铁和镍对光合细菌生长和产氢的影响

基于金属元素在生物体功能发挥中的作用以及它们参与光合细菌光合放氢的重要性,着重进行了铁和镍对沼泽红假单胞菌（Rhodopseudomonas palustris）Z菌株和一株红杆菌（Rhodobactersp.）细胞生长、光合放氢和光合色素合成影响的研究。结果表明,高浓度Fe3+可显著提高两菌株光放氢能力和生物合成能力,最适浓度的Fe3+可使其产氢能力分别达对照组的1.32倍和2.8倍,产氢得率分别为360.6mL/g和385.9 mL/g,生物量分别为对照组的1.42倍和1.54倍。9μmol/L Ni2+的添加可使两菌株产氢能力分别达对照组的1.48倍和1.96倍,产氢得率分别为429.7mL/g和456.3 mL/g。而当Ni2+浓度为12μmol/L时,两菌株的产氢活性受到不同程度的抑制,产氢得率分别降低46.7%和19.4%。在铁浓度相同时,添加6μmol/L Ni2+能明显促进两菌株的生长。而当Ni2+浓度大于6μmol/L时,细胞生长受到抑制。Fe3+和Ni2+对Rhodobactersp.菌株类胡萝卜色素有显著影响。研究结果显示, 426nm色素峰随铁浓度的增加和镍的添加而消失,同时,产氢活性提高。     

红假单胞菌H菌株生长细胞光照放氢条件的研究

本文报道红假单胞菌属（Ｒｈｏｄｏｐｓｅｕｄｏｍｏｎａｓ）Ｈ菌株光照放氢的主要条件。结果表明,Ｈ菌株除利用苹果酸外,还可以利用葡萄糖、丁酸钠、乳酸钠等底物光照放氢,尤以葡萄糖产氢量较高。Ｈ菌株光照放氢的适宜ｐＨ为７．０,与细胞生长最适ｐＨ值一致。可以明显地抑制生长细胞放氢,在气相完全为Ｎ_２条件下,Ｈ菌株仍能以较高速率光照放氢。Ｈ菌株光照放氢的适宜温度为３０℃。空气能抑制光照放氢,加入一定浓度的Ｎａ_２Ｓ可以缓解这种抑制作用。     

蜜环菌胞外漆酶的合成、纯化及性质研究

研究了蜜环菌胞外漆酶合成条件和酶学性质。实验表明,培养基初始pH5.5、培养温度25℃有利于菌株产酶;与麦芽糖、山梨糖和半乳糖相比,纤维二糖和棉子糖作为碳源时漆酶产量更高;有机氮源比无机氮源有利于漆酶合成。泥炭提取液可显著诱导漆酶生成,当其含量为50%时,菌株漆酶最高产量是对照组的7倍。在蜜环菌发酵上清液中检测到3个漆酶同功酶组分,其主要活性（约占75%）组份漆酶A经 (NH₄)₂SO₄沉淀、制备级PAGE电泳和阴离子交换柱层析被分离纯化至电泳均一,SDSPAGE法测得酶亚基分子量59kD,凝胶过滤色谱法测定活性酶分子量58kD。纯化的漆酶A等电点pI为4.0,氧化愈创木酚的最适反应pH为5.6,最适温度为60℃,在60℃和65℃时半衰期分别为45min和36.8min,在pH5.2～7.2范围内稳定性较好。100mmol/L Cl^-对该酶有显著抑制作用,1mmol/L SO^2-₄ 对漆酶有激活作用,1mmol/L NaN₃可完全抑制酶活性,10 mmol/L EDTA对漆酶活没有明显影响,1mmol/L Cu²⁺对漆酶有激活作用。以愈创木酚为底物时,测得酶的K_m=1.026mmol/L,V_max=5μmol/(min·mg);以ABTS为底物时,测得其K_m=0.22mmol/L,V_max=69μmol/(min·mg)。     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

一株产虾青素的黄杆菌CF-60的研究

从土壤中分离到一株黄杆菌（Ｆｌａｖｏｂａｃｔｅｒｉｕｍｓｐｐ）Ｃ_Ｆ－６０,该菌的生长需Ｍｇ^２＋存在,ＭｇＳＯ_４·７Ｈ_２Ｏ的最适浓度为０．２％;蛋白胨是该菌株生长的最好氮源,它不能利用无机氮。种龄超过９６ｈ的菌体不能在新鲜培养基中生长。经５４ｈ的２Ｌ恒化器发酵,生物量达６．８ｇ／Ｌ,色素产量为１０．６ｍｇ／Ｌ。该菌产生的类胡萝卜素成分简单,主要成分的含量为９０．３％,该成分经初步鉴定是分子结构中含有羰基和羟     

