Lycopene production from synthetic medium by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and 2896 (−) in a stirred-tank fermenter

The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V_\texttip = 0. 9 2 6- 2. 1 6 1  \textm/\texts V_{\text{tip}} = 0. 9 2 6- 2. 1 6 1 \, {\text{m}}/{\text{s}} ) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (−) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.     

Enhanced shear protection and increased production of an anti-tumor polysaccharide by Agaricus blazei in xanthan-supplemented cultures

Xanthan supplementation provided shear protection and stimulated polysaccharide production by Agaricus blazei. In xanthan-free cultures, the optimal cell yield, 0.63 g biomass g^–1 glucose, and product yield, 0.19 g polysaccharide g^–1 glucose, were, respectively, when the critical impeller tip speed was 50.3 cm s^–1 and 100.5 cm s^–1. Furthermore, the critical impeller tip speed of cell yield shifted from 50.3 cm s^–1 to 100.5 cm s^–1 with the supplementation of 1 g xanthan l^–1. Maximum specific product yield, namely 0.74 g polysaccharide g^–1 biomass, was achieved with inlet air supply of 3% O₂ and impeller tip speed of 100.5 cm s^–1.     

Morphology,cell growth,and polysaccharide production of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> in liquid suspension culture at different agitation rates

Noscoc flagelliforme is a terrestrial macroscopic cyanobacterium with high economic value. Free-living cells that were separated from a natural colony of N. flagelliforme were cultivated in a 20-L photobioreactor for 16 days at five agitation rates with impeller tip speeds at 0.3, 0., 0.8, 1.0, and 1.5 m·s⁻¹. With different impeller tip speeds there were significant differences in the cell growth and polysaccharide production, and different types of cell colonies appeared because of different shear forces caused by agitation. At harvest time, cell concentrations with tip speeds of 0.8 and 1.0 m·s⁻¹ were clearly higher than those with the other three tip speeds, but dry cell weights with the tip speeds of 0.3, 0.5, 0.8, and 1.0 m·s⁻¹ were almost the same. The highest RPS (polysaccharide that released into liquid medium) production was obtained with the tip speeds of 0.8 and 1.0 m·s⁻¹, while the highest EPS (polysaccharide that formed capsule or slime layer) production was obtained with the tip speed of 0.5 m·s⁻¹. The tip speed of 1.5 m·s⁻¹ was harmful for both cell growth and polysaccharide production, indicating that an appropriate shear force was needed in the liquid suspension culture of N. flagelliforme.     

Improved performance in γ-polyglutamic acid production by Bacillus subtilis LX on industrial scale by impeller retrofitting and its unstructured microbial growth kinetics model

Abstract

We conducted industrial scale γ-polyglutamic acid (γ-PGA) production by Bacillus subtilis (B. subtilis) LX and modeled its microbial growth kinetics based on a logistic regression. We found that the use of a three-layer impeller including a lower semicircular disc impeller and two-layers of six-wide-leaf impellers were able to both increase γ-PGA yields and decrease fermentation time as compared with two-layer Rushton impellers. Indeed, our results revealed that the optimal γ-PGA yield (20.67?±?2.19?g/L) was obtained after 40?hr in the impeller retrofitted fermenter, and this yield was 29.7% higher than that in Rushton impellers fixed fermenter. The microbial growth kinetics of B. subtilis LX in this system were established, and the model was consistent with the experimental data (R² = 0.924) suggesting that it was suitable for describing the microbial growth kinetics underlying γ-PGA production on an industrial scale. In addition, biomass yield (Y_x/s-glucose), γ-PGA yield (Y_p/s-glucose), γ-PGA yield (Y_{p/s-glutamate}), and the correlation between γ-PGA production and B. subtilis LX (Y_p/x) were found to be 0.043, 0.133, 0.743, and 3.090?g/g, respectively, in the impeller retrofitted fermenter, as compared with 0.036, 0.103, 0.629, and 2.819?g/g, respectively, in the two-layer Rushton impeller fermenter.     

