Supplementation of alanine improves biomass accumulation and lipid production of Chlorella pyrenoidosa by increasing the respiratory and metabolic processes

The function of exogenous alanine(Ala)in regulating biomass accumulation,lipid production,photosynthesis,and respiration in Chlorella pyrenoidosa was studied.Result shows that the supplementation of Ala increased C.pyrenoidosa biomass and lipid production in an 8-d batch culture.The concentration of 10 mmol/L of Ala was optimum and increased the microalgal cell biomass and lipid content by 39.3%and 21.4%,respectively,compared with that in the control(0-mmol/L Ala).Ala supplementation reduced photosynthetic activity while boosting respiratory activity and pyruvate levels,indicating that C.pyrenoidosa used exogenous Ala for biomass accumulation through the respiratory metabolic process.The accelerated respiratory metabolism due to Ala supplementation elevated the substrate pool and improved the lipogenic gene expression,promoting lipid production at last.This study provided a novel method for increasing biomass accumulation and lipid production and elucidated the role of Ala in regulating lipid production.

微塑料和紫外线吸收剂对小球藻(Chlorella sp.)的联合毒性效应

微塑料和紫外线吸收剂作为难降解、持久性强的新型污染物,近年来已成为了国内外环境问题的研究热点。然而,这2类广泛存在于水体中的污染物对海洋微藻的联合毒性仍不明确。本研究添加直径1μm的聚苯乙烯微塑料(polystyrene micro plastics,PS-MPs,10 mg·L^(-1))至不同浓度(50μg·L^(-1)和500μg·L^(-1))的有机紫外线吸收剂阿伏苯宗(butyl methoxydibenzoylmethane,BMDM)海水溶液中,对小球藻(Chlorella sp.)进行7 d的胁迫实验,从生长抑制率、表面形貌和物理特征、光合色素含量、氧化应激水平以及胞外产物(EPS)含量等指标揭示2种污染物对小球藻的联合毒性效应。结果显示:50μg·L^(-1)和500μg·L^(-1)2种浓度的BMDM单一胁迫时,小球藻的最高抑制率分别为26.15%和42.05%,而2种浓度的BMDM与PS-MPs复合胁迫时,小球藻的最高抑制率则分别为15.38%和17.95%,BMDM单一胁迫对小球藻的抑制率高于复合胁迫。原子力显微镜(AFM)对细胞表面的观察发现,与BMDM单一胁迫相比,复合胁迫的小球藻表面凹陷较少,褶皱程度较低。同时,小球藻的表面黏度以及粗糙度均呈现降低的趋势。还原型谷胱甘肽(GSH)含量较低,氧化应激反应减弱。此外,与BMDM单一胁迫相比,复合胁迫的小球藻EPS合成量显著减少,藻胆蛋白(PBPs)的含量较低,而小球藻的细胞密度较高。研究结果表明,PS-MPs和BMDM的联合作用对小球藻的生长表现拮抗效应,即PS-MPs的添加能降低BMDM对小球藻的毒性作用。

Inoculation of Chlorella and Food Waste Improves the Physio-Morphological Features of Red Pepper by Regulating Activating Antioxidant Defense System

Food waste is recognized as a valuable source for potential agricultural applications to supply organic matter and nutrients to arable soil.However,the information on the combined application of food waste and the plant growth-promoting bacterial strain,Chlorella,related to plant metabolic features and sodium chloride content in arable soil is limited.The present study was conducted to investigate the exogenous application of food waste along with Chlorella,which improved the physio-morphological features of red pepper.Our results revealed that this combination enhanced the organic matter in the soil,ultimately improving the fertility rate of the soil,and the physio-morphological features,such as chlorophyll a content(24.5±0.7),root(7.8±0.7)cm and shoot length(12.1±0.7)cm,fresh weight(2.1±0.05)g,dry weight(0.19±0.05)g,mineral contents,and hormonal concentration(ABA by up to 2 folds).The combined treatment also minimized free radicals via the activation of the intrinsic antioxidant series cascade and electrolyte leakage.Our findings showed that adding Chlorella and food wastes improved growth characteristics and can be used as a green bio-fertilizer for sustainable agriculture.

