Utilization of methanol by rhodospirillaceae

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophila strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or formaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.     

Identification and physiological characterization of the nitrogen fixing bacterium Corynebacterium autotrophicum GZ 29

The coryneform hydrogen bacterium strain GZ 29, assigned to Corynebacterium autotrophicum fixed molecular nitrogen under autotrophic (H₂, CO₂) as well as under heterotrophic (sucrose) conditions. Physiological parameters of nitrogen fixation were measured under heterotrophic conditions. The optimal dissolved oxygen concentration for cells grown in a fermenter with N₂ was rather low (0.14 mg O₂/l) compared with cells grown in the presence of NH ₄ ⁺ (4.45 mg O₂/l). C. autotrophicum GZ 29 had a doubling time of 3.7 h at 30°C with N₂ as N-source and sucrose as carbon source and at optimal pO₂. Acetylene reduction reached values of 12 nmoles of ethylene produced/minxmg protein. Although the oxygen concentration in the growing culture was kept constant, the optimal dissolved oxygen tension for the acetylene reduction assay shifted to higher pO₂-values. The overall efficiency of nitrogen fixation amounted to 22 mg N fixed/g sucrose consumed; it reached a maximal value of 65 mg N fixed/g sucrose consumed at the beginning of the exponential growth phase. Intact cells reduced acetylene even under anaerobic test conditions; further anaerobic metabolic activity could not be ascertained so far.     

Diazotrophic growth of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata under microaerobic conditions in the dark

Diazotrophy of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata was not obligatorily linked to photosynthesis. In the dark R. acidophila grew with dinitrogen as sole nitrogen source at a dissolved oxygen tension of 15 Torr (= 2.0 kPa); the doubling time was 8 h. Acetylene reduction by whole cells was more sensitive to oxygen in the light than in the dark. 16.5 mg N₂ were fixed per g lactic acid consumed. R. capsulata synthesized nitrogenase and fixed dinitrogen in the dark at a dissolved oxygen tension of less than one Torr (= 0.13 kPa). The doubling time of this bacterium was 16 h and 10.5 mg N₂ were fixed per g lactic acid consumed.Abbreviation kPa kilopascal     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

正交实验优选光合细菌混合菌群产氢的最佳条件

对光合细菌混合菌群产氢影响因子进行了实验研究。通过单因素实验和正交实验, 系统考察了碳源、氮源、碳源浓度、氮源浓度、初始pH值、光照方式、接种量等因素对产氢量的影响, 实验得出最佳工艺条件为: 采用3号菌群, 碳源为葡萄糖, 碳源浓度为3 g/L, 氮源为尿素, 氮源浓度为9 g/L, 接种量为10%, pH值为8.5, 光照方式为12 h光照-12 h黑暗交替光照, 培养温度为30°C。菌种、碳源、碳源浓度、氮源是影响产氢量的重要因素。     

Nutritional and cultural conditions for production of poly-3-hydroxybutyric acid byAzotobacter chroococcum

Free-living nitrogen-fixing bacteria have been identified as a potential source of poly-3-hydroxybutyric acid (PHB). Systematic study of this ability of N₂-fixing organisms has lead to the isolation of an efficient strain, identified asAzotobacter chroococcum. Nutritional requirements and cultural conditions for optimal production of PHB by this strain under laboratory conditions were determined. In N-free liquid medium containing 2% glucose, the strain accumulated PHB up to 68% of its cell dry mass. Glucose and mannitol were found to be the best carbon sources, while organic nitrogen compounds were preferred as nitrogen source. Maximum yield (3.3 g/L) was obtained with 0.2% bactopeptone supplementation. Inorganic phosphate at a concentration suboptimal for growth had some growth-promoting effect. Under oxygen limiting conditions, biomass production was enhanced but a different response was obtained for PHB production.     

