OBSERVATIONS ON PHOTOHETEROTROPHY IN A MARINE DIATOM1

An unidentified species of the diatom genus Cocconeis has been isolated from the sediments of Biscayne Bay, Florida. The organism is capable of utilizing a range of organic substrates, including lactate, in the light but not in the dark, as shown by growth studies. Information is included on changes in cell carbon, nitrogen, and chlorophyll a during growth in batch culture. Data obtained on the kinetics of uptake of lactate and glucose raise questions on the possible ecological significance of photoheterotrophy among marine microalgae, particularly those in estuaries.     

Heterotrophic nutrition of the marine pennate diatom Navicula pavillardi Hustedt.

Navicula pavillardi Hustedt, a marine, littoral, pennate diatom, can grow in the dark on glutamate or on the complex organic supplements tryptone or yeast extract. Growth on glutamate in the dark took place without an initial lag phase, whereas growth on tryptone began only after a 2-day lag phase that could be abolished by the simultaneous presence of glucose. Lactate inhibited growth in the dark on glutamate, but not photoautotrophic growth. Relatively low concentrations of glutamine inhibited photoautotrophic growth. The observed doubling time for heterotrophic growth on glutamate or tryptone was about 70 h, compared with a doubling time of 24 h under optimal photoautotrophic conditions. Glucose did not decrease the doubling time in the dark on tryptone. The assimilation efficiency for glutamate was 41%. The estimated necessary uptake rate for glutamate to account for the observed heterotrophic doubling time on glutamate was close to those measured with isotope techniques. The kinetic parameters for glutamate uptake, which followed Michelis-Menten kinetics, were Ks = 0.018 mM, and Vmax = 7.0 X 10(-10) mumol per cell per minute. Although several amino acids served as sole nitrogen sources for photoautotrophic growth and were demonstrated by the use of isotope techniques to enter the cells, they could not be used as substrates for growth in the dark. Glucose was not taken up to a significant extent except by cells grown in the presence of tryptone. Lactate was taken up only by dark-grown cells. Results of preliminary studies on the metabolic fate of several uniformly labeled amino acids are presented.     

Ability of casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461

The ability of casamino acids and vitamin-assay casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461 was examined in a medium containing glucose or corn syrup as the carbon source relative to yeast extract supplementation. When glucose or corn syrup served as the carbon source, the presence of yeast extract in the growth medium stimulated gellan production by strain ATCC 31461 on casamino acids. Using vitamin-assay casamino acids as the nitrogen source, the addition of vitamins lowered gellan synthesis by glucose-grown cells regardless of yeast extract supplementation while gellan elaboration by corn syrup-grown strain ATCC 31461 cells could only be increased by supplementing vitamins into medium lacking yeast extract. Independent of carbon source, the absence of yeast extract in the medium reduced biomass production. Biomass production by the strain grown on either carbon source was increased by supplementing vitamins in the medium containing yeast extract.     

Utilization of light for the assimilation of organic matter in Chlorella sp. VJ79

Chlorella sp. strain VJ79 was isolated from a total heterotrophic count of a wastewater collector. It grows autotrophically, heterotrophically, and mixotrophically on a variety of organic substrates. Glucose and serine promote a mixotrophic growth from which the yield is higher than the sum of autotrophic and heterotrophic yields, but serine assimilation requires light. The interaction of glucose and light was studied in proliferating and nonproliferating cells by respirometry (IRGA and Warburg) and growth experiments. Glucose inhibits the photosynthetic CO(2) fixation ten-fold and modifies the pigmentary system as it does in heterotrophic cultures. Light inhibits glucose uptake and assimilation, but under mixotrophic conditions maximal utilization of glucose is obtained. Mutants defective in autotrophic growth were isolated by mutagenesis with nitrosoguanidine. They show a degenerated pigmentary system and a mixotrophic growth yield equal to that of the heterotrophic growth. The analysis of the mixotrophic system shows that light energy, dissipated during autotrophic growth, is used under mixotrophic conditions. From the increase in growth, the increase in photosynthetic efficiency can be calculated as ca. sixfold.     

