Flocculation of Chlorella vulgaris by Lactobacillus casei

Summary The flocculation of Chlorella vulgaris by Lactobacillus casei was studied to determine whether the latter could act as a suitable flocculant for the removal of Chlorella from algal ponds. The flocculating activity of the Lactobacilli was caused by the bacterial cells themselves, and not by diffusible products of bacterial metabolism. Diffusible products of algal metabolism inhibited flocculation. For algae resuspended in water, the best flocculation occurred at pH values less than 3.5 where the charges on the bacterial and algal cells were opposite. For flocculation at least one bacterium was required for every algal cell; in terms of cell concentrations,10 mg/l of bacteria were required to flocculate an algal suspension of 1,000 mg/l. The mechanism of flocculation implied by the results is that positively charged cells of L. casei adsorb to the surface of negatively charged cells of C. vulgaris neutralizing the charge and thus destabilizing the algal suspension. Because of the low pH required and because diffusible products of algal metabolism inhibit the flocculation, it is unlikely that L. casei could be usefully employed as a flocculant of Chlorella from algal ponds.     

叶绿素合成缺陷型小球藻突变株的选育及其蛋白产能和品质评价

本研究旨在利用常压室温等离子体(atmospheric pressure room temperature plasma, ARTP)诱变技术构建叶绿素合成缺陷型凯式小球藻突变株,筛选出极低叶绿素、适用于发酵生产蛋白质的新藻种。首先经优化诱变处理时间后建立了野生型兼养细胞的致死率曲线,在高于95%致死率条件下处理对数早期兼养细胞,基于可视化藻落颜色变化初筛获得4株突变株。随后在摇瓶中异养培养突变株,系统评价了蛋白生产性能,发现在含有30 g/L葡萄糖和5 g/L NaNO₃的Basal培养基中,突变株P. ks4表现最优,蛋白含量及产率分别为39.25%干重及1.15g/(L·d),氨基酸评分达101.34,叶绿素a含量下降98.78%且不含叶绿素b,含有叶黄素0.62 mg/g而使藻体呈金黄色。本研究为微藻替代蛋白的发酵生产提供了高性能、高品质的新种质P. ks 4。     

The Occurrence of the Glucose-inducible Transport Systems for Glucose,Proline, and Arginine in Different Species of Chlorella

Incubation of the green alga Chlorella vulgaris (strain K, Tanner and Kandler, 1967) with glucose leads to the induction of a glucose transport system and of two amino acid transport systems. Because it was not clear whether the regulation of 3 different transport systems by glucose is specific to our strain of Chlorella or whether it is a general property of the genus Chlorella, 11 other free living and symbiotic Chlorella species and strains were tested for glucose-inducible glucose, arginine and proline transport. It was found that nearly all Chlorella species possess glucose and amino acid uptake systems. Often they were constitutive, although in some species they were induced or stimulated by glucose. According to the transport activities of the different Chlorella species and strains, a physiological classification of Chlorella was constructed, resulting in 3 groups: the C. fusca vacuolata, the C. vulgaris and the symbiotic Chlorella group. Our Chlorella (strain K) obviously belongs to the C. vulgaris group and forms a link to symbiotic Chlorella strains. This suggests that the possession of the glucose-regulated transport systems is of advantage for Chlorella in symbiotic situations, whereas the constitutive systems are useful for free living Chlorella.     

Generic concept in Chlorella‐related coccoid green algae (Chlorophyta,Trebouxiophyceae)

Using a combined set of sequences of SSU and ITS regions of nuclear‐encoded ribosomal DNA, the concept of the experimental algal genus Chlorella was evaluated. Conventionally in the genus Chlorella, only coccoid, solitary algae with spherical morphology that do not possess any mucilaginous envelope were included. All Chlorella species reproduce asexually by autospores. However, phylogenetic analyses showed that within the clade of ‘true’Chlorella species (Chlorella vulgaris, C. lobophora, and C. sorokiniana), taxa with a mucilaginous envelope and colonial lifeform have also evolved. These algae, formerly designated as Dictyosphaerium, are considered as members of the genus Chlorella. In close relationship to Chlorella, five different genera were supported by the phylogenetic analyses: Micractinium (spherical cells, colonial, with bristles), Didymogenes (ellipsoidal cells, two‐celled coenobia, with or without two spines per cell), Actinastrum (ellipsoidal cells within star‐shaped coenobia), Meyerella (spherical cells, solitary, without pyrenoids), and Hegewaldia (spherical cells, colonial, with or without bristles, oogamous propagation). Based on the secondary structures of SSU and ITS rDNA sequences, molecular signatures are provided for each genus of the Chlorella clade.     

