An novel immobilization method of Saccharomyces cerevisiae to sorghum bagasse for ethanol production

Natural sorghum bagasse without any treatment was used to immobilize Saccharomyces cerevisiae at 0.6+/-0.2g dry cell weight (DCW)/g dry sorghum bagasse weight (DSW) through solid-state or semi-solid state incubation. The scanning electron microscopy (SEM) of the carriers revealed the friendship between yeast cells and sorghum bagasse are adsorption and embedding. The ethanol productivity of the immobilized cells was 2.24 times higher than the free cells. In repeated batch fermentation with an initial sugar concentration of 200g/L, nearly 100% total sugar was consumed after 16 h. The ethanol yield and productivity were 4.9 g/g consumed sugar on average and 5.72 g/(Lh), respectively. The immobilized cell reactor was operated over a period of 20 days without breakage of the carriers, while the free cell concentration in the effluent remained less than 5 g/L thoughout the fermentation. The maximum ethanol productivity of 16.68 g/(Lh) appeared at the dilution rate of 0.3h(-1).     

VK3诱导悬浮培养的胡萝卜细胞凋亡

Menadione (VK3), a quinone that undergoes redox cycles leading to the formation of superoxide radicals, was found to induce cell death in suspension culture of carrot cells. The effect of menadione was in a dose-dependent manner. 100-800 mumol/L menadione caused 10-33 percent cell death. When concentration of menadione reached 1 mmol/L, 100 percent of cell death was observed. DNA cleavage, a hallmark of apoptosis was further studied. DNA ladders were observed in cells treated with 600 and 800 mumol/L menadione but not with lower concentration treatments where only very low percentage of cell death was found. There was no DNA ladders in the cells treated with 1 mmol/L menadion indicating that necrosis may occur. In situ detection of nuclear DNA fragmentation by TUNEL reaction revealed fragmented nuclear DNA in cells treated with 100-800 mumol/L menadion but not in cells treated with 1 mmol/L menadione.     

Effects of methanol on cell growth and lipid production from mixotrophic cultivation of Chlorella sp.

The marine microalga Chlorella sp. was cultivated under mixotrophic conditions using methanol as an organic carbon source, which may also act to maintain the sterility of the medium for long-term outdoor cultivation. The optimal methanol concentration was determined to be 1% (v/v) for both cell growth and lipid production when supplying 5% CO₂ with 450 μE/m²/sec of continuous illumination. Under these conditions, the maximal cell biomass and total lipid production were 4.2 g dry wt/L and 17.5% (w/w), respectively, compared to 2.2 g dry wt/L and 12.5% (w/w) from autotrophic growth. Cell growth was inhibited at methanol concentrations above 1% (v/v) due to increased toxicity, whereas 1% methanol alone sustained 1.0 g dry wt/L and 4.8% total lipid production. We found that methanol was preferentially consumed during the initial period of cultivation, and carbon dioxide was consumed when the methanol was depleted. A 12:12 h (light:dark) cyclic illumination period produced favorable cell growth (3.6 g dry wt/L). Higher lipid production was observed with cyclic illumination than with continuous illumination (18.6% (w/w) vs 17.5% (w/w)), and better lipid production was also obtained under mixotrophic rather than autotrophic conditions. Interestingly, under mixotrophic conditions with 12:12 (h) cyclic illumination, high proportions of C_16:0, C_18:0, and C_18:1 were observed, which are beneficial for biodiesel production. These results strongly indicate that the carbon source is important for controlling both lipid composition and cell growth under mixotrophic conditions, and they suggest that methanol could be utilized to scale up production to an open pond type system for outdoor cultivation where light illumination changes periodically.     

Optimization of enzymatic synthesis of L-ascorbyl palmitate by solvent engineering and statistical experimental designs

Statistical experimental designs combined with solvent engineering for optimization of enzymatic synthesis of L-ascorbyl palmitate were developed. First, the composition of the solvent for co-dissolving polar and apolar substrates was determined. The co-solvent mixture of tert-pentanol: DMSO at a ratio of 9:1 (v/v) and the optimal biocatalyst were obtained. Then, the Plackett-Burman design was implemented to screen the variables that significantly influence the conversion. The method of steepest ascent was used to approach the proximity of optimum. After determining the Plackett-Burman and steepest ascent designs, the optimum values were determined by central composite design under response surface methodology. The statistical analysis showed that the optimum reaction conditions (temperature 50°C, enzyme concentration 5.8 g/L, and substrate molar ratio 11:1, stirring rate 160 rpm, amount of molecular sieve 50 g/L, time 18 h) led to the maximum conversion (66.44%) and production concentration (20.63 g/L). A very satisfactory conversion (64.74%) and production concentration (20.13 g/L) could be achieved in short time (6 h).     

