Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves

An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 0.2 m KNO₃ (with or without phosphate buffer, pH 7.5) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark. Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals.     

The role of cyclic photophosphorylation in vivo

When cyclic photophosphorylation is inhibited in Chlorella vulgaris cells by carbonylcyanide-trifluoromethoxy phenylhy-drazone, photosynthetic CO₂-fixation under anaerobic conditions exhibits a distinct lag. Under the same conditions, the light-dependent formation of ribulose diphosphate shows also this lag. It is concluded that cyclic photophosphorylation is required to fill up the pools of phosphorylated intermediates of the Calvin cycle at a time when noncyclic photophosphorylation cannot yet efficiently operate. Under aerobic conditions, the initial energy demand can be accommodated by respiratory ATP or cyclic photophosphorylation or both. Evidence for stoichiometric participation of cyclic photophosphorylation in photosynthesis is still lacking.     

The ability of aquatic macrophytes to increase root porosity and radial oxygen loss determines their resistance to sediment anoxia

Radial oxygen loss (ROL) has been suggested to be a major process to protect plants exposed to root anaerobic stress. In the present study, we aimed to test the importance of root porosity and radial oxygen loss on the aquatic macrophyte resistance to sediment anoxia. We expected that species living in eutrophic environments characterized by anaerobic conditions in sediments exhibited higher root porosity and radial oxygen loss than species restrained to oligotrophic environments. In this way, we compared the responses to sediment anoxia of two hydrophyte species growing under meso-eutrophic conditions in the field (Myriophyllum spicatum L. and Vallisneria spiralis L.) and three species growing under oligotrophic conditions (Potamogeton coloratus Horne, Elodea canadensis Michx and Sparganium emersum Michx.). Under laboratory conditions, ROL, root porosity, plant metabolism (aerobic respiration, photosynthesis, root fermentative activity) and plant growth were analysed after 3?months of acclimation in anaerobic sediments and compared with control values obtained from aerobic sediments. The results showed that two meso-eutrophic species (M. spicatum and V. spiralis) survived in anaerobic sediments and maintained similar photosynthesis rates than those measured under aerobic conditions. In contrast, the three oligotrophic species (P. coloratus, E. canadensis and S. emersum) suffered net biomass loss and depressed their photosynthesis rates under anaerobic conditions. All variables associated with plant tolerance to anaerobic conditions (maintenance of photosynthesis, aerobic respiration and growth rate, and limitation of root fermentative activity) were positively linked to root porosity and ROL. According to our hypothesis, species that could survive to anaerobic conditions were the species able to increase their root porosity and ROL under these conditions. Thus, in ecological studies, it would be useful to use the root porosity and ROL plasticity as biological traits in order to model the distribution of macrophytes in river floodplains.     

Assimilation of [N]Nitrate and [N]Nitrite in Leaves of Five Plant Species under Light and Dark Conditions

Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resolving this controversy, the light requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of [¹⁵N]nitrate and [¹⁵N]nitrite to ammonium and amino-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of [¹⁵N]nitrate as a percentage of the light rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimilation of [¹⁵N]nitrite as a percentage of the light rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explains some of the contradictory results in the literature, but additional explanations must be sought to fully resolve the controversy.

In dark anaerobic conditions, the assimilation of [¹⁵N]nitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of [¹⁵N]nitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditions. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of [¹⁵N]nitrite assimilation.

     

Photoactivation of Chlorophyll Synthesis and Cytochrome Oxidase Activity in Anaerobically Germinated Seedlings of Echinochloa crusgalli var. Oryzicola

When seeds of Echinochloa crusgalli var. oryzicola are germinated in dark anaerobic conditions (99.995% N₂), the seedlings do not have detectable protochlorophyll(ide). Two hours after exposure to light aerobic conditions, they begin to synthesize chlorophyll. The lag in greening is shorter in seedlings exposed to light for 24 hours before exposure to air. Seedlings maintained in light anaerobic conditions exhibit no lag in greening upon transfer to an aerobic environment. Preillumination of anaerobically grown seedlings does not result in any chlorophyll accumulation. Phytochrome is probably the receptor for photoactivation of chlorophyll synthesis, since activation is achieved by red light alone, but not by far red light or red plus far red light. The cytochrome oxidase activity in anaerobically germinated seedlings is 30% of the normal level found in aerobically grown seedlings. Preillumination was also found to activate the ability of anaerobically germinated seedlings to increase their cytochrome oxidase activity upon exposure to air.     

