia sp. DNT and Pseudomonas aeruginosa demonstrated that expression of Vitreoscilla hemoglobin in heterologous hosts can enhance biodegradation of several aromatic compounds as well as an organophosphorus compound. These studies concentrated for the most part on enhancement of endogenous catabolic capabilities of the hosts; the presence of vgb/VHb enhanced both growth and biodegradation. The initial studies were followed by experiments in systems which more closely approximated conditions that would exist in field applications. These included soil columns, continuous flow reactors and membrane bioreactors. The latter work also enabled calculation of the effects of the presence of vgb/VHb on kinetic parameters such as growth rate, substrate and oxygen utilization rate, and degradation rate of pollutants, etc. Although not always the case, for the most part, and particularly in bioreactors, the advantages due to vgb/VHb were greater under conditions of limited aeration or hypoxic conditions.     

Efficacy of commercial products in enhancing oil biodegradation in closed laboratory reactors

Summary A laboratory screening protocol was designed and conducted to test the efficacy of eight commercial bacterial cultures and two non-bacterial products in enhancing the biodegradation of weathered Alaska North Slope crude oil in closed flasks. Three lines of evidence were used to support the decision to progress to field testing in Prince William Sound: rapid onset and high rate of oxygen uptake, substantial growth of oil degraders, and significant degradation of the aliphatic and aromatic hydrocarbon fractions of the weathered Alaska North Slope crude oil. A product had to enhance biodegradation greater than that achieved with excess mineral nutrients. Experiments were conducted in closed respirometer flasks and shake flasks, using seawater from Prince William Sound and weathered crude oil from a contaminated beach. Analysis of the data resulted in selection of two of the ten products for field testing. Both were bacterial products. Findings suggested that the indigenous Alaskan microorganisms were primarily responsible for the biodegradation in the closed flasks and respirometer vessels.     

Growth dynamics of Artemisia annua hairy roots in three culture systems

The transient growth of Artemisia annua hairy roots was compared for cultures grown in shake flasks and in bubble column and mist reactors. Instantaneous growth rates were obtained by numerically differentiating the transient biomass measurements. Specific sugar consumption rates showed good agreement with literature values. From the growth rate and sugar consumption rate, the specific yield and maintenance coefficient for sugar were determined for all three culture systems. These values were statistically indistinguishable for roots grown in shake flasks and bubble columns. In contrast, the values for roots grown in bubble columns and mist reactors were statistically different, suggesting that sugar utilization by roots grown in these two systems may be different. By measuring respiration rates in the bubble column reactor we also determined the actual biomass yield and maintenance coefficient for O(2) and CO(2). Together with an elemental analysis of the roots, this allowed us to obtain a reasonable carbon balance.     

Lipase production byGeotrichum candidum from sauerkraut brine

Sauerkraut brine could serve as a substrate for the production of extracellular lipase (EC 3.1.1.3) byGeotrichum candidum ATCC 34614. The fungus produced the highest specific activity (more than 140 units/mg protein) when cultivated in shake flasks for 72 h at 30°C in the brine, with a small amount of olive oil (0.3%, v/v) as an enzyme inducer. Lipase activity was recovered from the culture filtrate by fractionation with acetone and the yield was as high as 59%.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.     

The performance of immobilized glucose isomerase supported by shrimp chitin in various types of reactors

Five different types of reactors were employed for glucose isomerization using shrimp shell as the support on which to immobilize the glucose isomerase. The Michaelis-Menten constants and effective diffusivity of glucose in the immobilized enzyme bed were experimentally determined and used in a theoretical analysis of the radial-flow reactor. The fractional conversions of the radial-flow, fluidizedbed, and packed-bed reactors with the same -residence time were found experimentally to be almost the same. This result reveals that the use of radial-flow and fluidized-bed reactors for this immobilized enzyme system is highly feasible.     

