Utilization of pretreated molasses for serine alkaline protease production with recombinant bacillus species

Recombinant Bacillus amyloliquefaciens, Bacillus cereus, Bacillus subtilis , and Bacillus licheniformis were used for the production of serine alkaline protease (SAP) utilizing chemically and/or physically pretreated molasses. The highest enzyme activity was obtained with r- Bacillus subtilis , with the complex medium involving physically treated molasses having 20 kg m m 3 initial sucrose concentration in small-scale, agitation- and heating rate-controlled bioreactors at t=63 h. Effects of oxygen transfer were investigated in 3.5 dm 3 laboratory bioreactors under two different agitation rates with r- B. subtilis . Increase in the oxygen transfer rate increased the observed activity and caused the cultivation time of maximum activity shift to the earlier stages of the fermentation. At Q/V=0.5 vvm and N=750 min m 1 , SAP activity reached 2250U cm m 3 at t=36 h.     

Fermentation and oxygen transfer characteristics in serine alkaline protease production by recombinant Bacillus subtilis in molasses‐based complex medium

Serine alkaline protease (SAP) production in a complex medium based on physically pretreated molasses by recombinant Bacillus subtilis carrying pHV1431::subC gene is described. The effects of oxygen transfer were investigated in 3.5 dm³ bioreactor systems with controls for agitation rate, dissolved oxygen, pH, temperature, and foam formation under two different agitation rates, ie N = 500 and 750 min^?1, and four different air flow rates, ie Q/V_R = 0.2, 0.5, 0.7, and 1.0 vvm, at a molasses concentration equivalent to initial sucrose concentration (C_So) of 20 kg m^?3. The yield values (Y_X/S, Y_X/O, Y_S/O) and maintenance coefficient of oxygen (m_O), were calculated. m_O decreased with the increase in the air‐inlet rate. Increase in oxygen transfer rate increased the rate of growth and SAP activity, and affected the cultivation time to achieve maximum expression of SAP activity. At Q/V_R = 0.5 vvm and N = 750 min^?1, SAP activity reached 2250 U cm^?3 at t = 36 h. The oxygen transfer coefficient (K_La) and oxygen uptake rate (?r_O) were measured throughout the fermentations and their variation with the oxygen transfer conditions determined. New correlations for the calculation of K_La and ?r_O are proposed. Copyright © 2004 Society of Chemical Industry     

枯草芽孢杆菌出芽培养条件优化

探索了提高枯草芽孢杆菌产生芽孢的方法,使用单批一次投料培养法研究了培养基成分、p H值、培养过程中通气量及装液体积比对菌体产芽孢的影响。试验结果说明:枯草芽孢杆菌最适生长和产生芽孢的碳源是糖蜜,最适氮源是酵母浸粉;二价锰离子和无机盐对菌体的生长和产芽孢有很大影响。菌体生长和产芽孢的最适培养基组成:糖蜜1.0%,酵母粉0.8%,(K2HPO4+KH2PO4)0.5‰,Mn SO4 0.2‰;产芽孢的最适条件:开始培养p H值为7.0,最适装液量为20%(体积比)。在5L半自动发酵罐中,溶氧大于70%,发酵24 h后,芽孢数量为5.1×1011CFU/m L,出芽率为98.0%。     

The effects of a biosurfactant on oxygen transfer in a cyclone column reactor

A laboratory-scale cyclone column reactor was tested to determine how its oxygen transfer characteristics were affected by surfactants in the liquid medium. The volumetric oxygen transfer coefficient was greatly decreased by small quantities of the synthetic surfactants dodecyltrimethylammonium bromide and sodium dodecylsulfate, and the biosurfactant surfactin produced by Bacillus subtilis (ATCC 21332). Since the gas holdup fraction was generally increased due to foaming, the effectiveness of the surfactants was probably due to an increase in the interfacial film resistance. B. subtilis was grown in the cyclone column to 0.6 g dm^?3 with a significant level of surfactin produced while maintaining at least 75% oxygen saturation in the broth. Process optimization and scale-up of surfactin production will have to consider oxygen transfer as a key parameter.     

