酒色着色菌利用产酸克雷伯氏菌发酵废液产氢

研究了酒色着色菌(Chromatiumvinosum DSM185)利用产酸克雷伯氏菌(Klebsiellaoxytoca HP1)发酵产氢废液进行光发酵和暗发酵产氢的可行性,以达到对产氢底物的充分利用和对产氢废液的进一步处理。研究结果表明C.vinosum可以利用K.oxytoca的发酵废液进行光发酵产氢和暗发酵产氢。C.vinosum发酵产氢后废液中残余还原糖和主要有机酸(丁酸)的含量明显降低,发酵产氢的最佳pH为6.5,添加0.1%(W/W)NH4Cl能促进产氢。在光照条件下丁酸利用率可达54.38%,产氢量达36.97mL/mg;在黑暗条件下丁酸利用率可达36.01%,产氢量达37.50mL/mg。     

酒色着色菌利用产酸克雷伯氏菌发酵废液产氢

研究了酒色着色菌(Chromatium vinosum DSM185)利用产酸克雷伯氏菌（Klebsiella oxytoca HP1）发酵产氢废液进行光发酵和暗发酵产氢的可行性,以达到对产氢底物的充分利用和对产氢废液的进一步处理。研究结果表明C.vinosum可以利用K.oxytoca的发酵废液进行光发酵产氢和暗发酵产氢。C.vinosum发酵产氢后废液中残余还原糖和主要有机酸（丁酸）的含量明显降低,发酵产氢的最佳pH为6.5,添加0.1%(W/W)NH₄Cl能促进产氢。在光照条件下丁酸利用率可达54.38%,产氢量达36.97 mL/mg;在黑暗条件下丁酸利用率可达36.01%,产氢量达37.50mL/mg。     

种泥预处理和发酵温度对暗发酵产氢的影响研究进展

生物质暗发酵产氢不仅可以处理有机废物,同时可以获得清洁能源,实现了废物资源化利用。然而产氢种泥中大量耗氢菌的存在会导致暗发酵氢气产量低等问题,因此种泥预处理是暗发酵产氢的必需条件。随着暗发酵产氢基质的多样化,产氢种泥的预处理方法也不断发展。对近十年来产氢种泥预处理方法的发展进行了综述,并且结合发酵温度,讨论了种泥预处理方法和发酵温度两方面条件对暗发酵产氢的影响,并对该研究方向提出了展望,以期为后续暗发酵产氢的进一步研究提供参考。     

种泥预处理和发酵温度对暗发酵产氢的影响研究进展

生物质暗发酵产氢不仅可以处理有机废物,同时可以获得清洁能源,实现了废物资源化利用。然而产氢种泥中大量耗氢菌的存在会导致暗发酵氢气产量低等问题,因此种泥预处理是暗发酵产氢的必需条件。随着暗发酵产氢基质的多样化,产氢种泥的预处理方法也不断发展。本文对近十年来产氢种泥预处理方法的发展进行了综述,并且结合发酵温度,讨论了种泥预处理方法和发酵温度两方面条件对暗发酵产氢的影响,并对该研究方向提出了展望,以期为后续暗发酵产氢的进一步研究提供参考。     

转基因莱茵衣藻hemHc-lbac 和根瘤菌共培养提高产氢培养条件的优化

转基因莱茵衣藻hemHc-lbac(transgenic Chlamydomonas reinhardtii hemHc-lbac)以不同比例与日本慢生大豆根瘤菌(Bradyrhizobium japonicum)混合, 在不同光照条件下进行产氢培养, 以确定产氢的最优条件和探索产氢提高的机理。结果表明藻菌共培养的最优产氢条件为25 ℃、光照30 μE⋅m–2⋅s–1、生长至饱和期的菌和藻体积比为1: 80, 产氢量达到最大, 约为278 μmol⋅mg–1Chl, 是对照组80 μmol⋅mg–1Chl 的3.5 倍。藻菌共培养提高产氢量的主要原因是体系中氧气浓度的降低而使氢化酶活性提高、以及衣藻生物量的增加。该研究为利用藻菌共培养及转基因的方法提高微藻光合生物制氢效率提供了重要实验基础。     

