Experimental techniques,data treatment for studying the dynamics of ethanol production/consumption in baker's yeast

The dynamics of ethanol production/consumption in baker's yeast were studied under feed- rate controlled conditions. The yeast was grown on molasses in an 8-l fed-batch reactor and experiments were done at cell concentrations between 5 and 65 g l^?1. Small changes in the feed rate were made around a feed rate corresponding to the critical growth rate, at which the yeast cell metabolism switches between ethanol consumption and production. A membrane gas sensor was used for on-line measurement of the ethanol concentration in the broth. The measured ethanol signal was used for control and the system was excited through changes in the regulator set-point. The closed-loop experiments ensured that feed variations were within the critical range, and thus facilitated reproducible experiments. Data were fitted to a second-order difference equation by statistical methods. Results were compared with a theoretically derived model. The process gain could be understood in terms of the underlying stoichiometry by using the “bottleneck” view of yeast glucose metabolism. The process time constant was found to be longer than is implied by a simple Monod relation between glucose uptake rate and concentration.     

Kinetics of Growth and Enhanced Sophorolipids Production by Candida bombicola Using a Low-Cost Fermentative Medium

In this study, effect of various parameters on sophorolipid (SL) production by the yeast Candida bombicola was investigated for the enhancing of its production by employing L18 orthogonal array design of experiments. At optimum conditions of sugarcane molasses 50 g l⁻¹, soybean oil 50 g l⁻¹, inoculum size 5% (v/v), temperature 30 °C, inoculum age 2 days, and agitation 200 rpm, the yeast produced almost equal amounts of the product in batch shake flasks and in a 3-l fermentor without any pH control (45 and 47 g l⁻¹, respectively). However, the yield increased to 60 g l⁻¹ in the fermentor under controlled pH environment. Time course of SL production, yeast biomass growth, and utilization of sugarcane molasses and soybean oil at these optimized conditions were fitted to existing kinetic models reported in the literature. Estimated kinetic parameters from these models suggested that conventional medium containing glucose can very well be replaced with the present low-cost fermentative medium.     

Optimization of Surfactin Production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Isolate BS5

Bacillus subtilis BS5 is a soil isolate that produces promising yield of surfactin biosurfactant in mineral salts medium (MSM). It was found that cellular growth and surfactin production in MSM were greatly affected by the environmental fermentation conditions and the medium components (carbon and nitrogen sources and minerals). Optimum environmental conditions for high surfactin production on the shake flask level were found to be a slightly acidic initial pH (6.5-6.8), an incubation temperature of 30 degrees C, a 90% volumetric aeration percentage, and an inoculum size of 2% v/v. For media components, it was found that the optimum carbon source was molasses (160 ml/l), whereas the optimum nitrogen source was NaNO(3) (5 g/l) and the optimum trace elements were ZnSO(4).7H(2)O (0.16 g/l), FeCl(3).6H(2)O (0.27 g/l), and MnSO(4).H(2)O (0.017 g/l). A modified MSM (molasses MSM), combining the optimum medium components, was formulated and resulted in threefold increase in surfactin productivity that reached 1.12 g/l. No plasmid could be detected in the tested isolate, revealing that biosurfactant production by B. subtilis isolate BS5 is chromosomally mediated but not plasmid-mediated.     

Overproduction of riboflavin by anArthrobacter sp. mutant resistant to 5-fluorouracil

Effects of pH and dissolved oxygen concentration on batchwise riboflavin production by a 5-fluorouracil (5-FU)-resistant mutant ofArthrobacter sp. were investigated. The reaction was carried out in a jar fermentor. The optimal pH of culture medium was around 7.3. Dissolved oxygen concentration was almost constant during fermentation at 600 rpm of agitation rate. Production of riboflavin reached a maximum of 160 mg/L after 70 h fermentation under the agitation rate of 600 rpm, aeration rate of 1.0 L/min, and pH 7.0.     