Responses of Two Coastal Algae (Skeletonema costatum and Chlorella vulgaris) to Changes in Light and Iron Levels1

Iron (Fe) is essential for phytoplankton growth and photosynthesis, and is proposed to be an important factor regulating algal blooms under replete major nutrients in coastal environments. Here, Skeletonema costatum, a typical red-tide diatom species, and Chlorella vulgaris, a widely distributed Chlorella, were chosen to examine carbon fixation and Fe uptake by coastal algae under dark and light conditions with different Fe levels. The cellular carbon fixation and intracellular Fe uptake were measured via ¹⁴C and ⁵⁵Fe tracer assay, respectively. Cell growth, cell size, and chlorophyll-α concentration were measured to investigate the algal physiological variation in different treatments. Our results showed that cellular Fe uptake proceeds under dark and the uptake rates were comparable to or even higher than those in the light for both algal species. Fe requirements per unit carbon fixation were also higher in the dark resulting in higher Fe: C ratios. During the experimental period, high Fe addition significantly enhanced cellular carbon fixation and Fe uptake. Compared to C. vulgaris, S. costatum was the common dominant bloom species because of its lower Fe demand but higher Fe uptake rate. This study provides some of the first measurements of Fe quotas in coastal phytoplankton cells, and implies that light and Fe concentrations may influence the phytoplankton community succession when blooms occur in coastal ecosystems.     

Cyanide formation in preparations from Chlorella and New Zealand spinach leaves: Effect of added amino acids

Summary As part of an effort to identify the natural precursor(s) of HCN in the alga Chlorella vulgaris Beijerinck, and in leaves of New Zealand spinach (Tetragonia expansa, Murr.), HCN release was measured after addition of various amino acids to illuminated algal extracts and grana preparations. Histidine is particularly effective as an HCN precursor, both with Chlorella extracts and leaf grana. With the algal extracts, d-histidine is about ten times more effective than l-histidine and histamine, whereas the two isomers (and histamine) are about equally effective with leaf grana. In the presence of leaf grana plus added Mn²⁺ and peroxidase, l-tyrosine and l-cysteine like-wise cause HCN formation; but these amino acids cause little or no HCN formation in the presence of Chlorella extracts. A stimulation of HCN production by l-histidine was observed with intact Chlorella cells. Because of the limitations of the assay method, the possibility can not be excluded that other substances than histidine may also lead to HCN generation in Chlorella vulgaris, but the results show that histidine has an important role in HCN generation by this species.Abbreviation POD peroxidase     

Microalgal Biomass and Lipid Production on Dairy Effluent Using a Novel Microalga, <Emphasis Type="Italic">Chlorella</Emphasis> sp. Isolated from Dairy Wastewater

The present study focused on cost-effective production of microalgal biomass and lipid production on dairy effluent. The novel microalga, Chlorella sp. isolated from the dairy effluent showed high growth and lipid production on the undiluted and two-fold diluted dairy effluent which were four to five times higher than those of Chlorella vulgaris (control). The high growth of Chlorella sp. was thought to be possibly due to its heterotrophic growth capacity, high turbidity, COD, nutrients and trace elements. In contrast, C. vulgaris showed poor heterotrophic and photoautotrophic growth under the highly turbid conditions of dairy effluent. Both Chlorella sp. and C. vulgaris showed similar total FAME (mg FAME/g algal cells). The fatty acid composition analysis revealed that both Chlorella sp. and C. vulgaris possessed major C18 and C20 fatty acids which will be used for biodiesel production. Overall, the novel microalga, Chlorella sp. isolated from the dairy effluent showed high potential for cost-effective algal cultivation and lipid production on dairy effluent without any modification of process.     

Rates of photosynthesis of two lichens and of their isolated algal components

Abstract

The rate of photosynthesis of two lichen species (Peltigera leucophlebia and Ramalina farinacea) was found to be 30 to 40% that of spinach leaf dises and 20% that of the free-living alga Chlorella when the results were expressed on a per mg chlorophyll basis. When the algae were isolated from the thalli, the rate of photosynthesis per mg chlorophyll increased for Ramalina farinacea and decreased for Peltigera leucophlebia. Product analysis indicated that the products of photosynthesis depended on the association of the alga with the fungus: algae isolated from the thalli showed a «shift» in products from sugars and sugar alcohols. to compounds such as organic acids. The results suggest that a symbiotic relationship with a fungus alters both the rate and products of algal photosynthesis.     

