利用啤酒废水小球藻异养培养

摘要：【目的】利用小球藻异养培养技术处理啤酒废水,旨在为啤酒废水资源化利用和降低小球藻生产成本提供一个途径。【方法】在含有10 g/L葡萄糖的基本培养基进行异养小球藻高效藻株的筛选,并用于啤酒废水的资源化处理。【结果】从5株小球藻中得到2株适合高密度异养培养的藻株（Chlorella pyrenoidosa 15-2070 和 Chlorella vulgaris 15-2075）,在啤酒废水的资源化处理过程中这2株小球藻得到非常接近的试验结果。利用由废水配制含10 g/L葡萄糖的基本培养液培养Chlorella pyrenoidosa 15-2070获得了5.3 g/L藻细胞;并且在此过程中,啤酒废水得到有效利用,几种主要污染物最高去除率为：CODcr,92.2 %;BOD5,95.1 %;NO3--N,98.5 %;NH4+-N,92.3 %。【结论】啤酒废水中的重要环境污染物在培养小球藻的过程中可以得到有效地清除,并从中可以获得具有商业价值的小球藻细胞。     

Symbiotic association in Chlorella culture

Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.     

An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

Uptake and accumulation of exogenous docosahexaenoic acid by Chlorella

Tuna oil or its hydrolysate was added to a culture of Chlorella for its nutritional fortification as a feed for rotifer. Exogenous docosahexaenoic acid (DHA) in its free form was taken up by the cells of Chlorella vulgaris strain K-22 and by other strains, but tuna oil was not taken up by the cells. Accumulated DHA was found by electron microscopy in the cells in oil droplets. All strains of Chlorella used in these experiments took up exogenous DHA into the cells. It seems that the structure of the cell wall did not affect the uptake of DHA into the Chlorella cells.     

4株沙漠小球藻对几种常用抗生素的敏感性研究

目的:对分离自新疆沙漠的4株小球藻GTD8A1、GTD4A-1、GTD7C-2和TLD6B进行几种常用基因工程抗生素的敏感性研究,以期筛选出沙漠小球藻基因工程中选择标记的抗生素。方法:运用藻株在液体培养过程中藻体颜色变化和血球板细胞计数的方法,系统性研究沙漠小球藻对几种常用基因工程抗生素的敏感性。结果:4株沙漠小球藻对卡那霉素和链霉素都非常敏感,敏感浓度(细胞致死浓度,下同)均为25μg/m L;对氯霉素也很敏感,GTD8A1、GTD7C-2和TLD6B的敏感浓度也均为25μg/m L,GTD4A-1的敏感浓度为100μg/m L;4株沙漠小球藻对氨苄西林和头孢霉素的敏感性都不是很明显,在一定的浓度范围,氨苄西林和头孢霉素对藻细胞的生长还具有促进作用,当氨苄西林和头孢霉素的浓度很高时才表现出一定的抑制作用。结论:卡那霉素和链霉素可作为沙漠小球藻基因工程选择标记中的2种抗生素,为今后建立其遗传转化系统奠定了基础。     

THE HOST RANGE OF THE CHLORELLAVOROUS BACTERIUM ("VAMPIROVIBRIO CHLORELLVORUS")

The chlorellavorous bacterium . "Vampirovibrio chlorellavorus" Gromov et Mamkaeva, grows only by killing and consuming the cell contents of species of the green alga Chlorella Beijerinck. Of the 76 algal strains examined in this study, the bacterium attacks all 31 strains of the species C. vulgaris. C. sorokiniana, and C. kessleri, but attacks only two of 39 strains of nine other Chlorella species. Neither of two Prototheca strains was susceptible to attack. This narrow host specificity may be related to cell surface properties .     

Asymmetric reduction of ethyl 2-methyl 3-oxobutanoate by Chlorella

Chlorella pyrenoidosa Chick reduced ethyl 2-methyl 3-oxobutanoate to the corresponding alcohols with the diastereomer (anti/syn) ratio of 53/47. The enantiomer excesses of anti-(2S, 3S)- and syn-(2S, 3R)-hydroxy esters were 89 and > 99ee% respectively. C. vulgaris and C. regularis afforded predominantly the syn-isomer, contrary to C. pyrenoidosa. The differences in the activity of reducing ethyl 2-methyl 3-oxobutanoate were observed among three strains of Chlorella. Addition of 2% metal salts slightly increased the chemical yield of the hydroxy ester.     

