基于近红外光谱实时监测乙醇发酵过程多组分参数

生物量、葡萄糖浓度和乙醇浓度是乙醇发酵过程的重要参数,传统的方法通常对发酵液取样作离线测量,不仅需要采用多种仪器进行测试分析,而且耗时耗力,成为实时过程调控和优化的障碍。文中针对这些重要过程参数提出了一个基于近红外光谱技术的原位实时检测方法。通过采用浸入式近红外光谱仪对发酵溶液进行原位测量,基于多输出最小二乘支持向量机回归(MLS-SVR)方法建立了利用近红外光谱同时分析葡萄糖浓度、生物量和乙醇浓度的多输出预测模型。实验结果表明,该方法能实时准确地检测乙醇发酵过程中的葡萄糖浓度、生物量和乙醇浓度,而且相对于现有的偏最小二乘法(PLS)分别对各组分建模和预测,能明显提高测量准确性和可靠性。     

pta基因敲除对L-色氨酸发酵的影响

[目的]探究pta基因缺失对大肠杆菌发酵生产L-色氨酸的影响.[方法]运用Red重组技术敲除pta基因,构建pta缺失株E.coli TRTH△pta.利用30 L发酵罐进行分批补料发酵试验,考察重组菌E.coliTRTHApta发酵0生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、发酵液中NH4+浓度及变化....     

反相高效液相色谱法测定肠球菌FQ15发酵液中的有机酸

目的研究肠球菌FQ15发酵液中的有机酸在不同发酵时间的变化趋势。方法建立反相高效液相色谱法测定此株益生菌发酵液中有机酸的主要成分及其在发酵不同时间的变化趋势。结果丙酸、丙酮酸、乳酸、乙酸的浓度都是在菌体生长处于衰亡期时达到最大值,乳酸随发酵时间的延长浓度明显下降,而乙酸在发酵后期含量呈上升趋势。结论此方法重现性好,精密度高,为研究微生物合成中有机酸种类及变化趋势提供了一种可供参考的快捷分析手段。     

酿酒酵母和异常毕赤酵母混菌发酵对白酒液态发酵效率和风味物质的影响

[目的]通过酿酒酵母(Saccharomyces cerevisiae)和异常毕赤酵母(Pichia anomala)在麸皮汁培养基中的混菌发酵,以增加发酵液的风味酯含量并保证发酵效率.[方法]采用两种酵母混合接种、顺序接种混菌发酵方式,以酵母单独接种发酵作对照,测定酵母的发酵性能和发酵液中乙酸乙酯含量,并对发酵结束时风味物质进行半定量;利用无细胞系统,分析两种酵母之间的相互作用.[结果]采用顺序接种混菌发酵方式,避免S.cerevisiae 对P.anomala的生长竞争性抑制,使两种酵母均能获得较高的生物量;发酵结束时,乙醇浓度为20.17 g/L,比酿酒酵母单菌种发酵时降低了9.14％;但乙酸乙酯含量达到0.74 g/L,比异常毕赤酵母单菌种发酵时提高了80％;发酵液风味物质的测定结果表明,酿酒酵母与异常毕赤酵母的混合发酵能够形成更多的酯类物质,总酸和高级醇含量却相对较低,有效改善了发酵液的风味特性;在混菌发酵时,碳源是影响酿酒酵母繁殖的重要因素,但酵母的代谢物对异常毕赤酵母产生明显的抑制作用.[结论]混菌发酵,为丰富发酵产物的风味复杂性和增强风格的独特性提供了一条有效的途径.     

火菇属野生菌株发酵液对栽培菌株的化感效应

为探究火菇属Flammulina野生菌株发酵液对栽培菌株的影响,以火菇属野生柳生金针菇F. rossica F52、野生金针菇F. filiformis F39和栽培金针菇F. filiformis F47为供试菌株,采用固体平板培养和液体发酵的方法,对比了添加不同体积分数火菇属野生菌株发酵液对栽培菌株平板中菌丝长速、液体发酵中菌丝生物量、干质量及漆酶和淀粉酶活力的影响。结果表明：不同体积分数野生金针菇菌株F39发酵液对栽培菌株F47菌丝长速有抑制作用,对发酵液中菌丝生物量无显著影响,当添加发酵液体积分数为50%时可显著提高菌株F47发酵液中漆酶活力,当体积分数为40%时,可显著提高菌株F47发酵液中淀粉酶的活力（P<0.05）;添加不同体积分数的野生柳生金针菇菌株F52发酵液对菌株F47除淀粉酶外其他指标均影响显著,添加体积分数为30%的发酵液对栽培金针菇F47菌丝长速具有显著的促进作用,当添加发酵液体积分数为40%时,对菌株F47液体发酵中菌丝干质量和漆酶活力均有显著促进作用（P<0.05）。证明了化感效应不仅存在于属间,在同属下的种间也存在。     

