实验室模拟高负荷SPAC厌氧反应器运行

采用模拟废水, 对新型高负荷螺旋式自循环(Spiral automatic circulation, SPAC)厌氧反应器的运行性能进行了实验室模拟研究。结果表明: 在30oC, 水力停留时间(HRT)为12 h, 进水COD浓度从8000 mg/L升至20 000 mg/L的条件下, 反应器的COD去除率为91.1%~95.7%, 平均去除率为93.6％。在进水浓度为20 000 mg/L, HRT由5.95 h缩短至1.57 h的工况下, COD去除率从96.0%降低至78.7%, 反应器达到最高容积负荷率306 g COD/(L·d), 最大容积COD去除率240 g/(L·d), 最高容积产气率131 L/(L·d)。该反应器对基质浓度的连续提升具有良好的适应能力。进水COD浓度由8000 mg/L提升至20 000 mg/L时, 出水COD浓度一直处在较低水平(平均为852?mg/L), 容积COD去除率和容积产气率分别提高162%和119%。该反应器对HRT的连续缩短也有良好的适应能力。HRT由5.95 h缩短至1.57 h时,反应器容积COD去除率和容积产气率分别升高191%和195%。     

循环利用重组大肠杆菌细胞转化合成丁二酸

研究了回收丁二酸发酵液中的大肠杆菌进行细胞转化的可行性,以转化率和生产效率为指标,考察了不同菌体浓度、底物浓度、pH调节剂对细胞转化的影响。发酵结果表明大肠杆菌可以在仅含有葡萄糖和pH调节剂的水环境中转化生产丁二酸,并确定了最佳的转化条件为:细胞浓度(OD600)50,底物浓度40g/L,缓冲盐为MgCO3。基于优化好的条件,在7L发酵罐中进行重复批次转化,第1次转化的转化率和生产效率分别达到91%和3.22g/(L·h),第2次转化的生产效率和转化率达到了86%和2.04g/(L·h),第3次转化的转化率和生产效率分别达到了83%和1.82g/(L·h)。     

高、低氮浓度对2株真眼点藻的生长和油脂积累的影响

【目的】研究氮浓度对真眼点藻纲(Eustigmatophyceae)的2株高产油微藻大真眼点藻(Eustigmatos magnus,EM)和波氏真眼点藻(Eustigmatos polyphem,EP)的细胞形态、生长、总脂含量、脂质组成和脂肪酸组成与含量的时序变化规律。【方法】利用高氮(18.0 mmol/L NO3?-N)和低氮(3.6 mmol/L NO3?-N)浓度培养微藻。【结果】形态观察结果表明,大真眼点藻(E. magnus)和波氏真眼点藻(E. polyphem)营养细胞具有1个周生的裂叶状叶绿体,细胞质中有液泡,内含能够振动的颗粒物,以及一个较为明显的红色色素体;生殖方式通过形成2个D形或4个四角形的似亲孢子;随着培养周期的延伸和营养盐的消耗,细胞中油体逐步形成,其数量不断增加,体积不断增大。实验结果表明,初始氮浓度对2种微藻的总脂积累及生长均有显著影响(P<0.05),低氮浓度下2种微藻的生物质浓度分别为9.0 g/L和8.5 g/L,均低于高氮浓度下的生物质浓度。而低氮浓度下2种微藻的总脂、中性脂和总脂肪酸的含量以及总脂、中性脂与总脂肪酸的单位体积产率均明显高于高氮浓度组,其最高值分别为：59.10%、51.90%、46.95%和0.28、0.24、0.22 g/(L·d) (EM);64.20%、56.80%、50.01%和0.32、0.28、0.25 g/(L·d) (EP)。脂肪酸分析结果表明,两种微藻的脂肪酸主要成分均为棕榈酸(C16:0)、棕榈油酸(C16:1)、油酸(C18:1)和二十碳五烯酸(C20:5,EPA),四者的总含量(占总脂肪酸)分别达到85.83%和85.48%,其中棕榈油酸的含量最高。【结论】低氮浓度胁迫有利于大真眼点藻和波氏真眼点藻细胞内油脂的积累,两种微藻均为适合于生产生物柴油的油脂生产藻株。     