Chlorella across latitudes: investigating biochemical composition and antioxidant activities for biotechnological applications

With the present day rise of interest in acquiring sustainability in the pharmaceutical industry,there has been an emphasis on finding natural resources to replace the use of synthetic compounds used in products.Microalgae have garnered significant attention owing to their natural and sustainable capability to produce a diverse array of bioactive compounds.Therefore,this study aims to evaluate the biochemical composition and antioxidant properties of Chlorella strains from a tropical region(Chlorella UMACC 051 and Chlorella UMACC 038)and a polar region(Chlorella UMACC 250 and Chlorella UMACC 234).The cultures were grown for 10 d.At the end of the experiment,the specific growth rate,chlorophyll-a content,carotenoid content,biomass,and biochemical composition such as carbohydrate,protein and lipid content were determined.In addition,the phytochemical properties were determined using a total phenolic assay while the antioxidant activities were determined using 2,2-diphenyl-1-picrylhydrazyl(DPPH).Of all Chlorella strains tested,the tropical Chlorella UMACC 051 showed the fastest growth rate and biomass while the polar Chlorella UMACC 234 contained the highest pigment content and tropical Chlorella UMACC 038 has the highest total phenolic content.The biochemical composition analysis showed all strains have a high lipid content ranging from 45.36%to 60.30%dry weight.All Chlorella strains exhibited a small amount of antioxidant activity(15.42%to 30.15%)and total phenolic content ranging from 1.91±0.04 to 4.43±0.10 mg GAE·g–1 dry weight.The results indicated that polar Chlorella UMACC 234 has the most potential in containing significant amounts of bioactive compounds.

Effect of Light Intensity and pH on Cell Density Assessed by Spectrophotometry for the Unicellular Algae Chlorella vulgaris

In this study, an effective environment for Chlorella vulgaris growth is sought after. As a substitute source of food and feed, increasing the cell density of Chlorella culture is one of the keys to ensuring sustainability. It can be showed from different studies that optimum light intensity and pH could increase cell density. In this study, the effects of light and pH on the growth rate of C. vulgaris were observed in photobioreactor. A specific wavelength (682 nm) was determined by UV-Vis Spectrophotometry to carry out the further analysis. The light intensities were set at 7409, 9261 and 11,113 lux;pH values were set at 7, 8 and 9 respectively. The experimental results depicted the light intensity of 9261 lux as the best due to the higher number of cells (48.56 × 106 cells/mL) obtained using this intensity. In terms of pH, without pH control, cell numbers were found to be highest under the light intensity of 9261 lux. When pH was controlled, it was found that under the optimum light intensity, pH control between 7.0 and 7.5 was the optimum range for the growth of C. vulgaris. Moreover, this method of study may possibly be a promising source of low cost culture for Chlorella vulgaris.

Effect of Chlorella Intake on the Development of Atherosclerosis and Spontaneous Thrombolytic Activity

Background and Purpose: Thrombotic disease is a leading cause of death in industrialized countries. The development of atherosclerosis is a major underlying pathogenesis. Atherosclerotic lesions are largely related to abnormalities in lipid metabolism, and improvement of dietary habits is of great significance. Chlorella is a unicellular organism belonging to the green algae family, and is consumed worldwide as a functional food for the purpose of health promotion due to its excellent nutritional balance including high quality protein. In this study, we investigated the effects of long-term consumption of Chlorella as a food on the development of atherosclerosis and its ability to dissolve thrombi caused by the disruption of the atherosclerotic layer as a functional study of Chlorella. Methods: ApoE−/− and Ldlr−/− double-knockout mice were fed a chlorella-supplemented experimental diet for 14 weeks. The Entire aorta method was used to measure atherosclerosis development, and the area of sclerotic vessels was evaluated as a percentage of the total area of vessels. In addition, mRNA levels of lipid metabolism-related proteins in the liver and blood vessels were analyzed, as well as blood lipoprotein analysis. Spontaneous thrombolytic activity was measured by measuring the change in volume over time of thrombus formed in microvessel running over the cremaster muscle of the mice using the He-Ne laser-induced thrombus model. Results: There was no significant difference between the two groups in atherosclerosis development compared to the placebo group. However, a significant decrease in SREBP-1 mRNA level and a significant increase in mRNA levels of LXR and CPY71a were observed in the chlorella group. Cholesterol and TG levels in each lipoprotein fraction did not differ between the two groups. On the other hand, thrombolysis in vivo was not significantly different between the two groups in terms of thrombus volume at 60 minutes after thrombus formation. However, a trend toward decreased PAI-1 and TAFI mRNA expression levels was observed in the chlorella group. Conclusion: Intake of chlorella as a food suggested an effect on cholesterol catabolism, increased bile acid synthesis, improved lipid metabolism, and inhibited the development of atherosclerosis. Furthermore, it was suggested that chlorella may suppress the expression of fibrinolytic inhibitory factor and enhance thrombolytic activity.

