厌氧产氢菌的分离筛选、诱变选育及其产氢特性的研究

本文从四川绵竹酒厂、成都市龙泉长安垃圾填埋场以及四川大学荷花池底的厌氧污泥中先后分离得到63株厌氧产氢菌，其中H-8、H-61、HC-10等16株产氢细菌产氢能力较高，HC-10的产氢能力最高，最大产氢量和最大产氢速率分别达到2840 ml H2/L培养基和25.39 mmol H2/g drycell·h，对HC-10进行生理生化鉴定和分子生物学鉴定，判定其为clostridium sp.，对HC-10的产氢条件进行了研究，结果表明，该菌的最适生长温度为35 ℃，最适生长初始pH为7，以葡萄糖为最佳碳源，以蛋白胨为最佳氮源，不利用无机氮源，其产氢发酵液相产物以乙醇和乙酸为主，其发酵类型属于乙醇型发酵。此外，以酒糟废液作为底物，进行了菌株HC-10的生物强化试验，研究表明，投加了HC-10的强化系统其产氢量比对照高出40.32%。 同时为了获得厌氧产氢菌的高效突变株，分别以产氢菌H-8和H-61为原始菌株进行微波诱变处理，对微波诱变参数进行了优化，考察了突变株的遗传稳定性、产氢特性及耐酸性。菌株H-8经过微波诱变得到5株高产氢突变株HW7、HW33、HW181、HW184、HW195，经多次传代表明HW195是稳定的高产突变株。突变株HW195具有较好的耐酸性，在pH值为2.8时仍能生长。通过间歇发酵实验，其最大产氢量和最大产氢速率分别达到2460 mL/L培养基和27.97 mmol H2/g drycell·h，比原始菌分别提高了50.75%和41.7%。菌株H-61经过微波诱变后选育得到的突变株HW-18，其最大产氢量和最大产氢速率分别达到2190 mL/L培养基和25.86 mmol H2/g drycell·h，比原始菌分别提高了23.03%和31.00%。 为了对比各种诱变方式对产氢菌产氢能力的影响，以厌氧产氢菌H-61为原始菌株，先后经亚硝基胍(NTG)、紫外(UV)诱变，选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下，其产氢量为3024 mL/L培养基，比原始菌株提高了69.89%；其最大产氢速率为33.19 mmol H2/g drycell·h，比原始菌株提高了68.14%。经过多次传代实验，稳定性良好。其发酵末端产物以乙醇和乙酸为主，属于典型乙醇型发酵。其最适产氢初始pH为6.5，最适生长温度为36 ℃，以蔗糖为最佳碳源。与原始菌株相比，突变株HCM-23的产氢特性发生了改变，如生长延滞期延长，可利用无机氮源等。 From anaerobic activated sludge, 16 strains of hydrogen producing bacteria were newly isolated. One of them named as HC-10 had the highest hydrogen producing capability, under the batch fermentative hydrogen production condition, the maximal hydrogen yield and hydrogen production rate was 2840 mL/L culture and 25.39 mmol H2/g drycell·h. It was identified as clostridium sp.HC-10 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen producing of strain HC-10 were achieved as: initial pH 7.0, temperature 35 ℃, glucose as the favorite substrate, Moreover, using distiller's solubles wastewater as substrate, HC-10 strain was added in the biohydrogen producing system to research the bioaugmentation effection. The results showed that the hydrogen production of bioaugmentation system was 40.32% higher than the noaugmentation system. An anaerobic, hydrogen producing strain H-8 was irradiated by microwave to optimize the microwave mutagenesis condition, and to test the heredity, hydrogen-producing potential and aciduric of the mutants. An aciduric mutant named as HW195 with steady hydrogen-producing capability was obtained, which can grow at pH 2.8. Its capability of hydrogen production was tested in the batch culture experiments. The maximum hydrogen yield and hydrogen production rate was 2460 mL/L culture and 29.97 mmol H2/g drycell·h, which was 50.7% and 41.7% higher than those of the initial strain, respectively. When used the strain H-61 as original strain, a mutant named as HW18 was obtained. The maximum hydrogen yield and hydrogen production rate was 2190 mL/L culture and 25.86 mmol H2/g drycell·h, which was 23.03% and 31.00% higher than those of the initial strain, respectively. The results demonstrated that microwave mutagenesis could be used in the field of hydrogen producing microorganism. The hydrogen producing strain H-61 was used as an original strain which was induced by NTG and UV for increasing and the hydrogen production capability. One of the highest efficient H2-producing mutants was named as HCM-23 with its stable hydrogen production capability. which was tested in the batch culture experiments. With the condition of 10 g/L glucose, its cumulative hydrogen yield and hydrogen production rate was 3024 mL/L culture and 33.19 mmol H2/g drycell·h, 69.89％and 68.14％ higher than that of the original strain, respectively. The terminal liquid product compositions showed that the mutant HCM-23 fermentation was ethanol type, while the original strain H-61 fermentation was butyric acid type. Varieties of parameters of hydrogen production fermentation were studied, including time, carbon source, nitrogen source, glucose concentration, glucose utilization, initial pH and incubation temperature had been studied, indicated the optimum condition of hydrogen production for the mutant HCM-23 as initial pH6.5, temperature 36 ℃, and the favorite substrate was sucrose. The hydrogen production characters of the mutant and the original strain were different, such as, the growth lag phase and the utilization of inorganic nitrogen source, etc. This work shows a good application potential of NTG-UV combined mutation in the biohydrogen production. And the hydrogen production mechanism and metabolic pathway should be explored furthermore.