固定载体卧式厌氧反应器处理糖蜜废水的快速启动

为高效处理高浓度有机废水而设计了固定载体卧式厌氧反应器R1和R2, 它是厌氧折流板反应器(ABR)的改进, 以活性炭纤维作为生物膜载体固定并充当反应器的折流板, 在实验室规模上对R1和R2处理糖蜜废水进行快速启动运行。HRT和ORL是影响R1和R2稳定高效运行及启动的2个重要工艺参数。实验证明: HRT为2 d时, 反应器运行最佳。在第30天时, R1的COD去除率达到84.88%, R2达到81.72%。随着进水ORL由1.25 kg/(m3·d)提升到10 kg/(m3·d), 沼气容积产气率由0.35 L/(L·d)逐渐增加到4.98 L/(L·d)。进水pH值为3.9?4.5之间, 整个启动运行过程中, 未调节pH值, R1和R2的出水pH值均在6.7?7.6之间, 2个反应器均有较强的抗酸能力, R1的pH波动更为平缓。在整个实验过程中, 污泥流失量小, 没有发生堵塞现象, 在处理酸性高浓度有机废水时, 2个反应器均表现出较强的抗负荷冲击能力。     

产GL-7-ACA酰化酶重组菌的构建及高表达研究

戊二酰基-7-氨基头孢烷酸(GL-7-ACA)酰化酶是7-氨基头孢烷酸(7-ACA)两步酶法生产中的关键酶。成功构建组成型表达的产GL-7-ACA酰化酶重组大肠杆菌JM105/pMKC-ACY,并对其高表达条件进行了研究,得到了组成简单、廉价的国产培养基配方及操作简便、易于实现工业化的发酵工艺。在优化条件下,上罐补料高密度发酵的酶活高达6668.9U/L,是优化前的12.4倍,产率最高可达275.5U/(L.h),达到了工业生产的要求。     

丝孢菌Monodictys asperospera (Cooke & Massee) Ellis漆酶的分离纯化及其酶学性质

本研究首次发现Monodictyx asperospera(Cooke&Massee)Ellis具有较好的产漆酶能力.粗酶液经硫酸铵盐析、DEAE-纤维素层析及丙烯葡聚糖凝胶S-300层析纯化,纯化倍数为8.1,回收率为5.7%.漆酶分子量约为77kD,最适反应温度为55℃,最适反应pH6.0,以丁香醛连氮为底物时Km为0.163mm0l/L,Vmax为0.194 mmol(L·min),含糖量为18.14%,Cu2+对漆酶有明显抑制作用.     

In Vivo Estrogenicity of Nonylphenol and Its Ethoxylates in the Canadian Environment

The relative estrogenicity of nonylphenol and its ethoxylates has not been clearly demonstrated in the literature, despite the importance of this information for interpreting the environmental risk of these chemicals. There appears to be a discrepancy between the relative acute/chronic toxicity and estrogenicity reported in previous studies. These studies have suggested that the relatively higher concentrations of nonylphenol polyethoxylate metabolites (NP_nEO, NP_nEC) in municipal effluents may represent a risk to the environment. However, there is considerable uncertainty associated with the estimates of relative estrogenicity of these metabolites.

Plasma vitellogenin (Vg) was measured in rainbow trout (Oncorhynchus mykiss) after a 21-d flow-through exposure to concentrations of 1–250 μ g · L^{? 1} nonylphenol (NP), 1–280 μ g · L^{? 1} nonylphenol 1-ethoxylate (NP1EO), or 24–1450 μ g · L^{? 1} nonylphenol 1-ethoxycarboxylate (NP1EC). All three chemicals induced plasma Vg to varying degrees, their relative estrogenicity being NP > NP1EO > > NP1EC. Measurements of the relative potency of NP1EO and NP1EC compared to NP, yielded ratios of 0.22 and 0.03, respectively. These values are in general agreement with relative acute and chronic toxicity data in the literature. A re-evaluation of the estrogenicity of the biodegradation products of nonylphenol polyethoxylates in Canadian sewage treatment plant effluents was performed, using the relative estrogenicity determined in this study, and revealed that the contribution of alkylphenol polyethoxylates to effluent estrogenicity is significant but less than previously estimated. The ability of these chemicals, however, to act in concert with other estrogenic compounds such as 17β -estradiol, estrone, and 17α -ethinylestradiol, to provide a cumulative estrogenic exposure for the biota needs to be investigated.     