Effect of antisynaptosome antibodies on the metabolism of synaptosomal proteins

The authors studied the effect of intraventricular injection of antisynaptosomal gamma-globulin on the protein of synaptosomal proteins in the rat brain cortex. The effect was assesed according to the protein specific radioactivity 2 and 3 hours after injection into animals of 14C-protein hydrolysate of Chlorella. Injection of antisynaptosomal gamma-globulin provoked an increase and reduction in the specific radioactivity of the proteins under study 2 and 3 hours, respectively, after labeled precursor injection. It is assumed that interaction of synaptosomal antibodies with protein synaptosomal antigens produces intense decay of antigens, leading to activation of their synthesis.     

高产油小球藻的筛选及其油脂分析

小球藻广泛分布于各种生境,特别是淡水环境中,适应性强。其同化产物主要是淀粉,但在环境胁迫条件下可显著积累中性脂,其脂肪酸类型主要为C16和C18,适合作为生物柴油的原料。我们从中国部分地区水体中分离纯化到若干株小球藻,通过薄层层析比较分析了21株产油小球藻的油脂含量,筛选到一株三酰基甘油含量较高的藻株Chlorella sp.NMX37N。其适宜生长温区为15—35℃,在25℃时生长速率最快,比生长速率为0.53/d,生长的最适光强为250μmol photons/(m2.s)。批量培养实验显示,藻细胞的三酰基甘油含量随培养时间延长而增加,并在培养的稳定期达到最大值,此时培养液中氮基本被耗尽。在批量培养条件下培养Chlorella sp.NMX37N约40d,藻细胞中总脂含量可达到33%左右,与此相比通过两步培养方式,将培养至对数后期(约20d)的藻细胞缺氮处理48h后,得到的总脂产率相当。通过两步培养方式可以大大缩短培养时间,使得该藻细胞快速有效积累油脂。另外,气相色谱分析显示,该藻的总脂和三酰基甘油的脂肪酸均以C16∶0和C18∶2为主,占总脂肪酸的70%以上,且不含C20以上的长链脂肪酸,可以作为优质的生物柴油原料。     

Infection of Alga-Free Paramecium bursaria with Strains of Chlorella, Scenedesmus, and a Yeast

SYNOPSIS. Twenty-one different stocks of Paramecittm bursaria , belonging t o 4 separate varieties (syngens), whose endosymbiotic chorellae had been removed, were tested for reinfection by several strains of Chorella , some previously isolated from P. bursaria , and others free-living. In addition, infection of P. bursaria by a single strain of the green alga Scenedesmus sp., and an unidentified strain of yeast was attempted. Most combinations involving Chlorella yielded infected paramecia, and all those with Scenedesmus or the yeast did so. The failures with Chlorella were attributed to low infectibility of the stocks of Paramecium concerned, rather than to inability of the Chlorella to survive inside the paramecia. Little evidence was found that the strains of P. bursaria differed genetically in ability to maintain the symbiotic organisms.     

Physiologische und biochemische Beiträge zur Taxonomie der Gattung Chlorella

Zusammenfassung Die 15 verfügbaren Stämme von Chlorella vulgaris f. tertia Fott et Nováková (früher als Chlorella III, zwei auxotrophe Stämme als Chlorella VI, bezeichnet), die im Gegensatz zu Chlorella vulgaris Beijerinck Hydrogenase enthalten, wurden auf Thermophilie geprüft. Sie erwiesen sich sämtlich als thermophil und erreichen die obere Grenze des Wachstums bei 38–42°C. Damit erscheinen die beiden klassischen Hochtemperatur-Chlorellen (die Stämme 7-11-05 und 1-9-30 von Sorokin) als Angehörige eines ziemlich weit verbreiteten thermophilen Taxons. Unter den 19 verfügbaren Stämmen von Chlorella vulgaris wurden demgegenüber keine Hochtemperatur-Stämme gefunden. Auch bei C. homosphaera, C. fusca var. rubescens, C. luteoviridis, C. zofingiensis, C. sacharophila, C. minutissima und C. protothecoides konnten wir keine thermophilen Stämme nachweisen, während sich bei C. fusca und C. kessleri eine leichte Tendenz zur Thermophilie zeigte. Chlorella vulgaris f. tertia ist somit anscheinend ein Taxon ökologischer Spezialisten, die zu einer Existenz bei erhöhten Temperaturen, unter anaeroben Bedingungen oder auch als Symbionten in Tieren (zwei Stämme sind Zoochlorellen aus dem Süßwasser-Schwamm Spongilla) befähight sind. Wir nehmen an, daß es sich dabei um eine primitive, phylogenetisch alte Gruppe von Organismen handelt, aus der sich die morphologisch gleiche, typische Chlorella vulgaris durch den Verlust von Hydrogenase und Thermophilie entwickelt haben könnte.

