产油微藻的分离筛选与鉴定

微藻所含的油脂是制备生物柴油的理想原料。为了筛选具有产油能力的微藻,作者从自然界水体中分离出8株微藻,对他们的生长特性和产油能力进行评价,旨在筛选出生长速度快、生物量高、油脂含量高的优良藻种。实验结果得到一种油脂产率较高的CC-B3藻株,它的总脂质量分数为36.30%,生物量达到2.43 g/L,油脂产率为62.9 mg/(L·d)。符合优良藻种的条件,是一株高效产油微藻。通过18s r DNA鉴定,该藻株为斜生栅藻(Scenedesmus obliquus),构建了该藻株的进化树。     

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。     

共培养微藻Monoraphidium sp. eh1和Monoraphidium sp. eh3提高油脂产率和沉降率

为了提高微藻油脂产率,降低其采收成本,对比了微藻Monoraphidium sp. eh1和Monoraphidium sp. eh3单独培养和共培养方式的影响,同时对于培养结束后提升微藻沉降率的方法进行了研究。结果表明:微藻Monoraphidium sp. eh1和Monoraphidium sp. eh3单独培养的油脂产率分别为17. 45 mg/(L·d)和15. 35 mg/(L·d),而共培养油脂产率可达到38. 82 mg/(L·d);通过共培养后调节藻液pH至7. 0的方法,30 min沉降率可达到95. 38%,与添加絮凝剂的沉降效果相当,相较于单独培养的eh1、eh3的沉降率10. 01%和53. 60%有显著提升。共培养技术结合调节pH的方式,可以为提高微藻油脂产率和降低采收成本提供一种具有可行性的解决方案。     

培养条件对眼点拟微绿球藻油脂含量和EPA产率的影响

眼点拟微绿球藻(N.oculata)具有生长速度快、含油量高等优点,且其脂肪酸组成中含有大量二十碳五烯酸(EPA),因此它是微藻生物柴油的良好原料。以眼点拟微绿球藻为对象,研究了培养时间、盐度、pH值等培养条件对眼点拟微绿球藻油脂含量和EPA产率的影响。试验表明:选择稳定5 d为眼点拟微绿球藻的最佳采收时间,油脂含量为378.1 mg/g干藻,EPA产率为5.5 mg/(L·d);当盐度为16.7时,眼点拟微绿球藻的油脂含量和EPA产率均能达到最高,油脂含量为506.0 mg/g干藻,EPA产率为7.37 mg/(L·d);在适合眼点拟微绿球藻生长的pH值(8.0~10.0)下,保持整个培养过程中pH值为8.0,油脂含量和EPA产率均较高,油脂含量为471.7 mg/g干藻,EPA产率为7.45 mg/(L·d)。     

3种不同培养基对斜生栅藻生长和油脂积累的影响

以斜生栅藻FACHB-12为原料,采用BG11、F/2和SE 3种培养基对其培养。通过对生物量、生长速率、油脂含量、油脂组分以及脂肪酸组成的测定,比较不同培养基对斜生栅藻生长和油脂积累的影响。结果表明:BG11培养基更有利于斜生栅藻的生长,而F/2培养基更适合油脂以及中性脂的积累;斜生栅藻在BG11培养基中比生长速率为0.095,培养15 d生物量为0.36 g/L,叶绿素a含量达到1.23 mg/L;而在F/2培养基中油脂含量可达细胞干重的24.75%,其中中性脂占总脂的36.64%,产率达0.98 mg/(L·d);并且在F/2培养基中斜生栅藻脂肪酸组成以C16∶0和C18∶1为主,含量分别占总脂肪酸的25.84%和54.47%,更适合作为制备生物柴油的原料。     

响应面优化产油沙漠链带藻(Desmodesmus sp.XJ842)的培养基组成北大核心CSCD

以一株从新疆荒漠地区筛选出来的沙漠产油微藻为实验对象,经过形态学和分子生物学手段(18s rRNA及ITS测序)鉴定为链带藻属(Desmodesmus sp.),并命名为XJ842。为提高其油脂产率,采用响应面对其培养基组成(碳源、氮源和磷源)进行优化。单因素实验分析结果显示,其最适产油的碳源为Na2CO3,最适氮源为Na NO_3。运用Box-Behnken中心组合实验以及Design-Expert分析软件建立了以总脂含量和油脂产率为响应值的响应面方程,得到最适合产油的Na2CO3浓度为1.98 mmol/L,NaNO_3浓度为48.00 mmol/L,K_2HPO_4浓度为0.46 mmol/L。在最优培养基条件下,Desmodesmus sp.XJ842总脂含量达到67.73%,油脂产率达到29.27 mg/(L·d)。优化后的总脂含量、油脂产率比优化前的分别提高了2.08倍和2.61倍。优化后Desmodesmus sp.XJ842油中饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的88.41%,且亚麻酸的含量小于12%,符合欧洲生物柴油标准EN14214,具有生产生物柴油的潜力。     