Identification of a denitrifying bacterium and verification of its anaerobic ammonium oxidation ability

A strain D3 of denitrifying bacterium was isolated from an anammox reactor,and identi-fied as Pseudomonas mendocina based on the morphological and physiological assay,Vitek test,Biolog test,(G C) mol% content,and 16S rDNA phylogenetic analysis.As a typical denitrifying bac-terium,strain D3 achieved the maximal nitrate reduction rate of 26.2 mg/(L·d) at the nitrate concen-tration of 88.5 mg N/L.The optimal pH and growth temperature were 7.84 and 34.9℃,respectively.Strain D3 was able to oxidize ammonia under anaerobic condition.The maximum nitrate and ammo-nium utilization rates were 6.37 mg/(L·d) and 3.34 mg/(L·d) ,respectively,and the consumption ratio of ammonia to nitrate was 1:1.91.Electron microscopic observation revealed peculiar cell inclusions in strain D3.Because of its relation to anammox activity,strain D3 was presumed to be anammoxosome.The present investigation proved that denitrifying bacteria have the anammox ability,and the results have engorged the range of anammox populations.     

Identification of a denitrifying bacterium and verification of its anaerobic ammonium oxidation ability

A strain D₃ of denitrifying bacterium was isolated from an anammox reactor, and identified as Pseudomonas mendocina based on the morphological and physiological assay, Vitek test, Biolog test, (G+C) mol% content, and 16S rDNA phylogenetic analysis. As a typical denitrifying bacterium, strain D₃ achieved the maximal nitrate reduction rate of 26.2 mg/(L·d) at the nitrate concentration of 88.5 mg N/L. The optimal pH and growth temperature were 7.84 and 34.9°C, respectively. Strain D₃ was able to oxidize ammonia under anaerobic condition. The maximum nitrate and ammonium utilization rates were 6.37 mg/(L·d) and 3.34 mg/(L·d), respectively, and the consumption ratio of ammonia to nitrate was 1:1.91. Electron microscopic observation revealed peculiar cell in clusions in strain D₃. Because of its relation to anammox activity, strain D₃ was presumed to be anammoxosome. The present investigation proved that denitrifying bacteria have the anammox ability, and the results have engorged the range of anammox populations.     

化学改性甘蔗渣对固定化细胞发酵产丁醇的影响

旨在研究化学改性的甘蔗渣作为固定化载体对丙酮丁醇梭菌Clostridium acetobutylicum XY16发酵制备生物丁醇的影响。首先利用不同浓度的聚乙烯亚胺(PEI)和1 g/L戊二醛(GA)对甘蔗渣表面进行化学改性,增强甘蔗渣对Clostridium acetobutylicum XY16的附载能力。经4 g/L聚乙烯亚胺和1 g/L戊二醛改性的甘蔗渣(添加量10 g/L)应用到固定化批次发酵中,发酵36 h后丁醇和总溶剂浓度最高,分别达到了12.24 g/L和21.67 g/L,同时溶剂的生产速率达到0.60 g/(L·h),生产速率比游离细胞和未改性甘蔗渣固定化细胞分批发酵分别提高了130.8%和66.7%。在此基础上对改性甘蔗渣固定化的细胞进行6次重复批次发酵,丁醇和总溶剂的产量稳定,溶剂生产速率逐渐提高至0.83 g/(L·h),同时转化率也提高至0.42 g/g。     