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Physiological and biochemical contributions to the taxonomy of the genus Chlorella VII. The thermophily of Chlorella vulgaris f. tertia Fott et Nováková

Summary The 15 available strains of Chlorella vulgaris f. tertia Fott et Nováková (previously designated as Chlorella III, two auxotrophic strains as Chlorella VI) contain hydrogenase, in contrast to Chlorella vulgaris Beijerinck. In addition, all strains of C. vulgaris f. tertia were now found to be thermophilic, i.e., able to grow at up to 38–42°C. Thus the two classical high-temperature Chlorellae (strains 7-11-05 and 1-9-30 of Sorokin) appear as members of a rather common thermophilic taxon. Among the 19 available strains of Chlorella vulgaris, by contrast, no hightemperature strains were observed. Likewise, we did not find any thermophilic strains of C. homosphaera, C. fusca var. rubescens, C. luteoviridis, C. zofingiensis, C. saccharophila, C. minutissima, and C. protothecoides. C. fusca and C. kessleri, on the other hand, show a slight tendency towards thermophily. Chlorella vulgaris f. tertia therefore seems to be a taxon of ecological specialists, equipped for the existence at elevated temperatures, in anaerobic environments, or as symbionts in animals (two strains are Zoochlorellae isolated from the fresh-water sponge, Spongilla). We assume that it represents a primitive, phylogenetically old group of organisms. The morphologically identical, typical Chlorella vulgaris might have evolved from it through loss of hydrogenase and thermophily.

     

Glutamate dehydrogenases of unicellular green algae: effects of nitrate and ammonium in vivo]

The constitution and control by the inorganic nitrogen source of glutamate dehydrogenases of some unicellular green algae have been studied. The Ankistrodesmus braunii and Scenedesmus obliquus cells contain two different glutamate dehydrogenases, one of which is NADP-specific, the other is active with both NAD and NADP. Their synthesis does not depend on the nitrogen source. The activity of NADP-specific glutamate dehydrogenase increases sharply during nitrogen starvation. In Chlorella pyrenoidosa 82 and Ch. ellipsoidea only one constitutive double specific glutamate dehydrogenase is observed. Its activity does not change depending on the nitrogen nutrition conditions. In the cells of the thermophylic Chlorella strain Chlorella sp. K. ammomium induces a de novo synthesis of NADP-specific glutamate dehydrogenase in addition to the constitutive double specific glutamate dehydrogenase. Thus, the algae tested contain constitutive double specific glutamate dehydrogenase. The NADP-specific enzyme is absent in two Chlorella strains, is constitutive in A. braunii and S. obliquus, and is ammonium-inducible in three thermophylic Chlorella strains.     

Composition and positional distribution of fatty acids in polar lipids from Chlorella ellipsoidea differing in chilling susceptibility and frost hardiness

The composition and positional distribution of fatty acids in the polar lipids from 4 strains of Chlorella differing in chilling susceptibility and frost hardiness were analyzed by enzymatic hydrolysis and gas-liquid chromatography. Analysis of the polar lipids from chilling-sensitive, chilling-resistant and chilling-sensitive revertant strains of Chlorella ellipsoidea IAM C-102 showed that the sum of palmitic and trans -3-hexadecenoic acid in phosphatidylglycerol (PG) is about 60% for the sensitive strains and 53% for the resistant strain. The sum of dipalmitoyl and 1-palmitoyl-2-( trans -3-hexadecenoyl) PG as estimated from the positional distribution of their fatty acids, is about 10% in the case of each of the three strains. The contents of unsaturated fatty acids in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were higher in the resistant than in the sensitive strain. This suggests that unsaturation of fatty acids in not only PG but also PC and PE is involved in chilling sensitivity of Chlorella . On the other hand, lipid changes during the development of frost hardiness of C. ellipsoidea IAM C-27, a frost hardy strain, were examined. The results showed that fatty acids in most lipid classes are unsaturated in the hardening process but their degree of unsaturation is not greatly different from that of the chilling-resistant strain, suggesting that not only unsaturation of fatty acids in lipids but also other factors are necessary for the development of frost hardiness.     

Species-specificity of the lytic activity of the cell wall in the genus Chlorella

Cell wall lytic activity was compared among strains IAM C-27, C-87, SAG 211-1c, -1d, -9a, -8b, -8c, -8l, -11f, -8k, -11g, and -11h/9 of the genus Chlorella . The optimal pH was alkaline in strains with glucosamine as the characteristic group of the rigid wall, and acidic in strains characterised by glucan groups. The lytic enzymes of strains in the former type of algae lyzed the cell wall mainly to soluble high molecular oligosaccharides. The lytic activity of the Chlorella cell wall thus appears species- and strain-speicific.     