Effect of chlorate,molybdate, and shikimic acid on Salmonella enterica serovar Typhimurium in aerobic and anaerobic cultures

Experiments were conducted to determine factors that affect sensitivity of Salmonella enterica serovar Typhimurium to sodium chlorate (5 mM). In our first experiment, cultures grown without chlorate grew more rapidly than those with chlorate. An extended lag before logarithmic growth was observed in anaerobic but not aerobic cultures containing chlorate. Chlorate inhibition of growth during aerobic culture began later than that observed in anaerobic cultures but persisted once inhibition was apparent. Conversely, anaerobic cultures appeared to adapt to chlorate after approximately 10 h of incubation, exhibiting rapid compensatory growth. In anaerobic chlorate-containing cultures, 20% of total viable counts were resistant to chlorate by 6 h and had propagated to 100% resistance (>10⁹ CFU mL^?1) by 24 h. In the aerobic chlorate-containing cultures, 12.9% of colonies had detectable resistance to chlorate by 6 h, but only 1% retained detectable resistance at 24 h, likely because these cultures had opportunity to respire on oxygen and were thus not enriched via the selective pressure of chlorate. In another study, treatment with shikimic acid (0.34 mM), molybdate (1 mM) or their combination had little effect on aerobic or anaerobic growth of Salmonella in the absence of added chlorate. As observed in our earlier study, chlorate resistance was not detected in any cultures without added chlorate. Chlorate resistant Salmonella were recovered at equivalent numbers regardless of treatment after 8 h of aerobic or anaerobic culture with added chlorate; however, by 24 h incubation chlorate sensitivity was completely restored to aerobic but not anaerobic cultures treated with shikimic acid or molybdate but not their combination. Results indicate that anaerobic adaptation of S. Typhimurium to sodium chlorate during pure culture is likely due to the selective propagation of low numbers of cells exhibiting spontaneous resistance to chlorate and this resistance is not reversible by molybdenum supplementation.     

Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactors

The development of high‐performance photobioreactors equipped with automatic systems for non‐invasive real‐time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light‐induced fluorescence rise and the dark relaxation of the flash‐induced fluorescence yield (Qa^? ? re‐oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas‐tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long‐term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress‐sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real‐time monitoring of algal photosynthesis in photobioreactors.     

Effect of glutamate on the degradation of pentachlorophenol by Flavobacterium sp.

Summary The degradation of pentachlorophenol (PCP) by a Flavobacterium sp. was investigated by inoculating induced cells into cultures containing PCP alone or PCP and glutamate as carbon sources. Using PCP as the sole carbon source, the degradation activity increased with PCP concentration. However, a lag phase was observed and this lag was more pronounced at higher PCP concentrations. Exposure of cells to higher PCP concentrations during induction did not reduce the lag. The presence of glutamate reduced the lag in PCP degradation. Such an elimination of the lag phase appears to be due to maintaining cell viability with the presence of glutamate. Offprint requests to: W.-S. Hu     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

Errors in measurements of aquatic macrophyte gas exchange due to oxygen storage in internal airspaces

Dissolved oxygen measurements of respiratory and photosynthetic gas exchanges of the aquatic angiosperm Egeria densa Planch. and vesicles of the macroalga Carpophyllum maschalocarpum (Turn.) Grev. were made using a Clark-type oxygen electrode in a well-stirred closed chamber. Comparisons were made of dark—light and light—dark transients and light and dark oxygen exchanges for intact material and the same material with the internal airspaces flooded.For Egeria, there was an apparently greater gas exchange (up to 17% for photosynthesis and 50% for respiration) for infiltrated material. The duration of transient lags following light and dark treatments remained unchanged by infiltration, indicating that the storage error could not be detected from lag duration. The storage error during photosynthesis agreed closely with theoretical predictions based on oxygen solubility, but additional factors contribute to the dark error. Carpophyllum results were unpredictable from solubility calculations as the thick vesicle walls restricted exchange between the internal and external phases, allowing internal oxygen storage only at high external partial pressures.The widespread assumption that short lag phases indicate negligible internal oxygen storage in the gas spaces is questioned on the basis of these results. Solutions to the storage error are proposed.     

Microbial degradation of pentachlorophenol

Pentachlorophenol (PCP) was the most prevalent wood preservative for many years worldwide. Its widespread use had led to contamination of various environments. Traditional methods of PCP clean-up include storage in land-fill sites, incineration and abiotic degradation processes such as photodecomposition. Some aerobic and anaerobic microorganisms can degrade PCP under a variety of conditions. Axenic bacterial cultures, Flavobacterium sp., Rhodococcus sp., Arthrobacter sp., Pseudomonas sp., Sphingomonas sp., and Mycobacterium sp., and fungal cultures, Phanerochaete sp. and Trametes sp. exhibit varying rates and extent of PCP degradation. This paper provides some general information on properties of PCP and reviews the influence of nutrient amendment, temperature and pH on PCP degradation by various aerobic and anaerobic microorganisms. Where information is available, proposed degradation pathways, intermediates and enzymes are reviewed.     