Evaluation of a hollow fiber oxygenator for use in bubble—free mammalian cell bioreactors

The oxygen transfer capabilities of a hollow fiber oxygenator for intended use in bubble-free aerated bioreactors have been evaluated experimentally. The oxygenator (480 mL), which is the main component of an artificial heart/lung unit used routinely in cardiopulmonary bypass procedures, was assessed for use in a recycle stream to determine if the oxygen requirements in bubble-free hybridoma cell bioreactors could be supported on a large scale. Oxygen transfer to simulated medium in a 15 L bioreactor was found to depend primarily on the liquid recirculation rate (1 to 5 L/min) and was not seriously affected by the air flowrate in the oxygenator (0.5 to 15 L/min). Based on the experimentally determined mass transfer coefficient across the membrane, it was found that the oxygenator could support a 100 L fermentor at an average cell density of 2xl0⁶ cells/mL with a specific uptake rate of 4.8 mgO₂/(10⁹ cells - h).Furthermore, if oxygen enriched air were to be used in the oxygenator, a single unit could support cell densities beyond 8xl0⁶cells/mL in a 100 L working volume fermentor.     

枯草芽孢杆菌与水体中U(Ⅵ)的作用机制

通过静态吸附-解吸实验,研究了枯草芽孢杆菌对U(Ⅵ)的吸附热力学和动力学,U(Ⅵ)的解吸和菌体内P的释放过程,利用扫描电镜、能谱(SEM-EDS)和红外光谱(FTIR)分析了作用机理。结果表明:当pH=5时,在1 L 50 mg·L^-1的铀溶液中,投加枯草芽孢杆菌1.384 mg(干重,DW),2 h后,铀的去除率和吸附量分别为85.34%和308.31 mg·g^-1(DW)。随着作用时间从2 h延长至48 h,铀的解吸率从52.13%下降至36.25%,菌体内释放到溶液中的P浓度从 0.12 mg·L^-1增加到0.40 mg·L^-1。枯草芽孢杆菌对水体中U(Ⅵ)的吸附行为可以用Langmuir吸附等温模型和准二级动力学方程较好地描述。作用过程是放热,可自发进行。SEM-EDS表明枯草芽孢杆菌与水体中U(Ⅵ)作用后,菌体内部断裂呈短节状,铀沉积在细胞表面及周围,无明显晶体产物生成,FTIR图谱出现UO₂²⁺特征吸收峰,氨基、磷酸基团参与枯草芽孢杆菌与水体中U(Ⅵ)的作用。     

质芽孢杆菌PM13菌株培养基的优化

对胶质芽孢杆菌PM13菌株进行了培养基成分的优化,考察了培养基各组分对菌体生长和发酵液粘度的影响. 由单因素、正交和均匀实验结果可知,碳源和氮源种类的影响最显著,优化的培养基配方为(g/L)：糖蜜3.67,淀粉5.5,豆粕粉7.0, CaCO3 8.5, K2HPO4 2.0, MgSO4×7H2O 1.4, NaCl 0.2. 在此配方下,胶质芽孢杆菌芽孢生成量达2.2×108 cfu/mL,且发酵液稠度系数显著降低到0.028 Pa×sn,可采用常规生物反应器进行规模化发酵.     

枯草芽孢杆菌对土壤呼吸作用和脲酶活性的影响

通过模拟实验研究了微生物农药枯草芽孢杆菌对黑土的呼吸作用和脲酶活性的生态毒理效应.结果表明:枯草芽孢杆菌各质量分数处理均表现为对土壤呼吸作用的刺激效应,并且土壤巾枯草芽孢杆菌质量分数越大,对土壤呼吸强度的刺激作用越大,其中最高质量分数(3200 mg/kg干土)处理在第42天时达到最大刺激强度,刺激率为69.1%.与对照相比,除第1天外,所有处理(32～3200 mg/kg干土)对土壤脲酶均表现出刺激效应,其中最高质量分数(3200 mg/kg干土)处理在第28天脲酶活性上升到最高,刺激率达到101.1%.     

