Growth and carbohydrate metabolism of sulfobacilli]

The moderately thermophilic acidophilic bacteria Sulfobacillus thermosulfidooxidans, strain 1269, S. thermosulfidooxidans subsp. "asporogenes," strain 41, and the thermotolerant strain S. thermosulfidooxidans subsp. "thermotolerans" K1 prefer mixotrophic growth conditions (the concomitant presence of ferrous iron, thiosulfate, and organic compounds in the medium). In heterotrophic and autotrophic growth conditions, these sulfobacilli can grow over only a few culture transfers. In cell-free extracts of these sulfobacilli, key enzymes of the Embden-Meyerhof-Parnas, pentose-phosphate, and Entner-Doudoroff pathways were found. The role of a particular pathway depended on the cultivation conditions. All of the enzymes assayed were most active under mixotrophic conditions in the presence of Fe2+ and glucose, suggesting the operation of all of the three major pathways of carbohydrate metabolism under these conditions. However, the operation of the Entner-Doudoroff pathway in strain 41 was restricted under mixotrophic conditions. After the first culture transfer from mixotrophic to heterotrophic conditions, the utilization of glucose occurred only via the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. After the first culture transfer from mixotrophic to autotrophic conditions, the activity of carbohydrate metabolism enzymes decreased in all of the strains studied; in strain K1, only the glycolytic pathway remained operative. The high activity of fructose-bisphosphate aldolase, remaining in strain 41 cells under these conditions, suggests the involvement of this enzyme in the reactions of the Calvin cycle or of gluconeogenesis.     

Mixotrophic Growth of Hydrogenomonas eutropha

Mixotrophic growth conditions were established by the addition of lactate to cultures of Hydrogenomonas eutropha growing autotrophically in a gaseous environment of H(2), O(2), and CO(2) (6:2:1). The specific growth rate of mixotrophic cultures was double that of the autotrophic cultures, and lactate disappearance paralleled growth. Growth yields in mixotrophic cultures were significantly greater than those in heterotrophic cultures for equal quantities of lactate consumed. The magnitude of the increase in yield was directly proportional to the absolute growth rate at the time of lactate addition to the starting autotrophic culture and to the time under mixotrophic conditions. The specific activities of hydrogenase and ribulose diphosphate carboxylase decreased during mixotrophic growth; the total activities increased somewhat. The results suggested that the complete autotrophic and heterotrophic physiologies functioned simultaneously under mixotrophic contions.     

Oxidative Enzymes and Pathways of Hexose and Triose Metabolism in Chlorella

Cell-free preparations of Chlorella pyrenoidosa Chick, van Niel's strain, were assayed for oxidative enzymes, utilizing isotopic and spectrophotometric techniques. The enzyme activity of heterotrophic and autotrophic cells was compared. The study was divided into categories, one concerned with the spectrophotometric detection of enzymes involved in the initial reactions of glycolysis and the hexose monophosphate shunt, and the other with the direct oxidation of glucose as compared with that oxidized via glycolysis. The reduction of pyridine nucleotides in crude extracts was studied with glucose, glucose-6-phosphate, 6-phosphogluconate, and fructose-1-6-diphosphate as substrates. Enzymes detected in both heterotrophic and autotrophic cells were hexokinase, fructose-diphosphate-aldolase, NAD-linked 3-phosphoglyceraldchyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and a NADP-linked 3-phosphoglyceraldchyde dehydrogenase. In addition to isotopic studies designed to make an appraisal of the hexose monophosphate shunt, a comparison of the rate of reduction of NADP by glucose-6-phosphate and 6-phosphogluconate in relation to the reduction of NAD by 3-phosphoglyceraldehyde was made in light- and dark-grown cells. The rate of reduction of NADP appeared to be lowered in the light-grown cells, suggesting, as did also the isotopic studies, that the hexose monophosphate shunt is less active in autotrophic metabolism than in heterotrophic metabolism.     

EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY HETEROTROPHIC,AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE1

Heterotrophic growth of microalgae presents significant economic advantages over the more common autotrophic cultivation. The efficiency of growth and nitrogen, phosphorus, and glucose uptake from synthetic wastewater was compared under heterotrophic, autotrophic, and mixotrophic regimes of Chlorella vulgaris Beij. immobilized in alginate beads, either alone or with the bacterium Azospirillum brasilense. Heterotrophic cultivation of C. vulgaris growing alone was superior to autotrophic cultivation. The added bacteria enhanced growth only under autotrophic and mixotrophic cultivations. Uptake of ammonium by the culture, yield of cells per ammonium unit, and total volumetric productivity of the culture were the highest under heterotrophic conditions when the microalga grew without the bacterium. Uptake of phosphate was higher under autotrophic conditions and similar under the other two regimes. Positive influence of the addition of A. brasilense was found only when light was supplied (autotrophic and mixotrophic), where affinity to phosphate and yield per phosphate unit were the highest under heterotrophic conditions. The pH of the culture was significantly reduced in all regimes where glucose was consumed, similarly in heterotrophic and mixotrophic cultures. It was concluded that the heterotrophic regime, using glucose, is superior to autotrophic and mixotrophic regimes for the uptake of ammonium and phosphate. Addition of A. brasilense positively affects the nutrient uptake only in the two regimes supplied with light.     

Glycollate production and excretion byAlcaligenes eutrophus

Autotrophic cultures of the facultative chemolithotrophAlcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO₂ to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic conditions.Extracts from autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction ofd0ribulose 1,5-diphosphate carboxylase under the same conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - HPMS 2-pyridyl-hydroxymethane sulphonie acid - RuDP d-ribulose 1,5-diphosphate     

Factors Affecting the Synthesis and Degradation of Ribulose-1,5-Diphosphate Carboxylase in Hydrogenomonas facilis and Hydrogenomonas eutropha

Hydrogenomonas facilis and H. eutropha cultured in fructose medium retained high levels of ribulose-1,5-diphosphate carboxylase only when the following conditions were fulfilled: low aeration, FeCl(3) addition to fructose medium, and cell harvest at or prior to mid-exponential phase of growth. Repression of carboxylase synthesis was demonstrated under conditions of high oxygen tension during growth of H. eutropha on fructose. Upon depletion of fructose in the growth medium, carboxylase activity fell abruptly in both organisms. The decline could not be attributed to a repressive mechanism. Rapid inactivation of carboxylase was promoted by transfer of mid-exponential-phase H. eutropha to a basal salts medium lacking fructose. During severe fructose starvation, N(2), H(2), 80% H(2) to 20% air, 2,4-dinitrophenol, actinomycin D, streptomycin, bicarbonate, and magnesium ion deficiency spared carboxylase. Nitrogen starvation or chloramphenicol afforded no protection during severe starvation. In vitro inactivation was also demonstrated in crude cell-free extracts from nonstarved, fructose-grown H. eutropha. Substrate bicarbonate protected against this loss. Inactivation of the carboxylase could not be demonstrated either by starvation of autotrophically grown cells or in autotrophic extracts. Autotrophic extracts mixed with heterotrophic extracts lost their carboxylase activity, but mixing with heterotrophic extracts that had been heated to 50 C resulted in no loss of activity. Mechanisms are proposed to accommodate these observations.     

The regulation of hydrogenase formation as a differentiating character of strains of Paracoccus denitrificans

Paracoccus denitrificans strains Stanier 381 (DSM 65), Morris (DSM 413), and Vogt 11 (DSM 415) and eleven newly isolated strains were compared with respect to the localization of hydrogenase and its regulation. In all strains hydrogenase was found to be membrane-bound and not able to reduce pyridine nucleotides.The enzyme was inducible in strain 381 and was found only in cells grown with hydrogen as the sole hydrogen donor; in cells grown under mixotrophic or heterotrophic conditions the hydrogenase activity was zero.In all other strains hydrogenase was constitutive and was present in cells grown under autotrophic, mixotrophic and heterotrophic conditions. Under the latter conditions the specific hydrogenase activity was even higher than under mixotrophic conditions.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas: Regulation of Autotrophic Growth by Organic Substrates

The effects of a number of organic substrates on the autotrophic metabolism of Hydrogenomonas eutropha were examined. Dual substrate (mixotrophic) cultivation in the presence of hydrogen plus either fructose or alanine allowed autotrophic growth to begin immediately after the exhaustion of the organic substrate. On the other hand, the presence of acetate, pyruvate, or glutamate caused a lengthy lag to occur before autotrophic growth commenced. With acetate or pyruvate this lag (plateau) in the dicyclic growth curve was due to the repression of ribulose diphosphate carboxylase (RDPC) synthesis during mixotrophic growth. During heterotrophic growth with glutamate, RDPC was partially repressed; however, during mixotrophic growth, RDPC activity was high. Thus the delay of autotrophic growth was not due to a repression of RDPC by glutamate. The data suggest that glutamate interferes with autotrophic metabolism by repressing the incorporation of inorganic nitrogen. The repression of these vital autotrophic functions by acetate, pyruvate, and glutamate occurred both in the presence and absence of hydrogen, i.e., during both heterotrophic and mixotrophic cultivation. The derepression of the affected systems during the plateau phase of the dicyclic growth curves was demonstrated. Carbon dioxide assimilation by whole cells agreed well with the RDPC activity of extracts from cells grown under similar conditions.     