Perifusion of co-cultured hepatocytes: optimization of studies on drug metabolism and cytotoxicity in vitro

The combination of co-cultivation of hepatocytes and epithelial cell lines with a newly developed perifusion system was used for in vitro studies on drug metabolism and cytotoxicity. This approach improved the viability and enhanced the induction of the biotransforming capacity of the hepatocytes. As demonstrated for the induction of 7-ethoxyresorufin O-deethylase activity by 3-methylcholanthrene or benzanthracene, co-cultured hepatocytes in the perifusion system responded more sensitively to these inducers than without perifusion, most likely owing to stable (steady-state) concentrations of the inducers under the former conditions and rapidly declining concentrations under the latter conditions. The perifusion approach rendered it possible to determine the kinetics of drug metabolism during single or sequential incubations. After induction with 3-methylcholanthrene and phenobarbital, phase I metabolism of lonazolac to the monohydroxylated product in perifused co-cultures closely (87%) approached the values reported for the in vivo production, whereas in stationary co-cultures only 52% could be reached. Likewise, cytotoxic effects could be detected more precisely in the perifused co-cultures. If cells were pretreated with 0.2 mmol/L galactosamine for 3 h, perifusion with increasing concentrations of menadione differentially killed epithelial RL-ET-14 cells and hepatocytes at low and high concentrations, respectively, while in stationary co-cultures no differential effect was observed and only the higher concentrations were cytotoxic for both cells. Prevention by incubation with S-adenosylmethionine of menadione cytotoxicity up to a menadione concentration of 250 mol/L was seen only in the perifused co-cultures, whereas in stationary cultures only a slight shift of the cytotoxic concentration exerting 50% cell damage to higher values was noted. These results demonstrate the versatile application of perifused co-cultures for studies on drug metabolism including induction of cytochrome P450-dependent enzymes and steady-state kinetics of biotransformation, as well as cytotoxic and protective effects of different drugs.Abbreviations BA benzanthracene - CC₅₀ values cytotoxic concentration exerting 50% cell damage - EROD 7-ethoxyresorufin O-deethylase - LDH lactate dehydrogenase - LON lonazolac - MC 3-methylcholanthrene - PB phenobarbital     

Continuous ethanol fermentation using immobilized yeast cells

Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/L gel h and prolonged life stability for more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4 kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10%(v/v)ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.     

The enzymatic transformation of water-insoluble reactants in nonaqueous solvents. Conversion of cholesterol to cholest-4-ene-3-one by a Nocardia sp. Reprinted from Biotechnology and Bioengineering, Vol. XVII, Pages 815-826 (1975)

The rapid conversion of cholesterol to cholestenone by Nocardia in the presence of high proportions of water-immiscible solvent has been demonstrated. At high agitator speeds, the reaction rate was not limited by the rates of transfer of oxygen or cholesterol to the microorganisms. Using 100 g of thawed cells in 200 ml of carbon tetrachloride containing 16% (w/v) cholesterol, at 20 degrees C cholestenone was formed at 7 g/hr. Cells could be separated easily from the organic solvent and reused. After 7 runs (69 hr) the reaction rate had fallen only to half the value for the first run.     

