A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria

A simple effective and compact freeze-drying method involving skim milk 20% (w/v) and glutamate 5% or meso-inositol 5% or honey 10% or raffinose 5% for the long-term preservation of bacteria is described. As a case example more than 160 strains representing 36 species of nitrogen-fixing bacteria, 11 species of chemolithorutotrophic bacteria and five species of Aquaspirillum were successfully preserved. All tested strains proved viable and showed about 10–100% survival after freeze-drying and during 2–3 years of storage at +9°C. In such lyophilized cultures no loss in plasmids or other desirable characters was observed. The method is also suitable for the preservation of other fragile and difficult microorganisms as several other strains including bacteria with introduced plasmids could equally survive well and retained plasmids after lyophilization with this method.     

Arsenic respiration and detoxification by purple non-sulphur bacteria under anaerobic conditions

Two arsenic-resistant purple non-sulphur bacteria (PNSB), Q3B and Q3C, were isolated (from industrial contaminated site and paddy fields) and identified by SSU rRNA gene sequencing as Rhodospirillum and Rhodospirillaceae species, respectively. Maximum arsenic reduction by these PNSB was observed in anaerobic conditions. Rhodospirillum sp. Q3B showed 74.92% (v/v) arsenic reduction while Rhodospirillaceae sp. Q3C reduced arsenic up to 76.67% (v/v) in anaerobic conditions. Rhodospirillaceae sp. Q3C was found to contain highest carotenoid content up to 5.6 mg·g⁻¹. Under anaerobic conditions, the isolates were able to respire arsenic in the presence of lactate, citrate, and oxalate. Rhodospirillum sp. Q3B and Rhodospirillaceae sp. Q3C were also found to produce hydrogen gas. Such diverse bacteria can be useful tools for bioremediation purposes. These bacteria can be further exploited and optimized to treat wastewater containing arsenic along with bio-hydrogen production.     

A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions

A simple, effective and economical method for the long-term preservation of bacteria in liquid nitrogen under anaerobic conditions is described. As a case example anaerobic photosynthetic bacteria were successfully preserved. Gas tight small screw-cap glass ampoules with butyl rubber septa were used for freezing the specimen anaerobically. During experimental manipulations no anaerobic chamber or glove boxes were required. All teste cultures yielded high recoveries after repeated thawing and during storage. After freezing, survival recoveries of Rhodospirillaceae range from 70–100%, whereas with strict anaerobic strains of Chlorobiaceae and Chromatiaceae a maximum loss of 1–2 log₁₀ counts was observed. No further loss in viability occurred after 1–2 years of storage.The small size of the ampoules and the use of single ampoule for 15–20 repeated retrievals proved economical with respect to storage space and costs.The system is compact and suitable for the preservation of anaerobic phototropic bacteria and other fragile anaerobic microorganisms.     

Bacteriochlorophyll and Photosynthetic Reaction Centers in Rhizobium Strain BTAi 1

Rhizobium strain BTAi 1, which nodulates both stems and roots of Aeschynomene indica L., formed bacteriochlorophyll and photosynthetic reaction centers resembling those of purple photosynthetic bacteria when grown aerobically ex planta under a light-dark cycle. Bacteriochlorophyll formation was not observed under continuous dark or light growth conditions. The amount of pigment formed was similar to that previously found in aerobic photosynthetic bacteria. Stem nodules appear to fix nitrogen photosynthetically, as illumination of A. indica stem nodules with near-infrared light resulted in an enhanced rate of acetylene reduction. Near-infrared light did not enhance acetylene reduction when either A. indica or soybean root nodules were illuminated. The BTAi 1 isolate can be differentiated from members of the family Rhodospirillaceae by several criteria.     

Effective technological pectinases by Aspergillus carneus NRC1 utilizing the Egyptian orange juice industry scraps

The production of a notable and highly effective pectinase by the local fungal strain Aspergillus carneus NRC1 utilizing the abundant Egyptian orange peels and pulps (OPP) scraps excluded in the orange juice and canning industry was achieved in 5-day submerged fermentation (SMF) cultures, at temperature and pH ranges of 30–55 °C and 5.0–5.5, respectively. Fresh or thawed OPP (6%, w/v) were the most preferable sole carbon source. Pectinase activity was dramatically stimulated by ammonium sulphate as the sole nitrogen source, and at the same time strongly inhibited the production of the other tested enzymes, i.e., cellulases and hemicellulases. The lyophilized enzyme preparation was free from any ochra or aflatoxins. The optimum conditions of this methodology including enzyme and substrate (citrus pectin) concentrations were 40 mg ml^?1 and 7% (w/v), respectively, with pH and temperature of 4.0 and 55 °C, respectively.     

