Studies on Scenedesmus acutus growth. I effect of autotrophic and mixotrophic conditions on the growth of Scenedesmus acutus

Among sugars, glucose and mannose were found to be the most suitable substrates for mixotrophic growth, uptake of galactose and its influence on growth was negligible, and sucrose and fructose occupied intermediary positions. The optimum temperature for sugar uptake was 30 degrees C, both under light and in darkness. Enhancement in the photosynthetic oxygen-evolution rate, based on the utilization of substrates, was foremost in the presence of glucose, followed by mannose, sucrose, and fructose. Industrial by-products such as sugarcane molasses also were utilized to increase the algal growth under mixotrophic conditions. A maximum yield in biomass was obtained subsequent to the combined supply of sugarcane molasses with carbon dioxide to indoor as well as outdoor mixotrophic cultures. Doubling the carbon dioxide supply alone above a certain level, under autotrophic and mixotrophic outdoor conditions, did not produce a pronounced increase in the algal growth rate. The results on autotrophic and mixotrophic growth variations are discussed in the article.     

Studies on Scenedesmus acutus growth. The incorporation of 14C-glucose by Scenedesmus acutus under light and dark periods

Qualitative and quantitative estimations of alcohol-extractable compounds from (14)C-glucose-incorporated Scendesmus acutus cells were performed at various intervals of time. After two hours of incubation with (14)C-glucose in light, amino acids were synthesized at 65% and sucrose at 26% of the total input label. In the dark incorporation, 30% of the total radioactivity was found in amino acids and 46% in sucrose. Leucine and valine were detected only during the oxidation of glucose in light. The concentration of serine increased more in the presence of light, as compared to dark. These results on the oxidation of glucose are discussed in this article.     

葡萄糖对小球藻(Chloralla sp. HN08)光合作用和生长的影响

【目的】探讨葡萄糖作为外加碳源对热带海洋小球藻(Chloralla sp.HN08)生物质生产和脂、光合色素、碳水化合物及可溶性蛋白等细胞主要成份含量的影响。【方法】分析比较小球藻HN08在光合自养和兼养(添加10 g/L葡萄糖)2种营养方式下的生长速率、细胞密度、光合放氧速率、油脂相对含量,以及可溶性总糖、淀粉和可溶性蛋白的含量。【结果】结果表明,在光照条件下葡萄糖(10 g/L)能促进小球藻(Chloralla sp.HN08)生长,提高细胞终密度,而异养条件下藻细胞逐渐衰亡。兼养条件下,细胞相对生长速率及细胞终密度分别是自养条件下的6.8倍和1.3倍。兼养藻细胞中可溶性糖、淀粉、油脂含量显著高于(P0.05)光合自养细胞,然而可溶性蛋白质和光合色素含量显著低于(P0.05)光合自养细胞。添加葡萄糖的小球藻液的光饱和点和呼吸速率均高于光自养条件下的细胞,但2种培养条件下藻液的净光合速率无显著差异(P0.05)。【结论】光照条件下,添加葡萄糖可显著提高小球藻HN08相对生长速率和细胞终密度,促进油脂与淀粉的积累。     

有机碳源对三角褐指藻生长、胞内物质和脂肪酸组分的影响

研究了3种有机碳对三角褐指藻生长、胞内物质和脂肪酸组分的影响。结果表明, 三角褐指藻具有利用有机碳进行兼养生长的能力, 生长速率加快, 倍增时间缩短, 生物量显著提高, 100 mmol/L甘油兼养的生物量最高(713 mg/L), 是自养(460 mg/L)的1.60倍, 乙酸钠和葡萄糖兼养的生物量分别是自养的1.28倍和1.21倍。兼养下蛋白质含量较自养明显下降, 碳水化合物和总脂含量高于自养, 乙酸钠和甘油兼养的总脂含量分别是自养的1.43倍和1.20倍, 葡萄糖兼养的总脂含量与自养无明显差异。3种有机碳兼养的饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的比例增大, 多不饱和脂肪酸比例降低, EPA(eicosapentaenoic acid)比例降低, 乙酸钠兼养的胞内EPA含量(6.23%)和产量(36.59 mg/L)均高于自养, 分别是自养的1.10倍和1.40倍, 甘油和葡萄糖兼养的EPA含量和产量均低于自养。     