Enhancing the production of gamma-linolenic acid in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> by fed-batch fermentation using response surface methodology

Gamma-linolenic acid (GLA, C18:3Δ^6,9,12) is an n-6 polyunsaturated fatty acid (PUFA) that has been used for the alleviation and treatment of a number of symptoms and diseases. Increasing GLA demand has led to a search for alternative producers and potential strategies for GLA production. Based on the successful performance of Hansenula polymorpha, a methylotrophic yeast, as a “cell factory” for the production of valuable bioproducts, a bioprocess development approach was implemented for GLA production in the recombinant yeast carrying the mutated Δ⁶-desaturase gene of Mucor rouxii. Using a substrate-feeding strategy under glycerol-limited conditions, the physical-chemical variables during the fed-batch fermentation of the recombinant H. polymorpha were optimised for GLA production through response surface methodology using a Box-Behnken design. The medium composition, including yeast extract and trace elements, and dissolved oxygen tension (DOT) were targeted. We found that DOT was the most effective variable for enhancing GLA yield. These results also suggest that the optimum conditions for GLA production are 28 % saturation of DOT, 1 g L⁻¹ of yeast extract and 3.6 mL L⁻¹ of the Pichia trace metals 1 (PTM1).     

Biocalorimetry as a process analytical technology process analyser; robust in-line monitoring and control of aerobic fed-batch cultures of crabtree-negative yeast cells

Control of bioprocesses requires reliable and robust on- or in-line monitoring tools providing real-time information on process dynamics. Heat generation related to metabolic activity of living systems is currently gaining importance in bioprocess industry due to its non-invasive and essentially instantaneous characteristics. This study deals with monitoring and control of pure aerobic fed-batch cultures of three Crabtree-negative yeast strains, Kluyveromyces marxianus, Candida utilis and Pichia pastoris, based on in-line measured, metabolic heat flow signals. A high resolution biocalorimeter (BioRC1) was developed from a standard bench-scale heat flow calorimeter (RC1). The BioRC1 was equipped with in-line (dielectric spectroscopy, pH probe and dissolved oxygen probe) and at-line (exit gas analyser) sensors to characterise the growth behaviour of the yeast cells. Both metabolic heat flow and biomass profiles exhibited similar behaviour proving the significance of employing heat flow signal as a key-parameter for the system under investigation. A simple estimator for biomass concentration and specific growth rate was formulated based on heat flow values. In order to evaluate the potential of calorimetry as a reliable and powerful process monitoring tool, the robustness, reliability as well as the broad applicability of the developed estimators was assessed through comparison with off-line measurement techniques and showed promising results for general applicability with a wide range of bioprocesses.     

Lipase production by solid-state fermentation

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.     

Enhancement of 1,3-Dihydroxyacetone Production by a UV-induced Mutant of Gluconobacter oxydans with DO Control Strategy

The 1,3-dihydroxyacetone (DHA)-overproducing mutant of Gluconobacter oxydans was screened via UV mutagenesis to enhance the DHA production, and the DHA fermentation condition was optimized using the dissolved oxygen (DO) control strategy. The stable mutant G. oxydans ZJB11001 exhibits high DHA productivity and can tolerate high DHA concentrations. The optimal condition for DHA production by G. oxydans ZJB11001 in a 15-L fermentor required an initial medium containing 5 g/L yeast extract, 20 g/L glycerol, 0.5 g/L K(2)HPO(4), 0.1 g/L MgSO(4)·7H(2)O. The glycerol feeding rate was manually controlled to maintain the glycerol concentration at 5-10 g/L range. The culture pH was maintained at 6.0 within the first 20 h, and then adjusted to 5.0 until the end of the fermentation. The DO concentration increased from 20% to 30% after 24 h of fermentation, and then to 40% after 60 h of fermentation. The maximum DHA concentration of 209.6 ± 6.8 g/L was achieved after 72 h of fed-batch fermentation at 30 °C.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Cultivation of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in the Stirred Bioreactor with Different Feeding Strategies for CoQ<Subscript>10</Subscript> Production

The logistic growth model combined with the Luedeking-Piret equation was adopted in this study to model the batch production of CoQ₁₀ in the cultivation of Rhodobacter sphaeroides. The simulation results indicated that CoQ₁₀ production was a primary metabolite. As being a primary metabolite, a longer cell growing stage would tend to accumulate more biomass and lead to a higher CoQ₁₀ concentration being produced. In this context, a fed-batch operation by molasses feeding was performed to increase the biomass and subsequent CoQ₁₀ production. Three different molasses feeding strategies were operated in this study. Results suggested that the fed-batch operation with molasses controlled at 10 ± 1 g/l could increase the cell mass and CoQ₁₀ concentration to reach their maximum values of 18.6 g/l and 83.8 mg/l, respectively, nearly 2.2 times and 1.9 times their respective values obtained in the batch cultivation.     