Determining design and scale-up parameters for degradation of atrazine with suspended Pseudomonas sp. ADP in aqueous bioreactors

This work investigated the kinetic parameters of atrazine mineralization by suspended cells of Pseudomonas sp. ADP in both shake flasks and spherical stirred tank batch reactors (SSTR). The degradation of atrazine and growth of Pseudomonas sp. ADP were studied. Experiments were performed at different temperatures and stirring speeds in both reactors at varying initial concentrations of atrazine. Cell growth and atrazine concentration were monitored over time, and a Monod model with one limiting substrate was used to characterize the kinetic behavior. Temperature, stirring speed, and reactor type were all found to significantly affect the regressed Monod parameters. At 27 degrees C and 200 rpm, for the shaker flask experiments, mu max and Ks were determined to be 0.14 (+/-0.01) h-1 and 1.88 (+/-1.80) mg/L, respectively. At 37 degrees C, mu max and Ks increased to 0.25 (+/-0.05) h-1 and 9.59 (+/-6.55) mg/L, respectively. As expected, stirrer speed was also found to significantly alter the kinetic parameters. At 27 degrees C and 125 rpm, mu max and Ks were 0.04 (+/-0.002) h-1 and 3.72 (+/-1.05) mg/L, respectively, whereas at 37 degrees C and 125 rpm, mu max and Ks were 0.07 (+/-0.008) h-1 and 1.65 (+/-2.06) mg/L. In the SSTR the kinetic parameters mu max and Ks at room temperature were determined to be 0.12 (+/-0.009) h-1 and 2.18 (+/-0.47) mg/L, respectively. Although the mu max values for both types of reactors were similar, the shaker flask experiments resulted in considerable error. Error analysis on calculated values of Ks were found to impact estimates in atrazine concentration by as much as two orders of magnitude, depending on the reactor design, illustrating the importance of these factors in reactor scale-up.     

Optimization of lipase production byPseudomonas aeruginosa MB 5001 in batch cultivation

Summary The production ofPseudomonas aeruginosa MB 5001 extracellular lipase was optimized by batch cultivation employing shake flasks and 23-L bioreactors. This enzyme efficiently and selectively bioconverts dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)ethyl)phenyl)4,6-dithianonanedioate (diester) to its (S)-ester acid. Process development studies focused on the identification and optimization of the physicochemical parameters required to achieve maximum lipase production. Of the media evaluated, a peptonized milk-based medium was found to support excellent lipase production and stability. Medium composition and process parameters that supported optimal lipase production were different from those supporting maximum biomass formation. Of the parameters investigated, dissolved oxygen tension had the most significant and unexpected impact on lipase production. Elevated lipase production was achieved whenP. aeruginosa MB 5001 was cultivated in a dissolved oxygen limited environment. Overall, these process development studies resulted in a 100% increase in lipase production when compared to the original shake flask process employing skim milk.     

Novel airlift reactor fitting for hairy root cultures: developmental and performance studies

In vitro growth of Solanum chrysotrichum hairy roots was carried out in three different types of reactors: shake flasks, a glass-draught internal-loop 2-L basic design airlift reactor (BDR), and a novel modified mesh-draught with wire-helixes 2-L reactor (MR). In each of them, the growth patterns were different, as well as some of the dynamic parameters. The specific growth rates were 0.08, 0.067, and 0.112 d(-1) for shake flasks, BDR, and MR, respectively. In shake flasks and in the MR, growth followed first-order kinetics. In the MR without roots, superficial liquid velocity in the riser and downcomer ranged from 2.1 to 2.7 and 1.4 to 1.7 cm s(-1), respectively (nearly the same as the BDR values). After 42 days in culture, tissue density in the MR was twice that found in the BDR and about the same as that found in the shake flasks. At the tissue densities reached at 42 days, superficial liquid velocities in the MR and BDR downcomers were 4-5 and 7-8 times lower, and mixing times were 11 and 18 times longer than those observed without roots. Tissue densities measured at three points in the MR's downcomer and riser ranged from 10.21 to 12.17 and 4.94 to 5.24 gDW L(-1) respectively. Dynamic gas hold-up dropped faster when roots grew radially in the mesh-draught. In addition, root cultures were scaled-up in a 10-L MR reactor in which some geometric relations were maintained, such as the Q/V radio. Growth in 10-L MR followed first-order kinetics, but despite this, specific growth velocity was 0.09 d(-1) and overall tissue density diminished slightly with respect to that of the 2-L MR. Tissue inoculation, distribution, and harvest were more easily accomplished in the MRs.     

