利用甲硝唑及外加氧方法筛选耐氧产氢Klebsiella oxytoca HP1突变菌株

氢酶是生物制氢的关键酶, 大多数氢酶因对氧极敏感而易失活, 因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1%甲基磺酸乙酯对Klebsiella oxytoca HP1进行了两轮诱变, 经40 mmol/L 甲硝唑和21%氧联合处理1 h(第一轮诱变)或2 h(第二轮诱变)进行筛选。所得突变菌株经产氢测试, 结果在15%氧浓度条件下, 第一代突变菌株HP1-A15产氢活性为出发菌株Klebsiella oxytoca HP1的3.70倍, 在21%氧浓度条件下第二代突变菌株 HPA15-37产氢活性为HP1-A15菌株的2.75倍, 是出发菌株的11倍。突变菌株HP1-A15和 HPA15-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。     

利用甲硝唑及外加氧方法筛选耐氧产氢Klebsiella oxytoca HP1突变菌株

摘氢酶是生物制氢的关键酶,大多数氢酶因对氧极敏感而易失活,因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1％甲基磺酸乙酯对Klebsiella oxytoca HPl进行了两轮诱变,经40mmol／L甲硝唑和21％氧联合处理1h（第一轮诱变）或2h（第二轮诱变）进行筛选。所得突变菌株经产氢测试,结果在15％氧浓度条件下,第一代突变菌株HPl-A15产氢活性为出发菌株Klebsiella oxytoca HPl的3.70倍,在21％氧浓度条件下第二代突变菌株HPAl5-37产氢活性为HPl-A15菌株的2.75倍,是出发菌株的11倍。突变菌株HPl-A15和HPAl5-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。     

应用合成生物学策略改良光合蓝细菌

蓝细菌是一类能够直接利用光能和CO_2作为唯一能源和碳源进行生长的光合微生物。近年来,光合蓝细菌以其独特的优势作为"自养型细胞工厂"合成了多种燃料及化学品。以光合蓝细菌中的几种模式生物为例,总结近年来以蓝细菌为工程菌株合成生物燃料及化学品的研究进展,对目前蓝细菌菌株存在的固有问题进行分析,并提出应用合成生物学进行菌种改良的方案。     

固定化光合细菌产氢过程的基质利用动力学

研究了固定化荚膜红假单胞菌386、红假单胞菌D两菌株产氢过程基质利用的动力学特性。基质利用与产氢过程以不同的速率进行．琼脂包埋固定化细胞的产氢能力高于海藻酸钙固定化细胞,但最大产氢活性的进程迟于后者。固定化D菌株利用葡萄糖的动力学遵循一级反应,其反应常数K值为1．2×10　2h-1。宏观动力学分析表明,利用基质的产氢过程属于反应控制,扩散传质过程不构成控速步骤。386和D两个菌株固定化细胞生物反应器连续产氢系统,在以乳酸作基质时．平均产氢量分别可达到0．659L／d和0．457L／d,容积(液相)产氢率接近1．OL／L·d。     

生物产氢研究进展

氢能是一种清洁高效的能源。氢气可以利用工农业废料通过微生物发酵制取 ,是一种可再生燃料。文中介绍了厌氧菌、兼性厌氧菌、好氧菌、光合细菌和蓝细菌等产氢的微生物种类 ,以及它们的产氢机理。从光合细菌利用废料产氢的效率和产氢设备的研究来看 ,无疑具有很大的潜力。以产氢技术作为下一代能源开发创新的技术已引起国际社会的重视 ,具有广阔的市场前景。     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16SrDNA序列分析,初步鉴定菌株Z-16为Enterobactersp.,菌株C-32为Clostridiumsp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为:反应系统起始pH7·0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2·68molH2/mol蔗糖。菌株C-32的最适产氢条件为:反应系统起始pH8·0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2·71molH2/mol麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2·35和2·48molH2/mol葡萄糖。     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatumZ7产氢效率的因素,采用生理学方法对其进行了研究。结果表明:乙醇型发酵菌A.elongatumZ7的最适产氢温度为37℃,最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度>60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16S rDNA序列分析,初步鉴定菌株Z-16为Enterobacter sp.,菌株C-32为Clostridium sp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为：反应系统起始pH7.0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2.68mol H₂/mol蔗糖。菌株C-32的最适产氢条件为：反应系统起始pH 8.0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2.71mol H₂/mol 麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2.35和2.48mol H₂/mol葡萄糖。     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16S rDNA序列分析,初步鉴定菌株Z-16为Enterobacter sp.,菌株C-32为Clostridium sp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为：反应系统起始pH7.0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2.68mol H₂/mol蔗糖。菌株C-32的最适产氢条件为：反应系统起始pH 8.0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2.71mol H₂/mol 麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2.35和2.48mol H₂/mol葡萄糖。     

