Use of response surface methodology to optimize culture medium for production of lovastatin by Monascus ruber

Response surface methodology (RSM) was employed to study the effect of culture medium on the production of lovastatin in mixed solid-liquid state (or submerged) cultures by Monascus ruber. The maximal lovastatin yield (131 mg/L, average of three repeats) appeared at the region where the respective concentrations of rice powder, peptone, glycerin, and glucose were around 34.4 g/L, 10.8 g/L, 26.4 ml/L, and 129.2 g/L, respectively. The optimized medium resulted in a significant increase of lovastatin yield, as compared with that obtained by the fermentation of many other M. ruber species.     

Newly developed medium and strategy for bacterial cellulose production

Bacterial cellulose (BC) has unique properties, such as high crystallinity, a high degree of polymerisation, high tensile strength and high purity, compared with native cellulose. In this study, a previously determined BC production medium was improved in static culture, and the production cost was evaluated and compared with molasses and with other defined media, such as Hestrin–Schramm, Zhou, Yamanaka and Park, using Gluconacetobacter xylinus. In addition to this analysis, because the surface area/volume ratio is an important parameter in static culture, different surface area/volume ratios were analysed in the range of 0.2–1.46. The defined medium (M1A05P5) and culture type contained glucose (10 g/L), yeast extract (10 g/L), peptone (7 g/L), acetic acid (1.5 ml/L), and ethanol (5 ml/L), and the pH was adjusted to 5.0 in static culture. The highest productivity was observed in the M1A05P5 medium that was 5-fold higher than either molasses or Park's medium. Although the molasses medium was proposed as a cost-effective medium, the production price of BC was the lowest in the M1A05P5 medium. Therefore, the newly developed medium and strategy were highly promising candidates for the industrial-scale production of BC.     

The coupling between catabolism and anabolism of Methanobacterium thermoautotrophicum in H2- and iron-limited continuous cultures

The aim of the present work was to investigate whether uncoupling of catabolism from anabolism, which was often observed in heterotrophic microorganisms under energy-sufficient growth conditions, also occurs in the autotrophic bacterium Methanobacterium thermoautotrophicum. For this purpose, M. thermoautotrophicum was cultivated in continuous cultures that were limited by the trace element iron. The influences of both dilution rate and iron supply rate on the coupling between anabolism and catabolism were investigated. As compared to continuous cultures of M. thermoautotrophicum limited by the energy substrate H₂, a 5-fold decrease in the biomass concentration and a 3-fold decrease in H₂, CO₂, and CH₄ conversion rates were observed in iron-limited cultures. However, the specific substrate and product conversion rates increased as compared to the values determined in energy-limited cultures. Thus, iron limitation provoked an uncoupling of catabolism from anabolism. At a dilution rate of 0.096 h⁻¹ and at an iron concentration of 17 μM in the feed, the specific H₂ consumption rate was 100% higher than the rate determined under H₂-limiting conditions, whereas at a dilution rate of 0.168 h⁻¹, the values differed only by 5%. Uncoupling of catabolism from anabolism also increased dramatically when the iron supply rate was lowered but the dilution rate was kept constant. Thus, the extent of uncoupling is a function of both the dilution rate and the iron supply rate. It was found that the specific consumption rate of H₂ increased in parallel with the partial pressure of H₂ in the culture medium. This suggested that the catabolic activity of M. thermoautotrophicum was not stringently controlled at the enzymatic level and can be considerably stimulated by the excess of H₂ in the medium. Hypotheses as to the fate of the excess energy derived from uncoupled catabolism are discussed, but the physiological reason for the partial uncoupling between catabolism and anabolism remains yet to be clarified.     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.     

Statistical optimization of medium for the production of pyruvate oxidase by the recombinant Escherichia coli

Pyruvate oxidase (PyOD) is a very useful enzyme for clinical diagnostic applications and environmental monitor. Optimization of the fermentation medium for maximization of PyOD constitutively, production by Escherichia coli DH5α/pSMLPyOD was carried out. Response surface methodology (RSM) was used to optimize the medium constituents. A 2^6–2 fractional factorial design (first order model) was carried out to identify the significant effect of medium components towards PyOD production. Statistical analysis of results shows that yeast extract, ammonium sulfate and composite phosphate were significant factors on PyOD production. The optimized values of these three factors were obtained by RSM based on the result of a 2³ central composite rotatable design. Under these proposed optimized medium, the model predicted a PyOD activity of 610 U/L and via experimental rechecking the model, an activity of 670 U/L was attained.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

Medium optimization for the production of the secondary metabolite squalestatin S1 by a Phoma sp. combining orthogonal design and response surface methodology

In the present work, a combined statistical approach of orthogonal design (L₂₇(3¹³)), response surface techniques and polynomial regression were applied to optimize the composition and concentration of a liquid fermentation medium for the production of squalestatin S1 by a fungus (a Phoma species). Optimal conditions for maximal titres and productivity were determined based on 13 parameters at three different levels. Initially, a screening design methodology was used to evaluate the process variables, which were relevant to S1 titre and the response surfaces applied to find optimal regions for production. The sources of carbon and concentration, and their interactions with oily precursors were statistically significant factors. The combined orthogonal design and response surface methodology predicted optimal conditions for of 273 mg l⁻¹ of squalestatin S1. Confirmatory experiments of the optimal medium composition produced titres of 434 mg l⁻¹ in a 5-day fermentation at 25 °C. This represented a 60% improvement in the maximum titre predicted, and a two-fold higher productivity when compared with reported S1 yields of various fungal species. This combined statistical approach enables rapid identification and integration of key medium parameters for optimising secondary metabolite production and could be very useful in pharmaceutical screening programmes.     