Glycollate production and excretion by Alcaligenes eutrophus.

Autotrophic cultures of the facultative chemolithotroph Alcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO2 to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction of D-ribulose 1,5-diphosphate carboxylase under the same conditions.     

Control of autotrophic carbon assimilation in Alcaligenes eutrophus by inactivation and reactivation of phosphoribulokinase

Phosphoribulokinase in Alcaligenes eutrophus was partially inactivated when an autotrophic culture was shifted to heterotrophic growth with pyruvate as the sole source of carbon and energy. A similar response was observed on addition of various organic substrates to autotrophic cultures during the transition to mixotrophic growth. The extent of inactivation depended on the added substrate. Pyruvate or lactate caused the strongest inactivation among the tested substrates. Up to 75% of the phosphoribulokinase activity found in the autotrophic cells was lost within 30 min after supplementation of the cultures with either of these two substrates. This loss of enzyme activity was not the result of degradation of enzyme protein. Inactivation of phosphoribulokinase was accompanied by a decrease in the CO2 fixation rate of the cells. Reactivation of the enzyme occurred after exhaustion of pyruvate from the medium. Neither inactivation nor reactivation required de novo protein synthesis; however, continued energy conversion was necessary for the inactivation to occur. We suggest that the pyruvate metabolism of A. eutrophus is involved in these regulatory processes which act on phosphoribulokinase. They appear to contribute to the control of autotrophic CO2 assimilation in this organism.     

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.     

不同营养条件下原始小球藻对蒽的富集和降解研究

研究了自养与异养条件下原始小球藻对蒽的降解和富集能力 .结果表明 ,自养条件下 ,浓度为1 0mg·L-1的蒽有 48.18%被降解 ,其中 2 8.81%属于自然光降解 ,仅有 19.37%被原始小球藻降解 .而异养条件下的原始小球藻对浓度为 2 .5mg·L-1的蒽降解率达到 33.5 3%,说明异养原始小球藻不仅能耐受高浓度蒽 ,而且表现出比自养原始小球藻更强的蒽降解能力 .两种条件下 ,80 %以上残留的蒽都被富集到藻细胞中 .虽然自养条件下原始小球藻对蒽的生物富集系数达 90 6 4,远大于异养条件下的生物富集系数(1899) ,但异养条件下藻对蒽的绝对富集量 (2 0 2 .2 9μg)远远高于自养条件下的 6 9.6 87μg .     

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.     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

Comparison of Phosphoenolpyruvate-Carboxykinase from Autotrophically and Heterotrophically Grown Euglena and Its Role during Dark Anaerobiosis

Euglena gracilis (1224-5/9) contains phosphoenolpyruvate carboxykinase when grown autotrophic with CO₂ in the light. Its yield is higher when an additional carbon source like glucose has been added. The enzyme is lacking in cells provided with CO₂ alone and kept in the dark, whereas highest yields result if both glucose and CO₂ are provided together in the dark. The enzyme was purified by ammonium sulfate precipitation, gel filtration on Sephacryl S-300 and affinity chromatography on GMP-Sepharose. The latter step was most effective to protect the enzyme from inactivation. Its homogeneity was tested electrophoretically and immunologically. Enzymes from autotrophic and heterotrophically grown cells have identical pH optima and similar isoelectric points. The molecular weight was different: 761,000 for the enzyme from autotrophic and 550,000 for that from heterotrophic cells as determined by gel filtration. The subunit molecular weight of both enzymes is nearly the same. The kinetic data of the enzymes are slightly different. Glycolytic and tricarboxylic acid cycle intermediates are of limited influence on enzyme activity and inhibitory in unphysiological high concentrations. From Ouchterlony double immunodiffusion and enzyme-linked immunosorbent assay, it is evident that the enzyme is localized in the cytosol. With the latter quantification test the phosphoenolpyruvate carboxykinase protein content was found 10 times higher in heterotrophically grown cells than when cultivated under autotrophic conditions.     