Factors that Control the Species Composition of Freshwater Phytoplankton,with Special Attention to Nutrient Concentrations

Variations of species composition and population size of planktonic algae were studied in relation to the different nutrient levels in a eutrophic pond and an oligotrophic lake. The results obtained were discussed in comparison with the changes in photosynthesis and chlorophyll of several algal populations cultured under different nutrient conditions. As based on unialgal culture experiments two types of algae (the Chlorella-type and the Melosira-type) could be distinguished with regard to variations of photosynthesis and chlorophyll in response to different nutrient levels. Distribution of the Chlorella-type algae may be confined to eutrophic waters, while the Melosira-type algae can be distributed widely in waters from oligotrophic to eutrophic.     

Analysis of the agglutinating activity from unicellular algae

Agglutinating activity often varies both between and within the algal species assayed. However, it is difficulty to interpret such variation without further analysis. We report a statistical analysis of agglutinating activities against human, cow, sheep, and pig erythrocytes, using cell extracts from 43 taxa (strains) of freshwater microalgae. Most of the extracts agglutinated erythrocytes from at least one of the sources, but pig erythrocytes appeared to be most suitable for the detection of agglutination reactions. Chlorella cell extracts preferentially agglutinated human erythrocytes, whereas extracts of other taxa were less active against mammalian erythrocytes. Cluster analysis generated four distinct subclusters of taxa, characterized by different specificities for antigens or carbohydrate receptors on the erythrocytes. Principal component analysis further separated the agglutination characteristics of Chlamydomonas from Chlorella on the first two components. Specificity for pig erythrocytes accounted for most of the clustering or grouping of algal taxa in multivariate analysis. However, clustering or grouping patterns of Chlorella species on haemagglutinating activity resembled that based on DNA sequences, revealing a possible genetic connection of agglutinins and their biochemical characteristics in algal cells. Variability of agglutination reactions among the algae investigated is simplified and interpreted most easily using multivariate analysis.     

Phylogenetic position of someChlorella species within the chlorococcales based upon complete small-subunit ribosomal RNA sequences

Summary Complete small-subunit rRNA (16S-like rRNA) coding region sequences were determined for eight species of the Chlorococcales (Chlorophyceae). The genera investigated includePrototheca, Ankistrodesmus, Scenedesmus, and fiveChlorella species. Distance matrix methods were used to infer a phylogenetic tree that describes evolutionary relationships between several plant and green algal groups. The tree exhibits a bifurcation within the Chlorococcales consistent with the division into Oocystaceae and Scenedesmaceae, but three of the fiveChlorella species are more similar to other algae than toChlorella vulgaris. All of the sequences contain primary and secondary structural features that are characteristic of 16S-like rRNAs of chlorophytes and higher plants.Anikstrodesmus stipitatus, however, contains a 394-bp group I intervening sequence in its 16S-like rRNA coding region.     

Zur Taxonomie einer auxotrophen Chlorella aus Paramecium bursaria Ehrbg

Zusammenfassung Auf Grund der physiologischen Merkmale einer in Paramecium bursaria Ehrbg. auftretenden Chlorella ergibt sich eine systematische Zuordnung in den Formenkreis um Chlorella vulgaris f. tertia Fott et Nováková und Chlorella vulgaris var. vulgaris Beijerinck. Hiervon abweichende Befunde anderer Autoren werden diskutiert.

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On the taxonomy of an auxotrophic Chlorella isolated from Paramecium bursaria ehrbg

An auxotrophic Chlorella has been isolated from Paramecium bursaria Ehrbg. and cultivated in mass culture in an inorganic medium supplied with vitamins B₁ and B₁₂. With regard to its physiological properties it is not identical with either one of the so far known Chlorella species. It belongs, however, to the group of Chlorella vulgaris f. tertia Fott et Novaková and Chlorella vulgaris var. vulgaris Beijerinck.

     

CILIATE‐SYMBIONT SPECIFICITY OF FRESHWATER ENDOSYMBIOTIC CHLORELLA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

The nature of Chlorella symbioses in invertebrates and protists has attracted much interest, but the uncertain taxonomy of the algal partner has constrained a deeper ecological understanding of this symbiosis. We sequenced parts of the nuclear 18S rDNA, the internal transcribed spacer (ITS)‐1 region, and the chloroplast 16S rDNA of several Chlorella isolated from pelagic ciliate species of different lakes, Paramecium bursaria symbionts, and free‐living Chlorella to elucidate phylogenetic relationships of Chlorella‐like algae and to assess their host specificity. Sequence analyses resulted in well‐resolved phylogenetic trees providing strong statistical support for a homogenous ‘zoochlorellae’ group of different ciliate species from one lake, but clearly different Chlorella in one of those ciliate species occurring in another lake. The two Chlorella strains isolated from the same ciliate species, but from lakes having a 10‐fold difference in underwater UV transparency, also presented a distinct physiological trait, such as the ability to synthesize UV‐absorbing substances known as mycosporine‐like amino acids (MAAs). Algal symbionts of all P. bursaria strains of different origin resolved in one clade apart from the other ciliate symbionts but split into two distinct lineages, suggesting the existence of a biogeographic pattern. Overall, our results suggest a high degree of species specificity but also hint at the importance of physiological adaptation in symbiotic Chlorella.     

