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.     

分批异养培养小球藻光密度值与干重的关系

在小球藻的分批异养培养过程中,培养液的细胞浊度（ＯＤ）与细胞干重（ＤＷ）的关系受培养条件和培养过程的影响很大,在实验中,干重与光密度值的比值在０．５３～０．２８ｇ／ＯＤ·Ｌ间变化。因此在小球藻分批异养培养过程中,培养液藻生物量检测采用单一的ＤＷ与ＯＤ换算常数用ＯＤ来推算细胞的干重则会产生较大的误差。     

INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

ISOLATION AND CHARACTERIZATION OF CHLORELLA SOROKINIANA GXNN01 (CHLOROPHYTA) WITH THE PROPERTIES OF HETEROTROPHIC AND MICROAEROBIC GROWTH1

Heterotrophic and anaerobic microalgae are of significance in both basic research and industrial application. A microalga strain was isolated from a wastewater treatment pond and identified as Chlorella sorokiniana Shihira et W. R. Krauss GXNN01 in terms of morphology, physiology, and phylogeny. The strain grows rapidly in heterotrophic or mixotrophic conditions with addition of various carbon sources, and even in anaerobic conditions. The maximum growth rate reached 0.28 d^?1 when using d,l ‐malate as the carbon source, and the protein content of the microalgae was 75.32% in cell dry weight. The strain was shown to be capable of (1) utilizing d,l ‐malate only with light, (2) inhibiting photosynthesis in mixotrophic growth, and (3) growing in anaerobic conditions with regular photosynthesis and producing oxygen internally. This study demonstrates the influence of oxygen (aerobic vs. anaerobic) and metabolic regime (autotrophy, mixotrophy, heterotrophy) on the physiological state of the cell.     

不同营养方式对小球藻FACHB 484生长的影响及其非自养生长机理研究

系统研究了小球藻FACHB 484在含有葡萄糖的不同营养方式下的生长情况,并通过抑制试验探讨葡萄糖在小球藻FACHB 484光异养和兼养生长条件下所起的作用以及小球藻FACHB 484是否存在氧化呼吸系统的关键酶类。结果表明:小球藻FACHB 484可利用葡萄糖进行化能异养、光激活异养、光异养及兼养生长,其生长速率大小为:兼养光异养光激活异养化能异养光合自养。兼养培养的最大生物量和比生长速率分别是自养培养的8.6和3.4倍,其比生长速率接近于光合自养和光异养培养下的比生长速率之和。葡萄糖主要作为小球藻FACHB 484兼养和光异养培养的碳源,而能量主要源自光。小球藻FACHB 484存在氧化呼吸链代谢途径,其细胞中有琥珀酸脱氢酶和细胞色素氧化酶。       

CELL‐CELL INTERACTION IN THE EUKARYOTE‐PROKARYOTE MODEL OF THE MICROALGAE CHLORELLA VULGARIS AND THE BACTERIUM AZOSPIRILLUM BRASILENSE IMMOBILIZED IN POLYMER BEADS1

Cell‐cell interaction in the eukaryote‐prokaryote model of the unicellular, freshwater microalga Chlorella vulgaris Beij. and the plant growth‐promoting bacterium Azospirillum brasilense, when jointly immobilized in small polymer alginate beads, was evaluated by quantitative fluorescence in situ hybridization (FISH) combined with SEM. This step revealed significant changes, with an increase in the populations of both partners, cluster (mixed colonies) mode of colonization of the bead by the two microorganisms, increase in the size of microalgae‐bacterial clusters, movement of the motile bacteria cells toward the immotile microalgae cells within solid matrix, and formation of firm structures among the bacteria, microalgae cells, and the inert matrix that creates a biofilm. This biofilm was sufficiently strong to keep the two species attached to each other, even after eliminating the alginate support. This study showed that the common structural phenotypic interaction of Azospirillum with roots of higher plants, via fibrils and sheath material, is also formed and maintained during the interaction of this bacterium with the surface of rootless single‐cell microalgae.     

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.     