Influence of light on plastocyanin formation in the alga Scenedesmus acutus

Scenedesmus acutus, pregrown autotrophically, forms high levels of plastocyanin during an initiation period of 24 hr in the light, after cupric ions have been added to depleted cells. In the dark, no plastocyanin formation is observed. This is in contrast to heterotrophic cells which, over the same period and under identical conditions, yield about half the plastocyanin level regardless of whether they are incubated in the dark or in the light. The ATP level is high in all cases when substantial formation of plastocyanin takes place, while the ³⁵S-sulfolipid level, the marker of thylakoid formation, is at variance. After the 24-hr plastocyanin formation period, the ³⁵S-sulfolipid level is high in illuminated autotrophic cells as well as in dark-incubated heterotrophic ones, but low in illuminated heterotrophic cells, all of them having substantial plastocyanin contents. Additional experiments on light dependence of copper uptake, influence of light quality, occurrence of apoplastocyanins, provide a set of data to assume that the role of light in plastocyanin formation is primarily through photophosphorylation, the ATP presumably being necessary for processing of plastocyanin precursors to holoprotein.     

Effect of acetate on growth and ammonium uptake in the microalga Scenedesmus obliquus

The effect of acetate on growth and rate of ammonium uptake in Scenedesmus obliquus (UTEX 78) was investigated under light-limiting conditions. Addition of acetate to autotrophic cells with a growth constant of 0.71 day⁻¹ resulted in an increase in the growth rate (mixotrophy, k = 1.3 day⁻¹), and in the presence of acetate, growth occurred in the dark (heterophy, k = 0.44 day⁻¹). The rate of ammonium uptake in autotrophy (17.8 amol cell⁻¹ min⁻¹) was similar to that in heterotrophy (17.4 amol cell⁻¹ min⁻¹) but was 3.7 times lower than that in mixotrophy (65.9 amol cell⁻¹ min⁻¹). In general, mixotrophic cells showed optimum ammonium uptake at the acetate concentration at which they were grown. In autotrophy, uptake of ammonium leveled off at about 12.5 μ M while no saturation was observed in mixotrophic cells. An increase in the rate of uptake of ammonium was observed in autotrophic cells within 1 h after the addition of acetate. The activity of isocitrate lyase (EC 4.1.3.1), a key enzyme for the regulation of the glyoxylate cycle responsible for acetate catabolism, showed a 3.9-fold increase in activity after 24 h in the dark in the presence of acetate. The level of isocitrate lyase activity in cells grown for 24 h in the dark in the presence of 0–20 m M acetate also increased as a function of acetate concentration.     

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.     

A two-stage cultivation process for the growth enhancement of Chlorella vulgaris

This study proposes a two-stage cultivation process with an autotrophic growth followed by a mixotrophic process. The results indicated that a two-stage cultivation process using a daily dose of 3 g/L of glucose could achieve 7.4 g/L of biomass, which was about a 64 % increase over simple autotrophic cultivation. In the second stage of mixotrophic cultivation, glucose was regarded as a better carbon source for cell growth, than was glycerol. Linoleic acid (C18:2) would be the primary component in the two-stage cultivation as in the autotrophic cultivation. Even carbon source was provided in the second stage of mixotrophic cultivation; lower light intensity limited the mixotrophic growth, which indicated that photosynthesis still plays an important role in the second stage of mixotrophical cultivation. The final biomass was higher after this two-stage cultivation process, which made it suitable for application in the production scale-up of algal biomass.     

Hydrogenase and ribulose diphosphate carboxylase during autotrophic, heterotrophic, and mixotrophic growth of scotochromogenic mycobacteria.

Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence of carbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.     

Studies on Chlorella regularis, heterotrophic fast-growing strain II. Mixotrophic growth in relation to light intensity and acetate concentration

1) Chlorella regularis showed exponential growth under heterotrophiccondition (in the dark with acetate as the carbon source). Thegrowth rate depended on the acetate concentration. Under autotrophic condition (in light with an inorganic medium),growth proceeded exponentially then linearly, in a pattern typicalof unicellular algae. The effect of light intensity on the growthrates in the exponential and linear phases was examined. When the cells were cultured under mixotrophic condition (inlight with acetate), the growth rate was approximately the sameas the sum of the growth rates in the autotrophic and heterotrophiccultures. CMU (50 µM), a specific inhibitor of photosynthesis, causedcomplete suppression of autotrophic growth, but did not affectheterotrophic growth. When the inhibitor was added to the mixotrophicculture, growth decreased to the level of the heterotrophicculture. These facts indicate that the mixotrophic growth processes ofthe alga consist of autotrophic and heterotrophic processesthat can proceed noncompetitively with each other. (Received March 31, 1976; )     

