微生物耐受乙醇与丁醇机制及其在生物燃料生产与生物转化中的应用

生物法获取乙醇与丁醇过程中有机溶剂的毒性是生产菌重要环境胁迫因素之一,且当有机溶剂超过一定浓度时便会抑制微生物的生长,甚至引起微生物的死亡,因此提高工业微生物的有机溶剂耐受性对工业生产具有重要的意义。对微生物乙醇及丁醇耐受机制的研究可为选育具有较强溶剂耐受菌提供理论基础。本文系统介绍了微生物耐受乙醇与丁醇的机制,并对其在生物燃料生产及生物转化中面临的机遇与挑战等问题进行简要的评述。     

耐有机溶剂极端微生物的耐受机制及应用

耐有机溶剂微生物是一类新颖的极端微生物,直到20世纪80年代才被系统地研究.它们通过各种耐受机制,有效抵御或降低有机溶剂对其细胞产生的毒害作用.因此,在全细胞催化、环境污染治理等领域,耐有机溶剂极端微生物具有广阔的工业应用前景.此外,深入透彻地了解耐有机溶剂极端微生物的各种耐受机制,有助于利用基因工程技术改造和优化现有耐有机溶剂极端微生物的各种性能,进一步拓展其工业应用领域.本文将从囊泡外排、改变细胞膜磷脂结构和组成等4个方面概述近年来耐有机溶剂极端微生物的耐受机制研究新进展,并介绍它们在全细胞催化等领域的应用.     

有机溶剂耐受菌的研究进展

有机溶剂耐受菌是一类新的能与有害影响因素竞争并能在有机溶剂中茁壮生长的微生物。介绍了有机溶剂对细菌的毒性机理,有机溶剂耐受菌的获得方法,讨论了有机溶剂耐受菌的两种反应形式,并对有机溶剂耐受菌的应用进行了展望。     

中链脂肪酸毒性机制及耐受性菌株构建研究进展

中链脂肪酸(medium-chain fatty acids, MCFAs)作为一种重要的平台化学品,被广泛应用到能源、食品和医药等行业。工业微生物发酵生产MCFAs是一条绿色环保的路线,但MCFAs会对微生物细胞膜造成损伤,并导致细胞pH、渗透压失衡及氧化应激,从而严重抑制细胞的生长速率和生产能力。因此,构建MCFAs耐受性工业微生物菌株,有助于进一步提高MCFAs的生产效率。以大肠杆菌和酿酒酵母等工业微生物为例,首先简介了MCFAs对微生物细胞的毒性机制。其次综述了运用膜改造、转运体筛选等理性代谢工程手段构建MCFAs耐受性菌株的相关研究进展,并概括了运用适应性进化、代谢通量分析等方法系统性挖掘MCFAs耐受靶点进而增强菌株耐受性的研究进展。最后对后续提高工业微生物MCFAs耐受性和生产能力的研究方向进行了展望。     

耐有机溶剂微生物的耐受机制、改造策略和应用北大核心

耐有机溶剂微生物在生物燃料生产、全细胞催化、酶制剂的开发和生物修复等领域具有非常重要的作用。该文简要介绍了耐有机溶剂微生物及其耐受机制,并从基因过表达、基因敲除和全局转录机制三个方面总结了增强微生物有机溶剂耐受性的方法。通过对耐有机溶剂微生物应用的局限性进行分析,认为未来研究中应注重将微生物耐受表型和具体利用相结合,达到有机溶剂耐受和实际产出的最佳平衡状态,为耐有机溶剂工程菌的改造和选育提供参考。     

耐有机溶剂微生物的耐受机制、改造策略和应用

耐有机溶剂微生物在生物燃料生产、全细胞催化、酶制剂的开发和生物修复等领域具有非常重要的作用。该文简要介绍了耐有机溶剂微生物及其耐受机制,并从基因过表达、基因敲除和全局转录机制三个方面总结了增强微生物有机溶剂耐受性的方法。通过对耐有机溶剂微生物应用的局限性进行分析,认为未来研究中应注重将微生物耐受表型和具体利用相结合,达到有机溶剂耐受和实际产出的最佳平衡状态,为耐有机溶剂工程菌的改造和选育提供参考。     

利用分子生物学技术提高微生物有机溶剂耐受性的研究进展

耐有机溶剂微生物是一类能够在较高浓度有机溶剂中存活或者生长的微生物,其在非水相生物催化等领域表现出巨大的应用优势。与自然筛选、长期驯化和传统诱变等方法相比,利用分子生物学技术获得微生物有机溶剂耐受菌株是一种更为理性且高效的手段。主要综述了近年来利用分子生物学技术对耐性相关功能基因和转录因子进行改造,提高微生物有机溶剂耐受性的研究进展,并展望了有机溶剂耐受菌株的应用前景。     

