Taxonomic implications of Prototheca and Chlorella cell wall polysaccharide characterization

Summary Four polysaccharide fractions were obtained by acid and alkaline degradation of purified cell walls of Prototheca spp. and Chlorella spp. These fractions were further hydrolyzed and the component sugars identified. Five Prototheca strains and two Chlorella protothecoides strains have essentially similar polysaccharide compositions, which significantly differ from those of C. vulgaris and C. pyrenoidosa. This emphasizes the close affinity of C. protothecoides to Prototheca spp. not shared by other Chlorella spp.     

小球藻的营养及药用价值

微藻因具有丰富的营养价值和产油能力,因而已被广泛研究。小球藻属于单细胞绿藻,分布广泛,种类多达十余种,在食品、医药、饲料、能源和环保等多个领域具有广泛的应用价值。小球藻不仅可以光自养生长,还可以利用有机碳源进行异养生长。小球藻细胞壁坚厚,胞内含有丰富的蛋白质、必需氨基酸、多糖、色素、脂肪酸,并富含多种维生素,以及铁、钙、锌、钾等矿物元素,具有全面而均衡的营养价值。小球藻属中的蛋白核小球藻已被我国卫生部列为新资源食品。小球藻特有的促生长因子（CGF）具有提高免疫力、抗肿瘤等多种特殊功效,近年来研究证明小球藻在临床上可作为治疗多种疾病的辅助药物,被认为是绿色天然的营养保健食品。本文从小球藻的生物学特性、营养价值、药理和保健作用等方面进行了论述。     

Selective Medium for the Isolation of Prototheca

A medium was devised which permitted the selective isolation of Prototheca spp. from nature.     

Experimental role of the unicellular algae Prototheca and Chlorella (Chlorellaceae) in anti-cancer immunogenesis (murine BP8 sarcoma)]

Bacteria and Yeasts are able to induce, when inoculated into laboratory rodents, a general stimulation of defences by an immune process. We could estimate that unicellular algae would induce the same phenomenon because the chemical compounds of their walls are related to those of the precedings microorganisms specially to bacteria. Indeed, two Chlorellaceae, Prototheca segbwema and Chlorella pyrenoidosa are respectively protecting 78% and 82% CH3 mice against the sarcoma BP8 grafting.     

Nutritional effect of freshwater Chlorella on growth of the rotifer Brachionus plicatilis

Mass production of Brachionus plicatilis is usually accomplished by feeding so-called marine Chlorella (Nannochloropsis oculata) to the rotifers in marine fish hatcheries. If the marine Chlorella are in short supply, baker's yeast is usually used as a supplementary food. Recently, a condensed suspension of freshwater Chlorella (Chlorella vulgaris, k-22) was commercially developed as another supplementary food. We have evaluated the dietary value of this freshwater Chlorella for growth of the rotifer by means of individual and batch cultures. Rotifers cultured with the freshwater Chlorella suspension under almost bacteria-free conditions, showed very suppressed growth. However if the Chlorella was supplemented with vitamin B₁₂ by adding the vitamin solution into the suspension or by culturing the Chlorella in a medium containing vitamin B₁₂, the nutritional value of freshwater Chlorella was greatly improved and almost at the same level as that of marine Chlorella. Condensed Chlorella may therefore be effective as a supplementary food if vitamin B₁₂ is supplied.     

Nutritional value of Chlorella vulgaris: Effects of ultrasonication and electroporation on digestibility in rats

Three processed products derived from the green algae C. vulgaris were investigated: (1) spray-dried only (S-DA); (2) spray-dried and electroporated (ES-DA); (3) spray-dried; ultrasonicated treated (US-DA). A nitrogen-balance study was performed. Male growing Wistar rats, housed separately in metabolism cages, were fed the three algal products as the sole protein source at 150 mg N per 100 g of body weight. A control group of rats was fed with casein at a level to give the same protein nitrogen intake. The coefficients of total intestinal tract apparent crude protein digestibility for the different C. vulgaris products were: S-DA = 0.47 ± 0.127% (mean ± S.D.), ES-DA = 0.44 ± 0.075%, US-DA = 0.57 ± 0.137%. Protein efficiency ratio was 1.4 ± 0.3, 1.0 ± 0.5 and 2.1 ± 0.3, respectively. N-balance was 41.86 ± 32.8 mg, 31.3 ± 17.3 mg and 66.7 ± 30.1 mg, respectively. The biological value was 93 ± 9.5%, 93.6 ± 10%, and 101 ± 5%, respectively. The coefficient of total intestinal tract apparent crude protein digestibility and biological value of C. vulgaris was enhanced by ultrasonic treatment and reduced by electroporating, thus ultrasonication may be a helpful technological process in practical processing of green algae in food industry.     