Effect of Yeast Extract on Growth and Metabolism of H2-Utilizing Acetogenic Bacteria from the Human Colon

The aim of this work was to determine the effect of yeast extract and of its vitamin contents on autotrophic and heterotrophic growth and metabolism of four acetogenic bacteria from the human colon. Yeast extract exerted a stimulatory effect on autotrophic growth of the colonic acetogens, but concentration of this compound above 1–2 g. L⁻¹ in the medium did not enhance utilization of H₂/CO₂. Vitamins provided by yeast extract were shown to be essential cofactors of the reductive pathway of acetate synthesis except for one Clostridium strain. Yeast extract was also necessary to maintain heterotrophic growth and acetate synthesis from glucose in acetogenic species, except in the Streptococcus strain. In the absence of yeast extract, vitamins could efficiently restore glucose fermentation via acetate. The reductive and oxidative pathways of acetate synthesis might, therefore, depend on vitamin cofactors supplied by yeast extract in most of the human acetogenic bacteria. Non-vitaminic factors appeared also to be involved in the metabolism of some of these acetogenic species. Received: 6 March 1998 / Accepted: 3 April 1998     

HETEROTROPHY AND PHOTOHETEROTROPHY BY ANTARCTIC MICROALGAE: LIGHT-DEPENDENT INCORPORATION OF AMINO ACIDS AND GLUCOSE 1

Diatoms isolated from the benthic, planktonic and sea ice microbial communities in Mc Murdo Sound, Antarctica assimilated ambient concentrations of dissolved amino acids and glucose in both the light and dark. Uptake of amino acids but not glucose was influenced by the iucubation irradiance and amino acid uptake rates were up to 250 times greater than those of glucose. Amino acids were incorporated into proteins and other complex polymers and the rates of assimilation and patterns of polymer synthesis were similar to those of the light-saturated photosynthetic incorporation of inorganic carbon. This suggests that these diatoms can use exogenous amino acids to synthesize the essential macromolecules for heterotrophic growth. The assimilation of dissolved organic substrates could supplement light-limited growth during the austral spring and summer as well as potentially support the heterotrophic growth of these diatoms throughout the aphotic polar winter.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas I. Growth Yields and Patterns Under Dual Substrate Conditions

Auxotrophic mutants of Hydrogenomonas eutropha and H. facilis requiring utilizable amino acids were employed to demonstrate the simultaneous utilization of H(2) and an organic substrate for growth. The ratio of the cell yields under dual substrate conditions compared to heterotrophic conditions indicated the relative contributions of the autotrophic and heterotrophic systems to the growth of the organism. Wildtype H. eutropha grown under simultaneous conditions exhibited a dicyclic growth pattern, the first cycle representing either heterotrophic or simultaneous growth and the second cycle representing autotrophic growth. The duration of the changeover period was either very short with no plateau or long with a plateau up to 8 hr, depending upon the organic substrate. The growth rate under simultaneous conditions with some organic substrates was faster than either the autotrophic or heterotrophic rate, but was not the sum of the two rates. The data suggest that, in the presence of both organic and inorganic substrates, heterotrophic metabolism functions normally but autotrophic metabolism is partially repressed.     

Biogeochemical cycling of iron and sulphur in leaching environments

Abstract: Bacterial dissimilatory reduction of iron and sulphur in extremely acidic environments is described. Evidence for reduction at two disused mine sites is presented, within stratified 'acid streamers' growths and in sediments from an acid mine drainage stream. A high proportion (approx. 40%) of mesophilic heterotrophic acidophiles were found to be capable of reducing ferric iron (soluble and insoluble forms) under microaerophilic and anoxic conditions. Mixed cultures of Thiobacillus ferrooxidans and Acidiphilium -like isolate SJH displayed cycling of iron in shake flask and fermenter cultures. Oxido-reduction of iron in mixed cultures was determined by oxygen concentration and availability of organic substrates. Some moderately thermophilic iron-oxidis- ing bacteria were also shown to be capable of reducing ferric iron under conditions of limiting oxygen when grown in glycerol/yeast extract or elemental sulphur media. Cycling of iron was observed in pure cultures of these acidophiles. Sulphate-reducing bacteria isolated from acid streamers could be grown in acidified glycerol/yeast extract media (as low as pH 2.9), but not in media used conventionally for their laboratory culture. An endospore-forming, non-motile rod resembling Desulfotomaculum has been isolated. This bacterium has a wide pH spectrum, and appears to be acid-tolerant rather than acidophilic.     

Relationship between sporulation medium and germination ability of Mucor racemosus sporangiospores

Asexual sporangiospores of Mucor racemosus produced on a minimal sporulation medium (M spores) germinated only if glucose, mannose or a complex substrate such as peptone, yeast extract or Casamino acids was present. Once germinated, growth was supported by a wide range of substrates including amino acids, carbohydrates or organic acids. Sporangiospores produced on a nutritionally complex sporulation medium (C-spores) germinated on a wide range of carbon sources. C-spore phenotype was pleiotropic in that sporangiospores capable of germinating on cellobiose could always germinate on glutamate or xylose; but C-spores capable of germinating on xylose or glutamate did not always germinate on cellobiose. There was a hierarchy of substrates capable of initiating germination with glucose = mannose greater than xylose greater than glutamate greater than cellobiose. C-spores also differed from M-spores by initiating germination in the presence of the non-metabolizable glucose analogue 3-O-methylglucose. These results suggest that at least two sporangiospore phenotypes are produced depending upon the concentration and type of ingredients present in the sporulation medium.     