转基因衣藻lba及对照藻产氢培养条件的优化

通过对莱茵衣藻849及其转基因衣藻lba进行光照强度、细胞浓度和培养基中硫酸盐含量三因素三水平的正交实验,确定了两个藻种的最佳产氢条件,同时对转基因藻和849产氢培养条件下的光合放氧速率和pH进行了检测。实验结果表明,在25 ℃下,莱茵衣藻849和转基因衣藻lba的最佳产氢条件都为光照强度 60μmol/(m²·s),细胞浓度为叶绿素含量12.5μg/ml,培养基中硫酸盐含量0μmol/L。莱茵衣藻849和转基因衣藻lba的最高氢气产量分别达到了349μl/mg chlorophyll 和634μl/mg chlorophyll。在产氢条件下,转基因藻lba的净光合放氧速率比849低。结果为利用豆血红蛋白特性通过基因工程手段提高莱茵衣藻产氢量提供基础实验数据。     

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。     

固定化光合细菌利用有机物产氢的研究

应用固定化细胞技术包埋荚膜红假单胞菌(Rhodopseudomonas capsulata)菌株386．研究在光照下利用有机物产氢的特性。实验观察到,光照培养120小时,悬浮培养物的产氢量为68．2ml·比产氢速率为104．1ml H2/g(生物量)·h;用琼脂包埋后．其产氢能力得到改善,产氢量和比产氢速率分别达到128．4ml和l 9s．8mlH2/g·h。该菌株除可利用苹果酸外,还可利用葡萄糖、乳酸、丙酸等基质高效地产氢。基质浓度只有控制在适当水平时,才具有较高的基质转化产氢效率。此外．菌体生物量、菌龄、培养液pH、光照强度、光照／黑暗时间比以及温度对产氢过程均有不同程度的影响。     

厌氧生境体系中产氢产乙酸细菌的FISH定量解析

产氢产乙酸细菌是一类在有机物厌氧降解过程中起重要作用的细菌。以基于16S rRNA序列设计的特异性寡核苷酸探针为基础,优化FISH实验条件,确定该技术检测产氢产乙酸细菌的实验条件为样品固定19h、乙醇脱水5min,杂交缓冲液中甲酰胺浓度55%。运用建立的FISH技术检测了几种厌氧消化体系中产氢产乙酸细菌的数量,并与用传统MPN方法的结果进行了比较。结果表明,产氢产乙酸细菌分布广泛,废水处理UASB反应器和动物消化道,特别是反刍动物瘤胃中的产氢产乙酸细菌数量较高,其丰度分别为1.70×109 cells/mL样品,6.50×108 cells/mL样品。湖底沉积物中产氢产乙酸细菌数量较少,仅占整个微生物群落的0.4%,含量为1.20×108 cells/mL样品。     

氮、锰、硫缺乏对蛋白核小球藻Chlorella pyrenoidosa光合产氢及其生长的影响

本实验通过研究缺氮、缺锰和缺硫对蛋白核小球藻Chlorella pyrenoidosa产氢的影响,发现缺氮、缺锰及缺硫条件下该藻均能产氢,但在缺氮条件下产氢量最高,约为88.613μL H2/mgChla,分别是对照组、缺锰和缺硫实验组产氢量的4.61倍、1.92倍和3.63倍。通过对光合、呼吸及生长的比较研究,发现缺锰对该藻光合、呼吸及生长的影响要小于缺氮和缺硫;与正常培养条件相比,缺锰、缺硫抑制藻细胞的光合放氧和生长,对呼吸影响小,而缺氮不仅最大程度抑制光合放氧和生长,同时使呼吸作用增强,这为进一步优化该藻产氢条件及研究其产氢机制提供了线索。     

浑球红假单胞菌及其谷氨酸合成酶突变株氢酶活性和合成

浑球红假单胞菌野生型菌株的氢酶表达被有机碳、氮底物所抑制。在光照和黑暗时,氧浓度变化对氢酶的作用不同,但高氧浓度都阻遏氢酶的表达。微量Ni~(2+)能专一性地促进氢酶活性,固氮酶的产氢也可以调节氢酶的表达水平。该野生菌株的GOGAT突变株缺乏固氮酶和氢酶活性,在加入谷氨酰胺合成酶抑制剂MSX后,固氮酶和氢酶以相关联的方式合成出来,固氮酶产生的氢看来诱导了氢酶的合成。然而在固氮酶不表达的情况下,外源氢也可诱导氢酶的合成。     

Photoproduction of hydrogen from water in hydrogenase-containing algae.