Biodegradation of 2,4,5-trichlorophenol by aerobic microbial communities: biorecalcitrance,inhibition, and adaptation

Chlorinated aromatic compounds challenge our environment and wastewater treatment processes due to their biorecalcitrance and inhibition. In particular, 2,4,5-trichlorophenol (TCP) seems to demonstrate greater resistance to biodegradation than other trichlorophenols and is a known uncoupler of the electron transport chain, although little work addresses this compound specifically. Here, we investigate the biorecalcitrance, inhibition, and adaptation to 2,4,5-trichlorophenol by aerobic mixed microbial communities. We show that 2,4,5-trichlorophenol is strongly resistant to biodegradation at concentrations greater than 40 μM, demonstrates inhibition to respiration in direct proportion to 2,4,5-trichlorophenol concentration (with 50% inhibition projected near 85 μM 2,4,5-trichlorophenol), and does not sustain biomass in continuous reactors, even when all input 2,4,5-trichlorophenol is degraded. Communities showed consistent adaptation patterns to 2,4,5-trichlorophenol at concentrations of 10 μM and 20 μM, but these patterns diverged at concentrations greater than 40 μM. Finally, thermodynamic approximations were used to estimate the yield of 2,4,5-trichlorophenol as 0.165 gVSS/gCOD, a low value that partially explains why biodegradation of 2,4,5-trichlorophenol did not sustain the biomass. In particular, we estimated that the minimum concentration to support steady-state biomass (S _min) is approximately 180 μM, a value much larger than the 40-μM concentration that is strongly resistant to biodegradation. Thus, readily biodegradable concentrations of 2,4,5-trichlorophenol are too low to sustain the biomass that biodegrades it.     

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS-22.

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.     

Production of carbonyl reductase by Metschnikowia koreensis

A new strain of the yeast Metschnikowia koreensis was grown in shake flasks and a stirred bioreactor for the production of carbonyl reductase. The optimal conditions in the bioreactor for maximizing the biomass specific activity of the enzyme were found to be: a medium composed of glucose (20 g/L), peptone (5 g/L), yeast extract (5 g/L) and zinc sulfate (0.3g/L); the pH controlled at 7; the temperature controlled at 25 °C; an agitation speed of 500 rpm; and an aeration rate of 0.25 vvm. In the bioreactor, a biomass specific enzyme activity of 115.6 U/gDCW was obtained and the maximum biomass concentration was 15.3 gDCW/L. The biomass specific enzyme activity obtained in the optimized bioreactor culture was 11-fold higher than the best result achieved in shake flasks. The bioreactor culture afforded a 2.7-fold higher biomass concentration than could be attained in shake flasks.     

Hydromechanical stress in shake flasks: correlation for the maximum local energy dissipation rate

Shake flasks are widely used in biotechnological process research. Bioprocesses for which hydromechanical stress may become the rate controlling parameter include those where oils are applied as carbon sources, biotransformation of compounds with low solubility in the aqueous phase, or processes employing animal, plant, or filamentous microorganisms. In this study, the maximum local energy dissipation rate as the measure for hydromechanical stress is characterized in shake flasks by measuring the maximum stable drop size. The theoretical basis for the method is that the maximum stable drop diameter in a coalescence inhibited liquid/liquid dispersion is only a function of the maximum local energy dissipation rate and not of the dispersing apparatus. The maximum local energy dissipation rate is obtained by comparing the drop diameters in shake flasks to those in a stirred tank reactor. At the same volumetric power consumption, the maximum energy dissipation rate in shake flasks is about 10 times lower than in stirred tank reactors explaining the common observation of considerable differences in the morphology of hydromechanically sensitive cells between these two reactor types. At the same volumetric power consumption, the maximum local energy dissipation rate in baffled and in unbaffled shake flasks is very similar. A correlation is presented to quantify the maximum local energy dissipation rate in shake flasks as a function of the operating conditions. Non-negligible drop viscosity may be considered by known literature correlations. Further, from dispersion experiments a critical Reynolds number of about 60,000 is proposed for turbulent flow in unbaffled shake flasks.     