光合细菌Rhodopseudomonas产氢的影响因子实验研究

利用光合细菌Rhodopseudomonas 8株菌株研究初期活性、光照强度、C源种类、菌株差异对生物产氢的影响表明 ,不同菌株的C源利用性有较大差异 ,但对乳酸钠都有很好的利用性 .细菌的初期活性对产氢有一定程度的影响 ,稳定生长期的细菌比对数生长期的细菌产氢活性略高 .光照强度对产氢活性的影响明显 ,在光饱和度以下 ,光照强度大则产氢速率高 .不同菌种的产氢性能有效大差异 ,从上海地区有机污染环境中分离到的RhodopseudomonasB2 1 菌株在以乳酸钠 ( 50mmol·L- 1 )为C源、谷氨酸钠 ( 1 0mol·L- 1 )为N源 ,60 0 0Lx光照、30℃下 ,最大产氢速率达到 1 4.7ml·h- 1 ·g- 1 细胞干重 .     

Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production.

Escherichia coli can perform at least two modes of anaerobic hydrogen metabolism and expresses at least two types of hydrogenase activity. Respiratory hydrogen oxidation is catalysed by two 'uptake' hydrogenase isoenzymes, hydrogenase -1 and -2 (Hyd-1 and -2), and fermentative hydrogen production is catalysed by Hyd-3. Harnessing and enhancing the metabolic capability of E. coli to perform anaerobic mixed-acid fermentation is therefore an attractive approach for bio-hydrogen production from sugars. In this work, the effects of genetic modification of the genes encoding the uptake hydrogenases, as well as the importance of preculture conditions, on hydrogen production and fermentation balance were examined. In suspensions of resting cells pregrown aerobically with formate, deletions in Hyd-3 abolished hydrogen production, whereas the deletion of both uptake hydrogenases improved hydrogen production by 37% over the parent strain. Under fermentative conditions, respiratory H2 uptake activity was absent in strains lacking Hyd-2. The effect of a deletion in hycA on H2 production was found to be dependent upon environmental conditions, but H2 uptake was not significantly affected by this mutation.     

暗发酵-光发酵两阶段联合生物制氢技术研究进展

随着能源紧缺的日益加剧,以及化石燃料燃烧引起的环境问题逐渐突显,氢能作为一种清洁可再生能源越来越受到青睐。生物制氢与热化学及电化学制氢相比其反应条件温和、低耗、绿色,是一项非常有应用前景的技术。生物制氢从广义上可以分为暗发酵和光发酵产氢两种,其中暗发酵微生物可以利用有机废弃物产生氢气以及有机酸等副产物,光合细菌在光照和固氮酶的作用下可以将暗发酵产生的有机酸继续用于产氢,因此两种发酵产氢方式相结合可以提高有机废物的资源化效率。将近年来暗发酵-光发酵两阶段生物制氢技术进行整理分析,从其产氢机理、主要影响因素、暗发酵-光发酵产氢结合方式(两步法、混合培养产氢)几个方面进行阐述,最后指出该技术面临的挑战。     

Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species

Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the `oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing.     