Bioconversion of synthesis gas to hydrogen using a light-dependent photosynthetic bacterium,Rhodospirillum rubrum

Biological hydrogen production from synthesis gas was carried out in batch culture. The phototrophic anaerobic bacterium, Rhodospirillum rubrum was used to oxidize CO and water to CO₂ and hydrogen. The bacteria were grown under anaerobic conditions in liquid medium; also acetate was used as carbon source in presence of synthesis gas. Biological hydrogen production was catalysed by R. rubrum via the water–gas shift reaction. A light-dependent cell growth modelled with a desired rate of hydrogen production and CO uptake was determined. The effect of light intensity on microbial cell growth was also studied at 500, 1,000 and 1,500 m.cd. A complete conversion of CO to hydrogen and maximum light efficiency were obtained with an acetate concentration of 1 g/l and light intensity of 500 m.cd. Utilization of the carbon monoxide from the gas phase was often considered as a mass transfer limited process, which needed to diffuse through the gas–liquid interface and then further diffuse into liquid medium prior to reaction. The results from this study showed that maximum cell propagation and hydrogen production were achieved with a limited light intensity of 1,000 m.cd. It was also found that high-light intensity may interfere with cell metabolism. In low-light intensity and substrate concentration, no inhibition was observed, however at extreme conditions, non-competitive inhibition was identified. The adverse effect of high-light intensity was shown at 5,000 m.cd, where the CO conversion drastically dropped to as low as 21%. Maximum CO conversion of 98% and maximum yield of 86% with an acetate concentration of 1.5 g/l and a light intensity of 1,000 m.cd were achieved.     

Effects of temperature,light, carbon and nitrogen nutrition on reproduction in Phoma medicaginis

Reproduction by three isolates ofPhoma medicaginis growing on potato dextrose agar was studied. The formation of pycnidia was optimum at 30°C whereas the optimum for the formation of conidia was 20°C. Light consistently increased the numbers of pycnidia and conidia over those formed in darkness and more conidia were produced in light at 23°C than at 30°C. The effects of carbon and nitrogen sources on reproduction were studied using modified Richard's medium as the basal medium. Fourteen carbohydrates, 11 amino acids, 9 inorganic nitrogen sources and urea were evaluated by replacing the carbohydrate or nitrogen in Richard's medium with the test substance. Generally, the monosaccharides and disaccharides were about alike and superior to polysaccharides for the production of pycnidia. The carbon sources were about equally useful for production of conidia, but the polysaccharides were superior to the other two classes of carbon sources when the number of conidia/pycnidium was calculated. Generally, the formation of pycnidia and conidia was favored by nitrate more than by ammonium nitrogen sources. The average number of conidia/pycnidium was greatest, however, when the nitrogen source was NH₄NO₃. All amino acids tested appeared to be useful nitrogen sources for production of pycnidia but none were especially good for conidia production. L-isoleucine was superior to the other amino acids tested. Of three isolates used, Illinois 23 consistently produced more pycnidia and conidia that did isolates Minnesota 2 and Missouri 5. Usually the significant interactions between isolates and other treatments were due to a greater response by isolate Ill. 23. It was concluded that the reproduction ofP. medicaginis varies significantly with the isolates of the fungus, the environment, and nutrients as well as with interactions among these factors, and conclusions about the influence of a particular factor will depend on whether the formation of pycnidia, conidia or conidia/pycnidium is being studied.Paper No. 7425, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Productivity and selective accumulation of carotenoids of the novel extremophile microalga <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> grown with different carbon sources in batch systems

Cultivation of extremophile microorganisms has attracted interest due to their ability to accumulate high-value compounds. Chlamydomonas acidophila is an acidophile green microalga isolated by our group from Tinto River, an acidic river that flows down from the mining area in Huelva, Spain. This microalga accumulates high concentrations of lutein, a very well-known natural antioxidant. The aim of this study is to assess use of different carbon sources (CO₂, glucose, glycerol, starch, urea, and glycine) for efficient growth of and carotenoid production by C. acidophila. Our results reveal that growth of the microalga on different carbon sources resulted in different algal biomass productivities, urea being as efficient as CO₂ when used as sole carbon source (~20 g dry biomass m^–2 day^–1). Mixotrophic growth on glucose was also efficient in terms of biomass production (~14 g dry biomass m^–2 day^–1). In terms of carotenoid accumulation, mixotrophic growth on urea resulted in even higher productivity of carotenoids (mainly lutein, probably via α-carotene) than obtained with photoautotrophic cultures (70% versus 65% relative abundance of lutein, respectively). The accumulated lutein concentrations of C. acidophila reported in this work (about 10 g/kg dry weight, produced in batch systems) are among the highest reported for a microalga. Glycerol and glycine seem to enhance β-carotene biosynthesis, and when glycine is used as carbon source, zeaxanthin becomes the most accumulated carotenoid in the microalga. Strategies for production of lutein and zeaxanthin are suggested based on the obtained results.     