利用含纤维素原料发酵产氢的研究

本文从新鲜大熊猫粪便和实验室保存的沼气发酵富集物中筛选得到 4 株厌氧纤维素分解菌B5、C3、D3-2、D4-1，利用这4 株菌预处理秸秆，然后将预处理后的秸秆用本实验室保存的厌氧产氢菌来发酵进行生物产氢。同时还比较研究了：○1 用1% H2SO4、25% NH3 · H2O和12% NaOH对秸秆进行化学预处理；○2 用厌氧纤维素分解菌对秸秆进行生物预处理；○3 化学与生物组合预处理对秸秆发酵生物产氢的影响。实验结果表明：12% NaOH和生物组合预处理后的秸秆发酵产氢效果最好，其产氢量为21.04 mL g-1，是未经预处理秸秆的75 倍；最高氢气浓度为57.3%，是未经预处理秸秆的96 倍；其产氢的最适pH 为4.5 ~ 6.0，最佳底物浓度为45 ~ 55 g L-1；其发酵过程中的挥发性脂肪酸（VFAs）以乙酸和丁酸为主。 本实验筛选到的 4 株厌氧纤维素分解菌株中，B5 和D4-1 在降解纤维素的同时还具有直接以纤维素为底物产氢的功能，因此本文分别对菌株B5 和D4-1 以及二者的组合菌株B5+D4-1 直接利用秸秆为基质发酵生物产氢做了初步探索研究。结果发现：组合菌株发酵产氢的效果以及对秸秆纤维素和半纤维素的降解率要比单菌株好。菌株B5+D4-1 发酵，秸秆的产氢量为11.4 mL g-1，分别是B5 和D4-1 单菌株的1.6 倍和3.1 倍；组合菌株B5+D4-1 发酵的最大氢气浓度为31.6%，分别是B5 和D4-1 单菌株的1.3 倍和2.4 倍。在发酵过程中，组合菌株B5+D4-1 对秸秆纤维素和半纤维素的最高降解率分别为35.0%和11.8%，分别是菌株B5 的1.2 倍和1.1 倍，是菌株D4-1的1.5 倍和1.3 倍。菌株B5，D4-1 以及组合菌株B5+D4-1 发酵过程产生的挥发性脂肪酸（VFAs）均以乙酸为主。菌株B5 单独发酵过程中只检测到乙酸和丁酸，菌株D4-1 单独发酵以及组合菌株B5+D4-1 发酵过程检测到有乙醇、乙酸和丁酸。 The fermentative bio-hydrogen production by anaerobic hydrogen bacteria preserved in our laboratory from the straw which had been pretreated by four anaerobic cellulolytic decomposition strains of B5, C3, D3-2, D4-1 which were isolated and screened from giant panda’s excrement and biogas fermentation enrichments conserved in our laboratory was studied. Besides, the impact of chemical(1% H2SO4、25% NH3·H2O and 12% NaOH), biological (cellulolytic strains of B5, C3, D3-2, D4-1) and chemical-biological combination pretreatment on bio-hydrogen production from straw by fermentation was also comparatively studied. The experiments showed that the best results of bio-hydrogen production were obtained from the straw with 12% NaOH-biological combination pretreatment method, its capability of bio-hydrogen production was 21.04 mL g-1, which was 75 times higher than the straw without pretreatment; the maximum concentration of H2 was 57.3%, which was 96 times higher than the straw without pretreatment; its optimum pH range was 4.5 ~ 6.0, and its optimum range of substrate concentration was 45 ~ 55 g L-1; In the process of fermentation, the main composition of VFAs were acetate and