枯草芽孢杆菌对黑土中可培养微生物的生态效应

研究了微生物农药枯草芽孢杆菌对东北黑土中可培养微生物的生态影响.其动态变化表明:低质量分数枯草芽孢杆菌对细菌总数没有明显影响,较高质量分数枯草芽孢杆菌町促进细菌总数的显著增加,其中质量分数在3200 mg/kg时,枯草芽孢杆菌对细菌的刺激强度最高,为对照的11倍.枯草芽孢杆菌对土壤中的放线菌也有刺激作用,刺激强度最高时,放线菌数量可增至对照的8.3倍左右.枯草芽孢杆菌对真菌的敏感性较低,只有质量分数高达3200 mg/kg时,才对真菌产生明显刺激作用,最高刺激强度为同时期对照的29倍.     

催化亚临界水氧化法处理糖蜜酒精废液的研究

以铜、锰为活性成分和微波辐射法制备的γ-Al2O3为载体,制备了CuO/MnO2/γ-Al2O3复合催化剂,考察了成分配比、浸渍时间对催化剂催化活性的影响,研究了氧气分压、催化剂投加量、反应温度和反应时间对催化亚临界水氧化法处理糖蜜酒精废液过程的影响规律。结果表明:CuO/MnO2/γ-Al2O3催化剂是催化亚临界水氧化法处理糖蜜酒精废液的可选催化剂;当进水CODCr的质量浓度为10×104mg/L时,适宜的氧气供应量为理论需氧量的3.5～4.7倍,催化剂的投加量以10g/L为佳;在360℃下反应10min,处理水可达到《污水综合排放标准》GB9878-1996的一级标准。     

隔夜茶水抑菌活性的研究

采用滤纸片扩散法测定不同浓度的一道茶、二道茶在浸泡12 h后对大肠杆菌、枯草芽孢杆菌、黑曲霉、青霉四种微生物的抑制作用。一道茶、二道茶对大肠杆菌、枯草芽孢杆菌有较明显的抑制作用,一道茶的抑制作用大于二道茶,对黑曲霉、青霉没有抑制作用。一道隔夜茶水对大肠杆菌、枯草芽孢杆菌的最小抑菌浓度分别为0.063 g/mL、0.077 g/mL。本实验证实了隔夜茶水的抑菌作用,为其变废为宝提供理论依据。     

Separation of the protease enzymes of Bacillus licheniformis from the fermentation medium by crossflow ultrafiltration

The separation from fermentation medium of extracellular serine alkaline protease (SAP) enzyme produced by Bacillus licheniformis was investigated using a crossflow ultrafiltration system. SAP was separated from the high molecular weight neutral protease (NP) and amylase (AMY) enzymes and from the low molecular weight organic acids and amino acids in a crossflow ultrafiltration system with 30 000 Da and 10 000 Da MWCO polysulfone membranes, respectively. The effects of transmembrane pressure (TMP), recirculation velocity (v), and initial enzyme concentration (C_E) on the permeate flux, on the activities of SAP, NP and AMY enzymes, and on the recovery of SAP were investigated. High permeate flux was obtained at high recirculation velocity and TMP, but at low initial enzyme concentration. SAP enzyme recovery and activity measured in the system also showed alterations with hydrodynamic conditions. The best operation conditions for the separation of SAP from NP and AMY were TMP = 20 kPa, v = 0.50 ms⁻¹ and C_E = 0.28 gdm⁻³. The separation of SAP from the organic and amino acids was best performed at TMP = 100 kPa, v = 0.33 ms⁻¹ and C_E = 0.40 gdm⁻³. © 2000 Society of Chemical Industry     

醋糟酶解液的制备及其发酵生产枯草芽孢杆菌TS-02

采用水热处理技术对醋糟进行预处理,优化了醋糟的纤维素酶酶解条件,制得葡萄糖浓度27.00 g/L的醋糟酶解液. 以醋糟酶解液为基础培养基替代培养基中的葡萄糖,发酵生产枯草芽孢杆菌TS-02活菌制剂. 结果表明,在醋糟酶解液培养基中摇瓶发酵44 h时活菌数活菌数最高达4.64×1010个/mL, 7 L发酵罐中发酵周期为22 h,活菌数达6.16×1010个/mL,芽孢率达80%以上.     

