Biochemical Basis of Obligate Autotrophy in Blue-Green Algae and Thiobacilli

Differential rates of incorporation of sugars, organic acids, and amino acids during autotrophic growth of several blue-green algae and thiobacilli have been determined. In obligate autotrophs (both blue-green algae and thiobacilli), exogenously furnished organic compounds make a very small contribution to cellular carbon; acetate, the most readily incorporated compound of those studied, contributes about 10% of newly synthesized cellular carbon. In Thiobacillus intermedius, a facultative chemoautotroph, acetate contributes over 40% of newly synthesized cellular carbon, and succinate and glutamate almost 90%. In the obligate autotrophs, carbon from pyruvate, acetate, and glutamate is incorporated into restricted groups of cellular amino acids, and the patterns of incorporation in all five organisms are essentially identical. These patterns suggest that the tricarboxylic acid cycle is blocked at the level of alpha-ketoglutarate oxidation. Enzymatic analyses confirmed the absence of alpha-ketoglutarate dehydrogenase in the obligate autotrophs, and also revealed that they lacked reduced nicotinamide adenine dinucleotide oxidase, and had extremely low levels of malic and succinic dehydrogenase. These enzymatic deficiencies were not manifested by the two facultative chemoautotrophs examined. On the basis of the data obtained, an interpretation of obligate autotrophy in both physiological and evolutionary terms has been developed.     

Importance of Hydrogen Sulfide, Thiosulfate, and Methylmercaptan for Growth of Thiobacilli during Simulation of Concrete Corrosion

Biogenic sulfuric acid corrosion of concrete surfaces caused by thiobacilli was reproduced in simulation experiments. At 9 months after inoculation with thiobacilli, concrete blocks were severely corroded. The sulfur compounds hydrogen sulfide, thiosulfate, and methylmercaptan were tested for their corrosive action. With hydrogen sulfide, severe corrosion was noted. The flora was dominated by Thiobacillus thiooxidans. Thiosulfate led to medium corrosion and a dominance of Thiobacillus neapolitanus and Thiobacillus intermedius. Methylmercaptan resulted in negligible corrosion. A flora of heterotrophs and fungi grew on the blocks. This result implies that methylmercaptan cannot be degraded by thiobacilli.     

Growth, morphology and photosynthetic responses of Neochloris oleoabundans during cultivation in a mixotrophic brackish medium and subsequent starvation

The green microalga Neochloris oleoabundans is able to grow in both low and high salinity media and is largely studied for its capability to accumulate lipids under starvation. Moreover, N. oleoabundans is a mixotrophic alga, and then organic carbon addition can promote its growth. This research aims to study the morpho-physiological aspects, with a particular attention on the photosynthetic response, both during mixotrophic growth and starvation in brackish media, more sustainable than freshwater cultivation. In the first step, the alga was cultivated mixotrophically in a brackish medium added with an apple waste product; in the second one, cells were starved also to verify lipid induction. Results indicate that growth is highly promoted during the first week of mixotrophic cultivation, while photosynthetic pigments and lipids are over-produced during the following three weeks of cultivation. In parallel, in mixotrophic cultures the maximum PSII quantum yield was enhanced during the exponential phase of growth. Interesting changes affected the mixotrophic cultures with respect to the partitioning of absorbed light energy. Starvation of both 7-day-grown mixotrophic and autotrophic cultures caused growth inhibition, pigments and photosynthesis downshifting, and concomitantly promoted evident lipid synthesis.     

Bio-electrochemical removal of nitrate from water and wastewater--a review

Nitrates in different water and wastewater streams raised concerns due to severe impacts on human and animal health. Diverse methods are reported to remove nitrate from water streams which almost fail to entirely treat nitrate, except biological denitrification which is capable of reducing inorganic nitrate compounds to harmless nitrogen gas. Review of numerous studies in biological denitrification of nitrate containing water resources, aquaculture wastewaters and industrial wastewater confirmed the potential of this method and its flexibility towards the remediation of different concentrations of nitrate. The denitrifiers could be fed with organic and inorganic substrates which have different performances and subsequent advantages or disadvantages. Review of heterotrophic and autotrophic denitrifications with different food and energy sources concluded that autotrophic denitrifiers are more effective in denitrification. Autotrophs utilize carbon dioxide and hydrogen as the source of carbon substrate and electron donors, respectively. The application of this method in bio-electro reactors (BERs) has many advantages and is promising. However, this method is not so well established and documented. BERs provide proper environment for simultaneous hydrogen production on cathodes and appropriate consumption by immobilized autotrophs on these cathodes. This survey covers various designs and aspects of BERs and their performances.     