利用代谢工程技术提高工业微生物对胁迫的抗性

代谢工程是工业微生物菌种改造的平台技术,不仅可用于改变微生物细胞内的代谢流向,也可以用于改善工业微生物的生理功能。在工业生产过程中,微生物细胞会面临多种胁迫作用,这些胁迫诱导的基因调节作用,都有可能影响细胞的许多重要生理功能,从而影响生物转化过程的效率。从工业应用的观点出发,选择生产性能良好、对发酵过程中的主要胁迫因素有较强耐受性的菌株至关重要。以下评述了借鉴传统代谢工程技术和反向代谢工程技术来提高工业微生物对胁迫抗性的若干研究策略,提出了该领域目前存在的问题,以及利用代谢工程技术改善微生物胁迫抗性——即微生物生理功能工程的发展方向。     

耐受有机溶剂洋葱伯克霍尔德菌ZYB002全细胞脂肪酶酶学性质

一株对多种有机溶剂具有良好耐受能力的产脂肪酶菌株ZYB002经分子鉴定为洋葱伯克霍尔德菌。其产生的细胞结合脂肪酶最适温度为65°C,最适pH为8.0,在低于70°C和pH3-8.5的范围内,全细胞脂肪酶保持稳定。Ca2+、K+、Na+和NO3-等离子对脂肪酶活性有激活作用,而Zn2+有抑制效应。全细胞脂肪酶对正丁醇有较强的耐受能力,但曲拉通X-100对脂肪酶活性有强烈的抑制效应。洋葱伯克霍尔德菌ZYB002全细胞脂肪酶良好的碱稳定性、热稳定性和有机溶剂耐受性,表明该全细胞脂肪酶具有重要的工业应用潜力。     

裂褶菌cfcc7252菌株对孔雀石绿染料的高效降解

【目的】评价裂褶菌cfcc7252菌株降解孔雀石绿(Malachite Green,MG)的能力及其潜在的应用价值。【方法】采用单因子液体培养实验,研究了通气、p H、温度、碳源和氮源种类及浓度、金属离子、盐度、染料浓度对该菌降解效果的影响;采用平皿培养实验,利用植物种子萌发和微生物抑菌实验对降解产物进行毒性测试。【结果】研究结果表明,裂褶菌cfcc7252菌株在好氧和厌氧条件下均能高效降解MG。该菌在10.0 g/L葡萄糖,5.0 g/L酵母浸粉,0.01 mmol/L Zn2+,p H为4.0的液体培养基中培养36 h,对350 mg/L的MG降解率达67.8%;连续降解7次后,其降解率还能保持在95.4%以上。此外,该菌在盐度低于10.20%时,其对MG的降解率均达到98%以上。对植物、微生物的毒性测试结果表明,MG降解产物对红豆、豌豆等植物、金黄色葡萄球菌、枯草芽孢杆菌和铜绿假单胞杆菌等微生物基本没有毒性。【结论】裂褶菌cfcc7252菌株在处理以MG为主的染料废水时具有很强的应用潜力。     

Isolation of new toluene-tolerant marine strains of bacteria and characterization of their solvent-tolerance properties

Aims:  To isolate and characterize new marine bacteria capable of tolerating high concentrations of organic solvents, and to understand the toxic effects of these chemicals on marine bacteria. Methods and Results:  Five marine bacteria able to tolerate 0·1% (v/v) toluene were isolated and characterized on the basis of their growth and survival rates in the presence of different organic solvents. The toluene-tolerant marine bacteria identified in this study could not grow in the presence of 0·1% (v/v) of several organic solvents with a log P_ow higher than that of the toluene (which in theory should be less toxic than toluene). The mechanisms underlying solvent tolerance were explored. Conclusions:  Isolates of four different genera were identified as toluene-tolerant. Toxicity of a second phase of an organic solvent toward these isolates could not be predicted on the basis of the solvents’ log P_ow. Significance and Impact of the Study:  To improve the biodegradation rate of some water-insoluble compounds, double-phase bioreactors can be used. This type of bioreactor will require strains able to grow in a salt-containing environment and able to tolerate a second phase of an organic solvent.     

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.     

Solvent-tolerant bacteria in biocatalysis

The toxicity of fine chemicals to the producer organism is a problem in several biotechnological production processes. In several instances, an organic phase can be used to extract the toxic product from the aqueous phase during a fermentation. With the discovery of solvent-tolerant bacteria, more solvents can now be used in such two-liquid water–solvent systems. We are gaining new insights into the mechanisms of bacterial solvent tolerance, such as the active efflux of solvents from the cytoplasmic membrane and solvent-impermeable outer membranes.     