Scenedesmus obliquus and Chlorella vulgaris cells had active hydrogenase after dark anaerobic adaptation. Illumination of these algae with visible light led to an initial production of small quantities of hydrogen gas which soon ceased owing to production of oxygen by photolysis of water. The presence of oxygen-absorbing systems in a separate chamber, not in contact with the algae, gave only a slight stimulation of hydrogen production. Addition of sodium dithionite directly to the algae led to an extensive light-dependent production of hydrogen. This stimulation was due to oxygen removal by dithionite and not to its serving as an electron donor. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosystem II, abolished all hydrogen photoproduction. Hydrogen evolution was not accompanied by CO₂ production and little difference was noted between autotrophically and heterotrophically grown cells. Hydrogen was not produced in a photosystem II mutant of Scenedesmus even in the presence of dithionite, establishing that water was the source of hydrogen via photosystems II and I. Hydrogen production was stimulated by the presence of glucose and glucose oxidase as an oxygen-absorbing system. Oxygen inhibited hydrogen photoproduction, even if oxygen was undetectable in the gas phase, if the algal solution did not contain an oxygen absorber. It was demonstrated that under these conditions hydrogenase was still active and the inability to produce hydrogen was probably due to oxidation of the coupling electron carrier.     

Hydrogen production by <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> revisited: Rubisco as a biotechnological target

Hydrogen production by C. reinhardtii seems a promising alternative as a source of non-polluting biofuel. Hydrogen is generated as a result of combining free protons and electrons (supplied by ferredoxin) through the activity of an oxygen-sensitive hydrogenase. Thus, substantial hydrogen production is only observed in the light under anaerobic conditions. These require a reduced rate of photosynthetic oxygen evolution which is usually achieved by impairing photosystem II through sulphur starvation. Several approaches have been conducted to enhance and extend hydrogen production by addressing problems such as the mechanism of hydrogenase inhibition by oxygen, the stressing impact on the cells of the culture conditions, the use of starch as an alternate source of electrons under reduced photosynthetic activity, and the need of maintaining a balance between oxygen evolution and consumption. The photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) appears as suitable objective for biotechnological optimization of hydrogen production because of its relevance controlling the hydrogenase main competitor electron sink (the Calvin-Benson cycle), as well as starch accumulation and photorespiratory oxygen consumption. Possible strategies for increasing hydrogen generation based on alteration of Rubisco properties and/or catabolism through site-directed mutagenesis are discussed.     

The significance of hydrogenase activity for the energy metabolism of green algae: anaerobiosis favours ATP synthesis in cells of Chlorella with active hydrogenase