Enhancing Nutritional Quality of Silage by Fermentation with Lactobacillus plantarum

The present study was aimed to investigate the nutritive profiles, microbial counts and fermentation metabolites in rye, Italian rye-grass (IRG) and barley supplemented with Lactobacillus plantarum under the field condition, and its probiotic properties. After preparation of silage, the content of crude protein (CP), crude ash, acid detergent fiber (ADF), and neutral detergent fiber (NDF), microbes such as lactic acid bacteria (LAB), yeast and fungi counts, and fermentation metabolites lactic acid, acetic acid and butyric acid was assessed. Results indicated that the content of ADF and NDF were significantly varied between rye, IRG and barley mediated silages. The content of CP was increased in L. plantarum supplemented with IRG, but slightly decreased in rye and barley mediated silages. The maximum LAB count was recorded at 53.10 × 10⁷ cfu/g in rye, 16.18 × 10⁷ cfu/g in IRG and 2.63 × 10⁷ cfu/g in barley silages respectively. A considerable number of the yeasts were observed in the IRG silages than the rye silages (P < 0.05). The amount of lactic acid production is higher in L. plantarum supplemented silages as compared with control samples (P < 0.05). It was confirmed that higher amount of lactic acid produced only due to more number of LAB found in the silages. L. plantarum was able to survive at low pH and bile salt and the duodenum passage with the highest percentage of hydrophobicity. Furthermore, the strain was sensitive towards the antibiotics commonly used to maintain the microbes in food industrial setups. In conclusion, supplementation of L. plantarum is most beneficial in rye, IRG and barley silage preparations and probiotic characteristics of L. plantarum was an intrinsic feature for the application in the preparation of animal feeds and functional foods.     

Optimization of probiotic and lactic acid production by Lactobacillus plantarum in submerged bioreactor systems

Biomass and lactic acid production by a Lactobacillus plantarum strain isolated from Serrano cheese, a microorganism traditionally used in foods and recognized as a potent probiotic, was optimized. Optimization procedures were carried out in submerged batch bioreactors using cheese whey as the main carbon source. Sequential experimental Plackett–Burman designs followed by central composite design (CCD) were used to assess the influence of temperature, pH, stirring, aeration rate, and concentrations of lactose, peptone, and yeast extract on biomass and lactic acid production. Results showed that temperature, pH, aeration rate, lactose, and peptone were the most influential variables for biomass formation. Under optimized conditions, the CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g l⁻¹ (dw) of L. plantarum. At the central point of the CCD, a biomass of 10.2 g l⁻¹ (dw), with conversion rates of 0.10 g of cell g⁻¹ lactose and 1.08 g lactic acid g⁻¹ lactose (w/w), was obtained. These results provide useful information about the optimal cultivation conditions for growing L. plantarum in batch bioreactors in order to boost biomass to be used as industrial probiotic and to obtain high yields of conversion of lactose to lactic acid.     

Power input measurements in a production scale bioreactor

Summary Power input measurements are carried out in a production bioreactor with a liquid volume up to 25 m³. The results show that the cavity formation principle is applicable to reactors at this scale. It can also be observed that empirical correlations are not useful to predict gassed power input accurately. It is found that at gas flow rates for normal production conditions (N_Q =0.1), the gassed power input is about 30–40 % of the non gassed power input.Nomenclature C_p specific heat J/kgK - D impeller diameter m - D_b1 impeller blade diameter m - d baffle diameter m - Fr Froude number - - g gravitation m/s² - h impeller clearance m - H liquid height m - N stirrer speed s^-1 - N_p power number - - N_Q gas flow (aeration) number - - N_Q ^* critical gasflow number for 3 cavity formation - - P_o ungassed power consumption W - P_g gassed power consumption W - Q gas flow rate (273 K, 10⁵ N/m²) m³/s - Re Reynolds number - - T tankdiameter m temperature K - t time s - V liquid volume m³ - ^Vtip impeller tip speed m/s - V_s impeller correlated superficial gas flow rate m/s - W impeller blade width m - density kg/m³     

Production of biomass from untreated orange peel by Fusarium avenaceum

Summary The growth behaviour of Fusarium avenaceum (Sect. Roseum Wr.) in slurry fermentation systems using untreated orange peel as substrate was studied in a laboratory-fermenter scale to reproduce the results obtained in a shakenflask fermenter. The eventual effect of impeller speed on mechanical disruption of mycelial hyphae was then assessed by determining mycelial growth, total reducing sugars consumption, TOC reduction, carbon dioxide evolution and oxygen absorption rates. In particular, the main biomass yield coefficient, as well as the apparent specific growth rate, appeared to be independent of the impeller speed, at least within the experimental range of 450 and 900 min^–1 (equivalent to peripheral impeller speeds of 3.8–7.5 m sec^–1.     

The influence of bakers’ yeast cells on protein adsorption in anion exchange expanded bed chromatography

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.     

The disruption ofSaccharomyces cerevisiae cells and release of glucose 6-phosphate dehydrogenase (G6PDH) in a horizontal dyno bead mill operated in continuous recycling mode

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.     