Chlorella viruses in diverse fresh waters in North East England

Viruses, which infected strains of exsymbiotic Chlorella of European origin, were found in fresh waters in NE England. In contrast, an exsymbiotic Chlorella strain of N American origin, which is susceptible to viruses which are ubiquitous in US waters, was not infected by viruses from NE England waters.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

Mutagenic activity of the plant preparation KAC-81]

Mutagenic activity of vegetative preparation KAC-81 containing extracts of common wormwood and pine buds using crepis root seedlings, unicellular green alga of Chlorella indicator strains of Salmonellas TA 100, TA 1534, TA 1537, TA 50 and TA 98 as well as white mice and white rats has been studied. It is shown that the preparation caused a weak mutagenic effect on Crepis seedlings and Chlorella and no mutagenic effects on the indicator strains of salmonellas, in sex and somatic cells of mammals under single peroral action in maximally endured doses.     

Expression of plasmids coding for colonization factor antigen II (CFA/II) and enterotoxin production in Escherichia coli

Two plasmids transferred from enterotoxigenic Escherichia coli (ETEC) of serotype O6. H16 and biotypes A and C coded for mannose-resistant haemagglutination (MRHA) and production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Both plasmids were nonautotransferring being mobilized most efficiently by the R plasmid R100-1. They were similar in their genetic properties being incompatible with each other and plasmids of the Inc group FI. The wild-type strains produced the colonization factor antigen II (CFA/II) which was made up of different coli surface antigens (CS). The biotype A strains produced CS1 and CS3 while the biotype C strains produced CS2 and CS3. These three antigens have the ability to cause MRHA. When plasmids coding for MRHA were transferred to K12 strains, the degree of haemagglutination was markedly reduced and only CS3 was produced. When both plasmids were transferred back into biotype A strains, good MRHA was restored and the strains produced CS1 and CS3. In a biotype C strain CS2 and CS3 were formed. The production of the antigens was compared by enzyme-linked immunosorbent assay (ELISA). The strains were also examined by electron microscopy where it was found that CS1 and CS2 were fimbrial antigens while CS3 was not.     

Studies of sulfate utilization by algae: 8. The ubiquity of sulfate reduction to thiosulfate

Cell-free extracts from several microorganisms, when prepared by methods originally devised for Chlorella pyrenoidosa (Emerson strain 3) and incubated anaerobically with ATP, Mg²⁺, and 2, 3-dimercaptopropan-1-ol, are capable of reducing sulfate-³⁵S to thiosulfate. These microorganisms include, in addition to C. pyrenoidosa (Emerson strain 3), several other strains of C. pyrenoidosa, Chlorella protothecoides, Chlorella vulgaris, Anacystis sp., Chlamydomonas reinhardi, Escherichia coli, Salmonella typhimurium, and baker's yeast. Three of these organisms, E. coli, S. typhimurium, and baker's yeast, were previously reported by others to reduce sulfate to sulfite. Moreover, three mutant strains of S. typhimurium (Ba-25, Ce-363, and Bc-482) previously reported by other workers to be unable to reduce sulfate to sulfite also cannot form thiosulfate, and one mutant strain (Cd-68) reportedly able to form sulfite can also form thiosulfate. Taken together, this suggests that thiosulfate-forming activity may be a common feature of sulfate-reducing systems, and it may be present in enzymatic systems previously thought to be forming sulfite. Reasonably conclusive identification of thiosulfate is provided by ion exchange chromatography and by paper electrophoresis; the ambiguities associated with other analytical methods are discussed.     

Mouse spleen cell responses to trichomonal antigens in experimental Trichomonas vaginalis infection

Subcutaneous inoculation of live T. vaginalis into mice caused splenomegaly, particularly when using strains of parasites with low pathogenicity. The proliferative responses of spleen cells from uninfected mice, as measured by [3H] TdR uptake, showed that trichomonal antigens, whether from strains with high or low pathogenicity, have no mitogenic activity. Spleen cells from mice infected with trichomonads of low pathogenicity showed a proliferative response to trichomonal antigens that was maximal after 4 days incubation. The proliferative response of spleen cells from mice infected with trichomonads of high pathogenicity continued for at least 6 days in the presence of the antigen. Moreover, in the latter case there was a significantly greater uptake of [3H] TdR when cells were incubated with antigens of a highly pathogenic strain. These results support the view that although many antigens are common to strains with differing levels of pathogenicity, some antigens are more closely associated with strains that are more highly pathogenic. The strong proliferative response to these antigens may then be related to the clinical presentation of infection with these strains.