浑球红假单胞菌在暗处发酵生长时的固氮酶、吸氢酶以及放氢机制的研究

光合细菌浑球红假单胞菌(Rhodopesudomonas sphaeroides)能在黑暗厌氧条件下生长。首次发现发酵生长的浑球红假单胞菌有固氮酶和吸氧酶的活性。试验比较了在光营养、黑暗厌氧呼吸和好氧呼吸生长方式下该菌的放氢作用,认为黑暗厌氧呼吸过程中的放氢是氢酶催化的放氢。     

白腐菌产锰过氧化物酶条件的研究

白腐菌Phanerochaeta chrysosporium MIG. 383降解桉木时具有显著的选择性,30天内降解37．23％Klason木素,7.29％综纤维素。该菌株产胞外锰过氧化物酶,并在高碳低氧培养基中显示较高酶活。静置液体培养的优化培养条件是(L^-1)：10g葡萄糖,2mmol酒石酸铵,10mmol pH4.5醋酸钠缓冲液,1g吐温80,2gK₂PO₄,0.5g MgSO₄·7H₂O,0.1g CaCl₂·2H₂O,lmg VB₁,70ml微量元素混合液：最适产酶温度是37℃。上述条件下,该菌接种后静置培养4天,产锰过氧化物酶活达1840U／L,酶作用最适温度是37℃,最适DH是3.5。     

镧与钕对红假单胞菌的生长、类胡萝卜素生成及固氮活性的影响

本文报道了不同浓度的La³⁺和Nd³⁺对红假单胞菌(Rhodopseudomonas sp.)的细胞形态、生长、类胡萝卜素生成和固氮活性的影响。LaCl_3在25和50mg/L时对红假单胞菌的细胞生长有轻微刺激作用;当浓度高于75mg/L时有抑制作用,随浓度的提高而抑制作用增强,细胞缩小;NdCl_3在25和50mg/L时对该菌细胞生长有轻微抑制作用,高于75mg/L时抑制作用明显增强,细胞缩小。两种稀土元素在25和50mg/L时对该菌类胡萝卜素的生成有刺激作用,高于75mg/L时则有抑制作用。La³⁺在0～100mg/L,Nd³⁺在0～75mg/L时对固氮酶活性有刺激作用,La³⁺和Nd³⁺分别高于100mg/L和75mg/L时则有抑制作用,并随浓度的增高,抑制作用明显增强。     

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 elongatum Z7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. elongatum Z7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。     

不产氧光合细菌Rhodobacter sphaeroides产氢影响因子研究

对不产氧光合细菌球形红细菌Rhodobacter sphaeroides产氢的影响因子进行了初步研究。结果表明,处于不同生长期的球形红细菌接种后的产氢速率略有差异,稳定期的菌株的产氢能力相对较低。苹果酸钠、乳酸钠、丙酮酸钠和葡萄糖都是球形红细菌产氢的良好碳源,这表明球形红细菌具有利用食品工业等高浓度废水为底物产氢的可能性。以葡萄糖和谷氨酸钠为C源和N源产氢时,适宜的葡萄糖浓度在25~50mmol/L之间,谷氨酸钠浓度在2~10mmol/L之间。球形红细菌产氢的适宜pH值在7.0~8.0范围内,酸性环境明显不利于该菌的催化放氢,适宜的温度在30~35℃范围内。光照强度在5000~7000lx之间适合于产氢。球形红细菌的固氮酶活性和放氢活性之间呈正相关性。吸氢酶虽然可在无固氮酶和无放氢活性的状态下独立表达,但多数情况下仍受氢气浓度的调节。以氮气为氮源时,固氮酶活性和放氢活性较低,铵的浓度高于0.5mmol/L时,固氮酶活性完全受到抑制,进而抑制产氢。     