Automation of Biodegradation Tests

The feasibility of automating biodegradation tests was evaluated by comparing the results obtained using an Anachem SK233 automation system linked to an HPLC, and the traditional shake flask method using a shaker incubator and manual sampling. For the purpose of this study, several series of biodegradation kinetics were performed using a range of halogenated compounds which are common starting materials in organic synthesis. The first experiment involved two series of Fluorocinnamic Acid biodegradation kinetic tests performed over a period of 5 days. Several initial concentrations were used. Results obtained using the automated and manual methods were in very good agreement, with variations in absolute concentrations and kinetic constants ranging, between 10 % and 27 %. Half‐life values calculated from kinetic data ranged between 3.6 and 25.6 hours, depending on the initial organic compound concentrations (range 5 to 100 mg L^–1) and the activity of the activated sludge employed. In the second part of this study, the degradation of Bromobenzoic Acid, Chlorobenzoic Acid and Iodobenzoic Acid by activated sludge were investigated. Significant differences in the lag periods were observed. Differences in the hydrodynamics and the configuration of the vessels may contribute to the variations observed. However, it was found that for 4‐Iodobenzoic Acid, manual and automated methods yielded comparable first order kinetic values for the exponential growth bacterial cycle. 4‐Bromobenzoic Acid, 4‐Chlorobenzoic Acid and 4‐Iodobenzoic Acid rates of disappearance were estimated and complete removal on the shake flasks was observed after 72, 66 and 96 hours respectively. The results are very promising and open a lot of possibilities for future automation of a range of biodegradation and inhibition tests.     

Exopolysaccharide production by Acremonium persicinum in stirred-tank and air-lift fermentors

Summary Exopolysaccharide production by the fungus Acremonium persicinum was affected by the culture system used. The yields achieved in shake flasks were not obtained in a stirred tank reactor, except at very low stirring speeds (100 rpm). However when grown in an air-lift fermentor, exopolysaccharide levels were similar to those found with shake flask cultures. Results suggest that both dissolved oxygen tension and shear rate may determine the ability of this organism to synthesise this exopolysaccharide. Offprint requests to: R. J. Seviour     

Phytase production by Sporotrichum thermophile in a cost‐effective cane molasses medium in submerged fermentation and its application in bread

Aims: Phytase production by Sporotrichum thermophile in a cost‐effective cane molasses medium in submerged fermentation and its application in bread. Methods and Results: The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air‐lift fermenters. Among surfactants tested, Tweens (Tween‐20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X‐100 inhibited the enzyme production. The mould produced phytase optimally at a_w 0·95, and it declined sharply below this a_w value. The enzyme production was comparable in air‐lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca‐alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread. Conclusions: The phytase production by S. thermophile was enhanced in the presence of Tween‐80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca‐alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high‐soluble phosphate. Significance and Impact of the Study: The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air‐lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid.     

对-氯代苯胺(PCA)在填充床生物反应器中的降解作用

以氯代苯胺(PCA)为选择基质,用驯化技术从降解对二氯苯(p-DCB)的富集培养物中得到了以同化PCA为唯一碳源和氮源的混合微生物。将这种固定在填充床反应器中的微生物用于PCA的降解作用研究中。在该反应器里,PCA的生物降解遵循Logistic方程q=q_max/(1+e^α-βU_v).由方程求出了主要的动力学常数,K_s(半速率常数)和q_max(最大比基质降解速率).于PCA降解的同时,释放氯离子到培养基中。在水力停留时间3h, 进水PCA浓度为360mg·L^-1情况下,基质的体积降解率达到125mg·L^-1·h^-1;基质的百分去除率为91%.