ORGANIC CARBON SUPPLEMENTATION OF STERILIZED MUNICIPAL WASTEWATER IS ESSENTIAL FOR HETEROTROPHIC GROWTH AND REMOVING AMMONIUM BY THE MICROALGA CHLORELLA VULGARIS1

Heterotrophic growth of the microalga Chlorella vulgaris Beij. in synthetic as well as sterilized municipal wastewater of a nonindustrialized city was measured. The city wastewater contained high levels of ammonium and nitrate, medium levels of phosphate, and low levels of nitrite and organic molecules and could not support heterotrophic growth of C. vulgaris. Evaluation of 11 known carbon sources for this microalga that were added to standard synthetic wastewater containing the same levels of nitrogen and phosphorus as the municipal wastewater revealed that the best carbon sources for heterotrophic growth were Na‐acetate and d ‐glucose. These provided the highest growth rates and the largest removal of ammonium. Growth increased with concentration of the supplement to an optimum at 0.12 M Na‐acetate. This carbon source was consumed completely within 10 d of incubation. Higher concentrations inhibited the growth of C. vulgaris. The microalgal populations under heterotrophic growth conditions were one level of magnitude higher than that under autotrophic growth conditions that served as a comparison. No growth occurred in the dark in the absence of a carbon source. Na‐acetate was superior to d ‐glucose. In municipal wastewater, when Na‐acetate or d ‐glucose was added, C. vulgaris significantly enhanced ammonium removal under heterotrophic conditions, and its capacity was equal to ammonium removal under autotrophic growth conditions. This study showed that sterilized wastewater can be treated by C. vulgaris under heterotrophic conditions if supplemented with the appropriate organic carbon source for the microalgae.     

Investigation of mixotrophic,heterotrophic, and autotrophic growth of Chlorella vulgaris under agricultural waste medium

Growth of Chlorella vulgaris and its lipid production were investigated under autotrophic, heterotrophic, and mixotrophic conditions. Cheap agricultural waste molasses and corn steep liquor from industries were used as carbon and nitrogen sources, respectively. Chlorella vulgaris grew remarkably under this agricultural waste medium, which resulted in a reduction in the final cost of the biodiesel production. Maximum dry weight of 2.62 g L^?1 was obtained in mixotrophic growth with the highest lipid concentration of 0.86 g L^?1. These biomass and lipid concentrations were, respectively, 140% and 170% higher than autotrophic growth and 300% and 1200% higher than heterotrophic growth. In mixotrophic growth, independent or simultaneous occurrence of autotrophic and heterotrophic metabolisms was investigated. The growth of the microalgae was observed to take place first heterotrophically to a minimum substrate concentration with a little fraction in growth under autotrophic metabolism, and then the cells grew more autotrophically. It was found that mixotrophic growth was not a simple combination of heterotrophic and autotrophic growth.     

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.     

Biomass and lipid productivities of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> under autotrophic,heterotrophic and mixotrophic growth conditions

Biomass and lipid productivities of Chlorella vulgaris under different growth conditions were investigated. While autotrophic growth did provide higher cellular lipid content (38%), the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l⁻¹) and lipid productivity (54 mg l⁻¹ day⁻¹) were attained using glucose at 1% (w/v) whereas higher concentrations were inhibitory. Growth of C. vulgaris on glycerol had a similar dose effects as those from glucose. Overall, C. vulgaris is mixotrophic.     