Shifting N and P limitation along a north-south gradient of mangrove estuaries in South Florida

The heterotrophic utilization of organic substrates by diatoms is likely an important survival strategy when light levels are too low for photosynthesis. The objectives of this study were: (1) to determine if heterotrophic utilization of a large array of organic compounds by eight common freshwater benthic diatom taxa was light-dependent, and (2) to determine if organic substrate utilization patterns differed between dark-grown diatoms and bacteria as a possible means of reducing competition by niche separation. Eight light- and dark-grown diatom taxa and five bacterial species were incubated in 96-well Biolog^® Microtiter plates with each well containing 1 of 95 different organic substrates. Oxidation rates of each organic substrate were measured through time. There was a substantial increase in the number of organic substrates oxidized by diatoms grown in the dark compared to their light-grown counterparts, indicating that the transport systems for these molecules may be light activated. Therefore, diatoms likely only utilize these metabolically expensive uptake mechanisms when they are necessary for survival, or when substrates are plentiful. A principal components analysis indicated discernible differences in the types of organic-C substrates utilized by dark-grown diatoms and bacteria. Although bacteria were able to oxidize a more diverse array of organic substrates including carboxylic acids and large polymers, diatoms appeared to more readily utilize the complex carbohydrates. By oxidizing different organic substrates than bacteria, heterotrophically metabolizing diatoms may be reducing direct competition and enhancing coexistence with bacteria.     

Microbial characterization of thermophilicArchaea isolated from the Guaymas Basin hydrothermal vent

Extremely thermophilic bacteria were isolated from sediments collected at the Guaymas Basin hydrothermal vent located in the Gulf of California. One isolate, (FC89) is a hydrogenotrophic methanogen with an optimal growth temperature of 85°C; this isolate appears to be closely related to the previously describedMethanococcus jannaschii. Thermophilic isolates TY and TYS are heterotrophic, sulfur-reducing archaea that differ from other thermophilic heterotrophic strains in physiological and molecular properties. Both heterotrophic isolates fermented carbohydrates and proteinaceous substrates; acetate was the primary product of carbohydrate fermentation, whereas acetate and a mix of organic acids were primary products of proteinaceous substrate fermentation. A detailed microbiological characterization of the isolates and a profile of fermentable substrates and fermentation products are described.     

pH-Conditional, Ammonia Assimilation-Deficient Mutants of Hydrogenomonas eutropha: Isolation and Growth Characteristics

Two mutants of the facultative autotroph Hydrogenomonas eutropha were isolated by using a modified penicillin selection method. The mutation involved was unusual in that its effect on cellular growth was conditional with regard to extracellular pH and the type of substrate employed. Growth of both mutants was abnormal under autotrophic conditions and during heterotrophic cultivation in the presence of organic substrates which lacked an amino group. Abnormal growth was characterized by linear growth rates which were low at pH 6.0 and moderate at pH 7.2. In contrast, growth of the mutants was normal on most amino acids. Those substrates yielding abnormal growth were oxidized at normal rates by the mutants, indicating the mutation did not impair their uptake or metabolism. The data suggest that the mutants are defective in their ability to assimilate inorganic nitrogen into organic forms, and this defect is strongly influenced by pH.     

Physiological characteristics of cyanobacteria in pulp and paper waste-treatment systems

An investigation was made into the physiological characteristics of the extensive cyanobacterial communities in pulp and paper secondary waste-treatment systems, including the capacity of isolates to biodegrade organic contaminants in these systems. Although pulp and paper waste-treatment systems were found to be severely light-limited, photosynthesis-irradiance curves indicated that shade-adapted cyanobacterial communities could fix conspicuous amounts of inorganic carbon via photosynthesis. Of 21 cyanobacterial strains isolated from pulp and paper waste-treatment systems located in 4 countries, all except one were capable of glucose uptake in the light, and 19 also showed uptake in the dark. In the 6 species tested, glucose and acetate addition stimulated growth in low light in all except one species. Aphanocapsa rivularis grew equally well on glucose and acetate in the dark but Pseudanabaena sp. grew well on acetate, and minimally on glucose. Growth stimulation by glucose and acetate in these two strains was greater in low than in high light. Pseudanabaena sp. and Phormidium animale both accumulated 2,4-dichlorophenol and 3-chlorobenzoate but showed minimal mineralization to CO₂. None of the four species tested could accumulate or degrade phenol or dichloroacetate. It is concluded that, depending on the light conditions, cyanobacteria contribute organic carbon in photosynthesis, and/or remove small organic molecules during mixotrophic and heterotrophic growth but are not important degraders of contaminants in these waste treatment systems.     