Influence of carbon source on growth,biochemical composition and pigmentation of Ankistrodesmus convolutus

The unicellular chlorophyte Ankistrodesmus convolutus Corda was grown in the light using inorganic medium (Bold's Basal Medium, BBM) and BBM enriched with 0.1% w/v of glucose, sodium acetate, sodium citrate or sodium bicarbonate. Glucose supported the highest specific growth rate (μ = 0.93 d^-1) and gave the highest biomass (453 mg dry weight L^-1) at the time of harvest. Of four glucose concentrations (0.05, 0.1, 0.25, 0.5% w/v), best growth was attained at 0.1% w/v. At 0.5% w/v glucose, the cells had high carbohydrates but low lipids and proteins. The relative amounts of 16:0, 18:0, 18:1 and 18:2 increased at the expense of 18:3(n-3) in the carbon-supplemented cultures and at glucose concentrations higher than 0.1% w/v. Cultures grown on glucose had less chlorophyll and carotenoid contents than cultures grown on other carbon sources. Chlorophyll and carotenoid contents decreased with increasing glucose concentrations in the medium.     

A modified method for the cultivation of phototrophic bacteria

Simplified anaerobic media and a convenient method for the cultivation of Rhodospirillaceae, Chlorobiaceae, Chloroflexaceae and Chromatiaceae are described. The modified conditions assure almost complete anaerobiosis for media, growth and maintenance.Strains representing several species of Rhodospirillaceae, Chlorobiaceae and Chromatiaceae were successfully grown within relatively short times with full pigmentation, indicating that the new media and cultivation conditions were most suited for photoautotrophic growth.     

Effect of darkness on isoperoxidases in tobacco tissue cultures

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (C_n) appeared in the medium within 2 days after the cells were placed in the dark. C_n was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). C_n increased and new anodic isoperoxidases A_a, A_b, A_d and A_e appeared in the media as the cells approached death in darkness.     

Expression of the Mucus Adhesion Gene Mub, Surface Layer Protein Slp and Adhesion-Like Factor EF-TU of Lactobacillus acidophilus ATCC 4356 Under Digestive Stress Conditions, as Monitored with Real-Time PCR

Expression of the mucus adhesion gene Mub, surface layer protein Slp and adhesion-like factor EF-Tu by Lactobacillus acidophilus ATCC 4356 grown in the presence of mucin, bile and pancreatin and at low pH was studied using real-time PCR. None of the genes were up-regulated under increasing concentrations of mucin, while Slp and EF-Tu were up-regulated in the presence of bile and pancreatin at normal concentrations (0.3%, w/v) and under stress conditions (1.0%, w/v).     

Factors Affecting Phage D29 Infection: A Tool to Investigate Different Growth States of Mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay.     

Induction and Regulation of Expression of a Low-CO2-Induced Mitochondrial Carbonic Anhydrase in Chlamydomonas reinhardtii

The time course of and the influence of light intensity and light quality on the induction of a mitochondrial carbonic anhydrase (CA) in the unicellular green alga Chlamydomonas reinhardtii was characterized using western and northern blots. This CA was expressed only under low-CO₂ conditions (ambient air). In asynchronously grown cells, the mRNA was detected 15 min after transfer from air containing 5% CO₂ to ambient air, and the 21-kD polypeptide was detected on western blots after 1 h. When transferred back to air containing 5% CO₂, the mRNA disappeared within 1 h and the polypeptide was degraded within 3 d. Photosynthesis was required for the induction in asynchronous cultures. The induction increased with light up to 500 μmol m⁻² s⁻¹, where saturation occurred. In cells grown synchronously, however, expression of the mitochondrial CA was also detected in darkness. Under such conditions the expression followed a circadian rhythm, with mRNA appearing in the dark 30 min before the light was turned on. Algae left in darkness continued this rhythm for several days.     

Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor

Cells of the obligately lithotrophic species Nitrosomonas europaea and Nitrosomonas eutropha were able to nitrify and denitrify at the same time when grown under oxygen limitation. In addition to oxygen, nitrite was used as an electron acceptor. The simultaneous nitrification and denitrification resulted in significant formation of the gaseous N-compounds nitrous oxide and dinitrogen, causing significant nitrogen loss. In mixed cultures of N. europaea and various chemoorganotrophic bacteria, the nitrogen loss was strongly influenced by the partners growing under oxygen limitation. Under anoxic conditions, pure cultures of N. eutropha were able to denitrify with molecular hydrogen as electron donor and nitrite as the only electron acceptor in a sulfide-reduced complex medium. The increase of cell numbers was directly coupled to nitrite reduction. Nitrous oxide and dinitrogen were the only detectable end products. In pure cultures of N. eutropha and mixed cultures of N. eutropha and Enterobacter aerogenes, ammonium and nitrite disappeared slowly at a molar ratio of about one when oxygen was absent. However, under these conditions cell growth was not measurable.     

NMR-based metabonomic analysis on effect of light on production of antioxidant phenolic compounds in submerged cultures of Inonotus obliquus

This study was designed to investigate the light effect on biosynthesis of antioxidant phenolic compounds by Inonotus obliquus grown in submerged cultures using ¹H NMR spectroscopy combining multivariate pattern recognition strategies. I. obliquus were exposed to a range of light conditions and resultant data were compared to those from field-grown sclerotia and the mycelia grown in daylight. Daylight illumination inhibited biosynthesis of davallialactone and phelligridins and other hispidin analogs. Continuous darkness enhanced the formation of phelligridins, davallialactone and inoscavins. Phelligridins and davallialactone also occurred in the mycelia grown in blue and red light with levels lower than those found in darkness. In addition, polyphenols synthesized under daylight conditions showed less potential antioxidant activity than those determined with other light regimes. These findings demonstrate that light regulates biosynthesis of polyphenols in I. obliquus and their subsequent antioxidant activities, and ¹H NMR-based metabolic profiling is a cost-effective approach for evaluating light effects on fungal metabolisms.     

Photoregulation of Phosphoenolpyruvate Carboxylase in Salsola soda L. and Other C(4) Plants

Photoactivation of phosphoenolpyruvate carboxylase was found to occur in several, though not all, C₄ species examined; Salsola soda L. was used for a detailed study of this effect of light.

Activity differences between light and darkness are maximized when glycerol (25% v/v) is included in the extraction medium and in the absence of mercaptoethanol. In plants grown in the growth chamber, the night-form of the enzyme, in addition to low activity, shows a positive cooperativity (with phosphoenolpyruvate), which is gradually abolished by light of increasing intensities. This allosteric behavior is absent in plants adapted to a high light environment. Activation and deactivation, under light and darkness respectively, are quite fast, suggesting post-translational regulation. The photoactivation appears to depend on photosynthetic electron flow, since it is saturated at high photon fluxes (around 1000 microeinsteins per square meter per second) and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

     

Rapid recovery of photosynthetic cell suspension cultures following heterotrophic bleaching

Summary Seven suspension-cultured lines of five different species (Amaranthus powellii Datura innoxia, Glycine max, Gossypium hirsutum, andNicotiana tabacum × Nicotiana glutinosa fusion hybrid), which had been grown under photomixotrophic conditions, were placed under heterotrophic conditions (darkness and media with 3% sucrose or starch) where the chlorophyll levels declined to near zero. After three transfers over a 70-d period, the cells were placed back into photomixotrophic or photoautotrophic conditions where regreening occurred rapidly and continued growth was observed. This rapid adaptation to photosynthetic conditions contrasts with the original initiation process for these cultures, which required many months and an apparent selection since many of the original cells died. Thus, these seven photosynthetic cell suspension cultures appear to be different from the original cultures due possible to genetic or adaptive changes.     

Aerobiosis and the hemoprotein content of photosynthetic bacteria

Summary Rhodospirillum rubrum and Rhodopseudomonas spheroides, grown under various degrees of illumination, aeration, and iron deprivation, have been assayed for their content of cytochrome c, RHP, catalase, total iron, bacteriochlorophyll, and carotenoids.Concentrations of bacteriochlorophyll and carotenoids were consistent with the findings of Cohen-Bazire et al. (1957).Total iron content, which ranged from 0.017 to 0.04% of the dry weight, reflected the content of the principal hemoproteins but exceeded the amount of iron in these hemoproteins.The catalase content of R. rubrum, on a dry weight basis, was 0.0005% for cells grown anaerobically in the light, and 0.0028% for cells grown in darkness with vigorous aeration; that of Rps. spheroides was 0.006% and 0.25%, respectively. The catalase content in both species rose with increasingly vigorous aeration.Cytochrome c in both species, and RHP in R. rubrum, attained the same levels in cells grown under vigorous aeration as in cells grown anaerobically in the light. In cells grown under limited aeration the levels of these substances were about 50% higher. In Rps. spheroides the RHP content was greatest in cells grown anaerobically, falling under gentle aeration and declining further under more vigorous aeration.Iron deficiency caused a decrease in the catalase content of cells grown anaerobically in the light but not in cells grown aerobically. The content of cytochrome c and of RHP was diminished by iron depletion in aerobic cultures, but not in anaerobic cultures.operated by Union Carbide Corporation for the U.S.Atomic Rnergy Commission.     

