木薯发酵产丁醇的研究

对丙酮丁醇梭菌发酵木薯产溶剂进行研究,分别考察了N源、木薯含量、酶处理条件和培养基pH对发酵产丁醇的影响。结果表明：最佳的产丁醇发酵培养基为木薯粉120g／L,乙酸铵6g／L;木薯粉先用高温淀粉酶按酶量20U／g、90℃水解60min,再糊化30min;发酵初始pH为6．0,发酵96h。在此条件下,5L发酵罐中丁醇产量达到13．5g／L,总溶剂达到22．8g／L。     

乙、丁酸添加条件下丁醇发酵图论模型的构建

有机酸代谢途径在丁醇发酵过程中具有重要的作用,对细胞内碳流的分配和产物的合成影响显著。在7 L厌氧发酵罐中,进行了间歇添加乙酸或丁酸的发酵实验。结果表明,乙、丁酸的添加显著提高了总溶剂的生产效率,分别提高了47.1%和39.2%;此外,丁醇/丙酮比在添加丁酸的批次中提高了21.7%,在添加乙酸的批次中降低了16.2%;厌氧瓶中的发酵实验也证实了以上结果。有机酸代谢计算的结果表明,乙、丁酸的添加基本上阻断了相应有机酸闭环的吸收途径。基于相关报道和代谢计算结果,构建了针对乙、丁酸添加批次的图论模型,并利用该模型对不同发酵条件下的溶剂浓度和丁醇/丙酮比进行了计算。结果表明,该模型很好地预测了实验结果,合理地构建了乙、丁酸添加批次的信号传递线图。     

玉米黄浆水在丙酮-丁醇厌氧梭菌发酵中的应用

为降低丙酮-丁醇厌氧梭菌发酵生产丁醇的成本,研究了不同添加量玉米黄浆水对发酵的影响。与葡萄糖培养基相比,在发酵培养基中添加少量玉米黄浆水对发酵产量无显著影响。当添加体积分数为25％的玉米黄浆水时,丙酮、丁醇和乙醇的最终质量浓度分别是0．31、2．70和8．00g/L,总溶剂量为11．01g／L。通过成本核算,每生产1kg溶剂,添加体积分数25％的玉米黄浆水可比葡萄糖培养基节约成本2．11元。     

丙酮丁醇发酵菌的分子遗传改造

丙酮丁醇梭菌及拜氏梭菌是重要的ABE（丙酮、丁醇和乙醇）工业生产菌株,其发酵产物中的丙酮和丁醇均为重要的化工原料,汽车发动机试验证明丁醇还是一种性能优于乙醇的极具潜力的生物燃料和燃料添加剂。随着新生物技术的不断发展及工业生产的需求,遗传工程改造不断应用于丙酮丁醇生产菌株。在前人研究及工业实践的基础上,对丙酮丁醇生产菌株的遗传特性及其分子遗传改造取得的进展进行了详细概述。     

添加有机酸对Clostridium acetobutylicum合成丙酮和丁醇的影响

为提高丙酮-丁醇梭菌厌氧发酵生产丙酮和丁醇的能力,在发酵过程中添加有机酸（乙酸和丁酸）,考察其对菌体生长、溶剂合成影响。实验表明：当添加1.5 g/L乙酸时能够促进菌体的生长,促进丙酮的合成,在600 nm处的最大OD值比参照值高出18.4%,丙酮的最终质量分数提高了21.05%,但不能促进丁醇的合成;当添加1.0g/L丁酸时能够促进菌体生长,促进丁醇的合成,在600 nm处的最大OD比参照值高22.29%,丁醇的最终质量分数比对照组提高了24.32%,但不能促进丙酮的合成。     

果糖及葡萄糖混合物为底物的丙酮丁醇发酵

旨在以果糖和葡萄糖混合物模拟能源作物菊芋块茎水解液发酵生产丁醇。在培养基初始pH 5.5,发酵过程不控制pH的混合糖发酵中,出现了发酵提前终止现象,终点残糖浓度达23.26 g/L,而丁醇产量仅5.51 g/L。进一步对比混合糖及葡萄糖、果糖不控制pH的发酵结果表明,导致这一现象的原因可能是有机酸毒性太大和pH太低。全程控制pH的混合糖发酵结果表明,高pH条件有利于提高糖利用率,但产酸多,丁醇产量较低;而低pH条件下发酵残糖较多,但丁醇产量相对较高。基于此,文中采用阶段性pH调控策略,即将发酵初期的pH控     