Synergistic effects between silibinin and antibiotics on methicillin-resistant Staphylococcus aureus isolated from clinical specimens

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous microorganism, and creates serious medical problems. It causes many types of infections in humans and often acquires multi-drug resistance. In this study, silibinin was evaluated against 20 clinical isolates of MRSA, either alone or in combination with ampicillin or oxacillin, using a checkerboard assay. The silibinin exhibited good activity against isolates of MRSA, and MRSA ATCC33952 and MSSA ATCC25923, with minimum inhibitory concentrations/minimum bactericidal concentrations (MICs/MBCs) ranging between 2-8/4-16 μg/mL, for ampicillin 2-1024/2-2048 μg/mL, and for oxacillin 0.25-32/0.5-64 μg/mL. The range of MIC(50) and MIC(90) were 0.5-4 μg/mL and 2-8 μg/mL, respectively. The MICs/MBCs for the combination of silibinin plus oxacillin or ampicillin were reduced by ≥4-fold against the MRSA isolates tested, demonstrating a synergistic effect, as defined by a fractional inhibitory concentration index (FICI) of ≤0.5. Furthermore, a time-kill study evaluating the growth of the tested bacteria showed that growth was completely attenuated after 2-5 h of treatment with the 1/2 MIC of silibinin, regardless of whether it was administered alone or with oxacillin (1/2 MIC) or ampicillin (1/2 MIC). In conclusion, silibinin exerted synergistic effects when administered with oxacillin or ampicillin and the antibacterial activity and resistant regulation of silibinin against clinical isolates of MRSA might be useful in controlling MRSA infections.     

The enzymatic transformation of water-insoluble reactants in nonaqueous solvents. Conversion of cholesterol to cholest-4-ene-3-one by a Ncardia sp.

The rapid conversion of cholesterol to cholestenone by Nocardia in the presence of high proportions of water-immiscible solvent has been demonstrated. At high agitator speeds, the reaction rate was not limited by the rates of transfer of oxygen or cholesterol to the microorganisms. Using 100 g of thawed cells in 200 ml of carbon tetrachloride containing 16% (w/v) cholesterol, at 20°C cholestenone was formed at 7 g/hr. Cells could be separated easily from the organic solvent and reused. After 7 runs (69 hr) the reaction rate had fallen only to half the value for the first run.     

Effect of dehydrogenation/hydrogenation on the linear and nonlinear optical properties of Li@porphyrins

In the present work, Li@porphyrins and their derivatives were designed in order to explore the effect of dehydrogenation/hydrogenation on linear and nonlinear optical properties. Their stable structures were obtained by the M06-2X method. Moreover, the M06-2X method showed that dehydrogenation/hydrogenation has greatly influences polarizabilities (α (0) values) and hyperpolarizabilities (β (tot) and γ (tot) values): α (0) values ranged from 331 to 389 au, β (tot) values from 0 to 2465 au, and γ (tot) values from -21.2?×?10(4) to 21.4?×?10(4) au. This new knowledge of the effect of dehydrogenation/hydrogenation on nonlinear optical properties may prove beneficial to the design and development of high-performance porphyrin materials.     

Asymmetric reduction of o-chloroacetophenone with Candida pseudotropicalis 104

The asymmetric reduction of o-chloroacetophenone 1 with Candida pseudotropicalis 104 produced the corresponding (S)-1-(2-chloro-phenyl)-ethanol 2 with the enantiomeric excess (ee >99%) without addition of any cosolvent. The cell could tolerate high ketone 1 concentration of 233.8 mmol/L (i.e., 36 g/L) with considerable reduction activity in this method. The product 2 concentration achieved 38.9 and 58.4 mmol/L with cells of 40 and 60 g(DCW) (dry cell weight)/L, respectively, in 24 h. The optimum reaction time, the effect of substrate concentration, cosubstrate type and concentration, and cell concentration in the reaction were investigated in this paper.     

白细胞介素-6在工程菌分批培养中的高效表达及其纯化

在确定了培养基及pH值的基础上,进一步观察了升温诱导过程中有机酸的产生及其对工程菌E.coli DH5α(pHV-hIL-6)生长和rIL-6表达的影响。当有机酸浓度低于70mmol/L以下时,菌密度达到干重2～3.5g/L之间收菌,rIL-6的表达水平为25％～32％;当有机酸浓度达到70mmol/L以上时,工程菌的生长不受影响,而rIL-6的表达明显受抑制。产生的有机酸以乙酸为主。收集菌体后,经过破菌,分离提纯的包涵体,其rIL-6的纯度可达到70％。用GuHCl缓冲液溶解包涵体,样品稀释后经过Q Sepharose F F柱纯化,可得到纯度达95％以上的rIL-6。采用依赖IL-6的小鼠杂交瘤细胞系7TD1及MTT比色法测定生物活性,rIL-6的比活性为2×10~8U/mg。     

丙烯酸对十六碳二元酸发酵的影响和16L罐扩试

本文报道丙烯酸对发酵生产十六碳二元酸(DC_(16))的影响,加入0.1%丙烯酸,DC_(16)产量提高20—30%.在16L自动控制罐上,在最佳条件下,加入20%(v/v)正十六烷(nC_(16)),发酵5天,DC_(16)高达120g/L,nC_(16)的转化率高达79%.     