Production of succinic acid and lactic acid by Corynebacterium crenatum under anaerobic conditions

A new succinic acid and lactic acid production bioprocess by Corynebacterium crenatum was investigated in mineral medium under anaerobic conditions. Corynebacterium crenatum cells with sustained acid production ability and high acid volumetric productivity harvested from the glutamic acid fermentation broth were used to produce succinic acid and lactic acid. Compared with the first cycle, succinic acid production in the third cycle increased 120% and reached 43.4 g/L in 10 h during cell-recycling repeated fermentations. The volumetric productivities of succinic acid and lactic acid could maintain above 4.2 g/(L·h) and 3.1 g/(L·h), respectively, for at least 100 h. Moreover, wheat bran hydrolysates could be used for succinic acid and lactic acid production by the recycled C. crenatum cells. The final succinic acid concentration reached 43.6 g/L with a volumetric productivity of 4.36 g/(L·h); at the same time, 32 g/L lactic acid was produced.     

微生态制剂对海水养殖系统硝化功能建立过程的影响

比较分析投加不同微生态制剂的海水养殖系统硝化功能建立的过程,为实际应用提供依据。利用海水素构建4个海水养殖系统,通过投加硝化细菌、光合细菌、枯草芽胞杆菌3种微生态制剂以及纤维毛球作为生物膜载体,比较分析不同养殖系统硝化功能的建立过程及硝化强度差异。投加硝化细菌+光合细菌和硝化细菌+枯草芽胞杆菌系统硝化功能建立时间分别为108 h和96 h,氨氮初始质量浓度为6 mg/L时,氨氧化强度分别为1.69 mg/(L·d)和1.36 mg/(L·d);添加纤维毛球的生物膜系统与生物絮团系统硝化功能建立时间分别为96 h和120 h,氨氮初始质量浓度为6 mg/L时,氨氧化强度分别为1.36 mg/(L·d)和0.98 mg/(L·d);投加碳源系统和对照系统硝化功能建立时间分别为84 h和96 h,氨氮初始质量浓度为6 mg/L时,氨氧化强度分别为1.18 mg/(L·d)和1.36 mg/(L·d)。硝化细菌+枯草芽胞杆菌系统硝化功能建立时间更短,但系统硝化强度低于硝化细菌+光合细菌系统;生物膜系统硝化强度高于生物絮团系统且硝化功能建立更快;添加碳源能够加快系统硝化功能建立过程,但降低了硝化细菌+枯草芽胞杆菌系统的硝化强度。     

亚硝酸盐型同步厌氧生物脱氮除硫工艺的运行性能

采用上流式厌氧污泥床(UASB)反应器研究了亚硝酸盐型同步厌氧生物脱氮除硫工艺的性能。该工艺具有很高的硫化物和亚硝酸盐转化潜能,最大容积硫化物去除率和容积硝酸盐去除率分别为13.4kg/(m3·d)和2.3kg/(m3·d);所能耐受的最大进水硫化物和亚硝酸盐浓度分别为880mg/L和252.7mg/L;最适进水硫化物和亚硝酸盐浓度分别为460mg/L和132.3mg/L,最适水力停留时间为4h。硫化物和亚硝酸盐的表观半抑制浓度分别为403.9mg/L和120.8mg/L,两者之间的联合毒性为拮抗作用。     

马肝醇脱氢酶催化有机硅酮不对称还原反应动力学

探讨了马肝醇脱氢酶(HLADH)催化三甲基硅乙酮及其碳结构类似物不对称还原反应动力学.结果表明,在酶浓度低于150 mg/L时,底物浓度与反应初速度的关系符合米氏动力学方程;HLADH催化三甲基硅乙酮不对称还原反应的K_m、v_max和E_a分别为2.67 mmol/L、0.118 mmol/(L·min·mg)和37 kJ/mol, 其碳结构类似物的相应值分别为3.56 mmol/L、0.084 mmol/(L·min·mg)和61 kJ/mol.     