In cells of the green alga Chlorella fusca, which contain active hydrogenase(s), the concentration of ATP, NADH and NADPH were measured during a 5 h period of anaerobiosis in the dark and upon subsequent illumination with high light intensities (770 W/m²), conditions which favour optimal hydrogen photoproduction.ATP concentrations were also determined in cells of Chlorella fusca, whose hydrogenase was inactivated prior to illumination, and in cells of Chlorella vulgaris which do not contain hydrogenase. In the dark, the ATP concentration increased slightly during anaerobiosis in cells with active hydrogenase. This increase in ATP concentration was accompanied by an increase of NADH and a decrease of NADPH content.Upon illumination, the ATP content increased in cells with an active hydrogenase, whereas the NADH content decreased. The rate of phosphorylation was twice that observed in cells without active hydrogenase.This ATP synthesis in the light was not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (10 mol/l) nor by carbonylcyanide-3-chlorophenyl-hydrazone (CCCP) (1 mol/l) but was diminished by 500 mol/l dibromothymoquinone (DBMIB) and 6 mol/l carbonylcyanide-3-chlorophenyl-hydrazone (CCCP).It was concluded that an active hydrogenase can support ATP production under anaerobic conditions in the dark as well as in the light. NADH might serve in vivo as electron donor for a fermentative production of hydrogen in the light.Possible mechanisms underlying ATP production under anaerobiosis and hydrogen productive conditions are discussed.Abbreviations CCCP Carbonylcyanide-3-chlorophenyl-hydrazone - DBMIB dibromothymoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide-p-trifluormethoxyphenyl-hydrazone - HEPES N-2-hydroxyethylpiperazin-N-2-ethan-sulfonic acid - PSI II, photosystem I, II respectively - PQ plastoquinone     

Lichen Symbiosis: Nature's High Yielding Machines for Induced Hydrogen Production

Hydrogen is a promising future energy source. Although the ability of green algae to produce hydrogen has long been recognized (since 1939) and several biotechnological applications have been attempted, the greatest obstacle, being the O₂-sensitivity of the hydrogenase enzyme, has not yet been overcome. In the present contribution, 75 years after the first report on algal hydrogen production, taking advantage of a natural mechanism of oxygen balance, we demonstrate high hydrogen yields by lichens. Lichens have been selected as the ideal organisms in nature for hydrogen production, since they consist of a mycobiont and a photobiont in symbiosis. It has been hypothesized that the mycobiont’s and photobiont’s consumption of oxygen (increase of COX and AOX proteins of mitochondrial respiratory pathways and PTOX protein of chrolorespiration) establishes the required anoxic conditions for the activation of the phycobiont’s hydrogenase in a closed system. Our results clearly supported the above hypothesis, showing that lichens have the ability to activate appropriate bioenergetic pathways depending on the specific incubation conditions. Under light conditions, they successfully use the PSII-dependent and the PSII-independent pathways (decrease of D1 protein and parallel increase of PSaA protein) to transfer electrons to hydrogenase, while under dark conditions, lichens use the PFOR enzyme and the dark fermentative pathway to supply electrons to hydrogenase. These advantages of lichen symbiosis in combination with their ability to survive in extreme environments (while in a dry state) constitute them as unique and valuable hydrogen producing natural factories and pave the way for future biotechnological applications.     

Photoperiodic control of gonadal endocrine function in silver-gray foxes and its alteration during domestication

Studies have been made on the level of estradiol and progesterone in domesticated and nondomesticated female silver foxes under different photoperiodic regime. The duration of the "light--darkness" cycle was equal to 24 h. Total duration of the light period was equal to 9 1/2 h; illumination was natural for 7 h, being artificial for the rest 2 1/2 h in a dark part of a day. It was demonstrated that additional illumination affects the hormonal level and sexual activity, the effect being dependent on the particular time of illumination. Nocturnal illumination significantly increased the level of sex hormones in the blood, domesticated animals being more sensitive as compared to nondomesticated ones.     

光照对光生物反应器中微藻高密度光自养培养的影响

光生物反应器是实现微藻高密度培养的重要装置,其设计的关键技术之一是选择合适的光照方式。根据国内外近十年来的相关研究成果,重点介绍了入射光性质(光源、光强、光质和光暗循环)和光能分布对微藻生长的影响,评述了用于微藻高密度培养的光照技术,展望了进一步的研究方向,为高效光生物反应器的设计和优化提供参考。     

Disruption of the uptake hydrogenase gene, but not of the bidirectional hydrogenase gene, leads to enhanced photobiological hydrogen production by the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120