气升式光生物反应器中Chlorella sp.优化培养与能量计算

高油脂含量的微藻能够有效利用太阳能生产生物油脂,从而实现无机碳转化为有机碳的过程.如何降低微藻培养过程的能量消耗,是实现含油微藻规模化生产生物柴油的关键.通过对气升式光生物反应器中Chlorella sp.培养条件的优化,以及不同培养条件的能量计算和比较,发现当使用侧光光纤作为内置光源时可以提高光能利用率,在适宜的通气速率（0.6 L/min）条件下,可获得单位能耗的最大生物产量4.40 g/（W·d）,较同等条件下外置光源时产量提高了8~25倍.

Isolation of Chlorella vulgaris and Its DNA Extraction Methods

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.

不同抗生素和防腐剂对小球藻Chlorella sorokiniana细胞生长的影响

【目的】分析不同抗生素和防腐剂对小球藻Chlorella sorokiniana细胞生长的影响,筛选出适宜的抗生素和防腐剂种类及其剂量,为建立小球藻C.sorokiniana无菌培养体系提供参考依据。【方法】在HSM培养基中分别添加10、20、40、60、80和100μg/mL抗生素,包括氯霉素、链霉素、青霉素、庆大霉素和头孢噻肟,以及山梨酸钾(50、100、150和200μg/mL)、乳酸钠(0.5%、1.0%和2.0%)、富马酸二甲酯(0.1、0.5、1.0、5.0、10.0、25.0和50.0μg/mL)等防腐剂,培养3 d后测定小球藻C.sorokiniana的细胞密度、叶绿素含量及光系统Ⅱ最大光化学量子产量(Fv/F_m)。【结果】添加10~100μg/mL青霉素或10~80μg/mL头孢噻肟对小球藻C.sorokiniana细胞密度、叶绿素含量和Fv/F_m的影响均不显著(P>0.05,下同);添加100μg/mL头孢噻肟会抑制藻细胞生长,抑制率为18.89%,并引起叶绿素含量和Fv/F_m极显著下降(P<0.01,下同)。氯霉素、链霉素和庆大霉素等蛋白质合成抑制类抗生素对小球藻C.sorokiniana细胞的毒性较强,高于10μg/mL的链霉素和庆大霉素及高于20μg/mL的氯霉素均极显著抑制藻细胞生长,对应的叶绿素含量和Fv/F_m也极显著下降。3种防腐剂中,山梨酸钾对小球藻C.sorokiniana细胞生长无显著影响,添加200μg/mL山梨酸钾还会促进细胞叶绿素含量增加;乳酸钠对小球藻C.sorokiniana细胞有一定的毒性作用,其藻细胞密度、叶绿素含量和Fv/F_m随添加浓度增加而降低;添加0.1~10.0μg/mL富马酸二甲酯不影响小球藻C.sorokiniana的细胞密度及叶绿素含量,但Fv/F_m极显著下降。【结论】建立小球藻C.sorokiniana无菌培养体系时宜选用青霉素和头孢噻肟为抗生素、山梨酸钾为防腐剂,其推荐使用浓度分别为青霉素100μg/mL、头孢噻肟80μg/mL、山梨酸钾200μg/mL。

A(sⅢ)和A(sⅤ)对小球藻(Chlorella sp).的生长影响研究

砷是广泛存在于自然界中最常见的毒性污染物,包括土壤,沉积物,水体,大气层,甚至生物体,毒性极强。环境中砷的污染严重威胁人类的健康,已被视为一个全球性的公共卫生问题。为了研究砷对小球藻的生长影响,文章通过实验室培养方式,选取了6个不同浓度的As(Ⅲ()0.5、1.0、2.0、5.0、10.0、20.0 mg/L)和As(Ⅴ()1.0、5.0、10.0、20.0、50.0、100.0 mg/L)的培养处理,以在680 nm下小球藻的吸光度(OD680)作为衡量因子,对小球藻Chlorella sp(.100 ai)在不同浓度砷离子的培养液中的细胞密度进行了试验,整个实验共进行了7 d。结果表明,As(Ⅲ)和As(Ⅴ)浓度分别在10.0 mg/L和20.0 mg/L以上时抑制100 ai细胞生长,导致其密度下降。用概率统计方法计算得到A(sⅢ)对100 ai的96 h半数有效抑制浓度EC50值为25.79 mg/L。参照藻类生长抑制评价标准,A(sⅢ)对100 ai的毒性比A(sⅤ)大。