仙人掌提取物的抑菌作用

分别对食用仙人掌(OpuntiaMiloaAlta)和野生仙人掌 (OpuntiadilleniiHaw)提取物进行了抑菌作用、最低抑菌和杀菌条件的研究。实验结果表明:野生仙人掌提取物对大肠杆菌和枯草芽孢杆菌有明显的抑制作用,食用仙人掌提取物的抑菌作用效果不明显。野生仙人掌乙醇提取物 (odh)最小抑菌质量分数分别为:大肠杆菌w(odh) =2. 5%,枯草芽孢杆菌w(odh) =5%;最低杀菌质量分数分别为:大肠杆菌w(odh) =5%,枯草芽孢杆菌w(odh) =10%。此外,正交实验的最佳抑菌条件为:提取物w(odh) =10%,提取剂φ(乙醇) =85%,pH=4 5。     

Techniques for oxygen transfer measurement in bioreactors: a review

Oxygen is the most essential requirement for aerobic bioprocesses. The microbial growth in a bioreactor depends upon the oxygen transfer rate (OTR). The OTR is widely used to study the growth behavior of microbial and plant cell cultures. The mass transfer coefficient (k_La) determines the magnitude of the OTR. There are many techniques for measuring oxygen concentration and OTR in bioreactors. Zirconia, electrochemical, infrared, ultrasonic and laser cells are used to measure oxygen concentration in the liquid medium. Optical sensors are better alternatives to measure oxygen concentration in small bioreactors. Sulfite oxidation and gassing‐out methods with a Clark‐type electrode have been used for OTR measurements in bioreactors. Many new novel techniques have evolved recently for intermittent and continuous online measurement of OTR/k_La in various types of bioreactors. The present paper gives an overview of various measurement techniques and their limitations and/or suitability for measurement of OTR/k_La in various kinds of bioreactors, especially small bioreactors. Copyright © 2009 Society of Chemical Industry     

Beet molasses based exponential feeding strategy for thermostable glucose isomerase production by recombinant Escherichia coli BL21 (DE3)

BACKGROUND: The effects of pretreated beet molasses based feeding strategies on thermostable glucose isomerase (GI) production by recombinant Escherichia coli BL21 (DE3) pLysS were investigated. RESULTS: The thermostable GI encoding gene of Thermus thermophilus (xyl_A) was recombined with pRSETA vector, and the pRSETA::xyl_A obtained was transferred into E.coli BL21 (DE3) pLysS and used for GI production. The highest soluble GI activity was obtained at t = 30 h, as A = 16 400 U L^?1 (20.6 U mg^?1 protein) under molasses based fed‐batch operation, with a specific growth rate µ = 0.1 h^?1 (M‐0.1); on the other hand, the highest cell concentration was obtained at µ = 0.15 h^?1 operation as 9.6 g L^?1 at t = 32 h. The highest oxygen uptake was 4.57 mol m^?3 s^?1 at M‐0.1 operation. CONCLUSIONS: Molasses based fed‐batch operations were more successful in terms of cell concentration and thermostable enzyme production due to the existence of a natural sugar inducer, galactose, in the molasses composition. This study demonstrates the significance of proper feeding strategy development for over‐production of enzymes by recombinant E. coli strains. © 2012 Society of Chemical Industry     