响应面优化产油沙漠链带藻(Desmodesmus sp.XJ842)的培养基组成

以一株从新疆荒漠地区筛选出来的沙漠产油微藻为实验对象,经过形态学和分子生物学手段(18s rRNA及ITS测序)鉴定为链带藻属(Desmodesmus sp.),并命名为XJ842。为提高其油脂产率,采用响应面对其培养基组成(碳源、氮源和磷源)进行优化。单因素实验分析结果显示,其最适产油的碳源为Na2CO3,最适氮源为Na NO_3。运用Box-Behnken中心组合实验以及Design-Expert分析软件建立了以总脂含量和油脂产率为响应值的响应面方程,得到最适合产油的Na2CO3浓度为1.98 mmol/L,NaNO_3浓度为48.00 mmol/L,K_2HPO_4浓度为0.46 mmol/L。在最优培养基条件下,Desmodesmus sp.XJ842总脂含量达到67.73%,油脂产率达到29.27 mg/(L·d)。优化后的总脂含量、油脂产率比优化前的分别提高了2.08倍和2.61倍。优化后Desmodesmus sp.XJ842油中饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的88.41%,且亚麻酸的含量小于12%,符合欧洲生物柴油标准EN14214,具有生产生物柴油的潜力。     

淡水绿藻在培养基中生长及油脂积累研究

为筛选出最适合进行油脂生产的藻株及其较适生长和油脂积累的培养基,采用柱状光生物反应器进行一次性培养,对微藻Tetranephrisbrasiliensis DL12、Ankistrodesmusgracilis CJ09、Ankistrodesmus sp.CJ02及Desmodesmussubspicatus WC01在4种常用淡水绿藻培养基BG11、3N-BBM、SE及TAP中的生长和油脂积累进行研究。结果表明:3N-BBM有利于微藻的生长和生物量的积累,BG11有利于微藻细胞内油脂的积累;16组培养试验中,Tetranephrisbrasiliensis DL12在3N-BBM中的生物量产率((257.65±45.52)mg/(L·d))和油脂产率((80.34±14.19)mg/(L·d))最高,Ankistrodesmusgracilis CJ09在BG11中的油脂含量(42.72%±0.60%)最高。Tetranephrisbrasiliensis DL12最具油脂生产潜力,其最适生长和油脂积累的培养基为3N-BBM。     

产油微藻的筛选及油脂含量测定

为了促进微藻领域研究和微藻生物燃料的生产,从潍坊地区多种生境中分离、筛选产油微藻,并对产油微藻生长情况和油脂积累情况进行研究。结果表明:共分离出49株微藻,其中产油微藻19株;产油微藻的生物量为1.39~6.36 g/L,油脂含量为11.16%~45.62%;6株油脂产率大于90mg/(L·d)微藻在培养的12~15 d产油能力最高,6株微藻最佳收获时间在培养后的14 d。     

氮、磷对单针藻异养生长与油脂合成的影响

在异养条件下,应用Andrew方程考察了氮、磷营养盐对单针藻Monoraphidium sp.FXY-10生长的影响,并研究了氮、磷营养盐对单针藻油脂含量的影响。结果表明,磷的半饱和常数(KS,P)小于氮的半饱和常数(KS,N),说明磷对单针藻生长的影响大于氮。此外,在NaNO3质量浓度为1 g/L时,生物量产率(253.77 mg/(L·d))、油脂产率(110.36 mg/(L·d))、油脂含量(43.49%)达到最大值;在K2HPO4质量浓度为0.1 g/L时,生物量产率(323.54 mg/(L·d))、油脂产率(136.83 mg/(L·d))、油脂含量(42.29%)达到最大值。并且在氮、磷缺乏的情况下,油脂含量相对较高。     

利用甲醇厂CO_2尾气培养小球藻

利用甲醇厂所排放的高浓度CO2尾气培养小球藻,研究不同的通气方式、培养方式对小球藻生长的影响。实验结果表明,当通气速率为300 mL/min、CO2浓度为10%并以10 min/h的间歇通气方式培养时能够提高小球藻的生物量,生物量和产率分别为0.681 g/L和0.082 g/(L.d)。对氮源和磷源采用补料的方式进行培养时对小球藻生长具有促进作用,最高生物量和产率分别达到0.789 g/L和0.088 g/(L.d)。当培养基的更新率为20%时能够获得较高的生物量。利用醋酸进行兼性培养时,一次性添加50～125μL/L醋酸都可促进小球藻生长,每日添加10～25μL/L的醋酸时,可明显促进小球藻生长,最高生物量和产率分别为0.933 g/L和0.111 g/(L.d),分别是空气对照的1.3倍和1.4倍。说明利用甲醇厂高浓度CO2尾气培养小球藻提高生物量是可行的。     