Improved laminaribiose phosphorylase production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in a bioreactor: A comparative study of different cultivation methods

Laminaribiose phosphorylase (EC 2.4.1.31) catalyzes a reversible phosphorolysis reaction in which laminaribiose, a very high value sugar is produced. This enzyme is not being produced commercially therefore, to realize the most effective method for producing laminaribiose phosphorylase and obtaining as much activity units as possible per liter of culture, different cultivation methods of Euglena gracilis were compared. Heterotrophic and mixotrophic cultivations of Euglena gracilis in two different pHs, in flask and bioreactor were performed. The reverse phosphorolysis activity of laminaribiose phosphorylase produced under different cultivation methods was measured. The heterotrophic approach showed to be the more effective cultivation method as 47.6 IU/L was obtained compared to 27 IU/L in the mixotrophic one. The heterotrophic cultivation then was further investigated under two different pH values of the culture media. The culture at pH 6.8 resulted in 7.94 IU/L/day whereas only 4.06 was obtained for the culture at pH 4. Cultivation in a bioreactor resulted in a distinctive amount of 191.5 IU/L and an activity yield of 9.7 IU/g glucose compared to 5.4 in flask cultivation. Heterotrophic cultivation of Euglena gracilis in a bioreactor containing a culture media at pH 6.8 and controlled operation conditions showed enhanced laminaribiose phosphorylase activity production per liter and day of cultivation.     

Comparison of biomass production and total lipid content of freshwater green microalgae cultivated under various culture conditions

The growth and total lipid content of four green microalgae (Chlorella sp., Chlorella vulgaris CCAP211/11B, Botryococcus braunii FC124 and Scenedesmus obliquus R8) were investigated under different culture conditions. Among the various carbon sources tested, glucose produced the largest biomass or microalgae grown heterotrophically. It was found that 1 % (w/v) glucose was actively utilized by Chlorella sp., C. vulgaris CCAP211/11B and B. braunii FC124, whereas S. obliquus R8 preferred 2 % (w/v) glucose. No significant difference in biomass production was noted between heterotrophic and mixotrophic (heterotrophic with light illumination/exposure) growth conditions, however, less production was observed for autotrophic cultivation. Total lipid content in cells increased by approximately two-fold under mixotrophic cultivation with respect to heterotrophic and autotrophic cultivation. In addition, light intensity had an impact on microalgal growth and total lipid content. The highest total lipid content was observed at 100 μmol m^?2s^?1 for Chlorella sp. (22.5 %) and S. obliquus R8 (23.7 %) and 80 μmol m^?2s^?1 for C. vulgaris CCAP211/11B (20.1 %) and B. braunii FC124 (34.9 %).     

Effect of organic carbon,C:N ratio and light on the growth and lipid productivity of microalgae/cyanobacteria coculture

Current culture methods based on monocultures under phototrophic regimes are prone to contamination, predation, and collapse. Native cultures of multiple species are adapted to the local conditions and are more robust against contamination and predation. Growth, lipid and biomass productivity of a Louisiana native coculture of microalgae (Chlorella vulgaris) and cyanobacteria (Leptolyngbya sp.) in heterotrophic and mixotrophic regimes were investigated. Dextrose and sodium acetate at C:N ratios of 15:1 and 30:1 under heterotrophic (dark) and mixotrophic (400 μmol m^?2 s^?1) regimes were compared with autotrophic controls. The carbon source and C:N ratio impacted growth and biomass productivity. Mixotrophic cultures with sodium acetate (C:N 15:1) resulted in the highest mean biomass productivity (156 g m^?3 d^?1) and neutral lipid productivity (24.07 g m^?3 d^?1). The maximum net specific growth rate (U) was higher (0.97 d^?1) in mixotrophic cultures with dextrose (C:N 15:1) but could not be sustained resulting in lower total biomass than in mixotrophic cultures with acetate (C:N 15:1), with a U of 0.67 d^?1. The ability of the Louisiana coculture to use organic carbon for biomass and lipid production makes it a viable feedstock for biofuels and bioproducts.     