Growth interactions among blue-green (Anabaena Oscillarioides,Microcystis aeruginosa) and green (Chlorella sp.) algae

The growth interactions amongst the blue-green algal species Anabaena oscillarioides, Microcystis aeruginosa and the green alga, Chlorella sp. were studied both in mixed cultures and in filter cultures separated by a membrane filter in the two arms of an interaction U-tube. The role of nutrients especially phosphate upon the interaction has also been studied. Anabaena and Microcystis both inhibited the growth of Chlorella while Microcystis also inhibited the growth of Anabaena. The inhibitory effect of Microcystis was found to be dependent on high concentrations of the initial algal inocula and independent of the initial concentration of nutrients such as inorganic phosphate, indicating that the nature of the inhibition is probably due to the production of inhibitory extracellular products by Microcystis. On the other hand, the inhibitory effect of Anabaena on Chlorella is the consequence of nutrient competition with Anabaena competing more effectively for the available phosphate.     

Thermophilic,anaerobic co-digestion of microalgal biomass and cellulose for H<Subscript>2</Subscript> production

Microalgal biomass has been a focus in the sustainable energy field, especially biodiesel production. The purpose of this study was to assess the feasibility of treating microalgal biomass and cellulose by anaerobic digestion for H₂ production. A microbial consortium, TC60, known to degrade cellulose and other plant polymers, was enriched on a mixture of cellulose and green microalgal biomass of Dunaliella tertiolecta, a marine species, or Chlorella vulgaris, a freshwater species. After five enrichment steps at 60°C, hydrogen yields increased at least 10% under all conditions. Anaerobic digestion of D. tertiolecta and cellulose by TC60 produced 7.7 mmol H₂/g volatile solids (VS) which were higher than the levels (2.9–4.2 mmol/g VS) obtained with cellulose and C. vulgaris biomass. Both microalgal slurries contained satellite prokaryotes. The C. vulgaris slurry, without TC60 inoculation, generated H₂ levels on par with that of TC60 on cellulose alone. The biomass-fed anaerobic digestion resulted in large shifts in short chain fatty acid concentrations and increased ammonium levels. Growth and H₂ production increased when TC60 was grown on a combination of D. tertiolecta and cellulose due to nutrients released from algal cells via lysis. The results indicated that satellite heterotrophs from C. vulgaris produced H₂ but the Chlorella biomass was not substantially degraded by TC60. To date, this is the first study to examine H₂ production by anaerobic digestion of microalgal biomass. The results indicate that H₂ production is feasible but higher yields could be achieved by optimization of the bioprocess conditions including biomass pretreatment.     

Preferential feeding behaviour of Daphnia magna

Preferential feeding behaviour by Daphnia magna was shown when Daphnia were fed on a mixture of ¹⁴C-labelled algae (Chlorella vulgaris or Scenedesmus quadricauda) and ³H-labelled bacteria (Escherichia coli). Daphnia consumption of bacteria was equal or higher in the presence of algae. On the other hand, in the presence of bacteria, algal consumption decreased by 40–70% compared to algal consumption in the absence of bacteria. ¹⁴C radioactive uptake was in good agreement with the chlorophyll content and demonstrates the preferential feeding behaviour of Daphnia.     

Photosynthesis,photooxidation of chlorophyll and fluorescence of normal and manganese-deficient Chlorella with and without hydrogenase