限制葡萄糖、葡萄糖/乙酸双底物条件下自由控制丙丁梭菌ABE发酵丙酮浓度和丙酮/丁醇比

提出一种可以提高和自由控制丙丁梭菌ABE发酵丙酮浓度与丙酮/丁醇比的方法。（1）通过控制糖化酶用量、反应时间和温度调节玉米培养基初始葡萄糖浓度,使发酵进入到产溶剂期后,残留葡萄糖浓度降至接近于0 g/L的水平;（2）在葡萄糖受限的条件下,诱导丙丁梭菌合成分泌糖化酶,分解寡糖,将葡萄糖维持于低浓度,进而限制梭菌胞内糖酵解途径的碳代谢和NADH生成速度。与此同时,外添乙酸形成葡萄糖/乙酸双底物环境。在能量代谢基本不受破坏、丁醇未达到抑制浓度的条件下,适度抑制丁醇生产,有效地利用外添乙酸强化丙酮合成;（3）在外添乙酸的基础上,添加适量酿酒酵母,形成丙丁梭菌/酿酒酵母混合发酵体系,提高梭菌对高丁醇浓度的耐受能力。整个发酵体系可以将丙酮浓度和丙酮/丁醇比自由控制在5~12 g/L和0.5~1.0的水平,最大丙酮浓度和丙酮/丁醇比达到11.74 g/L和1.02,并可维持丁醇浓度于10~14 g/L的正常水平,充分满足工业ABE发酵对于丙酮和丁醇产品的不同需求。     

酵母浸粉刺激以木薯为原料的丁醇生产的发酵相转型

在7L静态厌氧发酵罐下,使用\"非粮\"作物木薯替代玉米淀粉开展丁醇发酵。无论是传统发酵还是油醇萃取发酵,木薯粉丁醇发酵的性能均远不及以玉米淀粉为原料时的水平,主要体现在发酵产酸相向溶剂生产相的转型严重迟延或无法转型、发酵时间长、丁醇生产效率低。实验结果表明,当发酵相转型迟延出现后,添加2.5g/L的酵母浸粉,可以刺激丁酸/乙酸向丁醇/丙酮的转化、转型延迟时间缩短10~30h左右。在此条件下,传统和萃取发酵方式下的丁醇总产量分别达到12.95g/L和29.81g/L,丁醇生产效率与使用玉米淀粉为原料时基本持平。     

丙酮丁醇梭菌半胱氨酸合成途径中铁氧还蛋白和胱硫醚-γ-裂解酶基因功能

【目的】探究丙酮丁醇梭菌半胱氨酸合成代谢途径上铁氧还蛋白和胱硫醚-γ-裂解酶基因的功能。【方法】使用ClosTron系统对半胱氨酸合成途径上的铁氧还蛋白基因(fer)和胱硫醚-γ-裂解酶基因(mccB)进行失活,得到突变株;在不同硫源的培养基中进行分批发酵,分析突变株的生长特点;通过pH控制,使用限磷的连续发酵方法将丙酮丁醇梭菌维持在产酸期和产溶剂期,分析野生型菌株和突变株在连续发酵中的生长情况。【结果】成功构建Δfer和ΔmccB突变株。在分批发酵中,敲除fer基因的突变株无法利用硫酸盐作为硫源,但添加亚硫酸盐或半胱氨酸可以使其恢复生长;在以半胱氨酸为唯一硫源进行分批发酵时,其终浓度1 mmol/L时不会影响野生型与Δfer突变株的生长,但高于1 mmol/L时生长均会受到抑制。在连续发酵中,Δfer突变株不能在产溶剂阶段生长,添加过量的半胱氨酸也不能恢复生长;敲除mccB基因的突变株仍能在添加甲硫氨酸的培养基中生长,但最大OD仅为野生型的57%;相较于野生型,ΔmccB突变株在产酸期和产溶剂期的生长均受到抑制。【结论】fer基因为半胱氨酸合成途径中硫酸盐还原为亚硫酸盐的关键基因,其控制合成的半胱氨酸不能完全由外源的半胱氨酸替代,敲除后对生长的抑制主要表现在连续发酵中的产溶剂阶段。mccB基因参与调控甲硫氨酸转化为半胱氨酸的过程,其敲除会影响甲硫氨酸到半胱氨酸的转化,但不会阻断该生物反应过程。     