ROLE OF GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHETASE IN CHLORELLA VULGARIS DURING ASSIMILATION OF AMMONIUM WHEN JOINTLY IMMOBILIZED WITH THE MICROALGAE‐GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM BRASILENSE1

Enzymatic activities of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) participating in the nitrogen metabolism and related ammonium absorption were assayed after the microalga Chlorella vulgaris Beij. was jointly immobilized with the microalgae‐growth‐promoting bacterium Azospirillum brasilense. At initial concentrations of 3, 6, and 10 mg · L^?1 NH₄⁺, joint immobilization enhances growth of C. vulgaris but does not affect ammonium absorption capacity of the microalga. However, at 8 mg · L^?1 NH₄⁺, joint immobilization enhanced ammonium absorption by the microalga without affecting the growth of the microalgal population. Correlations between absorption of ammonium per cell and per culture showed direct (negative and positive) linear correlations between these parameters and microalga populations at 3, 6, and 10 mg · L^?1 NH₄⁺, but not at 8 mg · L^?1 NH₄⁺, where the highest absorption of ammonium occurred. In all cultures, immobilized and jointly immobilized, having the four initial ammonium concentrations, enzymatic activities of Chlorella are affected by A. brasilense. Regardless of the initial concentration of ammonium, GS activity in C. vulgaris was always higher when jointly immobilized and determined on a per‐cell basis. When jointly immobilized, only at an initial concentration of 8 mg · L^?1 NH₄⁺ was GDH activity per cell higher.     

EFFECTS OF ARSENATE ON GROWTH OF NITROGEN-AND PHOSPHORUS-LIMITED CHLORELLA VULGARIS (CHLOROPHYCEAE) ISOLATES1

The effects of arsenate on the growth characteristics of five isolates of the freshwater alga, Chlorella vulgaris Beij., were examined. Two field isolates originated from arsenic-contaminated sites in Yukon, Canada and Kyushi, Japan; two reference isolates were obtained from the University of Texas Culture Collection. One isolate was selected for arsenic-tolerance in the laboratory. All five strains survived in culture solutions containing high arsenate concentrations. Arsenate (1–25 mM As) reduced photosynthesis and cell growth, as reflected by induced lag periods, slower growth rates, and lower stationary cell yields. Field isolates had shorter lag periods, higher growth rates, and enhanced cell yields compared to lab isolates when exposed to the same arsenic concentrations. Growth of the phosphorus-limited field strains was stimulated by the addition of arsenic. The cell yield of phosphorus-limited C. vulgaris Yukon, when treated with arsenic, was two times that of the phosphorus-limited control. This pattern was not evident when photosynthesis was used as a measure of cell response.     

普通小球藻生长与武汉东湖水体磷形态的相关研究

本文探讨了武汉东湖水体中总溶解磷（TSP）、溶解反应磷（SRP）、总反应磷（TRP）、溶解水解磷（SHP）和颗粒磷（PP）对普通小球藻生长的生物有效利用性。进行了普通小球藻生长量与各磷形态及总磷（TP）被利用浓度的一元相关分析,进一步讨论了这些一元相关方程的不足,并用多元回归分析建立了普通小球藻生长量（N）与所测几种磷形态被利用浓度（CTSP,CSRP,CTRP,CSHP,CPP）的相关模型。这些模型可以用于评价湖泊水体富营养化的程度及预测藻的生长潜力。     

小球藻LEA 家族蛋白Ccor1和Ccor2在抗盐胁迫中的作用

LEA蛋白是与植物抗逆反应相关的一类重要蛋白质,可以在发育的植物胚胎、处于干旱、寒冷、盐胁迫的植物组织中高水平表达。LEA蛋白最早发现于棉花子叶中,随后也在其他高等植物、藻类、真菌、蓝藻以及线虫中被发现。LEA蛋白代表一个庞大的基因家族,早期是根据     