Investigation of mixotrophic,heterotrophic, and autotrophic growth of Chlorella vulgaris under agricultural waste medium

Growth of Chlorella vulgaris and its lipid production were investigated under autotrophic, heterotrophic, and mixotrophic conditions. Cheap agricultural waste molasses and corn steep liquor from industries were used as carbon and nitrogen sources, respectively. Chlorella vulgaris grew remarkably under this agricultural waste medium, which resulted in a reduction in the final cost of the biodiesel production. Maximum dry weight of 2.62 g L^?1 was obtained in mixotrophic growth with the highest lipid concentration of 0.86 g L^?1. These biomass and lipid concentrations were, respectively, 140% and 170% higher than autotrophic growth and 300% and 1200% higher than heterotrophic growth. In mixotrophic growth, independent or simultaneous occurrence of autotrophic and heterotrophic metabolisms was investigated. The growth of the microalgae was observed to take place first heterotrophically to a minimum substrate concentration with a little fraction in growth under autotrophic metabolism, and then the cells grew more autotrophically. It was found that mixotrophic growth was not a simple combination of heterotrophic and autotrophic growth.     

小球藻两个品系在自养与异养条件下的生长、能荷与色素差异

Chlorella protothecoides中国品系(C品系)与美国品系(A品系)在自养培养基中生长正常。在高浓度葡萄糖、低浓度有机氮的异养生长条件下,C品系仍为绿色,细胞生长与繁殖缓慢,指数期生长速率降低,细胞内腺苷酸能荷值(AEC)下降,但细胞仍保留叶绿素;A品系细胞转变成乳黄色,细胞生长与繁殖加快,指数期生长速率是自养细胞的2倍,细胞内腺苷酸能荷值仍保持高水平,向异养条件转换后约24h,细胞内叶绿素基本消失,重新向自养条件转换后细胞内叶绿素又逐渐恢复。AEC值是判别微藻在异养条件下能量代谢与生理状态的良好指标。同一种小球藻不同品系间的上述差异可能与细胞内基因调控的差异相关联。     

Influence of organic compounds and light limitation on the growth rate of estuarine benthic diatoms

Ten species of benthic diatoms from the Eems-Dollard estuary were grown in axenic cultures under various combinations of irradiance and supply of organic substrates. Six species were capable of growth in the dark on yeast extract, casamino acids, or glucose. Four of these species grew best in the presence of glucose, whereas the growth of the other two species was supported only by yeast extract and casamino acids. The light limited growth rate of only those species that were also capable of heterotrophic growth in the dark was increased by organic substrates. The rate of this “mixed” growth together with the absence of a lag-phase upon change from autotrophic to heterotrophic conditions indicates the nutritional versatility of these diatom species. A positive relation between the organic matter content of the natural habitat and the heterotrophic capacities of the diatom species is suggested. All species with heterotrophic capacities were isolated from muddy sediments, whereas two species isolated from a sandflat seem to be obligately autotrophic. Also two species from muddy sediments apparently had no heterotrophic capacities. The cells of the six species with heterotrophic capacities differed from those of the four species without such capacities in their higher surface to volume ratio.     

Hydrogenase and ribulose diphosphate carboxylase during autotrophic, heterotrophic, and mixotrophic growth of scotochromogenic mycobacteria.

Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence of carbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.     

Independent regulation of synthesis of form I and form II ribulose bisphosphate carboxylase-oxygenase in Rhodopseudomonas sphaeroides.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBPC-O) activity was greatly enhanced when Rhodopseudomonas sphaeroides was grown in a mineral salts medium supplied with 1.5% CO2 in hydrogen. Analysis of cell extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that cells growing on 1.5% CO2 in H2 specifically accumulated RuBPC-O polypeptides. Quantitative immunological determinations revealed that accumulation of form I and form II RuBPC-O closely correlates with the increase of specific activity. However, the two enzymes appeared to be derepressed at different levels. Upon transfer from heterotrophic to autotrophic (1.5% CO2) growth conditions, the intracellular form I RuBPC-O concentration was augmented 17-fold, whereas the form II RuBPC-O content increased only fourfold. As a result, the form I-form II ratio changed from 0.5 to about 2.0. Since this change in the RuBPC-O ratio occurred in the early stage of growth, it suggests that form I RuBPC-O is required for growth under drastic CO2 limitation. The difference in the extent of derepression of form I and form II RuBPC-O also indicates that the synthesis of each enzyme is regulated somewhat independently of the other.