butyrate. Among the four strains of B5, C3, D3-2, D4-1, B5 and D4-1 have the function of hydrogen-producing by cellulose used as substrate when it decompose cellulose, so the preliminary exploration and research on fermentative bio-hydrogen production by B5, D4-1 and B5+D4-1 which directly used straw as substrate was carried out. The results showed that the combination strains of B5+D4-1 was strikingly better than either B5 or D4-1 strain in the fermentative hydrogen production. The hydrogen-production capability of B5+D4-1 was 11.4 mL g-1 which was respectively 1.6 times and 3.1times higher than B5 and D4-1; the maximum hydrogen concentration of B5+D4-1 was 31.6% which was respectively 1.3 times and 2.4 times higher than B5 and D4-1. In the process of fermentation, the maximum degradation rate of cellulose and hemicellulose in straw was respectively 35.0% and 11.8% by B5+D4-1, which was 1.2 times and 1.1 times higher than B5, and was 1.5 times and 1.3 times higher than D4-1 respectively. The Volatile Fattty Acids(VFAs) generated in the process of fermentation with strains of B5, D4-1 and B5+D4-1 were all mainly acetate. Acetate and butyrate were detected in the process of fermentation with B5, ethonal, acetate and butyrate were detected in the process of fermentation with D4-1 and B5+D4-1.

Fe改性工业Cu/Zn/Al催化剂甲醇水蒸气重整反应制氢的研究

中国科学院山西煤炭化学研究所

海洋发酵菌和光合细菌耦联制氢研究

近年来，生物制氢技术与废物综合治理技术相结合，在处理废物的同时获得清洁的能源。目前生物制氢技术发展的瓶颈问题是高效产氢菌种的获得。因此，本研究采用不同预处理方法对海洋污泥的产氢能力的影响进行了分析，同时分离获得多株海洋产酸产氢细菌和海洋光合产氢细菌。 采用不同预处理方法（包括酸处理、碱处理、热处理和硝酸盐处理）获得海洋产酸产氢混合菌种，且其对产氢能力的影响进行分析。结果表明在所有预处理方法中，热处理的最大产氢量最高；于是，我们研究了不同起始pH值（pH 4-10.0）和不同底物浓度对热处理产氢菌群的产氢能力的影响。结果表明热处理产氢菌群的最适起始pH值为8.0，该值与天然海水的pH值相近。这意味着该产酸产氢菌群可作为海洋有机废物处理的候选菌群。 通过富集获得两类海洋光合产氢菌群，它们分别可以利用发酵产氢的关键副产物—乙酸或丁酸作为产氢碳源。研究表明温度、光照强度、起始pH和乙酸或丁酸浓度皆对两类光合产氢菌群的生长和产氢能力皆有明显影响。此外，产酸产氢菌群和光合细菌菌群耦联放氢进行了初步研究，结果表明藕联放氢可以获得较高的产氢量和底物利用率。 建立了一种厌氧产氢细菌的快速筛选方法。采用该方法从已获得的暗发酵和光合高效产氢菌群中分别分离高效产氢菌株。结果分离纯化获得暗发酵产氢细菌61株和光合产氢细菌34株。其中12株暗发酵产氢细菌和7株光合产氢细菌产氢量相对较高。 利用海水养殖有机废水进行微生物制氢技术将微生物制氢和环境治理相结合，是集污水治理和废物利用为一体的综合技术，具有广阔的应用前景，本研究为海水养殖有机废水进行微生物制氢技术发展奠定了基础。