An Effective and Simplified Fed-Batch Strategy for Improved 2,3-Butanediol Production by <Emphasis Type="BoldItalic">Klebsiella oxytoca</Emphasis>

Substrate concentration in 2,3-butanediol (2,3-BD) fermentation could not be controlled well in traditional feeding strategies, such as constant, impulse, and exponential feeding strategies. In the present study, fermentative 2,3-BD production by Klebsiella oxytoca was investigated under different batch and fed-batch strategies. The glucose-feedback fed-batch strategy was proved to be not effective for economical 2,3-BD production for the inability of timely feeding, leading that the bacteria reused 2,3-BD as carbon source for cell growth. Based on the phenomena that the byproducing acids caused the pH declining and the requirement of maintaining the pH at a proper level for both cell growth and 2,3-BD accumulation, an improved strategy of pH-stat fed-batch culture with glucose and sodium hydrate fed at the same time was established. Thus, the residual glucose concentration could be controlled through the adjustment of pH automatically. At last, efficient 2,3-BD production was fulfilled under this fed-batch strategy, and the highest 2,3-BD concentration, productivity, and yield were 127.9 g/l, 1.78 g/(l•h), and 0.48 g/g (2,3-BD/glucose), respectively, compared to 98.5 g/l, 1.37 g/(l•h), and 0.43 g/g obtained in glucose-feedback fed-batch strategy. This feeding strategy was simple and easy to operate and could be feasible for industrial 2,3-BD production in the future.     

Environmental influences on diethyl phthalate biodegradation kinetics

The effects of temperature, dissolved oxygen level, and diethyl phthalate (DEP) concentration on the rates of DEP biodegradation have been investigated in shake flask and fermenter experiments, using aerobic and facultatively aerobic microorganisms. The aerobic strain followed Monod growth kinetics, and was negatively affected by temperatures lower than 25 °C and dissolved oxygen levels lower than 0.8 mg/L, whereas the specific DEP-degrading activity of the facultative strain was substrate inhibited under anaerobic conditions, higher at 15°C than 25°C under aerobic conditions, and unaffected by the dissolved oxygen level. Studies were also carried out in soil columns to identify additional factors that might be important for modeling DEP biodegradation.     

Pretreatment of Synthetic Dairy Wastewater Using the Sophorolipid-Producing Yeast <Emphasis Type="Italic">Candida bombicola</Emphasis>

The presence of high strength fats and oils in dairy industry wastewaters poses serious challenges for biological treatment systems, and, therefore, its pretreatment is necessary in order to remove them. In the present study, synthetic dairy wastewater prepared in the laboratory was pretreated using the sophorolipid-producing yeast Candida bombicola in a laboratory-scale bioreactor under batch, fed-batch, and continuous modes of operation. To support the yeast growth, the wastewater was supplemented with sugarcane molasses (1% w/v) and yeast extract (0.1% w/v). Results from the batch operated fermentor revealed complete utilization of fats present in the wastewater within 96 h with more than 93% COD removal efficiency. The yeast was, however, able to pretreat the wastewater more quickly and efficiently under fed-batch mode of operation than under batch operated condition in the same fermentor. Continuous experiments were carried out with a wastewater retention time of 28 h in the reactor; results showed very good performance of the system in complete utilization of fats and COD removal efficiency of more than 90%. The study proved the excellent potential of the biosurfactant-producing yeast in pretreating high-fat- and oil-containing dairy industry wastewater.     