Factors in single-cell protein production from pawpaw fruit pulp extract

Maximum production of single-;cell protein (SCP), biomass, chemicals or fuels from microorganisms demands the manipulation of environmental conditions. Consequently, single-;cell protein production was carried out using Candida sp. and pawpaw fruit pulp extract (substrate), under conditions of varying initial inoculum, different agitation rates, nitrogen sources, and heat treatments. The highest viable cell count of 8.00 ± 1.34 × 10¹⁰ colony forming units (cfu)/ml was obtained with substrate supplemented with ammonium sulphate and the least viable cell count of 7.10 ± 2.10 × 10⁶ cfu/ml was observed using urea treated substrate. An optimum viable cell count occurred with an initial inoculum of 5.60 × 10⁵ cfu/ml, conditions of non-sterilization and agitation at 200 r.p.m. Growth also peaked at 24, 48 and 72 h with varying treatments.     

Production and scale‐up of a monoclonal antibody against 17‐hydroxyprogesterone

The hybridoma 192 was used to produce a monoclonal antibody (MAb) against 17‐hydroxyprogesterone (17‐OHP), for possible use in screening for congenital adrenal hyperplasia (CAH). The factors influencing the MAb production were screened and optimized in a 2 L stirred bioreactor. The production was then scaled up to a 20 L bioreactor. All of the screened factors (aeration rate, stirring speed, dissolved oxygen concentration, pH, and temperature) were found to significantly affect production. Optimization using the response surface methodology identified the following optimal production conditions: 36.8°C, pH 7.4, stirring speed of 100 rpm, 30% dissolved oxygen concentration, and an aeration rate of 0.09 vvm. Under these conditions, the maximum viable cell density achieved was 1.34 ± 0.21 × 10⁶ cells mL^?1 and the specific growth rate was 0.036 ± 0.004 h^?1. The maximum MAb titer was 11.94 ± 4.81 μg mL^?1 with an average specific MAb production rate of 0.273 ± 0.135 pg cell^?1 h^?1. A constant impeller tip speed criterion was used for the scale‐up. The specific growth rate (0.040 h^?1) and the maximum viable cell density (1.89 × 10⁶ cells mL^?1) at the larger scale were better than the values achieved at the small scale, but the MAb titer in the 20 L bioreactor was 18% lower than in the smaller bioreactor. A change in the culture environment from the static conditions of a T‐flask to the stirred bioreactor culture did not affect the specificity of the MAb toward its antigen (17‐OHP) and did not compromise the structural integrity of the MAb. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Reactor properties of a high-speed bead mill for microbial cell rupture

Laboratory and pilot-plant high-speed bead mills of 0.6 and 5 liter capacity and consisting of four and five impellers in series, respectively, were used to follow the batch and continuous disruption of bakers' yeast (Saccharomyces cerevisiae). The mills are not scaled equivalents. Throughputs ranging from 1 × 10^?6m³/sec to 12 × 10^?6m³/sec for the 0.6 liter mill and from 16 × 10^?6m³/sec to 100 × 10^?6m³/sec for the 5 liter mill were used for continuous disruption studies. Variables studied included the effect of impeller tip speed, temperature, and packed yeast concentration (ranging from 15 to75% by weight packed yeast). Disruption kinetics, as measured by the release of soluble protein, followed a first-order rate equation, the rate constant being a function of impeller tip speed and yeast concentration. For continuous disruption studies the bead mills behaved as a series of continuous stirred-tank reactors, each impeller forming a reactor. In the smaller mill a considerable degree of backflow between the reactors was evident. For certain mixing conditions the maximum amount of releasable protein was dependent on the impeller geometry, construction material, and also the concentration of packed yeast. The relative power efficiencies of the two mills are discussed along with possible criteria for scaling of bead mills.     

A potential economical substrate for large-scale production of Bacillus thuringiensis var. kurstaki for caterpillar control

Bacillus thuringiensis var. kurstaki has been widely used in caterpillar control programs. Large-scale production of this bacterium is expensive because of the high cost of the raw materials used in the medium. In this study, we attempted to develop an economical medium, based on inexpensive, locally available raw materials using a 3-L fermenter. Parthenium hysterophorus L. extract based culture medium resulted in highest toxicity (LC₅₀ 14.628 µg mL–¹) against 7-day-old Spodoptera litura (Fab) larvae, spore count (4.1 × 10⁹ spores mL^–1) and biomass (4.9 g L^–1) within a short fermentation time of 36 h. It was 512 times cheaper than the nutrient broth (standard medium) used for B. thuringiensis production. Hence, this parthenium extract based culture medium was considered most economical with potential for the large-scale industrial production of B. thuringiensis.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Accumulation of organic acids in cultivations of Neisseria meningitidis C

Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 l min⁻¹. A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O₂) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O₂ control, varying glucose concentration from 1.0 to 3.0 g l⁻¹, nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O₂ control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O₂ control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.     