Nanoparticle and nanomineral production by fungi

Fungi show a variety of abilities in affecting metal speciation, toxicity, and mobility and mineral formation, dissolution or deterioration through several interacting biomechanical and biochemical mechanisms. A consequence of many metal-mineral interactions is the production of nanoparticles which may be in elemental, mineral or compound forms. Organisms may benefit from such nanomaterial formation through removal of metal toxicity, protection from environmental stress, and their redox properties since certain mycogenic nanoparticles can act as nanozymes mimicking enzymes such as peroxidase. With the development of nanotechnology, there is growing interest in the application of biological systems for nanomaterial production which may provide economic benefits and a lower damaging environmental effect than conventional chemical synthesis. Fungi offer some advantages since most are easily cultured under controlled conditions and well known for the secretion of metabolites and enzymes related to nanoparticle or nanomineral formation. Nanoparticles can be formed intracellularly or extracellularly, the latter being favourable for easy harvest, while the cell wall also provides abundant nucleation sites for their formation. In this article, we focus on the synthesis of nanoparticles and nanominerals by fungi, emphasizing the mechanisms involved, and highlight some possible applications of fungal nanomaterials in environmental biotechnology.     

Working conditions in hydrogen production: A social life cycle assessment

Social impacts of novel technology can, parallel to environmental and economic consequences, influence its sustainability. By analyzing the case of hydrogen production by advanced alkaline water electrolysis (AEL) from a life cycle perspective, this paper illustrates the social implications of the manufacturing of the electrolyzer and hydrogen production when installed in Germany, Austria, and Spain. This paper complements previous environmental and economic assessments, which selected this set of countries based on their different structures in electricity production. The paper uses a mixed method design to analyze the social impact for the workers along the process chain. Appropriate indicators related to working conditions are selected on the basis of the UN Agenda 2030 Sustainable Development Goals. The focus on workers is chosen as a first example to test the relatively new Product Social Impact Life Cycle Assessment (PSILCA) database version 2.0. The results of the quantitative assessment are then complemented and compared through an investigation of the underlying raw data and a qualitative literature analysis. Overall, advanced AEL is found to have least social impact along the German process chain, followed by the Spanish and the Austrian. All three process chains show impacts on global upstream processes. In order to reduce social impact and ultimately contribute to Sustainable Development, policymakers and industry need to work together to further improve certain aspects of working conditions in different locations, particularly within global upstream processes.     

Formation of Streptococcus pneumoniae non-phase-variable colony variants is due to increased mutation frequency present under biofilm growth conditions

In this report, we show that biofilm formation by Streptococcus pneumoniae serotype 19 gives rise to variants (the small mucoid variant [SMV] and the acapsular small-colony variant [SCV]) differing in capsule production, attachment, and biofilm formation compared to wild-type strains. All biofilm-derived variants harbored SNPs in cps19F. SCVs reverted to SMV, but no reversion to the wild-type phenotype was noted, indicating that these variants were distinct from opaque- and transparent-phase variants. The SCV-SMV reversion frequency was dependent on growth conditions and treatment with tetracycline. Increased reversion rates were coincident with antibiotic treatment, implicating oxidative stress as a trigger for the SCV-SMV switch. We, therefore, evaluated the role played by hydrogen peroxide, the oxidizing chemical, in the reversion and emergence of variants. Biofilms of S. pneumoniae TIGR4-ΔspxB, defective in hydrogen peroxide production, showed a significant reduction in variant formation. Similarly, supplementing the medium with catalase or sodium thiosulfate yielded a significant reduction in variants formed by wild-type biofilms. Resistance to rifampin, an indicator for mutation frequency, was found to increase approximately 55-fold in biofilms compared to planktonic cells for each of the three wild-type strains examined. In contrast, TIGR4-ΔspxB grown as a biofilm showed no increase in rifampin resistance compared to the same cells grown planktonically. Furthermore, addition of 2.5 and 10 mM hydrogen peroxide to planktonic cells resulted in a 12- and 160-fold increase in mutation frequency, respectively, and gave rise to variants similar in appearance, biofilm-related phenotypes, and distribution of biofilm-derived variants. The results suggest that hydrogen peroxide and environmental conditions specific to biofilms are responsible for the development of non-phase-variable colony variants.     