Statistical optimization of medium components and growth conditions by response surface methodology to enhance lipase production by Aspergillus carneus

A response surface approach has been used to study the production of an extracellular lipase from Aspergillus carneus, which has the property of immense industrial importance. Interactions were studied for five different variables (sunflower oil, glucose, peptone, agitation rate and incubation period), which were found influential for lipase production by one-at a time method. We report a 1.8-fold increase in production, with the final yield of 12.7 IU/ml in comparison to 7.2 U/ml obtained by one-at-a-time method. Using the statistical approach (response surface methodology (RSM)) the optimum values of these most influential parameters were as follows: sunflower oil (1%), glucose (0.8%), peptone (0.8%), agitation rate (200 rpm) and incubation period (96 h) at 37 °C. The subsequent verification experiment confirmed the validity of the model.     

Cell culture medium improvement by rigorous shuffling of components using media blending

A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.     

Semisynthetic culture medium for growth and dihydroxyacetone production by Gluconobacter oxydans

Only three vitamins (pantothenate, p-amino benzoic acid, nicotinic acid) and two amino acids (serine, glutamine) were required in the growth medium for Gluconobacter oxydans which allowed the concentration of yeast extract to be reduced to 5–10% of the previous concentration. When compared with data from cultivations with complex media, the new medium gave a lower yield (about 0.02 g biomass per g glycerol) and comparable growth rate (0.24 to 0.38 h–1) but a higher productivity (10.3 g dihydroxyacetone/gh).     

重组枯草芽孢杆菌发酵生产乳铁蛋白N叶工艺优化北大核心CSCD

为实现乳铁蛋白N叶的大规模制备,本研究对表达乳铁蛋白N叶的工程菌枯草芽孢杆菌pMA0911-D60Y/Y92D进行了发酵工艺的优化。确定了最佳的培养条件:以葡萄糖为最佳碳源,以胰蛋白胨为最佳氮源,在pH 7.0、温度28℃、发酵25.5 h条件下诱导表达目的蛋白,目的蛋白的IOD值高达68.03%。在10L发酵罐上对重组菌株的发酵条件进行优化,获得如下的最佳发酵工艺,即采用300 r/min转速,0-7 h时,在pH 7.5、30℃条件下培养菌体;7-25 h时,在pH 7.0、28℃条件下诱导表达目的蛋白。发酵结束后,收集细胞并破碎后取上清液用HisTrapHP亲和层析及SuperdexTM200(10/300GL)亲和层析法对细胞上清液进行纯化至均一条带,获得了纯度>94%的重组乳铁蛋白N叶,1 L菌体能制备23.5 mg纯蛋白。本研究为重组牛乳铁蛋白N-叶的高效制备奠定了基础。     

虫草素高产菌株的筛选及不同添加物对虫草素产量的影响研究

通过对14株蛹拟青霉菌株进行摇瓶液体发酵培养试验,筛选虫草素产量最高的蛹虫草菌株,并确定了虫草素的最佳收获期;比较8种营养添加物对虫草发酵过程中虫草素积累的影响,筛选出最佳的营养添加物,以提高液体发酵生产虫草素的产量。结果表明：蛹虫草CM001号菌株的虫草素产量最高,蛹虫草菌在第10天细胞量达到最大值20.44mg/mL,虫草素含量在第13天达到最大值73.81mg/L,70%以上的虫草素分布在发酵液中。其中分别添加腺苷、腺嘌呤、丙氨酸、L-天冬氨酸、甘氨酸5种物质,对虫草素合成的促进作用均较明显,均能有效提高蛹虫草液体发酵虫草素的产量,特别是添加1g/L的腺嘌呤能使10号菌株的虫草素产量提高7.09倍。     

A new approach to define optimized range of medium composition for enhancement of hairy root production in fed-batch process

This work proposes a new methodology to identify the best medium concentrations for fed-batch production of hairy root using Datura innoxia as a model. Firstly, the role of each component on the growth rate is investigated separately. Then, an experimental design allows refining the optimization studying the interactions between the major species. The result analysis let to define concentration range optimized for fed-batch process. The work novelties lie in two aspects. Firstly, concentrations have been kept constant during each run. Thus, biomass uptakes do not affect the optimization and the growth rate is maintained constant during the exponential phase. Secondly, the effects of salts are generally studied. In this work, the influences of each ion are investigated in order to avoid bias due to the counter-ion effects. Compared to the classical B₅ medium, the optimized medium shows a significant improvement leading to more than 80% increase of final biomass production.     