黄原胶降解菌BIT-BJ001培养条件的优化

黄原胶在采油工程中有重要的应用,但其难降解性质给采油工程带来很多问题。从塔里木油田胡杨木根部样品中分离得到1株黄原胶降解菌BIT-BJ001,对其发酵条件的研究表明,此黄原胶降解菌最适培养条件为:黄原胶0.3%,酵母粉0.5%,Na+浓度0.8%,Mg2+浓度0.8%,初始pH值为10,温度60℃。菌种BIT-BJ001降解黄原胶的能力与发酵时间、发酵液中还原糖浓度有关,发酵96 h,黄原胶降粘率达到最高,发酵液中还原糖浓度过高,将抑制菌株对黄原胶的降解。     

NH4+浓度对黄色短杆菌XV0505发酵生产L-缬氨酸的影响

以L-缬氨酸(L-Val)生产菌黄色短杆菌XV0505为供试菌株,以(NH4)2SO4为唯一添加N源,考察不同NH 4+浓度对发酵过程中菌体干质量、L-Val产量和葡萄糖消耗速率以及菌体内代谢流量的影响。研究表明:NH 4+浓度过高或不足都会影响发酵水平,降低L-Val的产量。合适的初始NH4+浓度为225 mmol/L,产酸期NH4+维持浓度为35 mmol/L时,有利菌体产酸。在此NH4+浓度下,在30 L发酵罐发酵60 h,发酵液中菌体生物量和L-Val质量浓度分别可达22.35和59.12 g/L。     

废糟液全循环条件下絮凝酵母乙醇连续发酵

丙酸是以玉米为原料自絮凝酵母乙醇连续发酵系统废糟液全循环过程中积累的主要抑制物。基于丙酸对酵母细胞抑制机理,开发了3种废糟液全循环条件下乙醇连续发酵工艺策略。首先根据高温导致丙酸生成的现象,去除了物料灭菌环节,使发酵液丙酸浓度显著降低,生物量和乙醇浓度分别提高了59.3%和7.4%。其次,以丙酸浓度达到半数抑制浓度(IC50)40 mmol/L为目标,通过拟合丙酸积累数据预测废糟液全循环的最长运行时间,发酵装置运行应控制在此时间范围内。再次,较低的环境pH值提高了丙酸毒性,而实验证明发酵液pH为5.5时,丙酸对细胞生长的抑制影响最小,因此控制发酵过程中的pH有利于弱化丙酸毒性。     

L-色氨酸基因工程菌发酵培养基配方优化研究

[目的]对L-色氨酸基因工程菌液态发酵的培养基进行优化。[方法]通过P-B试验,筛选出对基础发酵培养基的发酵液中L-色氨酸浓度影响显著的因素,进一步通过最陡爬坡试验、B-B试验对影响显著的因素进行优化。在此基础上,确定最佳的发酵培养基配方。[结果]酵母粉、Fe SO4·7H2O、KH2PO4对发酵液中L-色氨酸浓度的影响显著;最佳培养基配方为:Glucose 25.0 g/L,酵母粉4.5 g/L,(NH4)2SO49.0 g/L,Mg SO44.5 g/L,柠檬酸钠2.0 g/L,Fe SO4·7H2O 96.1 m g/L,KH2PO41.2 g/L。[结论]根据此配方进行验证实验,发酵液中L-色氨酸浓度可达2.25 g/L。     

活性干酵母SOD摇瓶发酵条件的研究

研究了营养与环境条件对耐高温酒精活性干酵母（ＴＨ－ＡＡＤＹ）ＳＯＤ摇瓶发酵的影响。实验结果表明,初糖浓度,金属离子、ＰＨ值、通气量（装液量）和培养时间等均对ＡＡＤＹ摇瓶发酵的生物量和ＳＯＤ含量有较大的影响。在实步优化的发酵条件下,细胞生物量为３９．６ｇ／Ｌ,菌体ＳＯＤ含量为１９４８Ｕ／ｇ,发酵液产酶能力为７．７万Ｕ／Ｌ。     

Methods for the quantification ofFrankia cell biomass

Six methods for the estimation of microbial biomass were compared for determination ofFrankia cell concentrations. Six strains ofFrankia were cultivated in stationary culture, harvested by centrifugation, washed with saline buffer and diluted to five standardized concentrations. These cell suspensions were then used to assess reliability of each of the biomass determination methods. The destructive total protein determination methods were the most sensitive and reliable. Two non-destructive methods, packed cell volume and turbidity measurement, were also accurate, and because of their simplicity hold advantage for routine growth measurements and inoculum dilutions. Dry weight determinations were inconsistent for the small cell masses used in this study. An ELISA procedure demonstrated reliability but little sensitivity.     