Morphological stability of Pimpinella anisum hairy root cultures and time-course study of their essential oils

The morphological stability of hairy root cultures of Pimpinella anisum was studied using cultures grown in four different media, both in darkness and under photoperiod conditions of 16 h light; they were first subcultured every eight days and then later on every three weeks. From the four media tested, the hairy root cultures grown in SH medium both in darkness and under photoperiod conditions showed a marked morphological stability with no de-differentiation or greening, when compared with the other culture systems. The time-course study of the essential oils, isolated by distillation-extraction and analysed by GC and GC-MS, showed only quantitative differences in the composition of the oils. A clear heterogeneity in the accumulation pattern was found with regard to the five dominant compounds (pregeijerene, geijerene, zingiberene, -bisabolene and trans-epoxypseudoisoeugenyl 2-methylbutyrate), i.e., those that appeared in a relative amount higher than 5% at least once during the time-course study of the oils from the eight culture systems.     

Tolerance for Prolonged Darkness of Three Phytoplankton Species, Microcystis aeruginosa (Cyanophyceae), Scenedesmus quadricauda (Chlorophyceae), and Melosira ambigua (Bacillariophyceae)

Phytoplankton community dynamics are affected not only by atural events such as overwintering but also by artificial events such as artificial circulation and related darkness conditions. In order to clarify the effect of tolerance for prolonged darkness on community succession, laboratory cultures of three phytoplankton taxa, Microcystis aeruginosa(Cyanophyceae), Scenedesmus quadricauda(Chlorophyceae), and Melosira ambigua(Bacillariophyceae) were carried out in darkness. The period of darkness was varied: 5, 10, 15, 20 days, and the control. Thereafter, all samples were reilluminated. After more than 10 days of darkness, M. aeruginosa decreased markedly with the length of the darkness period and was reduced to only 1% of the initial cell number after 20 days darkness. In contrast, S. quadricauda and M. ambigua retained their biomass even after 20 days of darkness. After restarting the light–dark cycle, however, all three species similarly increased exponentially and reached their maximum biomass levels. These results suggest that differences in tolerance for prolonged darkness may cause the succession of the phytoplankton under certain conditions.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Tepidibacillus fermentans gen. nov., sp. nov.: a moderately thermophilic anaerobic and microaerophilic bacterium from an underground gas storage

A novel moderately thermophilic bacterium, strain STGH^T, was isolated from Severo-Stavropolskoye underground gas storage (Russia). Cells of strain STGH^T were spore-forming motile straight rods 0.3 μm in diameter and 2.0–4.0 μm in length having a Gram-positive cell wall structure. The temperature range for growth was 36–65 °C, with an optimum at 50–52 °C. The pH range for growth was 5.5–8.0, with an optimum at pH 7.0–7.5. Growth of strain STGH^T was observed at NaCl concentrations ranging from 0 to 4.0 % (w/v) with an optimum at 1.0 % (w/v). Strain STGH^T grew anaerobically by reduction of nitrate, thiosulfate, S⁰ and AQDS using a number of complex proteinaceous compounds, organic acids and carbohydrates as electron donors. Nitrate was reduced to nitrite; thiosulfate and sulfur were reduced to sulfide. It also was able to ferment pyruvate, glucose, fructose, and maltose. The strain STGH^T did not grow under aerobic conditions during incubation with atmospheric concentration of oxygen but was able to microaerobic growth (up to 10 % of oxygen in gas phase). The G+C content of DNA of strain STGH^T was 34.8 mol%. 16S rRNA gene sequence analysis revealed that the isolated organism belongs to the class Bacilli. We propose to assign strain STGH^T to a new species of a novel genus Tepidibacillus fermentans gen. nov., sp.nov. The type strain is STGH^T (=DSM 23802^T, =VKM B-2671^T).