Effect of Aeration on Production of Anticancer Lignans by Cell Suspension Cultures of Linum album

The effects of aeration within the range of 0.2-0.5 vvm on transformed and high yielding cell cultures of Linum album were investigated in a 5-L stirred tank bioreactor equipped with low shear Setric impeller. The kinetics of cell growth, substrate utilization, and production of lignans, namely, podophyllotoxin and 6-methoxypodophyllotoxin, were established. Maximum biomass of 23.2 g/L and lignan accumulation levels of 176.3 mg/L podophyllotoxin and 10.86 mg/L 6-methoxypodophyllotoxin were obtained with initial air flow rate of 0.3 vvm. Specified oxygen demand of cells was estimated to be 1.35 g O(2)/g biomass. The optimum oxygen transfer coefficient was found to be 16.7 h(-1) (,) which corresponded to aeration rate of 0.3 vvm. The effect of minimum dissolved oxygen (DO) concentration was investigated with respect to biomass and lignan production by comparing identically aerated and agitated bioreactor cultivations at dissolved oxygen concentrations of 10%, 30%, and 50%. Cell growth and podophyllotoxin accumulation were not affected significantly at these DO levels, but 6-methoxypodophyllotoxin production was enhanced when cells were cultivated at 30% DO level. The maximum volumetric productivities of 18.2 mg/L day and 3.2 mg/L day for podophyllotoxin and 6-methoxypodophyllotoxin, respectively, were obtained. These results establish the key role of oxygen on mass scale production of anticancer lignans by cell cultures of L. album. It may serve as a suitable parameter for scale-up.     

Proficiency test of pH,conductivity and dissolved oxygen concentration field measurements in river water

In 2013, Proftest SYKE organised the first proficiency test (PT) in Finland for field measurements of temperature, conductivity, dissolved oxygen concentration, oxygen saturation and pH value in river water. The aim was to pilot the organisation of an in situ proficiency test—particularly, how to select the test location—and how to assess the homogeneity and stability of the measurement site and the water to be tested. The focus was also to evaluate the suitability of the common field sensors used for water analysis, as well as the comparability of the results between the instruments under field conditions. The overall application of quality assurance procedures was also surveyed. This paper deals with the results, findings and recommendations for the measurement of pH, conductivity and dissolved oxygen concentration. In total, nine participants with 16 sensors took part in the proficiency test. For the evaluation of the performance of each participant, z scores were calculated allowing 3 % to 8 % deviation from the assigned value. The standard deviation of the participant’s results was lower than organiser expected, and 80 % of the results for pH, 79 % for conductivity and 69 % for dissolved oxygen concentration were regarded as satisfactory. According to the results, the most challenging measurement was for dissolved oxygen with a Clark cell-type measurement principle based on electrochemical reaction. All sensors tested in the PT were less than 5 years, old and they were calibrated according to the manufacturer’s instructions. None of the participants had estimated measurement uncertainty for their sensor measurements. In addition, internal and external quality assurance protocols were usually lacking.     

Enhanced Production of Poly-γ-glutamic Acid by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> TISTR 1010 with Environmental Controls

Bacillus licheniformis TISTR 1010 was used for glutamic acid-independent production of poly-γ-glutamic acid (γ-PGA). A fed-batch production strategy was developed involving feedings of glucose, citric acid, and ammonium chloride at specified stages of the fermentation. With the dissolved oxygen concentration controlled at ≥50% of air saturation and the pH controlled at ~7.4, the fed-batch operation at 37 °C afforded a peak γ-PGA concentration of 39.9 ± 0.3 g L^?1 with a productivity of 0.926 ± 0.006 g L^?1 h^?1. The observed productivity was nearly threefold greater than previously reported for glutamic acid-independent production using the strain TISTR 1010. The molecular weight of γ-PGA was in the approximate range of 60 to 135 kDa.     