Anaerobic heterotrophic dark metabolism in the cyanobacterium Oscillatoria limnetica: Sulfur respiration and lactate fermentation

The cyanobacterium Oscillatoria limnetica, capable of anoxygenic photosynthesis in the light with sulfide as electron donor can anaerobically break down its intracellular polyglucose in the dark. In the absence of elemental sulfur, the organism carries out lactate fermentation; in its presence, anaerobic respiration occurs in which sulfur is reduced to sulfide. Induction of anoxygenic photosynthesis or synthesis of new proteins is not necessary for either process. Cells adapted in the dark to sulfur reduction are capable of anoxygenic photosynthesis during a subsequent light period, unless protein synthesis has been inhibited during the dark incubation period.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP Carbonylcyanide p-trifluoromethoxyphenylhydrazone - mgat milligramatom - OD optical density     

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [¹⁵N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O₂ inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.     

A comprehensive overview of bacteria and fungi used for pentachlorophenol biodegradation

Pentachlorophenol (PCP) is an extremely dangerous worldwide pollutant due to its high toxicity towards all organisms. It has been introduced into the environment mainly as a wood preservative, biocides and from the bleaching of paper or tissues. The use of PCP indiscriminate has led to the contamination of water and soil systems. Many countries have specific regulations, guidelines or procedures for the management and disposal of PCP but the most common methods are: adsorption with activate carbons, incineration in an approved and secure area, closed in sealed containers and biological degradation. PCP depletion can occur either by abiotic processes such as: absorption, volatilization and photo degradation or by biotic degradation. One of the main studies focused on remediation using plants, animals and microbial communities. Aerobic and anaerobic microorganisms can degrade PCP under a variety of conditions and at different PCP concentrations. Bacterial strains such as Pseudomonas sp., Sphingomonas sp., Arthrobacter sp., Mycobacterium sp., Flavobacterium sp., Serratia sp. and Bacillus sp., and fungal cultures as Trametes sp., Phanerochaete sp., Anthracophyllum sp., Armillaria sp., Bjerkandera sp., Ganoderma sp., Lentinula sp., Penicillium sp, Trichoderma sp., Rhizopus sp. and Plerotus sp. showed various rates and extent of PCP degradation. This review focuses on PCP degradation by various aerobic and anaerobic microorganisms with emphases on the biological and chemical aspects. Furthermore we will analyze intermediate products, processes and enzymes involved in the degradation of PCP in different environmental conditions and at various PCP concentrations.     

Remediation of PCE-contaminated groundwater from an industrial site in southern Italy: A laboratory-scale study

Batch reactors and microcosms were used to evaluate groundwater bioremediation potential of tetrachloroethene (PCE) in the presence of additional pollutants present at a site located in the Apulia Region (SE Italy). Reductive dechlorination of PCE was studied under anaerobic conditions by comparing the effectiveness of three inocula: (a) soil sampled at the contaminated site, (b) anaerobic sludge from a municipal wastewater plant, and (c) an enriched dehalogenating culture containing Dehalococcoides species. In order to enhance dehalogenation, reactors inoculated with sludge were also amended with selected electron donors. Aerobic reactors were also established to study oxidative degradation of vinyl chloride (VC), that may accumulate after incomplete dechlorination of PCE.Results showed that consortia derived from anaerobic sludge and amended with electron donors quantitatively and incompletely degraded PCE to cis-dichloroethylene, whereas in reactors augmented with a dehalogenating culture complete dechlorination of PCE occurred even in the presence of additional toxic contaminants. The presence of Dehalococcoides spp. in the dehalogenating culture and its absence in reactors inoculated with anaerobic sludge was confirmed using FISH community analyses. In all cases, prolonged incubation periods were necessary for dechlorination. On the other hand, oxidative degradation of VC in aerobic reactors occurred after short lag times.     