Growth of obligate autotrophic bacteria on glucose in a continuous flow-through apparatus

Nitrosomonas europaea, Nitrobacter agilis, Thiobacillus denitrificans, T. neapolitanus, and T. thioparus (all obligate autotrophic bacteria) have been grown in dialysis culture, on glucose salts media, in the absence of their specific inorganic energy source. Metabolic products for N. agilis grown on nitrite salts medium were identified as keto acids. Pyruvic acid inhibited this organism at 5 x 10(-5)m. Keto acids were not inhibitory for the thiobacilli grown on thiosulfate medium. However, when T. denitrificans was grown on glucose with dialysis, addition of 5 x 10(-4)m pyruvate inhibited growth. Thus, it appears pyruvate may be inhibitory for other autotrophs, as has been reported for T. thiooxidans.     

Microbial CO2 fixation potential in a tar-oil-contaminated porous aquifer

CO(2) fixation is one of the most important processes on the Earth's surface, but our current understanding of the occurrence and importance of chemolithoautotrophy in the terrestrial subsurface is poor. Groundwater ecosystems, especially at organically polluted sites, have all the requirements for autotrophic growth processes, and CO(2) fixation is thus suggested to contribute significantly to carbon flux in these environments. We explored the potential for autotrophic CO(2) fixation in microbial communities of a petroleum hydrocarbon-contaminated aquifer by detection of functional marker genes (cbbL, cbbM), encoding different forms of the key enzyme RubisCO of the Calvin-Benson-Bassham cycle. Quantification of (red-like) cbbL genes revealed highest numbers at the upper fringe of the contaminant plume and the capillary fringe where reduced sulphur and iron species are regularly oxidized in the course of groundwater table changes. Functional gene sequences retrieved from this area were most closely related to sequences of different thiobacilli. Moreover, several cultures could be enriched from fresh aquifer material, all of which are able to grow under chemolithoautotrophic conditions. A novel, nitrate-reducing, thiosulfate-oxidizing bacterial strain, recently described as Thiobacillus thiophilus D24TN(T) sp. nov., was shown to carry and transcribe RubisCO large-subunit genes of form I and II. Enzyme tests proved the actual activity of RubisCO in this strain.     

Classification of ethylene-producing bacteria in terms of biosynthetic pathways to ethylene

Two hundred and twenty-nine ethylene-producing strains of bacteria were identified among 757 bacterial strains which included 13 strains of chemolithotrophs. The ethylene-producing bacetria were classified into three groups, namely, l-methionine-dependent, 2-ketoglutarate-dependent and meat extract-dependent, with reference to their respective biosynthetic pathways to ethylene. Two hundred and twenty-five l-methionine-dependent strains were obtained, while the only 2-ketoglutarate-dependent strain was Pseudomonas syringae pv. phaseolicola PK2. Three strains of chemolithotrophs had ethylene-forming capacity, and Thiobacillus novellus IFO 12443 had a novel ethylene-forming system which was dependent upon the addition of meat extract into the culture medium. The ethylene-forming systems of two of the strains of Thiobacillus ferrooxidans have not yet been characterized. Several strains of non-ethylene-producing bacteria failed to produce ethylene, even when l-methionine was added to the culture medium. We examined the causes of their lack of ethylene-producing ability and judged that these strains are either NADH:Fe(III)EDTA oxidoreductase-less or methionine-uptake activity-less.     

Enzymes of Carbohydrate Metabolism in Thiobacillus species

A study was made of enzymes of carbohydrate metabolism in representative thiobacilli grown with and without glucose. The data show that Thiobacillus perometabolis possesses an inducible Entner-Doudoroff pathway and is thus similar to T. intermedius and T. ferrooxidans. T. novellus lacks this pathway. Instead, a non-cyclic pentose phosphate pathway along with the Krebs cycle is apparently the major route of glucose dissimilation in this organism. Glucose does not support or stimulate the growth of strains of T. neapolitanus, T. thioparus, and T. thiooxidans examined, nor does its presence in the growth medium greatly influence their enzymatic constitution. These obligately chemolithotrophic thiobacilli do not possess the Entner-Doudoroff pathway. Their nicotinamide adenine dinucleotide (NAD)-linked isocitrate dehydrogenase activity predominates over their nicotinamide adenine dinucleotide phosphate (NADP)-linked activity; the converse is true for the other thiobacilli. The data suggest that NAD-linked isocitrate dehydrogenase activity in thiobacilli is involved in biosynthetic reactions.     

Effect of phosphoglycerate mutase deficiency on heterotrophic and autotrophic carbon metabolism of Alcaligenes eutrophus.