Biosynthesis of short-chain-length-polyhydroxyalkanoates during the dual-nutrient-limited zone by Ralstonia eutropha

Biosynthesis of PHAs by Raltonia eutropha during the dual nutrient-limitation-zone was investigated with mixed organic acids as carbon sources and (NH₄)₂SO₄ as nitrogen source. Two different methods of maintaining the dual-nutrient-limitation zone were adopted by feeding mixed acids and (NH₄)₂SO₄ at determined rates into the fermentation cultures which were initially free of carbon sources (method A) or nitrogen sources (method B). The results indicate that, firstly, with the increase of the width of the dual-nutrient-limitation zone, the yield of short-chain-length-polyhydroxyalkanoates also increases and it suggests that most of the short-chain-length-polyhydroxyalkanoates were biosynthesized during the dual-nutrient-limitation zone. Secondly, in contrast with the dual-nutrient-limitation method of limiting the nitrogen source first (method B), the dual-nutrient-limitation method of limiting the carbon source first (method A) was more favourable for the production of short-chain-length-polyhydroxyalkanoates, and the maximum production of short-chain-length-polyhydroxyalkanoates of these two methods are 3.72 and 2.55 g/l, respectively.     

Chemoautotrophic growth of Rhodopseudomonas species with hydrogen and chemotrophic utilization of methanol and formate

A chemolithoautotrophic type of metabolism, which was hitherto unknown for purple nonsulfur bacteria, was demonstrated by growth experiments using Rhodopseudomonas capsulata Kb1 and Rhodopseudomonas acidophila 10050. These strains were able to grow in a mineral medium in the dark at the expense of H₂, O₂, and CO₂. A minimum doubling time of 9 h was obtained for R. capsulata under an atmosphere containing less than 15% oxygen; higher oxygen concentrations suppressed autotrophic but not chemoorganotrophic growth. Oxygen sensitivity of chemoautotrophically growing cells of R. acidophila was even more pronounced, whereas cells growing chemotrophically on methanol almost tolerated the oxygen concentration of air. Highest oxygen sensitivity of growth of R. acidophila was observed with formate as substrate. The growth yield of cultures grown semiaerobically in the dark on methanol was 0.23 g dry cell material per g methanol consumed.     

Extracellular lipase production by a sapwood-staining fungus,Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH₄)₂SO₄ gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH₄)₂SO₄and peptone as nitrogen source.The authors are with Forest Products Biotechnology, Department of Wood Science, Facully of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada     

Nitrogen fixation by hydrogen-utilizing bacteria

Seventeen strains of nitrogen-fixing bacteria, isolated from different habitats on hydrogen and carbon dioxide as well as on other substrates, morphologically resembled each other. All strains, including Mycobacterium flavum 301, grew autotrophically with hydrogen. The isolate strain 6 was sensitive to oxygen when dependent on N₂ as nitrogen source, a consequence of the sensitivity of its nitrogenase towards oxygen. At the same time, strain 6 was sensitive to hydrogen when growing autotrophically on N₂ as nitrogen source, but hydrogen did not affect acetylene reduction by these cells.Abbreviations MPN Most probable number - BS medium basal salts medium     

Anaerobic Elemental Sulfur Reduction by Fungus Fusarium oxysporum

Reduction of inorganic sulfur compounds by the fungus Fusarium oxysporum was examined. When transferred from a normoxic to an anoxic environment, F. oxysporum reduced elemental sulfur to hydrogen sulfide (H₂S). This reaction accompanied fungal growth and oxidation of the carbon source (ethanol) to acetate. Over 2-fold more of H₂S than of acetate was produced, which is the theoretical correlation for the oxidation of ethanol to acetate. NADH-dependent sulfur reductase (SR) activity was detected in cell-free extracts of the H₂S-producing fungus, and was found to be up-regulated under the anaerobic conditions. On the other hands both O₂ consumption by the cells and cytochrome c oxidase activity by the crude mitochondrial fractions decreased. These results indicate that H₂S production involving SR was due to a novel dissimilation mechanism of F. oxysporum, and that the fungus adapts to anaerobic conditions by replacing the energy-producing mechanism of O₂ respiration with sulfur reduction.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29