非水相体系中枯草杆菌蛋白酶-表面活性剂离子对的形成条件

枯草杆菌蛋白酶在水相pH值小于其等电点条件下带正电,能与异辛烷中带负电的阴离子表面活性剂二辛基丁二酸磺酸钠（AOT）通过静电引力结合形成离子对进入有机相。详细研究了两相接触方式、水相初始pH值、水相离子强度等因素对酶转移率的影响。采用120 r/min磁力搅拌使两相混合能得到较好的酶转移率,作用时间为6 h。水相初始pH值5.0有利于离子对的形成及酶蛋白的转移。水相中CaCl2的加入有利于消除两相混合过程中的乳化;但是过高的CaCl2浓度会减弱酶分子与AOT间的静电引力,不利于酶分子与AOT的结合。水相中酶浓度不变,随着有机相中AOT初始浓度的增加,酶的转移率呈现增长趋势。固定异辛烷中AOT浓度,随着水相中初始酶浓度的增加,转移至有机相中酶量先增加后减少。进入异辛烷的酶分子仍旧保持活性,能催化转酯化反应的进行,但随着反应时间的延长,酶的催化效率出现下降。     

Evaluation of antibacterial properties of triorganotin carboxylates containing functionalised ester groups in tests against some pathogenic bacteria

Bacterial screening employing the agar diffusion test on triphenyltin carboxylates containing various functional residues in the ester moiety revealed appreciable differences in their activities relative to triphenyltin acetate. Among these, [3-(Diethylphosphono)propionato] triphenyltin (1) and [N-cyclohexylcarbamoyl) glycinato] triphenyltin displayed activities comparable to tri-n-butyltin cinnamate (2) towards both Gram-positive and Gram-negative bacteria; the latter compound was the most active among the eleven triorganotin compounds tested, which included cyclopentyldiphenyltin hydroxide (3) and its methacrylate derivative. Applying the more quantitative plate count and optical density tests on compounds 1-3, it was shown that their inhibitory activity ranked in the order 2 > 3 >1. Significantly, 3 caused around 90% inhibition of both Eschechia coli (-) and Pseudomonas aeruginosa (-) when incubated for 24 h at 37+/-1 at the 10.0 mug/ mL concentration level. Compound 2 was less effective against P.aeruginosa than against E.coli. While the Gram-positive bacteria were all readily inhibited, Bacillus subtilis (+) appeared to the most susceptible among them towards the test compounds.     

枯草芽孢杆菌Dg5041液体发酵生产γ-聚谷氨酸

以枯草芽孢杆菌Dg5041为生产菌,对其摇瓶发酵生产γ–聚谷氨酸(γ-PGA)的工艺进行研究,通过单因素实验和正交实验获得了该菌的优化培养条件。优化的培养基组成为:葡萄糖35 g/L,酵母膏10 g/L,谷氨酸钠40 g/L,MgSO41.0 g/L,K2HPO4 2.0 g/L,MnSO40.5 g/L,pH 7.0,250 mL锥形瓶装液量50 mL。菌种在37℃,120 r/min培养24 h加入5%NaCl后继续培养24 h,γ-PGA产量达到18.54 g/L。研究结果表明,枯草芽孢杆菌Dg5041是一株很有潜力的γ-PGA高产菌。     

异噻唑啉酮类衍生物的合成及其抗菌效应

以苯并异噻唑-3-酮为原料合成了4种新型衍生物——3-氯丙酰氯-苯并异噻唑啉酮、丙酰氯-苯并异噻唑啉酮、丙烯酰氯-苯并异噻唑啉酮、甲基丙烯酰氯-苯并异噻唑啉酮,目标化合物的结构经过1HNMR和质谱(MS)分析确认。同时检测这4种化合物对几种常见微生物——大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)和金黄色葡萄球菌(Staphyloccus aurueus)的抗菌活性。结果表明,这4种化合物在较低浓度下(≤25 mg/L)对大肠杆菌和枯草芽孢杆菌的抑菌率均达到80%以上,而对金黄色葡萄球菌的抑菌效果较差,只有高浓度(≥50 mg/L)下才能明显抑制其生长。