EFFECT OF MALTOSE RELEASE ON UPTAKE AND ASSIMILATION OF AMMONIUM BY SYMBIOTIC CHLORELLA (CHLOROPHYTA)1

When incubated at pH 4–5, Chlorella freshly isolated from symbiosis with Hydra viridissima PALLAS 1766 (green hydra) release large amounts of photosynthetically fixed carbon in the form of maltose, and assimilation of inorganic N is inhibited. Physiological responses to N starvation of the cultured 3N813A strain of maltose-releasing Chlorella differed from those caused by 48 h of maltose release induced by low pH. N starvation increased rates of ammonium assimilation at pH 7.0 in light or darkness, and ammonium assimilation in darkness stimulated cell respiration. In contrast, cells pretreated at pH 5.0 to induce maltose release were unable to take up ammonium at pH 7.0 unless supplied with an external carbon source such as bicarbonate, acetate, or succinate, and rates of uptake were similar to control cells. Freshly isolated symbionts displayed a similar dependency. Rates of ammonium uptake by cells pretreated at pH 5.0 were reduced in darkness and did not stimulate cell respiration. N-starved cells supplied with ammonium also showed a large short-term increase in glutamine pools at the expense of glutamate, as might be expected if large amounts of ammonium were rapidly assimilated via glutamine synthetase/glutamate synthase, whereas after long-term maltose release cells showed only a small increase in glutamine when supplied with ammonium. Furthermore, maltose release caused a fall in pool sizes of a number of amino acids, including glutamine and glutamate, and also caused a decrease in pool sizes of 2-oxoglutarate and phospho-enol-pyruvate, which are required for ammonium assimilation into amino acids. Cells stimulated to synthesize and release maltose may be unable to assimilate ammonium and synthesize amino acids because of diversion of fixed carbon from N metabolism. We estimate that 40–50% affixed C is required for maximal maltose synthesis, whereas up to 30% fixed C is required for ammonium assimilation. These results are discussed in the context of host regulation of symbiotic algal growth.     

THE UPTAKE OF MANNITOL AND SORBITOL BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

Chlorella saccharophila (Krüger) Nadson takes up mannitol and sorbitol in the light and the dark. The rate of uptake is concentration dependent. is not affected by pH in the range pH 6.0 to 8.0 and ii not stimulated by light. Uptake is inhibited by the respiration inhibitor sodium azide (10^-2 M) but not by 3-(3,4-dichlorophenyl)-1,1-di-methyl urea (10^-6 M), an inhibitor of photosynthesis. Sorbitol. but not mannitol, stimulates the rate of dark respiration but both support the heterotrophic growth of the alga. Both compounds permeate the cells of C. miniata. and two strains of C. pyrenoidosa but do not support the heterotrophic growth of these algae. The cells of C. vulgaris are impermeable to both compounds.     

EFFECTS OF CO2 CONCENTRATION DURING GROWTH ON THE INTRACELLULAR STRUCTURE OF CHLORELLA AND SCENEDESMUS (CHLOROPHYTA)1

Effects of CO₂ concentration during growth on intracellular structure were studied with ftve species of Chlorella and Scenedesmus obliquus. Cells grown under ordinary air conditions (low-CO₂ cells) had a well developed pyrenoid surrounded by starch, while those grown under high CO₂ conditions (high-CO₂ cells) had a less developed pyrenoid or no detectable pyrenoid. Two mitochondria, one at each side of the neck of the projection of the chloroplast close to the pyrenoid, were found in low CO₂ cells of C. vulgaris 11h. Usually, lamellar stacks extended in parallel in the chloroplast of low-CO₂ cells of C. vulgaris 11h, while a grana-like structure was found in high-CO₂ cells. However, in C. pyrenoidosa, grana like structures were found more commonly in low-CO₂ cells than in high-CO₂ cells. These results suggest that development of pyrenoid starch is generally correlated with growth under low CO₂ conditions, whereas CO₂-effects on lamellar stacking are species dependent.     

CO2 ACCUMULATION BY CHLORELLA SACCHAROPHILA (CHLOROPHYCEAE) AT LOW EXTERNAL pH: EVIDENCE FOR ACTIVE TRANSPORT OF INORGANIC CARBON AT THE CHLOROPLAST ENVELOPE1

The CO₂ uptake characteristics of the acid-tolerant alga Chlorella saccharophila (Krüger) Nadson have been examined. These, together with direct estimations of internal inorganic C concentrations, indicate that air-adapted cells of this alga possess a CO₂ accumulation mechanism similar to that reported in other members of the Chlorophyceae and in Cyanophyceae. This accumulation mechanism operates at external pH values of 4 and less. Under these conditions CO₂ must be the carbon species crossing the plasmlemma, and since preliminary evidence suggests that the transport mechanism is active primary uniport of HCO₃^–, it is proposed that the site of the CO₂ accumulation mechanism is the chloroplast envelope.     