Medium optimization for the production of avermectin B1a by Streptomyces avermitilis 14-12A using response surface methodology

Response surface methodology was employed to optimize the composition of medium for the production of avermectin B1a by Streptomyces avermitilis 14-12A in shaker flask cultivation. Corn starch and yeast extract were found to have significant effects on avermectin B1a production by the Plackett–Burman design. The steepest ascent method was used to access the optimal region of the medium composition, followed by an application of response surface. The analysis revealed that the optimum values of the tested variables were 149.57 g/l corn starch and 8.92 g/l yeast extract. A production of 5128 mg/l, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of original level (3528 mg/l), 1.45-fold increase had been obtained.     

Assessment of olive-mill wastewater as a growth medium for lipase production by Candida cylindracea in bench-top reactor

Olive-mill wastewater (OMW) was investigated for its suitability to serve as a medium for lipase production by Candida cylindracea NRRL Y-17506. The OMW that best supported enzyme production was characterized by low COD and low total sugars content. In shake flask batch cultures, OMW supplementation with 2.4 g l⁻¹ NH₄Cl and 3 g l⁻¹ olive oil led to an enzyme activity of about 10 U ml⁻¹. The addition of glucose or malt extract and supplements containing organic N (e.g., peptone, yeast extract) either depressed or did not affect the enzyme production. Further experiments were then performed in a 3-l stirred tank reactor to assess the impact of medium pH and stirring speed on the yeast enzyme activity. The lipase activity was low (1.8 U ml⁻¹) when the pH was held constant at 6.5, significantly increased (18.7 U ml⁻¹) with uncontrolled pH and was maximum (20.4 U ml⁻¹) when the pH was let free to vary below 6.5. A stirring regime, that varied depending on the dissolved oxygen concentration in the medium, both prevented the occurrence of anoxic conditions during the exponential growth phase and enabled good lipase production (i.e., 21.6 U ml⁻¹) and mean volumetric productivity (i.e., 123.5 U l⁻¹ h⁻¹).     

Microplates with integrated oxygen sensing for medium optimization in animal cell culture

A new approach using microtiter plate cultivation with on-line measurement of dissolved oxygen (DO) was applied for medium optimization of mammalian cell culture. Applying dynamic liquid phase balance, oxygen uptake rates were calculated from the DO level and used as an indicator for culture viability. The developed method was successfully applied to optimization of the concentration of glucose, glutamine and inorganic salts for cultivation of a Chinese Hamster Ovary (CHO) cell line. Using 2³ full factorial central composite design, the optimum medium composition could be identified in one single run. The concentration of inorganic salts had a significant influence on cultivation. The developed method exhibits high potential to improve procedures of medium optimization for animal cell cultivation by allowing the investigation of large sets of potentially important variables in short time and with reduced effort.     

Statistical optimization of medium composition for aspergiolide A production by marine-derived fungus <Emphasis Type="Italic">Aspergillus glaucus</Emphasis>

Statistical methodologies were employed to optimize submerged culture medium for the production of a novel antineoplastic compound aspergiolide A by a marine-derived fungus Aspergillus glaucus HB1-19 for the first time. Orthogonal design was preformed to determine the initial composition. Then Plackett–Burman design was applied to evaluate the influence of related nutrients, and yeast extract paste, soybean powder and sodium glutamate were confirmed as critical factors in the medium. Response surface methodology (RSM) was finally taken as an effective approach to optimize the combination of the obtained three significant factors. The predicted maximal aspergiolide A production of 62.4 mg/L appeared at the region where the concentrations of sodium glutamate, soybean powder, and yeast extract paste were 2, 1, and 1.07 g/L, respectively. Under the proposed optimized conditions, the experimental aspergiolide A production reached 71.2 mg/L. The correlation between predicted value and measured value of these experiments proved the validity of the response model. After optimization, aspergiolide A production increased 4.22 times compared to that of the original medium. Elemental analysis was finally taken into consideration, and carbon–nitrogen ratio in the medium increased from 20.1:1 to 86.6:1. This great difference was inferred as the most important reason for production enhancement by metabolic pathway analysis.     

Medium optimization by combination of response surface methodology and desirability function: an application in glutamine production

An optimization strategy based on desirability function approach (DFA) together with response surface methodology (RSM) has been used to optimize production medium in L-glutamine fermentation. Fermentation problems often force to reach a compromise between different experimental variables in order to achieve the most suitable strategy applying in industrial production. The importance of the use of multi-objective optimization methods lies in the ability to cope with this kind of problems. A sequential RSM with different combinations of glucose and (NH₄)₂SO₄ was performed to attain the optimal medium (OM-1) in glutamine production. Based on the result of RSM and the evaluation of production cost, a more economical optimal medium (OM-2) was obtained with the aid of DFA. In DFA study, glutamate, the main by-product in glutamine fermentation as another response was considered. Compared with OM-1 in validated experiment, similar amounts of glutamine were obtained in OM-2 while the concentration of glutamate and the production cost decreased by 53.6 and 7.1%, respectively.