Isolation and characterization of Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium

Thermo-acidophilic prokaryotes isolated from geothermal sites in Yellowstone National Park were identified as novel α-Proteobacteria, distantly related (~93% 16S rRNA gene identity) to the mesophilic acidophile Acidisphaera rubrifaciens. One of these isolates (Y008) was shown to be more thermophilic than all previously characterized acidophilic proteobacteria, with a temperature optimum for growth between 50 and 55°C and a temperature maximum of 65°C. Growth was observed in media maintained at pH between 1.75 and 3.0 and was fastest at pH between 2.5 and 3.0. The G + C content of Y008 was 71.8±0.9 mol%. The acidophile was able to grow heterotrophically on a range of organic substrates, including various monosaccharides, alcohols and amino acids and phenol, though growth on single organic compounds required the provision of one or more growth factors. The isolate oxidized sulfur to sulfuric acid in media containing yeast extract, but was not capable of autotrophic growth with sulfur as energy source. Growth occurred under aerobic conditions and also in the absence of oxygen via anaerobic respiration using ferric iron as terminal electron acceptor. Based on these genotypic and phenotypic traits, it is proposed that Y008 represents the type species of Acidicaldus organivorus, gen. nov., sp. nov.     

Studies on metabolic activity of planktonic bacteria isolated from three lakes.

Studies were carried out on metabolic activity of 480 bacterial isolates on 9 substrates. All bacterial isolates studied except one utilized the casamino acids. Also with casamino acids the highest oxygen uptake values were obtained. Among the carbohydrates, glucose was most frequently utilized by the planktonic bacteria. Bacteria isolated from water of the various lakes differed in their metabolic activity as well as in the capability of utilizing the individual substrates. The capability of attacking by the bacteria the substrates applied depended largely upon the time of their isolation.     

Bacillus tusciae,a new species of thermoacidophilic,facultatively chemolithoautotrophic hydrogen oxidizing sporeformer from a geothermal area

A thermophilic, chemolithoautotrophic, hydrogen-oxidizing sporeformer has been isolated from ponds in a solfatara in the geothermal area of Tuscany (Italy). Some physicochemical parameters of the habitat were determined. The habitat was characterized by the presence of molecular hydrogen in the escaping gases, a very low content of phosphate and organic matter. Temperature and water level in the ponds varied widely. The organism formed oval, subterminal spores, which swelled distinctly the sporangium. Optimal growth occured between pH 4.2 and 4.8 at 55°C. It grew best under autotrophic conditions, but organic substrates including short chain fatty acids, amino acids and alcohols could also support heterotrophic growth. Sugars were not metabolized. The hydrogenase was soluble but did not reduce pyridine nucleotides.Based on its morphological and biochemical features, the organism belongs to the genus Bacillus, but differs from all the previously described species. It is therefore proposed as constituting a new species, Bacillus tusciae.Abbreviations SCC standard saline citrate buffer - 0.15 M NaCl 0.015 M trisodium citrate, pH 7.0 Dedicated to Prof. H.-G. Schlegel on the occasion of his 60th birthday     

ORGANIC CARBON SUPPLEMENTATION OF STERILIZED MUNICIPAL WASTEWATER IS ESSENTIAL FOR HETEROTROPHIC GROWTH AND REMOVING AMMONIUM BY THE MICROALGA CHLORELLA VULGARIS1

Heterotrophic growth of the microalga Chlorella vulgaris Beij. in synthetic as well as sterilized municipal wastewater of a nonindustrialized city was measured. The city wastewater contained high levels of ammonium and nitrate, medium levels of phosphate, and low levels of nitrite and organic molecules and could not support heterotrophic growth of C. vulgaris. Evaluation of 11 known carbon sources for this microalga that were added to standard synthetic wastewater containing the same levels of nitrogen and phosphorus as the municipal wastewater revealed that the best carbon sources for heterotrophic growth were Na‐acetate and d ‐glucose. These provided the highest growth rates and the largest removal of ammonium. Growth increased with concentration of the supplement to an optimum at 0.12 M Na‐acetate. This carbon source was consumed completely within 10 d of incubation. Higher concentrations inhibited the growth of C. vulgaris. The microalgal populations under heterotrophic growth conditions were one level of magnitude higher than that under autotrophic growth conditions that served as a comparison. No growth occurred in the dark in the absence of a carbon source. Na‐acetate was superior to d ‐glucose. In municipal wastewater, when Na‐acetate or d ‐glucose was added, C. vulgaris significantly enhanced ammonium removal under heterotrophic conditions, and its capacity was equal to ammonium removal under autotrophic growth conditions. This study showed that sterilized wastewater can be treated by C. vulgaris under heterotrophic conditions if supplemented with the appropriate organic carbon source for the microalgae.     