In order to determine the effects of the deletion of hydrogenase genes on nitrogenase-based photobiological H(2) productivity by heterocystous N(2)-fixing cyanobacteria, we have constructed three hydrogenase mutants from Anabaena sp. PCC 7120: hupL(-) (deficient in the uptake hydrogenase), hoxH(-) (deficient in the bidirectional hydrogenase), and hupL(-)/ hoxH(-) (deficient in both genes). The hupL(-) mutant produced H(2) at a rate four to seven times that of the wild-type under optimal conditions. The hoxH(-) mutant produced significantly lower amounts of H(2) and had slightly lower nitrogenase activity than wild-type. H(2) production by the hupL(-)/ hoxH(-) mutant was slightly lower than, but almost equal to, that of the hupL(-) mutant. The efficiency of light energy conversion to H(2) by the hupL(-) mutant at its highest H(2) production stage was 1.2% at an actinic visible light intensity of 10 W/m(2) (PAR) under argon atmosphere. These results indicate that deletion of the hupL gene could be employed as a source for further improvement of H(2) production in a nitrogenase-based photobiological H(2) production system.     

Pathways of hydrogen uptake in the cyanobacterium Nostoc muscorum

Two pathways of hydrogen uptake in Nostoc muscorum are apparent using either oxygen or nitrogen as electron acceptor. Hydrogen uptake (under argon with some oxygen as electron acceptor assayed in the dark; oxyhydrogen reaction) is found to be more active in dense, light-limited cultures than in thin cultures when light is not limiting. Addition of bicarbonate inhibits this hydrogen uptake, because photosynthesis is stimulated. In a cell-free hydrogenase assay, a 10-fold increase of the activity can be measured, after the cells having been kept under lightlimiting conditions. After incubation under light-saturating conditions, no hydrogen uptake is found, when filaments are assayed under argon plus some oxygen. Assaying these cells under a nitrogen atmosphere, a strong hydrogen uptake occurs. The corresponding cell-free hydrogenase assay exhibits low hydrogenase activity. Furthermore, the hydrogen uptake by intact filaments under nitrogen in the light apparently is correlated with nitrogenase activity. These studies give evidence that, under certain physiological conditions, hydrogen uptake of heterocysts proceeds directly via nitrogenase, with no hydrogenase involved.Abbreviations Chl chlorophyll - DCMU (diuron) 3-3,4-dichlorophenyl)-1,1-dimethylurea - pev packed cell volume     

Circadian effects of light no brighter than moonlight

In mammals, light entrains endogenous circadian pacemakers by inducing daily phase shifts via a photoreceptor mechanism recently discovered in retinal ganglion cells. Light that is comparable in intensity to moonlight is generally ineffective at inducing phase shifts or suppressing melatonin secretion, which has prompted the view that circadian photic sensitivity has been titrated so that the central pacemaker is unaffected by natural nighttime illumination. However, the authors have shown in several different entrainment paradigms that completely dark nights are not functionally equivalent to dimly lit nights, even when nighttime illumination is below putative thresholds for the circadian visual system. The present studies extend these findings. Dim illumination is shown here to be neither a strong zeitgeber, consistent with published fluence response curves, nor a potentiator of other zeitgebers. Nevertheless, dim light markedly alters the behavior of the free-running circadian pacemaker. Syrian hamsters were released from entrained conditions into constant darkness or dim narrowband green illumination (~0.01 lx, 1.3 x 10(-9) W/cm(2), peak lambda = 560 nm). Relative to complete darkness, constant dim light lengthened the period by ~0.3 h and altered the waveform of circadian rhythmicity. Among animals transferred from long day lengths (14 L:10 D) into constant conditions, dim illumination increased the duration of the active phase (alpha) by ~3 h relative to complete darkness. Short day entrainment (8 L:16 D) produced initially long alpha that increased further under constant dim light but decreased under complete darkness. In contrast, dim light pulses 2 h or longer produced effects on circadian phase and melatonin secretion that were small in magnitude. Furthermore, the amplitude of phase resetting to bright light and nonphotic stimuli was similar against dimly lit and dark backgrounds, indicating that the former does not directly amplify circadian inputs. Dim illumination markedly alters circadian waveform through effects on alpha, suggesting that dim light influences the coupling between oscillators theorized to program the beginning and end of subjective night. Physiological mechanisms responsible for conveying dim light stimuli to the pacemaker and implications for chronotherapeutics warrant further study.