THE BIOTRANSFORMATION,BIODEGRADATION, AND BIOREMEDIATION OF ORGANIC COMPOUNDS BY MICROALGAE1

Rapid growth in the biotechnological industry and production has put tremendous pressure on the biological methods that may be used according to the guidelines of green chemistry. However, despite continuing dramatic increases in published research on organic biotransformation by microorganisms, more research exists with microalgae. Our efforts in transforming chemicals such as organic compounds for the production of functionalized products help to lessen the environmental effects of organic synthesis. These biotransformations convert organic contaminants to obtain carbon or energy for growth or as cosubstrates. This review aims to focus on the potential of microalgae in transformation, conversion, remediation, accumulation, degradation, and synthesis of various organic compounds. However, these technologies have the ability to provide the most efficient and environmentally safe approach for inexpensive biotransforming of a variety of organic contaminants, which are most industrial residues. In addition, the recent advances in microalgal bioactivity were discussed.     

INHIBITION OF CHLORELLA (CHLOROPHYCEAE) GROWTH BY FATTY ACIDS, USING THE PAPER DISC METHOD1,2

Using the paper disc-agar plate method, a number of fatty and related acids have been tested for tested activity for inhibiting the growth of Chlorella pyrenoidosa Chick. Of the saturated acids, a peak in growth inhibiting activity wax observed in the C₇–C₁₂ range, where inhibition wax observed when solutions down to 0.02 M were applied to the discs. Most of the unsaturated acids tested showed greater inhibition than did the corresponding saturated acids. Acrylic acid showed detectable inhibition at 0.001 M concentration.     

DISTINCTION BETWEEN MULTIPLE ISOLATES OF CHLORELLA VULGARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE) AND TESTING FOR CONSPECIFICITY USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM AND ITS RDNA SEQUENCES1

Multiple strains of individual algal species are available from public culture collections, often with the same isolate being maintained in parallel at a number of collections under different culture regimes. To unravel genomic variation and to identify unique genotypes among such multiple strains, two approaches were used on a sample of 29 strains of Chlorella vulgaris Beijerinck, an alga of great value for applied research, from five culture collections. With the exception of two strains, internal transcribed spacer rDNA sequence data substantiated conspecificity of the studied strains and only minor sequence differences with the authentic “Beijerinck isolate” were observed. Amplified fragment length polymorphism (AFLP) detected considerable genomic variation when rDNA sequences were identical. Band detection and the construction of a binary matrix from AFLP patterns for phylogenetic analyses were fully automated, but comparison of similar patterns still required manual refinement. The AFLPs distinguished 11 unique genotypes and provided robust support for the presence of five cryptic species. This finding advocates the need to carefully record which strain has been used in any experiment or in applied research, because genomic variation may also correspond to differences in physiological/biochemical properties. No genomic differences could be detected between duplicate strains of the same isolate that were maintained by continuous subculturing over many decades or within those stored at ultralow temperatures.     

TRANSPORT OF INORGANIC CARBON AND THE ‘CO2 CONCENTRATING MECHANISM’ IN CHLORELLA EMERSONII (CHLOROPHYCEAE)1

Chlorella emersonii Shihira et Krauss var. emersonii exhibits ‘C₄-like’ gas exchange characteristics when grown at air levels of CO₂, but is ‘C₃-like’ when grown with extra CO₂. The total inorganic carbon concentration, and the free CO₂ concentration, averaged over the cell interior are higher in air-adapted cells than can be accounted for by passive CO₂ equilibration from the medium and the mean intracellular pH value. The ‘extra’ inorganic C in the air-grown cells probably cannot all be accounted for in terms of binding to proteins and requires an active transport process to account for it. The electrical potential of the cell interior becomes more negative when the ‘CO₂ concentrating mechanism’ is operative; this is most readily explained if the active step in inorganic C accumulation is primary active uniport of HCO₃^?. Since the ‘CO₂ concentrating mechanism’ can operate when CO₂ is the species crossing the outer permeation barrier, it is suggested that the site of active HCO₃^? transport in Chlorella (and other eukaryotes) is the chloroplast envelope, and the plasmalemma in cyanobacteria. This scheme explains the obligatory role of the de-repressed carbonic anhydrase in C₄-like photosynthesis in algae, but some other data support an explanation of C₄-like photosynthesis in terms of special properties of carbonic anhydrase as a carbon donor to RuBP carboxylase-oxygenase.