Summary Growth, chlorophyll content, chloroplast structure, photosynthesis, photooxidation of chlorophyll and fluorescence were studied in normal and manganese-deficient, closely related Chlorella strains with hydrogenase (Chlorella vulgaris f. tertia 211-8k) and without hydrogenase (Chlorella vulgaris 211-8m).Under Mn-deficient conditions, algae with hydrogenase grow slowly for several weeks without becoming chlorotic and without any major change in thylakoid structure, whereas those without hydrogenase show a rapid loss of most of their chlorophyll, followed by a breakdown of thylakoid structure. In algae without hydrogenase, the inhibition of photosynthesis by Mn deficiency is much less pronounced when photosynthesis is determined on a chlorophyll rather than on a cell-volume or dry-weight basis. Thus the little remaining chlorophyll of these algae is photosynthetically much more active than the chlorophyll of the Mn-deficient, non-chlorotic algae with hydrogenase.Photooxidation of chlorophyll under pure O₂ in very strong light is always accelerated by Mn deficiency.Only in algae containing hydrogenase is there under aerobic conditions a pronounced peak of fluorescence at the beginning of illumination. This is indicative of a high degree of reduction of Q, the primary electron acceptor of System II of photosynthesis. The fluorescence peak can be eliminated by either 1 min preillumination with far red (=717 nm) or by treatment in the dark for 1 hr with pure O₂. In algae without hydrogenase, in contrast, fluorescence intensity at the onset of illumination is lower than the steady-state level, indicating an oxidized state of Q. In these algae a high start of fluorescence can only be produced by prolonged anaerobic incubation. These results indicate that even under aerobic conditions algal hydrogenase, or a biochemical system very closely associated with it, has some residual activity which enables it to feed electrons into System II of photosynthesis, thereby keeping Q in a reduced state with correspondingly high fluorescence at the beginning of illumination. In organisms without hydrogenase, in contrast, this can only be achieved by prolonged anaerobiosis. In addition, the presence of hydrogenase seems to protect the chlorophyll against photooxidative destruction when the cells are under Mn deficiency.This paper is dedicated to Professor A. Pirson on the occasion of his 60th birthday.     

CHARACTERIZATION OF TWO CHLORELLA VULGARIS (CHLOROPHYCEAE) STRAINS ISOLATED FROM WASTEWATER OXIDATION PONDS1

We report here on the characterization and isolation of two ecotypes of Chlorella vulgaris Beyerinck that coexist in wastewater reservoirs. One ecotype (C1) contains high amounts of chlorophyll b, is capable of autotrophic growth, and can utilize only a few organic solutes for growth. The second ecotype (C2) contains low amounts of chlorophyll b, requires vitamin B₁₂, and can support its growth with a broad range of organic compounds. Of the two ecotypes, the latter showed slower growth rates when light was the sole source of energy. Cells of C2-type Chlorella attained higher photosynthetic activities than C1-type cells at saturating irradiances. However, their low chlorophyll b content and lower light utilization efficiency suggest that C2-type Chlorella contains relatively low amounts of light-harvesting antennae, a disadvantage in severely light-limited ecosystems like wastewater reservoirs. We hypothesize that the two Chlorella types coexist by adopting different lifestyles: C1-type cells rely largely on their photosynthetic potential for energy conservation and growth, whereas C2-type cells may exploit their heterotrophic properties for this purpose.     

Effect of mixed diets (cyanobacteria and green algae) on the population growth of the cladocerans <Emphasis Type="Italic">Ceriodaphnia dubia</Emphasis> and <Emphasis Type="Italic">Moina macrocopa</Emphasis>

Microcystis aeruginosa, a cosmopolitan form, is a colonial cyanobacterium, which is also common in many freshwater bodies in Mexico. In eutrophic water bodies cyanobacteria are often the main phytoplankton that co-exist with cladocerans. We evaluated the effect of mixed diets, comprising 0, 25, 50, 75, and 100% on dry weight basis of M. aeruginosa, and the rest of one of two green algal species (Chlorella vulgaris and Scenedesmus acutus), on the population growth of the cladocerans Ceriodaphnia dubia and Moina macrocopa. Regardless of the share of M. aeruginosa in the mixed diet, C. dubia fed Chlorella had a longer initial lag phase. However, in mixed diet with S. acutus, the lag phase of C. dubia increased with increasing proportion of M. aeruginosa. When raised on 100% M. aeruginosa, the population growth of C. dubia was lowered compared with 100% S. acutus or 100% C. vulgaris. Increased proportion of M. aeruginosa in the mixed diet also resulted in decreased abundance of M. macrocopa. Irrespective of diet type, M. macrocopa had a shorter lag phase than C. dubia. Depending on the diet type, the rate of population increase (r) of C. dubia varied from 0.07 to 0.26 d⁻¹ while that of M. macrocopa was higher (0.14–0.61 d⁻¹). For both cladoceran species, the lower r values were obtained when fed Microcystis. Our study showed that the strain of M. aeruginosa was not highly toxic to cause total elimination of either C. dubia or M. macrocopa. Addition of a green algal component to the diet improved the population growth rates of both cladoceran species.