Heterotrophic carbon metabolism and energy acquisition in Candidatus Thioglobus singularis strain PS1, a member of the SUP05 clade of marine Gammaproteobacteria

A hallmark of the SUP05 clade of marine Gammaproteobacteria is the ability to use energy obtained from reduced inorganic sulfur to fuel autotrophic fixation of carbon using RuBisCo. However, some SUP05 also have the genetic potential for heterotrophic growth, raising questions about the roles of SUP05 in the marine carbon cycle. We used genomic reconstructions, physiological growth experiments and proteomics to characterize central carbon and energy metabolism in Candidatus Thioglobus singularis strain PS1, a representative from the SUP05 clade that has the genetic potential for autotrophy and heterotrophy. Here, we show that the addition of individual organic compounds and 0.2 μm filtered diatom lysate significantly enhanced the growth of this bacterium. This positive growth response to organic substrates, combined with expression of a complete TCA cycle, heterotrophic pathways for carbon assimilation, and methylotrophic pathways for energy conversion demonstrate strain PS1's capacity for heterotrophic growth. Further, our inability to verify the expression of RuBisCO suggests that carbon fixation was not critical for growth. These results highlight the metabolic diversity of the SUP05 clade that harbours both primary producers and consumers of organic carbon in the oceans and expand our understanding of specific pathways of organic matter oxidation by the heterotrophic SUP05.     

Amino acids as main substrates for sulfate-reducing bacteria in surface sediment of a eutrophic bay

The inner part of Tokyo Bay, Japan, is highly eutrophicated as shown by the frequent occurrence of red tide. The bottom water is anoxic during warm seasons especially at artificially dredged sites. In the sediment slurries prepared from surface sediment samples collected from the dredged sites, substrate addition stimulated the consumption of sulfate during anaerobic incubation. Of the substrates added, the seston composed mainly of diatom stimulated consumption more than lactate and acetate. Its effect was nearly equal to that of casamino acids. Casamino acids and some amino acids also accelerated the rate of sulfate reduction measured by the tracer method in sediment samples more than lactate or acetate. Anaerobic incubation of the sediment slurry amended with casamino acids showed that the consumption of amino acids was retarded by the addition of molybdate (final concentration; 20 mM). In the slurry amended with only molybdate, glutamate was accumulated distinctively and linearly with time. Its accumulation rate in molar base was comparable to the rate of sulfate reduction. These results suggested that amino acids were the main substrates for sulfate-reducing bacteria (SRB) in the sediment. The MPN values of SRB in these sediment samples were often higher with the enumeration medium containing casamino acids instead of lactate. Furthermore, during a week incubation of sediment slurries amended with substrates, casamino acids and seston more greatly stimulated the growth of SRB enumerated by both media than lactate.     

Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria

Extremophilic organisms are gaining increasing interest because of their unique metabolic capacities and great biotechnological potential. The unicellular acidophilic and mesothermophilic red alga Galdieria sulphuraria (074G) can grow autotrophically in light as well as heterotrophically in the dark. In this paper, the effects of externally added glucose on primary and secondary photosynthetic reactions are assessed to elucidate mixotrophic capacities of the alga. Photosynthetic O2 evolution was quantified in an open system with a constant supply of CO2 to avoid rapid volatilization of dissolved inorganic carbon at low pH levels. In the presence of glucose, O2 evolution was repressed even in illuminated cells. Ratios of variable to maximum chlorophyll fluorescence (Fv/Fm) and 77 K fluorescence spectra indicated a reduced photochemical efficiency of photosystem II. The results were corroborated by strongly reduced levels of the photosystem II reaction centre protein D1. The downregulation of primary photosynthetic reactions was accompanied by reduced levels of the Calvin Cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Both effects depended on functional sugar uptake and are thus initiated by intracellular rather than extracellular glucose. Following glucose depletion, photosynthetic O2 evolution of illuminated cells commenced after 15 h and Rubisco levels again reached the levels of autotrophic cells. It is concluded that true mixotrophy, involving electron transport across both photosystems, does not occur in G. sulphuraria 074G, and that heterotrophic growth is favoured over autotrophic growth if sufficient organic carbon is available.