Zur mixotrophen Kultivation von Chlorella vulgaris in homokontinuerlicher Chemostatkultur

Cells of Chlorella vulgaris, BEIJ. Greifswald 9, were grown on autotrophic and mixotrophic conditions using glucose and acetate as organic substrates. It was shown that these C-sources applicated in a suitable range of concentrations increase the growth rate and the productivity of the algal cultures. The cells grown on mixotrophic conditions have a higher total pigment content and exhibit variations in the ratio chlorophyll a/chlorophyll b. In addition the contents of proteins, lipids, carbohydrates, and nucleic acids of the biomass were shown to be dependent on the kind of the organic substrate used.     

Das rhythmische Verhalten einer farblosen Mutante von Euglena gracilis

Summary A colorless mutant of Euglena gracilis shows a cireadian rhythmic mobility in darkness just as mixotrophic and autotrophic green forms do.Therefore the obligatory heterotrophic form is very suitable for testing to what extent the rhythmic behavior of the mixotrophic cells, in contrast to that of the autotrophic cells, is independent of photosynthesis.Like the green cells, the colorless cells are synchronized by a single transient from light to dark. About 12 hours after the beginning of the darkness the rhythmic mobility of the colorless cells attains the first maximum, whereas the green forms both show their first maximum 18 hours after the end of the light period, which is much more advantageous for a photosynthesizing organism.The free running period seems to be dependent on the temperature during anaerobic glycolysis and independent of temperature during respiration, just as it has been recently found out in the case of green forms. The type of energy supply changes with the age of the cultures.Respiration has no significance as energy source for the rhythm which continues under pure glycolytic conditions.A sudden increase of the constant temperature does not shift the phase. The same is true in the case of mixotrophic cells but not in the case of autotrophic cells. However, the rhythm is often first suppressed for several days.A lowering of the temperature is followed by two or three transients with about half the frequency, but it does not influence the phase as far as it can be extrapolated. The same has been shown to be true in the case of mixotrophic cells.The rhythmic behavior of the heterotrophic cells is very similar to that of the mixotrophic ones. However, the heterotrophic cells are very sensitive to changes in temperature, which are more compensated for in the mixotrophic cells, apparently by photosynthesis.     

Kinetics of Glucose Incorporation by Aphanocapsa 6714

Photoautotrophic metabolism of CO(2) was compared with glucose metabolism in the facultative unicellular blue-green alga, Aphanocapsa 6714. Glucose-fed cells incorporated more (14)C into phosphorylated sugar intermediates of the reductive and oxidative pentose phosphate cycles than autotrophic cells. The relative increases were: 140-fold in dark cells; 32-fold in dichlorophenylmethylurea (DCMU)-inhibited cells; and 16-fold in cells assumilating glucose during photosynthetic carbon reduction. On the other hand, incorporation of (14)C from glucose into 3-phosphoglycerate and the amino acid pools of glutamate and aspartate was reduced in dark cells. Rates of protein synthesis in dark and DCMU-inhibited cells were reduced 50 and 80% compared to photoautotrophic cells. In cells assimilating glucose during photosynthesis, rates of (14)C incorporation into the two amino acids and protein were the same as in photoautotrophic cells. Chase experiments, using an excess of (12)C-glucose and CO(2), revealed slow turnover of carbon in dark cells and intermediate turnover rates in DCMU-inhibited cells, when compared to cells assimilating glucose during photosynthesis.     

Germination of Rhizopus oligosporus Sporangiospores

The morphology of Rhizopus oligosporus (NRRL 2710) sporangiospores and their physiological requirements for germination were studied. Germination proceeded in two separable phases: phase I (swelling) and phase II (germ tube protrusion). The optimal conditions for germination were 42 degrees C and pH 4.0. Sporangiospores contained insufficient endogenous carbon for swelling or germination to occur in distilled water. Initial swelling during phase I occurred only in the presence of a suitable carbohydrate. Subsequent production of germ tubes during phase II required exogenous sources of both carbon and nitrogen. Spores germinated most rapidly in mixtures of amino acids; l-proline and l-alanine were the most effective. These amino acids, at concentrations as low as 10 M, supported germination when combined with glucose and McIlvaine (citric acid-phosphate) buffer. d-Glucose, d-xylose, and d-mannose were the most effective carbohydrates tested for promotion of germination.     

Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions

Chlorella pyrenoidosa was cultivated under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions. The influence of light on the carbon and energy metabolism of microalgae was investigated by the use of metabolic flux analysis. The respiratory activity of microalgae in the light was assessed from the autotrophic flux distribution. Results showed that the glycolytic pathway, tricarboxylic acid cycle and mitochondrial oxidative phosphorylation maintained high activities during illumination, indicating little effect of light on these pathways, while the flux through the pentose phosphate pathway during illumination was very small due to the light-mediated regulation. The theoretical yields of biomass on ATP decreased in the following order: heterotrophic culture>mixotrophic culture>autotrophic culture, and a significant amount of the available ATP was required for maintenance processes in microalgal cells. The energy conversion efficiency between the supplied energy to culture, the absorbed energy by cells and the free energy conserved in ATP were analyzed for the different cultures. Analysis showed that the heterotrophic culture generated more ATP from the supplied energy than the autotrophic and mixotrophic cultures. The maximum thermodynamic efficiency of ATP production from the absorbed energy, which was calculated from the metabolic fluxes at zero growth rate, was the highest in the heterotrophic culture and as low as 16% in the autotrophic culture. By evaluating the energy economy through the energy utilization efficiency, it was found that the biomass yield on the supplied energy was the lowest in the autotrophic cultivation, and the cyclic culture gave the most efficient utilization of energy for biomass production.     

Effects of methanol on cell growth and lipid production from mixotrophic cultivation of Chlorella sp.

The marine microalga Chlorella sp. was cultivated under mixotrophic conditions using methanol as an organic carbon source, which may also act to maintain the sterility of the medium for long-term outdoor cultivation. The optimal methanol concentration was determined to be 1% (v/v) for both cell growth and lipid production when supplying 5% CO₂ with 450 μE/m²/sec of continuous illumination. Under these conditions, the maximal cell biomass and total lipid production were 4.2 g dry wt/L and 17.5% (w/w), respectively, compared to 2.2 g dry wt/L and 12.5% (w/w) from autotrophic growth. Cell growth was inhibited at methanol concentrations above 1% (v/v) due to increased toxicity, whereas 1% methanol alone sustained 1.0 g dry wt/L and 4.8% total lipid production. We found that methanol was preferentially consumed during the initial period of cultivation, and carbon dioxide was consumed when the methanol was depleted. A 12:12 h (light:dark) cyclic illumination period produced favorable cell growth (3.6 g dry wt/L). Higher lipid production was observed with cyclic illumination than with continuous illumination (18.6% (w/w) vs 17.5% (w/w)), and better lipid production was also obtained under mixotrophic rather than autotrophic conditions. Interestingly, under mixotrophic conditions with 12:12 (h) cyclic illumination, high proportions of C_16:0, C_18:0, and C_18:1 were observed, which are beneficial for biodiesel production. These results strongly indicate that the carbon source is important for controlling both lipid composition and cell growth under mixotrophic conditions, and they suggest that methanol could be utilized to scale up production to an open pond type system for outdoor cultivation where light illumination changes periodically.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas: Regulation of Autotrophic Growth by Organic Substrates

The effects of a number of organic substrates on the autotrophic metabolism of Hydrogenomonas eutropha were examined. Dual substrate (mixotrophic) cultivation in the presence of hydrogen plus either fructose or alanine allowed autotrophic growth to begin immediately after the exhaustion of the organic substrate. On the other hand, the presence of acetate, pyruvate, or glutamate caused a lengthy lag to occur before autotrophic growth commenced. With acetate or pyruvate this lag (plateau) in the dicyclic growth curve was due to the repression of ribulose diphosphate carboxylase (RDPC) synthesis during mixotrophic growth. During heterotrophic growth with glutamate, RDPC was partially repressed; however, during mixotrophic growth, RDPC activity was high. Thus the delay of autotrophic growth was not due to a repression of RDPC by glutamate. The data suggest that glutamate interferes with autotrophic metabolism by repressing the incorporation of inorganic nitrogen. The repression of these vital autotrophic functions by acetate, pyruvate, and glutamate occurred both in the presence and absence of hydrogen, i.e., during both heterotrophic and mixotrophic cultivation. The derepression of the affected systems during the plateau phase of the dicyclic growth curves was demonstrated. Carbon dioxide assimilation by whole cells agreed well with the RDPC activity of extracts from cells grown under similar conditions.     

自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。