Evaluation of transformation capacity for degradation of ethylene chlorides by<Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

The transformation capacity (T_c) ofMethylosinus trichosporium OB3b in the degradation of ethylene chlorides was determined by measuring the decrease of soluble methane monooxygenase (sMMO) activity of resting cells in batch experiments. All measurements of sMMO activity were taken in the presence of 20 mM formate to avoid the deficiency of reducing power, and within 2 hrs to avoid the effect of natural inactivation from instability of the resting cells. The constant T_c values of 0.58±0.132 and 0.80±0.17 μmol/mg cell were obtained for trichloroethylene (TCE) and 1,2-dichloroethylene (cis andtrans-1,2-DCE), respectively, regardless of their concentrations. The transformation capacity measured by this method can be used to predict the amount of cells that should be stimulated inin-situ bioremediation.     

三峡库区消落带落羽杉与立柳林土壤微生物生物量碳氮磷动态变化

为探究库区消落带人工乔木植被恢复重建后土壤质量及肥力的变化特征,于2016年6月(T_1)、2016年9月(T_2)、2017年6月(T_3)及2017年9月(T_4)选择165—175 m高程落羽杉与立柳土壤为研究对象,并以裸地作为对照,测定土壤微生物生物量碳、氮、磷和相关理化性质。结果表明:(1)经历水淹(T_2—T_3)会使土壤微生物生物量处于较低水平,落干期(T_1—T_2、T_3—T_4)落羽杉与立柳人工植被恢复生长能显著提高土壤微生物生物量,对土壤微生物恢复具有重要意义。(2)落羽杉与立柳土壤微生物生物量碳、氮占土壤有机碳、全氮百分比在4个时期均显著高于裸地,表明落羽杉与立柳土壤微生物对土壤碳、氮库的贡献大于裸地;落羽杉土壤微生物生物量磷及其占全磷百分比在T_1和T_3处于极低水平,T_2和T_4处于较高水平,应注意磷元素的迁移。(3)土壤微生物生物量碳、氮、磷与土壤有机碳和全氮有极显著相关性,与土壤pH值呈不同程度的负相关。在三峡库区消落带进行落羽杉与立柳乔木植被恢复重建能显著提高土壤微生物生物量及土壤肥力,进一步证实开展科学的植被修复与重建值得提倡和肯定。     

Improved synchronous light scattering method for measuring baker’s yeast biomass using thickened suspensions

Measuring yeast biomass is important in the processes of microbial fermentations. It has been demonstrated that synchronous light scattering (SLS) signals could be applied for the quantification of model bioparticles such as Saccharomyces cerevisiae. In this study, an improved synchronous light scattering method was developed for yeast biomass estimation. The settlement of yeast cells during SLS signals measuring process was studied, and hydrolysis anionic polyacrylamide was added into yeast suspensions to increase the stability of the cells in liquid environment. By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, ?λ = 0), the SLS intensity was found to be proportional to the yeast concentration in the range from 0 to 4.9 × 10⁶ cell/mL (R ² = 0.9907), the detection limit is 8.1 × 10³ cell/mL. The developed method exhibited good stability and sensitivity in the recovery test and growth curve drawing process, demonstrating the potential of the method in practical application of biomass estimation.     

巨桉连栽对土壤微生物生物量和数量的影响

为探讨巨桉连栽对土壤微生物生物量和数量的影响,采用时空互换法,研究了马尾松(Pinus massoniana)林和不同连栽代次的巨桉(Eucalyptus grandis)人工林的微生物生物量、数量与土壤理化性质的关系。结果表明,巨桉一代林的土壤微生物生物量碳、生物量氮和土壤真菌、细菌数量与马尾松林的差异不显著,放线菌数量则显著增加。随巨桉连栽代次增加,土壤微生物生物量碳、生物量氮和土壤细菌、放线菌、真菌数量均递减。回归分析表明,土壤全磷能独立解释微生物生物量氮、细菌和真菌数量71.7%、86.1%和63.0%的变异,与总孔隙度共同解释微生物生物量碳87.9%的变异,与全氮共同解释放线菌数量89.6%的变异。可见,土壤全磷较大程度解释了微生物的变化。     

Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l^–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.     