干酪乳杆菌L-乳酸脱氢酶突变体在毕赤酵母中的表达及其不对称还原苯丙酮酸

为提高L-苯乳酸(L-phenyllactic acid,L-PLA)的生产效率,以干酪乳杆菌Lactobacillus casei L-乳酸脱氢酶突变体L-Lc LDH1Q88A/I229A为研究对象,实现其在毕赤酵母Pichia pastoris GS115中的分泌表达,并与葡萄糖脱氢酶SyGDH偶联,构建并优化体外辅酶循环体系,不对称还原苯丙酮酸(Phenylpyruvate,PPA)制备L-PLA。结果显示,毕赤酵母重组酶re Lc LDH1Q88A/I229A的表观分子量为36.8 kDa,比活力为270.5 U/mg,是原酶的42.9倍。在40℃,初始pH为5.0,底物PPA、辅酶NAD+和葡萄糖浓度分别为100、2和120mmol/L,SyGDH和re Lc LDH1Q88A/I229A添加量分别为1和10U/mL的最优条件下,L-PLA的产率可达99.8%,对映体过量(ee)值99.9%,时空产率和平均转化率分别高达9.5 g/(L·h)和257.0 g/(g·h)。结果表明,re Lc LDH1Q88A/I229A在不对称还原PPA制备L-PLA中生产效率高,具有工业应用的潜力。     

过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性

木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。     

分阶段pH调控提高2-酮基-L-古龙酸生产

为了提高酮古龙酸菌Ketogulonicigenium vulgare和巨大芽胞杆菌Bacillus megaterium生产2-酮基-L-古龙酸(2-KLG)的生产效率,分析了pH对K.vulgare和B.megaterium生长和产酸的影响,发现K.vulgare和B.megaterium的最适生长pH值分别为6.0和8.0,但是K.vulgare的糖酸转化活力在pH7.0时达到最大值,因此提出了三阶段pH控制策略(第一阶段:0～8h,pH8.0;第二阶段:8～20h,pH6.0;第三阶段:20h至发酵结束,pH7.0)以促进K.vulgare生长和2-KLG生产。结果表明,三阶段pH控制策略的实施进一步提高了2-KLG的产量(77.3g/L)、生产强度(1.38g/(L·h))和L-山梨糖消耗速率(1.42g/(L·h)),分别比恒定pH7.0时提高了9.7%、33.2%和25.7%。     

Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor

Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, k_cat/K_M) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was attained by inactivated laccase.     

Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from <Emphasis Type="Italic">Trametes multicolor</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d⁺, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d⁺ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d⁺ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d⁺ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h^-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.     

Extracellular ligninolytic enzymes by Lentinus polychrous Lév. under solid-state fermentation of potential agro-industrial wastes and their effectiveness in decolorization of synthetic dyes

Six agro-industrial wastes were evaluated as a support for ligninolytic enzyme production by the white-rot fungus Lentinus polychrous Lév. under solid-state fermentation. Enzyme production was markedly different according to the substrate used. Rice bran (RB) yielded the highest laccase activity of 1,449 U/L (after 21 days of culture) with specific activity of 4.4 U/g substrate. Rice bran supplemented with rice husk (RH) (2:1 by wt) showed high laccase activity of 1,425 U/L with specific activity of 10.0 U/g substrate (after 17 days of culture). The crude enzyme of the RH-RB culture also contained manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities in relative proportions of 1.9:1.4:1 of laccase:MnP:MIP, respectively. Zymogram studies showed the same isoenzyme pattern with these ligninolytic enzymes. The high enzyme production level and low substrate cost of SSF-L. polychrous Lév. suggest that it has potential for industrial applications. Our studies showed that the crude enzyme from this culture exhibited in vitro decolorization of Indigo Carmine. The highest efficiency of dye decolorization was observed under alkaline conditions (pH 9.0) at an initial dye concentration of 10 mg/L. The rather high pH conditions and high efficiency in Indigo Carmine decolorization make the enzyme further interest for the applications in treatment of waste water from the textile industry, which contains synthetic dyes.