Mixed reforming of heptane to syngas in the Ba0.5Sr0.5Co0.8Fe0.2O3 membrane reactor

Fuel cells are recognized as the most promising new power generation technology, but hydrogen supply is still a problem. In our previous work, we have developed a LiLaNiO/gamma-Al2O3 catalyst, which is excellent not only for partial oxidation of hydrocarbons, but also for steam reforming and autothermal reforming. However, the reaction needs pure oxygen or air as oxidant. We have developed a dense oxygen permeable membrane Ba0.5Sr0.5Co0.8Fe0.2O3 which has an oxygen permeation flux around 11.5 ml/cm(2) min at reaction conditions. Therefore, this work is to combine the oxygen permeable membrane with the catalyst LiLaNiO/gamma-Al2O3 in a membrane reactor for hydrogen production by mixed reforming of heptane. Under optimized reaction conditions, a heptane conversion of 100%, a CO selectivity of 91-93% and a H-2 selectivity of 95-97% have been achieved. (c) 2005 Elsevier B.V. All rights reserved.

Characterizations and activities of the nano-sized Ni/Al2O3 and Ni/La-Al2O3 catalysts for NH3 decomposition

A series of nano-sized Ni/Al2O3 and Ni/La-Al2O3 catalysts that possess high activities for NH3 decomposition have been successfully synthesized by a coprecipitation method. The catalytic performance was investigated under the atmospheric conditions and a significant enhancement in the activity after the introduction of La was observed. Aiming to study the influence of La promoter on the physicochemical properties, we characterized the catalysts by N-2 adsorption/desorption, XRD, H-2-TPR, chemisorption and TEM techniques. Physisorption results suggested a high specific surface area and XRD spectra showed that nickel particles are in a highly dispersed state. A combination of XRD, TEM and chemisorption showed that Ni-0 particles with the average size lower, than 5.0 nm are always obtained even though the Ni loading ranged widely from 4 to 63 %. Compared with the Ni/Al2O3 catalysts, the Ni/La-Al2O3 ones with an appropriate amount of promoter enjoy a more open mesoporous structure and higher dispersion of Ni. Reduction kinetic studies of prepared catalysts were investigated by temperature-programmed reduction (TPR) method and the fact that La additive partially destroyed the metastable Ni-Al mixed oxide phase was detailed. (c) 2005 Elsevier B.V. All rights reserved.