MoO_3-TiO_2纳米晶体复合材料光降解糖蜜(英文)

A MoO3-TiO2 nanocrystalline composite material was prepared by a simple solgel method.The synthesized material was charac-terized by X-ray diffraction,scanning electron microscopy with an electron dispersion spectroscopy,transmission electron microscopy,and Fourier transform infrared spectroscopy.Melanoidin is a dark brown pigment found in wastewater from the sugar industry and it pollutes water.This polluted water is generally referred to as molasses and it undergoes fermentation and is solely responsible for water,soil,and air pollution.The synthesized catalytic material was found to be effective in degrading molasses under UV-visible radiation.Analysis of treated and untreated molasses was carried out by measuring its color,chemical oxygen demand,biological oxygen demand,pH,and total dissolved solid.Results from these analyses indicate the effective photodegradation of the molasses.This methodology has several advantages such as high photocatalytic activity,non-toxicity,cleanliness,and reusability of the catalytic material.     

MoO3-TiO2纳米晶体复合材料光降解糖蜜（英文）

A MoO3-TiO2 nanocrystalline composite material was prepared by a simple solgel method.The synthesized material was charac-terized by X-ray diffraction,scanning electron microscopy with an electron dispersion spectroscopy,transmission electron microscopy,and Fourier transform infrared spectroscopy.Melanoidin is a dark brown pigment found in wastewater from the sugar industry and it pollutes water.This polluted water is generally referred to as molasses and it undergoes fermentation and is solely responsible for water,soil,and air pollution.The synthesized catalytic material was found to be effective in degrading molasses under UV-visible radiation.Analysis of treated and untreated molasses was carried out by measuring its color,chemical oxygen demand,biological oxygen demand,pH,and total dissolved solid.Results from these analyses indicate the effective photodegradation of the molasses.This methodology has several advantages such as high photocatalytic activity,non-toxicity,cleanliness,and reusability of the catalytic material.     

Optimization of Spore and Antifungal Lipopeptide Production During the Solid-state Fermentation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis strain TrigoCor 1448 was grown on wheat middlings in 0.5-l solid-state fermentation (SSF) bioreactors for the production of an antifungal biological control agent. Total antifungal activity was quantified using a 96-well microplate bioassay against the plant pathogen Fusarium oxysporum f. sp. melonis. The experimental design for process optimization consisted of a 2⁶⁻¹ fractional factorial design followed by a central composite face-centered design. Initial SSF parameters included in the optimization were aeration, fermentation length, pH buffering, peptone addition, nitrate addition, and incubator temperature. Central composite face-centered design parameters included incubator temperature, aeration rate, and initial moisture content (MC). Optimized fermentation conditions were determined with response surface models fitted for both spore concentration and activity of biological control product extracts. Models showed that activity measurements and spore production were most sensitive to substrate MC with highest levels of each response variable occurring at maximum moisture levels. Whereas maximum antifungal activity was seen in a limited area of the design space, spore production was fairly robust with near maximum levels occurring over a wider range of fermentation conditions. Optimization resulted in a 55% increase in inhibition and a 40% increase in spore production over nonoptimized conditions.     

Culture pH affects exopolysaccharide production in submerged mycelial culture of Ganoderma lucidum

In submerged culture of Ganoderma lucidum, the pH optimum for cell growth has been shown to be lower than that for exopolysaccharides (EPS) formation. Therefore, in the present study, a two-stage pH-control strategy was employed to maximize the productions of mycelial biomass and EPS. When compared, a batch culture without pH control had a maximum concentration of EPS and endopolysaccharides, which was much lower than those with pH control. Maximum mycelial growth (12.5 g/L) and EPS production (4.7 g/L) were achieved by shifting the controlled pH from 3.0 to 6.0 after day 4. The contrast between the controlled-pH process and uncontrolled pH was marked. By using various two-stage culture processes, it was also observed that culture pH has a significant affect on the yield of product, mycelial morphology, chemical composition, and molecular weight of EPS. A detailed observation of mycelial morphology revealed that the productive morphological form for EPS production was a dispersed pellet (controlled pH shifting from 3.0 to 6.0) rather than a compact pellet with a dense core area (controlled pH 4.5) or a feather-like pellet (controlled pH shifting from 6.0 to 3.0). Three different polysaccharides were obtained from each pH conditions, and their molecular weights and chemical compositions were significantly different.