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。     

SURVEY OF SELECTED SEA WEEDS FOR SIMULTANEOUS PHOTOPRODUCTION OF HYDROGEN AND OXYGEN1

Ten seaweed species were surveyed for simultaneous photoevolution of hydrogen and oxygen. In an attempt to induce hydrogenase activity (as measured by hydrogen photoproduction) the seaweeds were maintained under anaerobiosis in CO₂-free seawater for varying lengths of time. Although oxygen evolution was observed in every alga studied, hydrogen evolution was not observed. One conclusion of this research is that, in contrast to the microscopic algae, there is not a single example of a macroscopic alga for which the photoevolution of hydrogen has been observed, in spite of the fact that there are now at least nine macroscopic algal species known for which hydrogenase activity has been reported (either by dark hydrogen evolution or light-activated hydrogen uptake). These results are in conflict with the conventional view that algal hydrogenase can catalyze a multiplicity of reactions, one of which is the photoproduction of molecular hydrogen. Two possible explanations for the lack of hydrogen photoproduction in macroscopic algae are presented. It is postulated that electron acceptors other than carbon dioxide can take up reducing equivalents from Photosystem I to the measurable exclusion of hydrogen photoproduction. Alternatively, the hydrogenase system in macroscopic algae may be primarily a hydrogen-uptake system with respect to light-activated reactions. A simple kinetic argument based on recent measurements of the photosynthetic turnover times of simultaneous light-activated hydrogen and oxygen production is presented that supports the second explanation.     

Hydrogen evolution from dithionite and H2 photoproduction by hydrogenase incorporated into various hydrophobic matrices

The effects of surfactants, lipids and amphiphilic viologen mediators on H2 production from dithionite as well as on Ru(bpy) sensitized H2 photoproduction by hydrogenase from Thiocapsa roseopersicina was studied. Three systems which differed as to the nature of the hydrophobic matrix around the hydrogenase were tested. An enhanced hydrogenase activity was observed in the presence of surfactants, in the 1-6 mM concentration range. Hydrogenase showed a selectivity for the amphiphilic viologens, 2C7-diCl was the most efficient electron mediator in both reactions. H2 photoproduction seemed not to be feasible in the detergent-hydrogenase system because of intensive foaming. Hydrogenase incorporated into liposomes catalyzed H2 photoevolution efficiently but the rate was decreasing in time, though reversibly. Using intact bacterial cells instead of purified hydrogenase yielded stable H2 photoevolution for at least 12 hours. This system offers several advantages for potential practical applications.     

Hydrogen production from organic substrates in an aerobic nitrogen-fixing marine unicellular cyanobacterium Synechococcus sp. strain Miami BG 043511

Synechococus sp. strain Miami BG 043511 exhibits very high H(2) photoproduction from water, but the H(2) photoproduction capability is lost rapidly with the age of the batch culture. The decreases of the capability coincides with the decrease of cellular glucose (glycogen) content. However, H(2) photoproduction capability can be restored by the addition of organic substrates. Among 40 organic compounds tested, carbohydrates such as glucose, fructose, maltose, and sucrose were effective electron donors. Among organic acids tested, only pyruvate was an effective electron donor. Among alcohols tested, glycerol was a good electron donor. These results demonstrate that this unicellular cyanobacterium exhibits a wide substrate specificity for H(2) photoproduction but has a different substrate specificity compared to photosynthetic bacteria. The maximum rates of H(2) photoproduction from a 6-day-old batch culture with 25 mmol of pyruvate, glucose, maltose, sucrose, fructose, and glycerol were 1.11, 0.62, 0.50, 0.47, 0.30, and 0.39 micromoles per mg cell dry weight per hour respectively. Therefore, this cyanobacterium strain may have a potential significance in removing organic materials from the wastewater and simultaneously transforming them to H(2) gas, a pollution free energy. The activity of nitrogenase, which catalyzes hydrogen production, completely disappeared when intracellular glucose (glycogen) was used up, but it could be restored by the addition of organic substrates such as glucose and pyruvate. (c) 1994 John Wiley & Sons, Inc.     

Optimization of glutamate concentration and pH for H production from volatile fatty acids by Rhodopseudomonas capsulata

AIMS: This study attempted to employ response surface methodology (RSM) to evaluate the effects of glutamate concentration and pH on H(2) production from volatile fatty acids by Rhodopseudomonas capsulata. METHODS AND RESULTS: A mixture of acetate, propionate and butyrate was used as a carbon source for the H(2) production by R. capsulata. The H(2) yield and H(2) production rate were strongly affected by the glutamate concentration, pH and their interaction. The predicted maximum H(2) yield of 0.534 was obtained when glutamate concentration and pH were 6.56 mmol l(-1) and 7.29 respectively. On the contrary, the maximum H(2) production rate of 18.72 ml l(-1) h(-1) was achieved at a glutamate concentration of 7.01 mmol l(-1) and pH 7.31. CONCLUSIONS: Taking H(2) yield and H(2) production rate together into account, a glutamate concentration of 6.56-7.01 mmol l(-1) and pH of 7.29-7.31 should be selected for H(2) production from a mixture of acetate, propionate and butyrate by R. capsulata. SIGNIFICANCE AND IMPACT OF THE STUDY: The RSM was a useful tool for maximizing H(2) production by photosynthetic bacteria (PSB).     