三吲哚乙酸对Chlorella vulgaris XJB生长及产油能力的影响

三吲哚乙酸(IAA)具有促进细胞增殖的作用,将其添加到培养基中能够提升藻类细胞生长,可增强利用微藻发展生物柴油的能力。本文以自行筛选到的一株具有快速生长能力的微藻ChlorellavulgarisXJB为研究对象,考察了IAA添加时间和添加浓度对C.vulgaris XJB生长及产油能力的影响。实验研究结果表明,在微藻生长适应期,添加适宜浓度的IAA有助于藻体生物量的累积和油脂产率的提高,但对油脂含量的影响较小,当第0天添加0.08mg/L IAA时,细胞个数、生物量及油脂产率达到最大值,分别为20.2×106个/mL、338.9mg/L、15.2mg/(L/d),油脂含量为22.4%,此时生物量和油脂产率分别是未经IAA处理条件下的2.5倍和2.0倍。

Effects of Sonic Waves at Different Frequencies on Propagation of Chlorella pyrenoidosa

[Objective] The aim was to investigate the effects of sound wave on propagation of Chlorella pyrenoidosa to explore the optimal frequency for Chlorella pyrenoidosa.[Method] In the research,Chlorella pyrenoidosa was cultured for 7 d with sound waves at different frequencies and a control group was set to study effects of sound wave at different frequencies on growth of Chlorella pyrenoidosa.[Result] Growth of Chlorella pyrenoidosa was significantly improved by sound wave,especially for wave at 400 Hz.[Conclusion] Chlorella pyrenoidosa propagation would be promoted by sound wave at certain frequencies.

Study on the Removing Nitrogen and Phosphorus from Wastewater by Chlorella

[Objective] The aim was to research Chlorella properties in removing ni- trogen and phosphorus from wastewater. [Method] The effects of initial NH4＋-N and total P concentrations, N-to-P ratios, lightJdarkness ratios and pH on the removal of NH4＋-N and total P were evaluated. [Result] The results showed that Chlorella al- most removed NH4＋-N and total P at 100% as initial concentrations of NH4＋-N and total P were no more than 55 and 7 mg/L, respectively, whereas the removal ratio of NH4＋-N decreased to 90% with initial NH4＋-N concentration coming up to 75 mg/L. With N-to-P ratios of 5：1, 10：1 and 25：1, Chlorella completely removed NH4＋-N within 4 d, while the removal ratio of total P reached almost 100% within 7 d, with different N-to-P ratios. With L./D ratios of 24 h： 0 h and 12 h： 12 h as well as the initial concentrations of NH4＋-N at 30 mg/L and total P at 7 mg/L, the removing ra- tio of NH4＋-N and total P almost achieved 100% by Chlorella, and the removing ra- tio under L/D ratio of 24 h：0 h proved much faster. The optimal pH range for Chlorella to remove NH4＋-N and total P was 7-8. [Conclusion] The research pro- vides references for wastewater treatment in biological way and highly-efficient and environment-friendly treatment in future.

营养条件对Chlorella vulgaris生长的影响

以Chlorella vulgaris为研究对象,考察了培养基和有机碳源的影响。结果表明:在不同的营养方式中,混养的细胞密度和比生长速率最大,均超过自养和异养之和;改变培养液中的有机碳源种类,C.vulgaris的细胞密度最大分别达到2.92g/L(葡萄糖)、1.62g/L(乙酸钠)和1.05g/L(乙醇),且以葡萄糖为碳源时的比生长速率最大;当葡萄糖浓度从5g/L增加到30g/L时,C.vulgaris的比生长速率由0.0248h^(-1)降低至0.0186h^(-1),不过在培养晚期,C.vulgaris在10g/L葡萄糖时的细胞密度超过5g/L葡萄糖时的密度;当以10g/L葡萄糖作为C.vulgaris混养的有机碳源时,BG11培养基的细胞密度和比生长速率最大,分别达到3.41g/L和0.0257h^(-1),而在Basal培养基中,C.vulgaris培养液较稳定,藻细胞始终处于悬浮的状态,其细胞密度和比生长速率分别为3.25g/L和0.0204h^(-1)。