Photoheterotrophic Metabolism of Acrylamide by a Newly Isolated Strain of Rhodopseudomonas palustris

Acrylamide, a neurotoxin and suspected carcinogen, is produced by industrial processes and during the heating of foods. In this study, the microbial diversity of acrylamide metabolism has been expanded through the isolation and characterization of a new strain of Rhodopseudomonas palustris capable of growth with acrylamide under photoheterotrophic conditions. The newly isolated strain grew rapidly with acrylamide under photoheterotrophic conditions (doubling time of 10 to 12 h) but poorly under anaerobic dark or aerobic conditions. Acrylamide was rapidly deamidated to acrylate by strain Ac1, and the subsequent degradation of acrylate was the rate-limiting reaction in cell growth. Acrylamide metabolism by succinate-grown cultures occurred only after a lag period, and the induction of acrylamide-degrading activity was prevented by the presence of protein or RNA synthesis inhibitors. ¹³C nuclear magnetic resonance studies of [1,2,3-¹³C]acrylamide metabolism by actively growing cultures confirmed the rapid conversion of acrylamide to acrylate but failed to detect any subsequent intermediates of acrylate degradation. Using concentrated cell suspensions containing natural abundance succinate as an additional carbon source, [¹³C]acrylate consumption occurred with the production and then degradation of [¹³C]propionate. Although R. palustris strain Ac1 grew well and with comparable doubling times for each of acrylamide, acrylate, and propionate, R. palustris strain CGA009 was incapable of significant acrylamide- or acrylate-dependent growth over the same time course, but grew comparably with propionate. These results provide the first demonstration of anaerobic photoheterotrophic bacterial acrylamide catabolism and provide evidence for a new pathway for acrylate catabolism involving propionate as an intermediate.     

The interaction of nitrite with photosynthetic electron transport under anaerobic conditions

Chlorella cells were examined in a modulated oxygen polarograph under aerobic and anaerobic conditions. At light intensities below about 600 ergs · cm^?2 · s^?1 of 650 nm light, the oxygen yield and phase lag are lower under anaerobic conditions. Addition of 25 mM sodium nitrite increases both these parameters to values close to those found in the presence of oxygen. It is proposed that nitrite is reduced by Photosystem I thus diverting electrons from the cyclic electron transport pathway. The intersystem electron transport chain becomes more oxidized and this suppresses a backflow of electrons to the oxidizing side of Photosystem II, hence increasing the oxygen yield and the phase lag. The removal of oxygen from the bathing medium also alters the response of dark adapted _Chlorella to a series of saturating light flashes. In terms of the Kok model of Photosystem II (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) there is a large increase in the parameter α. Addition of nitrite reverses this change and virtually restores the response seen in the presence of oxygen. The deactivation of the S₂ state is greatly speeded up in the absence of oxygen but the addition of nitrite again reverses this.     

Aerobic and Anaerobic Biodegradation of Aged Pentachlorophenol by Indigenous Microorganisms

Pentachlorophenol (PCP) use as a general biocide, particularly for treating wood, has led to widespread environmental contamination. Biodegradation has emerged as the main mechanism for PCP degradation in soil and groundwater and a key strategy for remediation. Examining the microbial biodegrading potential for PCP at a contaminated site is crucial in determining its fate. Hundreds of studies have been published on PCP microbial degradation, but few have described the biodegradation of PCP that has been in contact with soils for many years. The bioavailability of “aged” hydrophobic organics is a significant concern. PCP- and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)-contaminated soil samples from several depths at a former wood treatment site were placed under varying conditions in the laboratory to determine the anaerobic and aerobic potential for biodegradation of chlorophenols at the site. PCP biodegradation occurred in both anaerobic and aerobic soil samples. Rapid aerobic degradation occurred in samples spiked with 2- and 4-chlorophenol, but not with 3-chlorophenol. Reductive dechlorination of PCP in anaerobic samples resulted in the accumulation of 3-chlorophenol. In most anaerobic replicates, 3-chlorophenol was degraded with the appearance of detectable, but not quantifiable amounts of phenol. These results indicate excellent potential for remediation at the site using the indigenous microorganisms under both aerobic and anaerobic conditions. However, a fraction of the PCP was unavailable for degradation.     

THE INDUCTION PERIOD IN PHOTOSYNTHESIS

1. Measurements on the photosynthesis of Cabomba caroliniana show an induction period at low and high light intensities and CO₂ concentrations. 2. The equation which describes the data for Cabomba also describes the data obtained by other investigators on different species. The phenomenon is thus shown to be similar in plants representative of three phyla. 3. A derivation of the induction period equation is made from a consideration of the cycle of light and dark processes known to occur in photosynthesis. The equation indicates that light intensity enters as the square, and that the same light reactions are involved as those which affect the stationary state rates. However, a different dark reaction appears to limit photosynthesis during the induction period.