Hydrogen and a bioflocculant could be produced simultaneously by anaerobic culture of Enterobacter sp. BY-29. For production of hydrogen and the bioflocculant by cell culture of the bacterium in batch cultures, cultivation at 37 °C in a medium containing glucose as a carbon source and Polypepton as a nitrogen source was found to be suitable. In continuous production of hydrogen and the bioflocculant by cell culture or immobilized cells of the bacterium, the hydrogen production rate and hydrogen yield by the immobilized cells on porous glass beads in stirred and column reactors were higher than those by the cell culture in a stirred reactor. However, production of the bioflocculant by the cell culture was superior to that by the immobilized cells in continuous production.     

Influence of carbon sources on the viability and resuscitation of Acetobacter senegalensis during high-temperature gluconic acid fermentation

Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l^− 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (q_s) and gluconate production (q_p) reduced progressively. Interestingly, gradual q_s and q_p reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles.

     

Effects of growth conditions of acetate utilization byRhodopseudomonas palustris isolated from a freshwater lake

Rhodopseudomonas palustris, a purple non-sulfur bacterium, was recently found throughout the water column in Lake Kinneret. It was demonstrated to be of a versatile nature, growing under both aerobic and anaerobic conditions at different light intensities. A comparison of C-acetate uptake byR. palustris andChlorobium phaeobacterioides, a green sulfur bacterium, showed that, under identical growth conditions, C-acetate assimilation byR. palustris was greater. Furthermore, C-acetate uptake forR. palustris was greater than C−CO₂ uptake at all light intensities. Depending on the prevailing conditions, acetate can be used byR. palustris as both an electron donor and carbon source. Malate synthase was used as an indicator of activity of the glyoxylic acid cycle. It was found that enzyme activity was higher (i.e., acetate was used mainly as a carbon source) under anaerobic conditions, in the dark, or in the absence of HCO ₃ ⁻ . Acetate was used preferably as an electron donor under photosynthetic microaerophillic conditions.     

The effect of anaerobiosis on the induced synthesis of β-galactosidase in non-growingEscherichia coli

Depending on conditions of aeration maltose and glucose were found to exhibit different effects on the inducible synthesis of β-galactosidase in aerobically grown cells ofEscherichia coli starving for an exogenous source of nitrogen; both saccharides repressed the synthesis of the enzyme under aerobic conditions, while the above-mentioned saccharides were essential for the enzyme synthesis under anaerobic conditions. The presence of maltose in the medium resulted in the repression of the enzyme synthesis in anaerobically grown cells starving for an exogenous nitrogen source under anaerobic conditions. The synthesis of β-galactosidase-specific messenger RNA was completely blocked and the synthesis of the enzyme proper considerably inhibited in aerobically grown cells incubated anaerobically in a medium without nitrogen and carbon sources.     

Hydrogen production from aromatic acids byRhodopseudomonas palustris

The capability of five strains of the phototrophic bacteriumRhodopseudomonas palustris to produce molecular hydrogen (H₂) from the aromatic acids benzoate,p-hydroxybenzoate, cinnamate and D- and L-mandelate was investigated. Optimal H₂ production was achieved when the strains were grown anaerobically in the light at 10,000 lx under nitrogen (N) limitation using 1 mM L-glutamate as an N source. In the presence of 2 mM benzoate or L-mandelate as carbon and electron sources, strain DSM 131 produced 45% H₂ of the maximal theoretical value and strain F2 32%, respectively. Increased H₂ production correlated with increased nitrogenase activities, but H₂ formation was not further stimulated by inhibition of the H₂ uptake (hup) hydrogenase with ethylenediaminetetraacetic acid (EDTA).