Growth and Carbohydrate Metabolism of Sulfobacilli

The moderately thermophilic acidophilic bacteria Sulfobacillus thermosulfidooxidans, strain 1269, S. thermosulfidooxidanssubsp. asporogenes, strain 41, and the thermotolerant strain S. thermosulfidooxidanssubsp. thermotolerans K1 prefer mixotrophic growth conditions (the concomitant presence of ferrous iron, thiosulfate, and organic compounds in the medium). In heterotrophic and autotrophic growth conditions, these sulfobacilli can grow over only a few culture transfers. In cell-free extracts of these sulfobacilli, key enzymes of the Embden–Meyerhof–Parnas, pentose-phosphate, and Entner–Doudoroff pathways were found. The role of a particular pathway depended on the cultivation conditions. All of the enzymes assayed were most active under mixotrophic conditions in the presence of Fe²⁺and glucose, suggesting the operation of all of the three major pathways of carbohydrate metabolism under these conditions. However, the operation of the Entner–Doudoroff pathway in strain 41 was restricted under mixotrophic conditions. After the first culture transfer from mixotrophic to heterotrophic conditions, the utilization of glucose occurred only via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. After the first culture transfer from mixotrophic to autotrophic conditions, the activity of carbohydrate metabolism enzymes decreased in all of the strains studied; in strain K1, only the glycolytic pathway remained operative. The high activity of fructose-bisphosphate aldolase, remaining in strain 41 cells under these conditions, suggests the involvement of this enzyme in the reactions of the Calvin cycle or of gluconeogenesis.     

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.     

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。     

AUTO-, HETERO-, AND MIXOTROPHIC GROWTH OF CHLAMYDOMONAS HUMICOLA (CMLOROIMIYCKAK) ON ACETATE1

Relative growth rate, isocitrate lyase activity, chlorophyll, protein, lipid, and soluble carbohydrate contents were investigated in Chlamydomonas humicola Lucksch during auto-, mixo-, and heterotrnphic growth. Mixotrophic cells have a relative growth rate of 1.66 d ^–1as compared to 0.78 d ^–1 and 0.21 d ^–1 for hetero- and autotrophic cells, respectively. Addition of acetate to autotrophic cells resulted in an increase in cell dry weight during the first day, followed by a rapid decrease and stabilization at 40 pg·cell ^–1. Cellular yield of mixotrophu cells, on a dry weight basis, was 6.6 times that of heterotrophic cells and 21.9 limes that of autotrophic ones. After 4 d, mixotrophic cells were characterized by higher chlorophyll (3.6% dry weight [d.w.]) and protein (58.6% d.w.) contents and lower lipid (4.8% d.w.) and soluble carbohydrate (1.3% d.w.) contents than those of autotrophic (2.6% d.w. chlorophyll, 31.0% d.w. protein, 10.2% d.w. lipid, and 6.5% d.w. soluble carbohydrate) and heterotrophic (1.5% d.w. chlorophyll, 36.9% d.w. protein, 5.6% d.w. lipid, and 6.0% d.w. soluble carbohydrate) cells. The ratio of chlorophyll a/b was highest in heterotrophic cells due to lower chlorophyll b content. Isocitrate lyase activity, a key enzyme in ecetate assimitation, could not be detected in autotrophic cells. Addition of 10 mM acetate to the culture medium of hetero- and mixotrophic cells resulted in increased isocitrate lyase activity with a maximum after 24 h, followed by a decline in activity over a 7-d period. After 7 d of growth, only 0.01 mM acetate was found in the culture medium of mixotrophic cells as compared to 3.2 mM in the medium of heterotrophic ones, from an initial concentration of 10 mM.     

Sorption of some ions by algae related to their trophic conditions

The unicellular algaeScenedesmus obliquus (125),Chlorella pyrenoidosa (82) andCoccomyxa solorinae saccatae (111) were studied with respect to the form of uptake of potassium, phosphate, calcium and zinc ions and to the energy sources involved: light under autotrophic conditions, glucose under mixotrophic or heterotrophic conditions (in light and in darkness or together with yeast extract as an auxotrophic substrate). We respected the trophic conditions of algae when preparing the experimental material (precultivation). The following conditions were reached:

1. (1)
   The three algae grow faster in a glucose medium under mixotrophic conditions and are capable of growing on it also heterotrophically:Ch. pyrenoidosa andSc. obliquus grow substantially better thanC. solorinae saccatae. The first two algae grow more intensively in a glucose medium containing yeast extract whileCoccomyxa does not. After cultivation under mixotrophic conditions the first two diminish endogenous respiration, the third raises it. Glucose stimulates respiration in the first two when grown autotrophically, while after mixotrophic cultivation the effect of glucose is suppressed inCh. pyrenoidosa and in the other two only after growth on glucose with yeast extract.     
   