Fermentative biohydrogen production: trends and perspectives

Biologically produced hydrogen (biohydrogen) is a valuable gas that is seen as a future energy carrier, since its utilization via combustion or fuel cells produces pure water. Heterotrophic fermentations for biohydrogen production are driven by a wide variety of microorganisms such as strict anaerobes, facultative anaerobes and aerobes kept under anoxic conditions. Substrates such as simple sugars, starch, cellulose, as well as diverse organic waste materials can be used for biohydrogen production. Various bioreactor types have been used and operated under batch and continuous conditions; substantial increases in hydrogen yields have been achieved through optimum design of the bioreactor and fermentation conditions. This review explores the research work carried out in fermentative hydrogen production using organic compounds as substrates. The review also presents the state of the art in novel molecular strategies to improve the hydrogen production.     

Photooxidative stress in plants

The light-dependent generation of active oxygen species is termed photooxidative stress. This can occur in two ways: (1) the donation of energy or electrons directly to oxygen as a result of photosynthetic activity; (2) exposure of tissues to ultraviolet irradiation. The light-dependent destruction of catalase compounds the problem. Although generally detrimental to metabolism, superoxide and hydrogen peroxide may serve useful functions if rigorously controlled and compartmentalised. During photosynthesis the formation of active oxygen species is minimised by a number of complex and refined regulatory mechanisms. When produced, active oxygen species are eliminated rapidly by efficient antioxidative systems. The chloroplast is able to use the production and destruction of hydrogen peroxide to regulate the thermal dissipation of excess excitation energy. This is an intrinsic feature of the regulation of photosynthetic electron transport. Photoinhibition and photooxidation only usually occur when plants are exposed to stress. Active oxygen species are part of the alarm-signalling processes in plants. These serve to modify metabolism and gene expression so that the plant can respond to adverse environmental conditions, invading organisms and ultraviolet irradiation. The capacity of the antioxidative defense system is often increased at such times but if the response is not sufficient, radical production will exceed scavenging and ultimately lead to the disruption of metabolism. Oxidative damage arises in high light principally when the latter is in synergy with additional stress factors such as chilling temperatures or pollution. Environmental stress can modify the photooxidative processes in various ways ranging from direct involvement in light-induced free radical formation to the inhibition of metabolism that renders previously optimal light levels excessive. It is in just such situations that the capacity for the production of active oxygen species can exceed that for scavenging by the antioxidative defense systems. The advent of plant transformation, however, may have placed within our grasp the possibility of engineering greater stress tolerance in plants by enhancement of the antioxidative defence system.     

Effects of Molybdate and Selenite on Formate and Nitrate Metabolism in Escherichia coli

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.     

Thermophilic biohydrogen production: how far are we?

Apart from being applied as an energy carrier, hydrogen is in increasing demand as a commodity. Currently, the majority of hydrogen (H₂) is produced from fossil fuels, but from an environmental perspective, sustainable H₂ production should be considered. One of the possible ways of hydrogen production is through fermentation, in particular, at elevated temperature, i.e. thermophilic biohydrogen production. This short review recapitulates the current status in thermophilic biohydrogen production through fermentation of commercially viable substrates produced from readily available renewable resources, such as agricultural residues. The route to commercially viable biohydrogen production is a multidisciplinary enterprise. Microbiological studies have pointed out certain desirable physiological characteristics in H₂-producing microorganisms. More process-oriented research has identified best applicable reactor types and cultivation conditions. Techno-economic and life cycle analyses have identified key process bottlenecks with respect to economic feasibility and its environmental impact. The review has further identified current limitations and gaps in the knowledge, and also deliberates directions for future research and development of thermophilic biohydrogen production.