Lipid accumulation and growth characteristics of Chlorella zofingiensis under different nitrate and phosphate concentrations

To evaluate the potential of the green agla Chlorella zofingiensis as a feedstock for biodiesel production, the effects of nitrogen and phosphate on lipid accumulation and growth of C.?zofingiensis were studied. The maximum specific growth rate (μ(max)) reached 2.15?day(-1) when the concentration of NaNO(3) and K(2)HPO(4)·3H(2)O was 1.0?g?L(-1) and 0.01?g?L(-1), respectively. The lipid contents of C.?zofingiensis grown in media deficient of nitrogen (65.1%) or phosphate (44.7%) were both higher than that obtained from cells grown in full medium (33.5%). The highest lipid productivity (87.1?mg?L(-1)?day(-1)) was also obtained from cells grown in nitrogen deficient media, indicating nitrogen deficiency was more effective than phosphate deficiency for inducing lipid accumulation in C.?zofingiensis. In addition, the feasibility of cultivating the alga in 60?L flat plate photobioreactors and 10?L bottles outdoors for biodiesel was also tested. It was found that C.?zofingiensis could adapt to fluctuating temperatures and irradiance of outdoors and the highest μ(max) and lipid productivity could reach 0.362?day(-1) and 26.6?mg?L(-1) day(-)(1) outdoors, respectively. The lipid production potential of C.?zofingiensis is projected to be 31.1?kg?ha(-1)?day(-1) in outdoor culture. These results suggested that C.?zofingiensis is a promising organism for feedstock production of biofuel and can be used in scaled up culture outdoors.     

微藻总脂含量快速测定的方法比较

快速、可靠、简便的总脂含量测定方法在富油藻种筛选、功能性藻油评估及微藻生物学研究中至关重要。本研究以三种微藻(微绿球藻Nannochloris sp.、普通小球藻Chlorella vulgaris、绿色巴夫藻Pavlova viridis)藻粉为原料,系统比较了四种方法(Bligh-Dyer法、改良Bligh-Dyer法、ASTM标准法和皂化法)用于测定微藻中总脂含量的差异,并以原位转酯法为对照,进一步分析了各方法总脂提取物中的脂肪酸含量及组成。改良Bligh-Dyer法和ASTM标准法总脂测定结果稳定,脂肪酸回收率最高,三种藻粉中分别为93.65%和93.74%、85.02%和85.77%、88.62%和88.84%;两者总脂含量及提取物中脂肪酸的组成和含量均无显著差异(p0.05)。显然,改良Bligh-Dyer法操作简便、分析时间短(1 h)、所需样品量少(0.25 g),可替代ASTM标准法,作为实验室快速准确测定微藻总脂含量的首选方法。     

有机碳源对单针藻细胞生长、油脂积累和光合作用的影响

研究不同有机碳源对单针藻(Monoraphidium sp.)细胞生长,油脂积累和光合作用的影响,探讨其细胞生长和油脂积累的最佳碳源浓度。结果表明单针藻具有利用有机碳源进行混合营养生长的能力,葡萄糖、蔗糖对其细胞生长、总脂含量和光合放氧速率具有明显的促进作用,甘氨酸能够促进细胞生长,但总脂含量下降,乙酸钠则表现为抑制作用;在BG-11培养基中加入5g/L葡萄糖后,细胞生物量、总脂产量和光合放氧速率分别为7.8、3.2g/L和240.3μmolO2·(mg·chla)-1·h-1,是同等光合自养条件下的5.6、8.0和1.3倍;单针藻细胞生长、总脂积累的最佳葡萄糖添加浓度为10g/L。     

Photosynthetic production of microalgal biomass in a raceway system under greenhouse conditions in Sendai city

The photosynthetic productivities of the marine microalgae Chlorophyta sp. and freshwater microalgae Chlorella sp. were investigated in a raceway system under greenhouse conditions in Sendai city. The system was constructed with a surface area of 0.986 m2 and equipped with paddle wheels (8 fins). The semicontinuous batch culture experiment was carried out for 4 months from June to October 1998 in Sendai city. The productivity of Chlorophyta sp. was ranged from 4 to 13 g.m(-2).d(-1) and the average was 8.2 g.m(-2).d(-1), which corresponded to the photosynthetic efficiency of 4.15% (PAR). The productivity of Chlorella sp. was ranged from 7 to 21 g.m(-2).d(-1) and the average was 13.2 g.m(-2).d(-1), which corresponded to the photosynthetic efficiency of 6.56% (PAR). These results indicate that stable microalgal cultivation with comparatively high photosynthetic efficiency could be obtained in a raceway system under greenhouse conditions in Sendai city located in the northern part of Japan.     