Animal cell culture in stirred bioreactors: Observations on scale-up

Scale-up of stirred tank bioreactors from 0.02 m³ to 0.3 m³ commercial plant is discussed for hybridoma suspension culture. Schemes for dissolved oxygen control with sparged air in serum containing media are described as well as mechanical breakage of foam in small and large bioreactors. Porous metal spargers (180?200×10^?6 m) are found to produce foams which are hard to control. Aeration with larger (> 0.001 m) multihole spargers is recommended. Combined cell damage due to foam formation and control, and possible damage at mechanical seals or submerged bearings, are found to have no measurable effect on cell growth relative to roller bottle production. Hybridomas are shown to withstand significant impeller tip speed (> 1 ms^?1) and fluid turbulence as evidenced by impeller Reynolds numbers in excess of 10⁵. The size of the energy dissipating terminal eddies is calculated to be > 10-fold that of the hybridoma cells. Specific fluid turnover rate is employed as the scale-up criterion.     

Production of lactic acid from hemicellulose extracts by <Emphasis Type="Italic">Bacillus coagulans</Emphasis> MXL-9

Bacillus coagulans MXL-9 was found capable of growing on pre-pulping hemicellulose extracts, utilizing all of the principle monosugars found in woody biomass. This organism is a moderate thermophile isolated from compost for its pentose-utilizing capabilities. It was found to have high tolerance for inhibitors such as acetic acid and sodium, which are present in pre-pulping hemicellulose extracts. Fermentation of 20 g/l xylose in the presence of 30 g/l acetic acid required a longer lag phase but overall lactic acid yield was not diminished. Similarly, fermentation of xylose in the presence of 20 g/l sodium increased the lag time but did not affect overall product yield, though 30 g/l sodium proved completely inhibitory. Fermentation of hot water-extracted Siberian larch containing 45 g/l total monosaccharides, mainly galactose and arabinose, produced 33 g/l lactic acid in 60 h and completely consumed all sugars. Small amounts of co-products were formed, including acetic acid, formic acid, and ethanol. Hemicellulose extract formed during autohydrolysis of mixed hardwoods contained mainly xylose and was converted into lactic acid with a 94% yield. Green liquor-extracted hardwood hemicellulose containing 10 g/l acetic acid and 6 g/l sodium was also completely converted into lactic acid at a 72% yield. The Bacillus coagulans MXL-9 strain was found to be well suited to production of lactic acid from lignocellulosic biomass due to its compatibility with conditions favorable to industrial enzymes and its ability to withstand inhibitors while rapidly consuming all pentose and hexose sugars of interest at high product yields.     

峡谷型喀斯特不同生态系统的土壤微生物数量及生物量特征

采用样地调查与室内分析相结合的方法,研究了峡谷型喀斯特水田、旱地、草地、灌丛、人工林、次生林6种生态系统不同深度土壤微生物数量、微生物生物量特征及其分形关系。结果表明:峡谷型喀斯特不同生态系统的土壤微生物数量及组成不同,微生物数量均以次生林最高,旱地最低,其组成数量均为细菌放线菌真菌,细菌是土壤微生物的主要类群,数量多达26.66×105—71.64×105cfu/g,占全部微生物比例为87.00%—95.50%,其次为放线菌数量,为1.45×105—3.78×105cfu/g,所占比例为4.21%—12.39%,真菌数量最小,为0.07×105—0.23×105cfu/g,所占比例仅为0.24%—0.61%,不足1%。不同生态系统土壤微生物生物量碳(MBC)、氮(MBN)、磷(MBP)的含量不同,次生林MBC与MBN最高,人工林MBP最高,旱地MBC最低,草地MBN与MBP最低;各生态系统均为MBCMBNMBP。不同生态系统的MBC/SOC、MBN/TN、MBP/TP分别为0.44%—0.97%、2.13%—3.13%、1.46%—2.13%,差异不显著;MBC/MBN在3.06—6.54之间,其中次生林极显著高于其他生态系统,其他生态系统差异不显著。不同生态系统土壤微生物数量及生物量均随土层加深而减少,且具有良好分形关系,均达到了极显著水平(P0.01)。探讨土壤微生物活性为提高石灰土土壤肥力、促进喀斯特植被迅速恢复提供依据。     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.