High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring

Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 58 degrees C. The 33HL produced H2 yield (HY) of up to 3.2 mol-H2/mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H2 production was unstable. In the ASBR, maintained with 33HL, the H2 production enhanced after the addition of 6 mg/L of FeSO4 x H2O resulting in HY up to 2.51 mol-H2/mol-glucose (H2 production rate (HPR) of 7.85 mmol/h/L). The H2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO4 supplementation, stable, high-rate H2 production was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/L) and HY of 1.54 mol-H2/mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affiliated with Thermobrachium celere (99.8-100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotearoense (98.5-99.6%) affiliated strains, producing H2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H2 production efficiency and ready granulation is considered very promising for H2 fermentation from carbohydrates.     

Optimization of activation conditions of Rhodobacter sphaeroides in hydrogen generation process

AIMS: To examine the effects of the culture age, illuminance intensity and changes in these parameters during activation on hydrogen generation process carried out by purple nonsulfur Rhodobacter sphaeroides bacteria. METHODS AND RESULTS: The following parameters were determined in all experiments: the amount of hydrogen evolved (measured using gas chromatography), biomass increase as dry mass, pH values and consumption of organic substance as chemical oxygen demand (COD). The medium used in the process of activation and hydrogen generation contained malic acid (15 mmol) and sodium glutamate (2 mmol). The optimum age of bacteria was 12-24 h and the best intensity of illuminance was found to be 5 cd sr m-2 on activation and 9 cd sr m-2 on hydrogen generation. These conditions provided hydrogen evolution of 1.39 l l-1 of the medium with the highest specific hydrogen production of 0.146 l H2 l-1 medium h-1 g-1 inoculum. An increase in the illuminance intensity resulted in a slight inhibition of the process. CONCLUSIONS: The activation stage of bacteria has a significant effect on the parameters of hydrogen photogeneration. The optimization of the activation stages allowed a shortening of the time of hydrogen generation and of the period after which hydrogen evolution starts. SIGNIFICANCE AND IMPACT OF THE STUDY: An innovative method of bacteria activation before the initiation of the hydrogen generation process has been used to optimize this process. The shortening of the process duration as well as the twice higher hydrogen yield can help in the designing of other systems (including also those operating under solar irradiation) in which R. sphaeroides bacteria are to be applied.     

Stably Sustained Hydrogen Production with High Molar Yield through a Combination of a Marine Green Alga and a Photosynthetic Bacterium

Stably sustained continuous production of hydrogen with high molar yield was achieved through a combination of dark fermentative hydrogen evolution by Chlamydomonas sp. strain MGA161 and hydrogen photoevolution by a marine photosynthetic bacterium W-1S in an alternating light-dark cycle as a model of the day-night cycle. The newly isolated strain W-1S could use acetic acid and ethanol excreted by strain MGA161 as electron donors for hydrogen photoevolution. The fermentation broth of strain MGA161 stimulated the hydrogen photoproduction of strain W-1S. This alga-bacterial combination had a high conversion yield of 8 mol H₂/mol of glucose of starch, with the possibility of improvement up to 10.5.     

H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: production and utilization of H2 by resting cells.

Photoproduction of H2 and activation of H2 for CO2 reduction (photoreduction) by Rhodopseudomonas capsulata are catalyzed by different enzyme systems. Formation of H2 from organic compounds is mediated by nitrogenase and is nto inhibited by an atmosphere of 99% H2. Cells grown photoheterotrophically on C4 dicarboxylic acids (with glutamate as N source) evolve H2 from the C4 acids and also from lactate and pyruvate; cells grown on C3 carbon sources, however, are inactive with the C4 acids, presumably because they lack inducible transport systems. Ammonia is known to inhibit N2 fixation by photosynthetic bacteria, and it also effectively prevents photoproduction of H2; these effects are due to inhibition and, in part, inactivation of nitrogenase. Biosynthesis of the latter, as measured by both H2 production and acetylene reduction assays, is markedly increased when cells are grown at high light intensity; synthesis of the photoreduction system, on the other hand, is not appreciably influenced by light intensity during photoheterotrophic growth. The photoreduction activity of cells grown on lactate + glutamate (which contain active nitrogenase) is greatly activated by NH4+, but this effect is not observed in cells grown with NH4+ as N source (nitrogenase repressed) or in a Nif- mutant that is unable to produce H2. Lactate, malate, and succinate, which are readily used as growth substrates by R. capsulata and are excellent H donors for photoproduction of H2, abolish photoreduction activity. The physiological significances of this phenomenon and of the reciprocal regulatory effects of NH4+ on H2 production and photoreduction are discussed.     

Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.