Biochemical profile of halophilous microalgae strains from high-andean extreme ecosystems (NE-Chile) using methodological validation approaches

The main goal of this work is to characterize the biochemical profile of three halophilous microalgal strains isolated from extreme ecosystems in northern Chile to assess their potential as possible sources of technological and commercial advantages. A procedure for the validation of a routine method for determining the total protein, carbohydrate, lipid and chlorophyll a (Chl-a) contents in Chlorella fusca, Dunaliella salina and Spirulina sp. has been developed. Detection limits of 10.8 μg/mg, 2.7 μg/mg and 5.6 μg/mg for total protein content and 3.5 μg/mg, 1.8 μg/mg and 8.8 μg/mg for total carbohydrate content were determined in C. fusca, D. salina and Spirulina sp., respectively. Most of the biological macromolecule levels measured in the microalgae were in accordance with the magnitude of previously reported data for other strains of the same taxa. However, lower levels than expected of lipids and Chl-a were measured in C. fusca and Spirulina sp., which may be associated with an imbalance between specific growth rates and the rate of macromolecule synthesis. The protein values measured in Spirulina sp. (52.3 ± 2.2 μg/mg DW) were close to the lower limit of the range reported in the literature for non-halophilous strains of the same genus. Except for the Chl-a quantification procedure, the analytical methods for macromolecules had a high degree of repeatability and reproducibility. The variability among repeated measurements of Chl-a was associated with auto-degradation processes during pigment extraction.     

黄腐酸对单针藻Monoraphidium sp.FXY-10油脂积累及me、pepc、accD基因表达量的影响

研究了黄腐酸对单针藻Monoraphidium sp.FXY-10生长、油脂含量、活性氧(ROS)水平及与油脂合成相关基因表达的影响。采用两阶段培养方法(第一阶段异养培养,第二阶段自养培养),并添加黄腐酸诱导。结果表明:在25 mg/L黄腐酸诱导下,6 d时藻细胞中油脂含量可达54.3%,是对照组的1.12倍,藻细胞内活性氧水平是对照组的1.46倍。此外,油脂合成相关酶基因与油脂合成存在相关性,黄腐酸诱导下,me、acc D、pepc基因表达量分别是对照组的1.32倍(4 d)、1.67倍(4 d)及1.71倍(6 d)。研究表明,黄腐酸提高油脂积累可能与活性氧水平及基因表达有关。     

Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis

Single cell oils (SCO) produced from oleaginous microorganisms are a potential alternative oil feedstock for biodiesel production. The worldwide production of glycerol, a 10% (w/w) byproduct produced in the transesterfication process of oils converted to biodiesel, is increasing as more biodiesel is being produced. For the purposes of cost reduction, crude glycerol was regarded as a suitable carbon source for the cultivation of Rhodotorula glutinis. In addition to using renewable crude glycerol, waste solution collected from the brewing company (called thin stillage) was adopted as a substitute to replace a costly nitrogen source used in the medium. The results of using mixture of crude glycerol and thin stillage indicated about a 27% increase in total biomass as compared to that of using crude glycerol with a standard medium. Using glycerol instead of glucose as the carbon source could also alter the lipid profile, resulting in an increase in linolenic acid (C18:2) to comprise over 20% of the total lipid. Successfully using renewable crude glycerol and thin stillage for the cultivation of oleaginous microorganisms could greatly enhance the economic competition of biodiesel produced from SCO.     

促进黄丝藻储能物质高效富集的氮调控策略

为了促进黄丝藻胞内储能物质的高效积累，研究了不同氮形态(NO^-₃、Urea和NH⁺₄)及初始氮浓度(1、3、6、9、18 mmol/L)对黄丝藻生长、碳水化合物和总脂积累的影响，在此基础上建立了基于氮调控促进黄丝藻生物量和储能物质同步高效积累的“低氮(初始氮浓度3 mmol/L)转高氮(初始氮浓度18 mmol/L)”两步法培养策略，并对两步法培养策略的培养时间进行优化。结果表明：与Urea和NH⁺₄比较，黄丝藻更倾向于利用NO^-₃,分别在3 mmol/L和18 mmol/L条件下获得最高生物量(3.69 g/L)和总脂含量(57.3%);在“低氮转高氮”两步法培养中，低氮培养时间6 d转高氮培养12 d可显著提高黄丝藻胞内储能物质的积累，储能物质产率最高，达到274.26 mg/(L·d),其中生物量、总脂产率和碳水化合物产率分别为6.37 g/L、152.92 mg/(L·d)和121.35 mg/(L·d)...