Cellular response mechanisms in Pseudomonas aeruginosa PseA during growth in organic solvents

Aims:  Solvent-tolerant bacteria have emerged as a new class of micro-organisms able to grow at high concentrations of toxic solvents. Such bacteria and their solvent-stable enzymes are perceived to be useful for biotransformations in nonaqueous media. In the present study, the solvent-responsive features of a lipase–producing, solvent-tolerant strain Pseudomonas aeruginosa PseA have been investigated to understand the cellular mechanisms followed under solvent-rich conditions.
Methods and Results:  The solvents, cyclohexane and tetradecane with differing log P -values (3·2 and 7·6 respectively), have been used as model systems. Effect of solvents on (i) the cell morphology and structure (ii) surface hydrophobicity and (iii) permeability of cell membrane have been examined using transmission electron microscopy, atomic force microscopy and other biochemical techniques. The results show that (i) less hydrophobic (low log P -value) solvent cyclohexane alters the cell membrane integrity and (ii) cells adapt to organic solvents by changing morphology, size, permeability and surface hydrophobicity. However, no such changes were observed in the cells grown in tetradecane.
Conclusions:  It may be concluded that P. aeruginosa PseA responds differently to solvents of different hydrophobicities. Bacterial cell membrane is more permeable to less hydrophobic solvents that eventually accumulate in the cytoplasm, while highly hydrophobic solvents have lesser tendency to access the membrane.
Significance and Impact of the Study:  To the best of our knowledge, these are first time observations that show that way of bacterial solvent adaptability depends on nature of solvent. Difference in cellular responses towards solvents of varying log P -values (hydrophobicity) might prove useful to search for a suitable solvent for carrying out whole-cell biocatalysis.     

Industrial potential of organic solvent tolerant bacteria

Most bacteria and their enzymes are destroyed or inactivated in the presence of organic solvents. Organic solvent tolerant bacteria are a relatively novel group of extremophilic microorganisms that combat these destructive effects and thrive in the presence of high concentrations of organic solvents as a result of various adaptations. These bacteria are being explored for their potential in industrial and environmental biotechnology, since their enzymes retain activity in the presence of toxic solvents. This property could be exploited to carry out bioremediation and biocatalysis in the presence of an organic phase. Because a large number of substrates used in industrial chemistry, such as steroids, are water-insoluble, their bioconversion rates are affected by poor dissolution in water. This problem can be overcome by carrying out the process in a biphasic organic-aqueous fermentation system, wherein the substrate is dissolved in the organic phase and provided to cells present in the aqueous phase. In bioprocessing of fine chemicals such as cis-diols and epoxides using such cultures, organic solvents can be used to extract a toxic product from the aqueous phase, thereby improving the efficiency of the process. Bacterial strains reported to grow on and utilize saturated concentrations of organic solvents such as toluene can revolutionize the removal of such pollutants. It is now known that enzymes display striking new properties in the presence of organic solvents. The role of solvent-stable enzymes in nonaqueous biocatalysis needs to be explored and could result in novel applications.     

Organic solvent adaptation of Gram positive bacteria: applications and biotechnological potentials

Organic-solvent-tolerant bacteria are considered extremophiles with different tolerance levels that change among species and strains, but also depend on the inherent toxicity of the solvent. Extensive studies to understand the mechanisms of organic solvent tolerance have been done in Gram-negative bacteria. On the contrary, the information on the solvent tolerance mechanisms in Gram-positive bacteria remains scarce. Possible shared mechanisms among Gram-(−) and Gram-(+) microorganisms include: energy-dependent active efflux pumps that export toxic organic solvents to the external medium; cis-to-trans isomerization of unsaturated membrane fatty acids and modifications in the membrane phospholipid headgroups; formation of vesicles loaded with toxic compounds; and changes in the biosynthesis rate of phospholipids to accelerate repair processes. However, additional physiological responses of Gram-(+) bacteria to organic solvents seem to be specific. The aim of the present work is to review the state of the art of responsible mechanisms for organic solvent tolerance in Gram-positive bacteria, and their industrial and environmental biotechnology potential.     

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium

Aims: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. Methods and Results: A solvent‐tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram‐positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p‐xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p‐xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron‐transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. Conclusions: The thermophilic, Gram‐positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. Significance and Impact of the Study: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.     

Growth of Gram-negative bacteria in the presence of organic solvents

The growth behavior of Gram-negative bacteria when exposed to high concentrations (50% v/v) of water-insoluble organic solvents was investigated. The solvents were chosen according to their polarity values as denoted by a logarithmically expressed parameter log P, where P is the partition coefficient of a given solvent in an equimolar mixture of octanol and water. The cell growth was measured by the number of colonies developed on a solid agar medium in direct contact with the solvents. All 31 strains tested showed characteristic growth patterns. The survival and subsequent growth of bacteria increased with the increase in the log P value and was found to be strain specific. For all the strains, 100% cell growth was reached from 0% within 0.1–0.4 log P units. Log P₅₀ values, defined as the log P values at which 50% of the cells form colonies, were determined for each bacterial strain. On the whole, Pseudomonas strains were found to be more resistant to apolar solvents than all other bacteria tested. This resistance was dependent not only on the polarities but also on the toxic nature of different organic solvents, the cell membrane components, and to a limited extent, the growth medium. A tenfold increase in the Mg²⁺ concentration in the growth medium enhanced the solvent resistance of E. coli but had no such effect on Pseudomonads. In general, different growth temperatures had no impact on the solvent resistance of the Gram-negative bacteria tested.