Methanol steam reforming in a compact plate-fin reformer for fuel-cell systems

A compact plate-fin reformer (PFR) consisting of closely spaced plate-fins, in which endothermic and exothermic reactions take place in alternate chambers, has been studied. In the PFR, which was based on a plate-fin heat exchanger, catalytic combustion of the reforming gas, as a simulation of the fuel cell anode off gas (AOG), supplied the necessary heat for the reforming reaction. One reforming chamber, which was for hydrogen production, was integrated with two vaporization chambers and two combustion chambers to constitute a single unit of PFR. The PFR is very compact, easy to be placed and scaled up. The effect of the ratio of H2O/CH3OH on the performance of the PFR has been investigated, and temperature distributions in different chambers were studied. Besides, the stationary behavior of the PFR was also investigated. Heat transfer of the reformer was enhanced by internal plate-fins as well as by external catalytic combustion, which offer both high methanol conversion ratio and low CO concentration. In addition, the fully integrated reformer exhibited good test stability. Based on the PFR, a scale-up reformer was designed and operated continuously for 1000 h, with high methanol conversion ratio and low CO concentration. (c) 2004 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Effects on the proton production from explosions of hydrogen clusters subjected to intense femtosecond laser fields

The dependence of the maximum and average energies of protons, which were produced in the interaction of an intense laser pulse (similar to 1 x 10(16) W cm(-2), 65 fs) with hydrogen clusters in a gas jet backed up to 80 bar at liquid nitrogen temperature (similar to 80 K), on the backing pressure has been studied. The general trend of the proton energy dependence on the square of the average cluster radius, which is determined by a calibrated Rayleigh scattering measurement, is similar to that described by theory under the single size approximation. Calculations are made to fit the experimental results under a simplified model by taking into account both a log-normal cluster size distribution and the laser intensity attenuation in the interaction volume. A very good agreement between the experimental proton energy spectra and the calculations is obtained in the high- energy part of the proton energy distributions, but a discrepancy of the fits is revealed in the low-energy part at higher backing pressures which are associated with denser flows. A possible mechanism which would be responsible for this discrepancy is discussed. Finally, from the fits, a variation of the cluster size distributions was revealed to be dependent on the gas backing pressure as well as on the evolving time of the gas flow of clusters.

Two-stage photo-biological production of hydrogen by marine green alga Platymonas subcordiformis

A marine green alga, Platymonas subcordiformis, was demonstrated to photobiologically evolve hydrogen (H-2) after the first stage of photosynthesis, when subjected to a two-phase incubation protocol in a second stage of H2 production: anaerobic incubation in the dark followed by the exposure to light illumination. The anaerobic incubation induced hydrogenase activity to catalyse H? evolution in the following phase of light illumination. H,) evolution strongly depended upon the duration of anaerobic incubation, deprivation of sulphur (S) from the medium and the medium pH. An optimal anaerobic incubation period of 32 h gave the maximum H2 evolution in the second phase in the absence of sulphur. Evolution of H,) was greatly enhanced by 13 times when S was deprived from the medium. This result suggests that S plays a critical role in the mediation of H-2 evolution from R subcordiformis. A 14-fold increase in H-2 production was obtained when the medium pH increased from 5 to 8; with a sharp decline at pH above eight. H-2 evolution was enhanced by 30-50% when supplementing the optimal concentrations of 25 mM acetate and 37.5 mM glucose. (C) 2003 Elsevier B.V. All rights reserved.

The experimental research for production of hydrogen from n-octane through partially oxidizing and steam reforming method

The reaction of producing hydrogen for fuel cell which used normal octane as gasoline or diesel oil reactant through catalytic partial oxidizing and steam reforming method has been researched in the fixed-bed reactor. A series of catalysts that mainly used nickel supported on Al2O3 have been studied. It showed that the activity of the catalyst was increased with the content of nickel by using only nickel supported on Al2O3. However, its activity was not obviously increased when the content of nickel was over 5 wt%. The conversion ratio of normal octane and hydrogen selectivity were higher at higher reaction temperature. The single noble catalyst of palladium had better stability compared with that of platinum catalyst although their activity and selectivity were similar during the experimental reaction temperature. The prepared bimetallic catalyst consisted mainly of nickel and little noble metal of palladium supported on Al2O3. It showed that this catalyst had higher activity and selectivity, especially at lower or higher reaction temperatures compared with single nickel or palladium catalyst, and better stability. ((C) 2001 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.