β-环糊精对甾体微生物羟化反应的影响

考察了β-环糊精(β-cyclodextrin, CD)对雄甾-4-烯-3,17-二酮(androst-4-ene-3,17-diorle,AD)在水中的溶解度及微生物对其11а羟化反应的影响,结果表明β-环糊精能显著提高底物AD在发酵培养基中的溶解度,增溶效果优于有机溶剂.在底物投料浓度0.2%(w/v)时,与4%无...     

Ethanol production from seaweed (Undaria pinnatifida) using yeast acclimated to specific sugars

Ethanol production from Undaria pinnatifida (Sea mustard, Miyuk) was performed using yeast acclimated to specific sugars. Pretreatment conditions were optimized by thermal acid hydrolysis and enzyme treatment to increase the monosaccharide yield. Pretreatment by thermal acid hydrolysis was carried out using seaweed powder at 8 ～ 17% (w/v) solid content with a treatment time of 30 ～ 60 min. Enzyme treatment was carried out with 1% (v/v) Viscozyme L (1.2 FGU/mL), 1% (v/v) Celluclast 1.5 L (8.5 EGU/mL), 1% (v/v) AMG 300 L (3.0 AGU/mL), and 1% (v/v) Termamyl 120 L (0.72 KNU/mL). All enzymes except Termamyl 120 L, which was applied during pretreatment, were treated at 45°C for 24 h following pretreatment. Optimal pretreatment and enzyme conditions were determined to be 75 mM H₂SO₄, 13% (w/v) slurry, and 2.88 KNU/mL Termamyl 120 L at 121°C for 60 min. A maximum monosaccharide concentration of 33.1 g/L with 50.1% theoretical yield was obtained. To increase the ethanol yield, Pichia angophorae KCTC 17574 was acclimated to a high concentration (120 g/L) of galactose and mannitol at 30oC for 24 h. Ethanol production of 12.98 g/L with 40.12% theoretical yield was obtained from U. pinnatifida through fermentation with 0.35 g dry cell weight/L P. angophorae KCTC 17574 acclimated to mannitol and galactose.     

Screening of iron- and zinc-enriched yeast strain and optimization of cultivation conditions

Saccharomyces cerevisiae LN-17 was selected from 26 kinds of primary yeast strains that belong to different genera and species. The iron- and zinc-enriched capability of strain LN-17 was higher than the others. The highest iron and zinc contents of the strain were obtained when the strain grew up under the following conditions: The strain was incubated (5%, v/v) in 50 mL wort medium (pH 6.0) with 100 mg/L Fe ion and 120 mg/L Zn ion. The medium was loaded into a 250-mL Erlenmeyer flask and shaken in a rotary shaker (200 rpm) at 30°C for 60 h. Ferrous sulfate and zinc sulfate were chosen as the source of Fe and Zn. The Fe and Zn contents of the dry cells were determined by atomic absorption spectrum analysis. Under the optimized cultivation conditions, the Fe and Zn contents reached 7.854 mg/g dry cells and 4.976 mg/g dry cells.     

Outdoor and indoor cultivation of Spirulina platensis in the extreme south of Brazil