Mutants of Alcaligenes eutrophus were isolated on the basis of their inability to grow on succinate as the sole source of carbon and energy. The mutants also failed to grow on other gluconeogenic substrates, including pyruvate, acetate, and citrate. Simultaneously, they had lost their capability for autotrophic growth. The mutants grew, but slower than the wild type, on fructose or gluconate. Growth retardation on gluconate was more pronounced. The mutants lacked phosphoglycerate mutase activity, and spontaneous revertants of normal growth phenotype had regained the activity. The physiological characteristics of the mutants indicate the role of phosphoglycerate mutase in heterotrophic and autotrophic carbon metabolism of A. eutrophus. Although the enzyme is necessary for gluconeogenesis during heterotrophic growth on three- or four-carbon substrates, its glycolytic function is not essential for the catabolism of fructose or gluconate via the Entner-Doudoroff pathway. The enzyme is required during autotrophic growth as a catalyst in the biosynthetic route leading from glycerate 3-phosphate to pyruvate. It is suggested that the mutants accomplish the complete degradation of fructose and gluconate mutase lesion. The catabolically produced triose phosphates are converted to fructose 6-phosphate which is rechanneled into the Entner-Doudoroff pathway. This carbon recycling mechanism operates less effectively in mutant cells growing on gluconate.     

Effects of organic matter on the growth of Thiobacillus intermedius

London, Jack (University of California, Los Angeles), and Sydney C. Rittenberg. Effects of organic matter on the growth of Thiobacillus intermedius. J. Bacteriol. 91:1062-1069. 1966.-Yeast extract, glucose, glutamate, and other organic materials stimulate the rate and extent of growth of Thiobacillus intermedius in thiosulfate broth. Growth did not occur in glucose or glutamate mineral salts medium in the absence of thiosulfate, although a stable variant was obtained which grows on yeast extract alone. Cells harvested from media supplemented with organic matter have a reduced rate of thiosulfate oxidation (20 to 30% of autotrophic), oxidize the organic supplement, and have an additive rate of oxidation in the presence of both the organic substrate and thiosulfate. Carboxydismutase synthesis is repressed, and the incorporation of bicarbonate carbon into cell material is almost completely eliminated by the presence of organic matter in the growth medium. It is concluded that the availability of organic matter eliminates the autotrophic assimilatory mechanisms of T. intermedius but not its autotrophic energy-generating system. The data are discussed in relation to the existence of "obligate" chemoautotrophic bacteria.     

Nonautotrophic Thiobacillus in Acid Mine Water

Nonautotrophic thiobacilli were isolated from the acidic water of a coal mine. Based on their mixotrophic physiology, the isolates are regarded as strains of Thiobacillus perometabolis.     

THE OXIDATION OF HYDROGEN SULFIDE BY BEGGIATOA

Knowledge of the conditions in which Beggiatoa is capable of autotrophic nutrition is incomplete. It is not known whether sulfur-free trichomes from heterotrophic cultures are able to return to the utilization of H₂S-oxidation. Devices were developed which permitted the supply of pure cultures of Beggiatoa, previously cultivated heterotrophically, with H₂S, O₂, and CO₂. Development in media devoid of organic nutrients was achieved, and subculturing under autotrophic conditions could be repeated indefinitely. The strains used behaved differently with respect to their tendency to grow autotrophically. The ability to dispense with organic substrates corresponds to the place in the groups to which they had previously been assigned. All the strains multiplied better when, under otherwise equal conditions, the inorganic medium was supplemented with acetate, very low concentrations of which were effective. This result may, however, be due to the selection of varieties by the isolation procedure. The mixotrophic tendency of our strains may not be a general feature of the genus. There are indications that the wider forms of Beggiatoa tend more toward autotrophic growth than the narrower ones.     

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.     

Molecular genetic studies on the thiobacilli and the development of improved biomining bacteria

Acidophilic autotrophic thiobacilli, which are able to oxidize metal and solubilize sulphide ores, are used industrially to leach metals from mineral ores. Genetic manipulation of the thiobacilli has the potential for the production of leaching bacteria with desirable characteristics for industry. In this review we examine the development of genetic systems in the thiobacilli and the present status of molecular genetics in the group.     

Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulphur-oxidising autotrophic eubacterium from a Western Australian hypersaline lake

The isolation of a novel obligately chemolithotrophic, halophilic and extremely halotolerant Thiobacillus from a hypersaline lake is described. Attempts to demonstrate sulphur- and ferrous iron-oxidizing chemolithotrophs in neighbouring hypersaline lakes were unsuccessful. The organism isolated differs from any other Thiobacillus species previously described and is formally named as Thiobacillus halophilus. It possesses ribulose bisphosphate carboxylase and grows chemolithoautotrophically on thiosulphate, tetrathionate and sulphur, oxidising them to sulphate. Kinetic constants for oxidation of sulphide, thiosulphate, trithionate and tetrathionate are presented. The organism is obligately halophilic, growing best with 0.8–1.0 M NaCl, and tolerating up to 4 M NaCl. Optimum growth was obtained at about 30° C and pH 7.0–7.3. It contains ubiquinone Q-8 and its DNA contains 45 mol % G+C. Organisms of this type might contribute significantly to the autotrophic fixation of carbon dioxide in some hypersaline extreme environments of the kind described.     