Extracellular production of L-ascorbic acid by Chlorella protothecoides,Prototheca species,and mutants of P. moriformis during aerobic culturing at low pH

Nine strains of Chlorella protothecoides and 43 strains representing the five species of Prototheca were screened in flask culture for their ability to synthesize L-ascorbic acid (AA). Ascorbic acid was detected in all strains, ranging from 4.8 to 0.38 mg AA g⁻¹ of dry cells. Organisms selected for further study grew well and maintained their AA productivity above a pH of 3.5. They can produce AA using a variety of carbon and nitrogen sources. Aerobic fermentation of selected strains resulted in extracellular accumulation of AA up to 76 mg l⁻¹. By classical mutagenesis and selection methods, we created mutants of Prototheca moriformis ATCC 75669 that produced greater quantities of AA than the wild-type strain (78.4 vs 21.9 mg AA g⁻¹ of cells). A process based on extracellular production could greatly reduce the cost of AA manufacture by eliminating the need for extraction of the AA from the cells. Journal of Industrial Microbiology & Biotechnology (2002) 29, 93–98 doi:10.1038/sj.jim.7000275 Received 04 December 2001/ Accepted in revised form 09 May 2002     

一种适用于PCR反应的酵母菌、无绿藻及丝状真菌DNA提取方法

目的 介绍一种从酵母、无绿藻及丝状真菌中提取DNA以用于PCR反应的方法。方法 所用菌种包括临床分离的未知菌株和保藏菌株共23株：未知酵母菌（5株）、真皮毛孢子菌（1株）、糠秕马拉色菌（1株）、季也蒙念珠菌（5株）、未知丝状真菌（6株）、无绿藻（1株）、烟曲霉（2株）、拟青霉菌（1株）、茎点霉（1株）。用溶细胞酶（lyticase）结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA,A260／A280检测纯度并计算质量浓度,用真菌通用引物ITS1／ITS4扩增真菌核糖体基因（rDNA）内转录间区ITS基因,经PCR扩增检验所提取的DNA质量。结果 成功提取所有23株真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论 用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒从酵母菌、无绿藻及丝状真菌提取的DNA可用于PCR反应。     

X-ray structure of Paramecium bursaria Chlorella virus arginine decarboxylase: insight into the structural basis for substrate specificity

The group IV pyridoxal-5'-phosphate (PLP)-dependent decarboxylases belong to the beta/alpha barrel structural family, and include enzymes with substrate specificity for a range of basic amino acids. A unique homolog of this family, the Paramecium bursaria Chlorella virus arginine decarboxylase (cvADC), shares about 40% amino acid sequence identity with the eukaryotic ornithine decarboxylases (ODCs). The X-ray structure of cvADC has been solved to 1.95 and 1.8 A resolution for the free and agmatine (product)-bound enzymes. The global structural differences between cvADC and eukaryotic ODC are minimal (rmsd of 1.2-1.4 A); however, the active site has significant structural rearrangements. The key "specificity element," is identified as the 310-helix that contains and positions substrate-binding residues such as E296 cvADC (D332 in T. brucei ODC). In comparison to the ODC structures, the 310-helix in cvADC is shifted over 2 A away from the PLP cofactor, thus accommodating the larger arginine substrate. Within the context of this conserved fold, the protein is designed to be flexible in the positioning and amino acid sequence of the 310-helix, providing a mechanism to evolve different substrate preferences within the family without large structural rearrangements. Also, in the structure, the "K148-loop" (homologous to the "K169-loop" of ODC) is observed in a closed, substrate-bound conformation for the first time. Apparently the K148 loop is a mobile loop, analogous to those observed in triose phosphate isomerase and tryptophan synthetase. In conjunction with prior structural studies these data predict that this loop adopts different conformations throughout the catalytic cycle, and that loop movement may be kinetically linked to the rate-limiting step of product release.     

Simple new test for presumptive differentiation between genus Candida and genus Prototheca

A differential test was made between genus Candida and genus Prototheca using a new and very simple differential test.A total of 59 strains of Candida and 78 strains of Prototheca were used. The basis of the test was the differential use of a disc carrying 60 mcg of Rybostamicin to which all the Candida were resistant and the Prototheca inhibited.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

Use of morpho-physiological characteristics for differentiation of the species of Prototheca

Fifty-two cultures of Prototheca spp. isolated from water and 7 isolates received from a culture collection were tested for their ability to assimilate carbon and nitrogen sources. Based upon these findings and on micromorphological features of the isolates a rapid method allowing differentiation of Prototheca spp. in culture is presented.     

The assimilation of acetate and propionate by Prototheca zopfi

1. The tricarboxylic acid and glyoxylate cycles are of major importance in the assimilation of acetate and propionate by Prototheca zopfii. The pattern of assimilation of [2-(14)C]acetate and [2-(14)C]propionate by whole cells growing with their respective substrates is similar except that, with propionate, beta-hydroxypropionate is the first labelled intermediate detected. 2. Carbon dioxide fixation is of little quantitative importance for the growth of this organism with propionate. 3. The yield of cells obtained/mole of acetate is similar to that obtained/mole of propionate and about half that obtained/mole of n-butyrate, these substrates acting as sole sources of carbon and energy.     

Growth of Prototheca isolates on n-hexadecane and mixed-hydrocarbon substrate

Prototheca zopfii, an achlorphyllous alga, was capable of using hydrocarbons as sole carbon and energy source. The ability of P. zopfii to use hydrocarbons did not correlate with source of isolation. Seventy-five percent of the P. zopfii cultures recovered from sewage, plants, or animals utilized hydrocarbons. Other Prototheca species and P. zopfii that did not utilize hydrocarbons were isolated simultaneously from several sources with isolates that did use hydrocarbons. Species type rather than source of isolation was the predominant factor that determined hydrocarbon utilization.