Water supplemented with 10% or 20% (v/v) of Zarrouk medium was used to cultivate Spirulina platensis in closed and open bioreactors under controlled conditions (30 degrees C, 32.5 micromol m(-2) s(-1), 12 h light/dark photoperiod) and in a greenhouse (9.4 to 46 degrees C, up to 2800 micromol m(-2) s(-1), variable day length photoperiod) using different initial biomass concentrations (X0) in the extreme south of Brazil (32.05 degrees S, 52.11 degrees W). Under controlled conditions the maximum specific growth rate (micromax) was 0.102 d(-1), the biomass doubling time (t(d)) was 6.8 d, the maximum dry biomass concentration (Xmax) was 1.94 g L(-1) and the maximum productivity (Pmax) was 0.059 g L(-)1 d(-1), while the corresponding values in the greenhouse experiments were micromax = 0.322 d(-1), t(d) = 2.2 d, Xmax = 1.73 g L(-1) and Pmax = 0.112 g L(-1) d(-1). Under controlled conditions the highest values for these parameters occurred when X0 = 0.15 g L(-1), while in the greenhouse X0 = 0.4 g L(-1) produced the highest values. These results show that the cultivation of S. platensis in greenhouses in the extreme south of Brazil is technically viable and that the S. platensis inoculum and the concentration of Zarrouk medium can be combined in such a way as to obtain growth and productivity parameters comparable, or superior, to those occurring in bioreactors under controlled conditions of temperature, illuminance and photoperiod.     

Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containing materials

Yarrowia lipolytica ACA-DC 50109 has been reported to be an oleaginous yeast and significant quantities of lipids were accumulated inside the yeast cells. In this study, the INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the expression plasmid pINA1317 and expressed in the cells of the oleaginous yeast. The activity of the inulinase with 6 × His tag secreted by the transformant Z31 obtained was found to be 41.7U mL(-1) after cell growth for 78 h. After optimization of the medium and cultivation conditions for single cell oil production, the transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g L(-1) within 78 h at the flask level. During the 2-L fermentation, the transformant could accumulate 48.3% (w/w) oil from inulin in its cells and cell dry weight was 13.3 g L(-1) within 78 h while the transformant could accumulate 50.6% (w/w) oil from extract of Jerusalem artichoke tubers in its cells and cell dry weight was 14.6 g L(-1) within 78 h. At the end of fermentation, most of the added sugar was utilized by the transformant cells. Over 91.5% of the fatty acids from the transformant cultivated in the extract of Jerusalem artichoke tubercles was C(16:0), C(18:1) and C(18:2), especially C(18:1) (58.5%).     

Influence of exponentially decreasing feeding rates on fed-batch ethanol fermentation of sugar cane blackstrap molasses

Summary The ethanol yield was not affected and the ethanol productivity was increased when exponentially decreasing feeding rates were used instead of constant feeding rates in fed batch ethanol fermentations. The influences of the initial sugar feeding rate on the ethanol productivity, on the constant ethanol production rate during the feeding phase and on the initial ethanol production specific rate are represented by Monod-like equations.Nomenclature F reactor feeding rate (L.h^–1) - F_o initial reactor feeding rate (L.h^–1) - K time constant; see equation (l) (h^–1) - M_E mass of ethanol in the fermentor (g) - M_s mass of TRS in the fermentor (g) - M_x mass of yeast cells (dry matter) in the fermentor (g) - P ethanol productivity (g.L^–1.h^–1) - R ethanol constant production rate during the feeding phase (g.h^–1) - s standard deviation - S_o TRS concentration in the feeding mash (g.L^–1) - t time (h) - T fermentor filling-up-time (h) - T time necessary to complete the fermentation (h) - TRS total reducing sugars calculated as glucose (g.L^–1) - V_o volume of the inoculum (L) - V_f final volume of medium in the fermentor (L) - X_o yeast concentration of the inoculum (dry matter) (g.L^–1) - ethanol yield (% of the theoretical value) - initial specific rate of ethanol production (h^–1)     

高生物量富硒酵母的选育及培养条件初步优化

通过筛选、单倍体分离、诱变和原生质体融合,从融合子中选育了一株高生物量富硒酵母菌株（编号为ZFF-28）,其细胞硒总含量分别是原始亲株ZY-67和ZY-198的2.8倍和2.0倍。通过单因素实验和正交试验设计,确定了优化培养条件：6%糖浓度的蔗糖糖蜜,添加0.5% (NH4)2SO4、0.1% H3PO4、60μg/mL Se,pH60～6.5,装液量50mL/250mL三角瓶,接种量10%,培养时间25h。在优化培养条件下,菌株ZFF_28的生物量可达8.2g/L,细胞中硒的含量达2050μg/g,硒总含量达到了16810μg/L,是培养条件优化前的1.3倍。细胞硒含量的91%为有机硒。