Chlorella Resistance to Dehydration Conditions and Protection Effect of Different Cryoprotecants on Chlorella Cells

[Objective] The aim was to study on the chlorella resistance to dehydration conditions and effect of different cryoprotecants on drying of chlorella cells.[Method]The protection effect of seven kinds of cryoprotecants on chlorella cells was analyzed.[Result]The protection effect of trehalose on chlorella cells was the best,and the survival rate of chlorella cells had improved by 50% with the applied trehalose concentration of 5%.[Conclusion]The result in this study had important guidance effect on the mutation breeding of chlorella.

Discussion on Problem of Fermentation Contamination of Heterotrophic Chlorella

The contamination reasons were analyzed form the viewpoints of strain preparation, air system, medium and equipment sterilization and pipeline configura- tion. Moreover, the contamination-preventing measures were defined. We also sum- marized the characteristics of bacteria that contaminated the fermentation, as well as checking methods and remedial measures.

小球藻Chlorella variabilis NC64A二酰甘油酰基转移酶基因的克隆表达与功能研究

为了揭示二酰甘油酰基转移酶(DGAT)在小球藻油脂合成过程中的作用,对单细胞小球藻Chlorella variabilis NC64A的二酰甘油酰基转移酶进行原核克隆表达及功能初步研究。结果表明,其编码序列为894bp,编码297个氨基酸,表达蛋白的表观分子量为33 k Da,p I 9.48。保守结构域分析表明,该蛋白属于Lysophospholipid acyltransferases(LPLATs)超家族,具有二酰甘油酰基转移酶活性,序列位点H68,L71,F76,R94,I97和GAA(144?146)组成特定的酰基受体结合口袋,能够结合酰基?酰基载体蛋白(ACP)或者酰基辅酶A上的酰基,催化三酰甘油合成的最后一步。表达蛋白与Ss PDAT和At PDAT的序列相似性分别为32%和24%,表明该蛋白可能具有磷脂酰甘油酰基转移酶(PDAT)活性,能利用磷脂上的酰基合成三酰甘油。因此,采用薄层层析方法以L-α-磷脂酰胆碱和1,2-二油酰-sn-甘油为底物,检测到其确实具有PDAT活性,表明限氮条件下NC64A中DGAT的PDAT活性可能促进了膜脂降解耦合三酰甘油的合成。

Genetic diversity analysis with ISSR PCR on green algae Chlorella vulgaris and Chlorella pyrenoidosa

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.

Effects of Fed-batch and Nitrogen-deficient Culture on Lipid Yield of Chlorella sp.

[Objective] This study was to realize high-density culture of Chlorella sp. as well as the culture with high lipid yield. [Method] Through analyzing the growth curve of Chlorella sp. cells, dextrose consumption curve, change curves of pH and dissolved oxygen, a fed-batch culture was conducted, followed by a nitrogen-deficient culture aiming at accumulating the lipids in Chlorella sp. cells when a high density of Chlorella sp. cells was obtained. [Result] After four batches of feeding were pro- vided, the biomass of Chlorella sp. reached up to 65.25 g/L, and the lipid content increased from 42.75% to 63.82% in Chlorella sp. cells, with the yield of 43.37 g/L in the following 12 hours of nitrogen-deficient culture. [Conclusion] Reasonable fed- batch can significantly improve the biomass of Chlorella sp., and the nitrogen-defi- cient culture further raises the lipid yield of Chlorella sp.

共培养微藻Monoraphidium sp.FXY-10与Chlorella sp.U4341提高油脂产率与沉降率

采用共培养技术,在异养条件下培养微藻Chlorella sp.U4341和Monoraphidium sp.FXY-10,提高两株微藻共培养的油脂产率;通过共培养方式促进微藻细胞的自絮凝,加速微藻细胞的沉降,以期降低微藻生物量的采收成本。结果表明:微藻Chlorella sp.U4341与Monoraphidium sp.FXY-10单独培养的油脂产率分别为272.07、268.54 mg/(L·d),而两株微藻共培养的油脂产率提高到315.60 mg/(L·d);两株微藻共培养后,自然沉降5 h,微藻细胞沉降率高于90%,远高于单独培养条件下的沉降率。因此,微藻的共培养技术有望成为解决微藻油脂产率低、采收成本高两大瓶颈问题的解决方案。