   

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.     

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。     

Biomass production in mixotrophic culture of Euglena gracilis under acidic condition and its growth energetics

When Euglena gracilis was grown in the heterotrophic condition with glucose and (NH₄)₂SO₄ as the carbon and nitrogen source, a high cell yield (4.28–4.48 g l^–1) was obtained and the culture pH decreased to 1.6–2. The biomass production in the heterotrophic culture was compared to those in the autotrophic and mixotrophic cultures. Autotrophic growth was 4.7–6.3% of the heterotrophic one, whereas about 15–19% higher growth was obtained in the mixotrophic culture. Moreover, good production of chlorophyll (39.4 mg l^–1) and carotenoids (13.8 mg l^–1) were attained in the mixotrophic culture, giving the highest fermenter productivity with respect to biomass as well as chlorophyll and carotenoids. Through an energetic analysis in the mixotrophic culture, it was estimated about 25–28% of the total ATP requirement is formed in the photochemical reactions. This resulted in an improved biomass production in the mixotrophic culture of E. gracilis.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

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。     

Growth and carbohydrate metabolism of sulfobacilli]

The moderately thermophilic acidophilic bacteria Sulfobacillus thermosulfidooxidans, strain 1269, S. thermosulfidooxidans subsp. "asporogenes," strain 41, and the thermotolerant strain S. thermosulfidooxidans subsp. "thermotolerans" K1 prefer mixotrophic growth conditions (the concomitant presence of ferrous iron, thiosulfate, and organic compounds in the medium). In heterotrophic and autotrophic growth conditions, these sulfobacilli can grow over only a few culture transfers. In cell-free extracts of these sulfobacilli, key enzymes of the Embden-Meyerhof-Parnas, pentose-phosphate, and Entner-Doudoroff pathways were found. The role of a particular pathway depended on the cultivation conditions. All of the enzymes assayed were most active under mixotrophic conditions in the presence of Fe2+ and glucose, suggesting the operation of all of the three major pathways of carbohydrate metabolism under these conditions. However, the operation of the Entner-Doudoroff pathway in strain 41 was restricted under mixotrophic conditions. After the first culture transfer from mixotrophic to heterotrophic conditions, the utilization of glucose occurred only via the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. After the first culture transfer from mixotrophic to autotrophic conditions, the activity of carbohydrate metabolism enzymes decreased in all of the strains studied; in strain K1, only the glycolytic pathway remained operative. The high activity of fructose-bisphosphate aldolase, remaining in strain 41 cells under these conditions, suggests the involvement of this enzyme in the reactions of the Calvin cycle or of gluconeogenesis.     

Mixotrophic organisms become more heterotrophic with rising temperature

The metabolic theory of ecology predicts that temperature affects heterotrophic processes more strongly than autotrophic processes. We hypothesized that this differential temperature response may shift mixotrophic organisms towards more heterotrophic nutrition with rising temperature. The hypothesis was tested in experiments with the mixotrophic chrysophyte Ochromonas sp., grown under autotrophic, mixotrophic and heterotrophic conditions. Our results show that (1) grazing rates on bacterial prey increased more strongly with temperature than photosynthetic electron transport rates, (2) heterotrophic growth rates increased exponentially with temperature over the entire range from 13 to 33 °C, while autotrophic growth rates reached a maximum at intermediate temperatures and (3) chlorophyll contents during mixotrophic growth decreased at high temperature. Hence, the contribution of photosynthesis to mixotrophic growth strongly decreased with temperature. These findings support the hypothesis that mixotrophs become more heterotrophic with rising temperature, which alters their functional role in food webs and the carbon cycle.     

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.