Characterization of Xanthobacter strains H4-14 and 25a and enzyme profiles after growth under autotrophic and heterotrophic conditions

All Xanthobacter strains studied are versatile autotrophic bacteria, able to grow on methanol and other substrates. Strain 25a, a yellow-pigmented, pleomorphic, Gram-negative bacterium, capable of autotrophic growth on methanol, formate, thiosulfate, and molecular hydrogen, was isolated from an enrichment culture inoculated with soil from a subtropical greenhouse. Subsequent studies showed that the organism also grows on a wide range of multicarbon substrates. Ammonia, nitrate and molecular nitrogen were used as nitrogen sources. The taxonomic relationship of strains H4-14 and 25a with previously described Xanthobacter strains was studied by numerical classification. Strain H4-14 was identified as a X. flavus strain, but the precise position of strain 25a remained uncertain. It probably belongs to a new species of the genus Xanthobacter. The levels of various enzymes involved in autotrophic and heterotrophic metabolism were determined following growth of strains H4-14 and 25a in batch and continuous cultures. The mechanisms involved in controlling ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis in Xanthobacter strains appear to be comparable to those observed for other autotrophic bacteria, namely repression by organic compounds and derepression by autotrophic energy sources, such as methanol and hydrogen.Abbreviations API appareils et procédés d'identification - CS citrate synthase - ED Entner-Doudoroff pathway - FBP fructose-1,6-bisphosphate - FDH formate dehydrogenase - HPS hexulose-6-phosphate synthase - ICDH isocitrate dehydrogenase - KDPG 2-keto-3-deoxy-6-phosphogluconate - MDH methanol dehydrogenase - PRK phosphoribulokinase - PQQ pyrrolo quinoline quinone - RuBisC/O ribulose-1,5-bisphosphate carboxylase/oxygenase - RuMP ribulose monophosphate     

极端嗜酸性专性化能自养硫细菌有机质代谢的研究进展

极端嗜酸性专性化能自养硫细菌具有独特的生理特性, 在农业、细菌冶金、含硫废水处理以及环境保护等方面发挥着重要作用, 但这类细菌在其特殊能源缺乏时不能代谢有机质, 生长缓慢, 代时长, 细胞得率低, 限制了它在实际生产中的应用效率。对其进行遗传改造, 构建能够利用有机质快速生长的基因工程菌, 将为这类细菌的工业化应用提供一条可行的途径。主要对极端嗜酸性专性化能自养硫细菌有机质代谢的研究进展进行了综述, 其中包括有机化合物的抑制作用、有机化合物的有限利用、中心代谢途径及物质的转运等, 还包括专性化能自养硫细菌有机质代谢遗传改造研究的最新进展。     

Phylogenetic analysis of the genera Thiobacillus and Thiomicrospira by 5S rRNA sequences.

5S rRNA nucleotide sequences from Thiobacillus neapolitanus, Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Thiobacillus intermedius, Thiobacillus perometabolis, Thiobacillus thioparus, Thiobacillus versutus, Thiobacillus novellus, Thiobacillus acidophilus, Thiomicrospira pelophila, Thiomicrospira sp. strain L-12, and Acidiphilium cryptum were determined. A phylogenetic tree, based upon comparison of these and other related 5S rRNA sequences, is presented. The results place the thiobacilli, Thiomicrospira spp., and Acidiphilium spp. in the "purple photosynthetic" bacterial grouping which also includes the enteric, vibrio, pseudomonad, and other familiar eubacterial groups in addition to the purple photosynthetic bacteria. The genus Thiobacillus is not an evolutionarily coherent grouping but rather spans the full breadth of the purple photosynthetic bacteria.     

Utilization of Glucose and the Effect of Organic Compounds on the Chemolithotroph Thiobacillus ferrooxidans

The utilization of glucose by the chemolithotroph Thiobacillus ferrooxidans results in a repression of the ability to oxidize iron, the substrate for autotrophic growth. An assay with resting cells was used to measure iron oxidation rates. Concomitant with the decreased iron oxidation rates, the enzyme responsible for carbon dioxide fixation, ribulose diphosphate (RuDP) carboxylase, was also repressed. Maximum iron oxidation rates precede peak RuDP carboxylase levels, consistent with the role of these processes in autotrophic metabolism in nonrepressed cells. The degree of iron oxidation repression depends on the organic substrate supplied, as does the level of RuDP carboxylase. The uptake of glucose parallels an increase in synthesis of glucose-6-phosphate dehydrogenase and the accumulation in cells of poly-beta-hydroxybutyrate. The organism is also capable of growing on glucose and other organic supplements in the absence of its inorganic energy source; growth rates depend on the organic substrate supplied.