利用以废弃毕赤酵母为氮源的丁酸发酵上清液和葡萄糖高效生产具有高丁醇/丙酮比特征的生物丁醇

【背景】生物丁醇是高效的液态燃料。丁醇发酵,也称丙酮-丁醇-乙醇(Acetone-Butanol-Ethanol,ABE)发酵,其发酵的产品是丁醇、丙酮和乙醇的混合物,主产物丁醇与主要副产物丙酮的质量比率(B/A比)约为2.0。【目的】丁酸是ABE发酵合成丁醇的重要前体物质,以丁酸/葡萄糖为双底物可以高效生产具有以高B/A比为特征的丁醇,提高ABE发酵产品的品质。【方法】7L厌氧发酵罐下,以玉米淀粉/废弃毕赤酵母处理液为原料得到的丁酸发酵上清液与葡萄糖溶液直接复配作为ABE发酵培养基,并按需要在发酵途中添加该上清液和浓缩葡萄糖溶液。【结果】与使用150 g/L玉米淀粉的传统ABE发酵相比,丁醇浓度保持在12.7-12.8 g/L的较高水平,B/A比从2.0大幅提高到4.4-5.0,丁醇对总碳源的得率从0.32-0.34提升至0.39-0.41 (摩尔基准),丁酸/葡萄糖质量消耗比高达37%-53%。【结论】ABE发酵性能的改善得益于丁酸发酵上清液中丁酸、寡糖和氨基酸等得到了有效利用,NADH利用效率大幅提高。该发酵策略节省了ABE发酵的原料和操作成本,大幅降低了丁酸发酵上清液中残存的寡糖浓度,还可根据市场供需和产品价格变化的状况实现发酵产品的多样性和生产操作的灵活性,具有良好的经济和环保意义。     

Evaluation of high butanol/acetone ratios in ABE fermentations with cassava by graph theory and NADH regeneration analysis

Higher butanol/acetone ratio is always desirable in ABE fermentation, and this ratio is closely associated with the complicated patterns of metabolic reactions and NADH generation rate. The patterns of acetate/butyrate formation and re-assimilation in multiple closed reaction loops, as well as NADH regeneration in ABE fermentation using different substrates varies. In this study, we evaluated butanol/acetone ratio in ABE fermentations utilizing cassava and corn based media by graph theory and NADH regeneration analysis. The theoretical calculations and experimental data revealed that a lower metabolic strength in butyrate loop and enhanced NADH generation rate were responsible for the achievement of higher butanol/acetone ratio when fermenting cassava based substrate. In traditional fermentations and extractive fermentations with oleyl alcohol/bio-diesel as the extractants when using cassava based substrate, butanol/acetone ratios reached 2.24, 2.84, and 2.19 with the increasing increments of 14.9, 61.4, and 6.8% respectively, while butanol productivities stayed at comparably high levels as compared with those of the fermentations when cultivating on corn based substrate.     

On adaptation: A reduction of the Kauffman-Levin model to a problem in graph theory and its consequences

It is shown that complex adaptations are best modelled as discrete processes represented on directed weighted graphs. Such a representation captures the idea that problems of adaptation in evolutionary biology are problems in a discrete space, something that the conventional representations using continuous adaptive landscapes does not. Further, this representation allows the utilization of well-known algorithms for the computation of several biologically interesting results such as the accessibility of one allele from another by a specified number of point mutations, the accessibility of alleles at a local maximum of fitness, the accessibility of the allele with the globally maximum fitness, etc. A reduction of a model due to Kauffman and Levin to such a representation is explicitly carried out and it is shown how this reduction clarifies the biological questions that are of interest.Thanks are due to William Wimsatt, James F. Crow, and the referees for Biology and Philosophy for comments on an earlier version of this paper. Remarks by members of the audience, especially Abner Shimony, of a seminar at Boston University, February 19, 1988, were also very helpful. The diagrams were prepared with the assistance of Tracy Lubas.     

Measurement of body size and abundance in tests of macroecological and food web theory

1. Mean body mass (W) and mean numerical (N) or biomass (B) abundance are frequently used as variables to describe populations and species in macroecological and food web studies. 2. We investigate how the use of mean W and mean N or B, rather than other measures of W and/or accounting for the properties of all individuals, can affect the outcome of tests of macroecological and food web theory. 3. Theoretical and empirical analyses demonstrate that mean W, W at maximum biomass (W(mb)), W when energy requirements are greatest (W(me)) and the W when a species uses the greatest proportion of the energy available to all species in a W class (W(mpe)) are not consistently related. 4. For a population at equilibrium, relationships between mean W and W(me) depend on the slope b of the relationship between trophic level and W. For marine fishes, data show that b varies widely among species and thus mean W is an unreliable indicator of the role of a species in the food web. 5. Two different approaches, 'cross-species' and 'all individuals' have been used to estimate slopes of abundance-body mass relationships and to test the energetic equivalence hypothesis and related theory. The approaches, based on relationships between (1) log(10) mean W and log(10) mean N or B, and (2) log(10) W and log(10) N or B of all individuals binned into log(10) W classes (size spectra), give different slopes and confidence intervals with the same data. 6. Our results show that the 'all individuals' approach has the potential to provide more powerful tests of the energetic equivalence hypothesis and role of energy availability in determining slopes, but new theory and empirical analysis are needed to explain distributions of species relative abundance at W. 7. Biases introduced when working with mean W in macroecological and food web studies are greatest when species have indeterminate growth, when relationships between W and trophic level are strong and when the range of species'W is narrow.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.