Dielectric measurement to monitor the growth and the physiological states of biological cells

Measurement of capacitance, also referred to as dielectric permittivity, is a new method of estimating the concentration of cells, monitoring the growth and detecting the physiological changes during the cultivation of organisms in various bioprocess. Several types of biological cells were studied, namely; Saccharomyces cerevisiae, Escherichia coli, Perilla frutescens (plant cells) and AFP-27 hybridoma cells. Generally, a linear correlation between cell capacitance (C) and other biomass measurement technique such as optical density (OD) and dry weight (DW) was obtained using the different types of cell suspension. Therefore, this method could be used to monitor the growth of the organism during the active growth. It could be conveniently used to make a rapid estimate of the cell concentration such as in plant cell suspension culture. The capacitance sensor could also be designed to be installed and autoclaved in-situ in a bioreactor and used for on-line monitoring of cell growth. On the other hand, distinct deviations in the capacitance value were observed in relation with the growth stage of the organism. This was observed in all the organisms studied but the type of deviation depends on the physiology of the organism. This variation in cell capacitance showed the possibility of using this method as a means to indicate changes in the physiological state of cells during cultivation. This capability would be very useful in designing control strategies that would depend on the physiological states in the bioprocess. Present address: Miles Inc., Berkeley, CA 94701 U.S.A.The authors sincerely appreciated the generosity of Dr. K. Mishima and Dr. A. Mimura of Kobe Steel Co., Japan. The useful discussions with M. Nakajima and technical assistance of J. Zhong and R. Pambayun were also acknowledged. The work in hybridoma cell culture was done through the collaboration with C. Perusich-Kussow and Prof. W. S. Hu, University of Minnesota, USA.     

梨形四膜虫衰老过程中DNA含量的变化

应用一种新型的细胞核内DNA含量测定方法──图像分析法,测定真核细胞梨形四膜虫衰老过程中DNA含量的变化.根据Beer-Lambert定律,以细胞核在不同生长期内的积分光密度的水平表示核内DNA含量的变化.该方法具有测量速度快,重复性好,操作简单,结果可靠等优点.实验结果表明:四膜虫在进入对数生长期时,DNA含量逐渐达到高峰,随着细胞逐渐老化,细胞分裂次数及核内DNA含量逐步减少.     

拉曼光谱分析技术在资源微生物发酵性能评价中的应用

微生物发酵过程是细胞新陈代谢进行物质转化的过程,为了提高目标产物的转化率,需要对微生物发酵动态特性进行实时分析,以便实时优化发酵过程。拉曼光谱(Raman spectroscopy)量化测试作为一种有应用前景的在线过程分析技术,可以在避免微生物污染的条件下,实现精准监测,进而用于优化控制微生物发酵过程。【目的】以运动发酵单胞菌(Zymomonas mobilis)为例,建立微生物发酵过程中葡萄糖、木糖、乙醇和乳酸浓度拉曼光谱预测模型,并进行准确性验证。【方法】采用浸入式在线拉曼探头,收集运动发酵单胞菌发酵过程中多个组分的拉曼光谱,采用偏最小二乘法对光谱信号进行预处理和多元数据分析,结合离线色谱分析数据,对拉曼光谱进行建模分析和浓度预测。【结果】针对运动发酵单胞菌,首先实现拉曼分析仪对单一产品乙醇发酵过程的精准检测,其次基于多元变量分析,建立葡萄糖、乙醇和乳酸浓度变化的预测模型,实现对发酵过程中各成分浓度变化的准确有效分析。【结论】成功建立了一种评价资源微生物尤其是工业菌株发酵液多种组分的拉曼光谱分析方法。该方法为运动发酵单胞菌等工业菌株利用多组分底物工业化生产不同产物的实时检测,以及其他微生物尤其工业菌株的选育和过程优化提供了新方法。     

Dynamics of the changes of electrophysical properties of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 cells at their binding with wheat germ agglutinin

The electrooptical properties of Azospirillum brasilense Sp7 cell suspensions, have been studied at a specific interaction with wheat germ agglutinin (WGA), using the dependences between the changes of optical densities of cell suspensions at the electric orientation of cells and the orienting field frequencies of 740, 1000, 1450, 2000, and 2800 kHz. It was shown that the electrooptical (EO) properties of cell suspensions changed at the interaction of A. brasilense Sp7 cells with WGA and that the EO signal value changed irrespective of the cultivation conditions. At the same time, the dynamics of the changes of the EO properties of microbial suspensions was different for microbial cells grown under different conditions. It may be evidence of the differences in the cell surface properties of microbial cells, and of the dependence, between bacterial response to lectin and growth conditions. The possibility of using the EO analysis of bacterial suspensions for the study of the high-specific binding of polypeptide molecular signals with the bacterial target cells and for assessment of the dynamics of this process has been demonstrated.