热带普通小球藻培养模式的筛选及其培养基的优化

【背景】从海南热带海区中分离得到一株微藻,其生长速度快、适应力强,经鉴定该微藻为普通小球藻。【目的】提高热带普通小球藻的生长速率。【方法】以"宁波大学3#微藻培养液配方"为基础培养液,分别添加有机碳(C6H12O6和CH3COONa)对热带普通小球藻进行自养、兼养及异养培养,获得促进热带普通小球藻快速生长的培养方式。在"宁波大学3#微藻培养液配方"的基础上对热带普通小球藻的兼养培养基配方进行优化,并用优化兼养培养基与"宁波大学3#微藻培养基"对比培养热带普通小球藻。【结果】添加6 g/L CH3COONa的兼养模式促进热带普通小球藻生长效果最好;优化的兼养培养基配方为:6 g/L CH3COONa,20 mg/L(NH4)2SO4-N,5 mg/L Na H2PO4-P,3 mg/L Fe SO4-Fe,1 mg/L Vitamin B1和0.000 5 mg/L Vitamin B12。对比培养实验结果显示,培养第6天,兼养培养液收获的生物量(细胞密度)达4.20×107 cells/m L,是"宁波大学3#配方微藻培养液"的2.30倍。【结论】兼养培养模式为热带普通小球藻的最佳培养模式,优化的兼养培养基极显著地提高了热带普通小球藻的生物量(P0.01)。     

氮胁迫对埃氏小球藻生长及油脂积累的影响

以生长快、可除污的埃氏小球藻株系SXND-25为试材,研究不同氮浓度培养条件对其生物量和油脂产量的影响,以期建立优化培养体系利用该株小球藻生产优质生物燃油。以硝酸钠为氮源、BG11培养基中的氮浓度为基准(1.5 g/L),设置氮浓度梯度对小球藻进行培养。通过光密度测定、尼罗红染色、转酯化法抽提油脂和GC分析,对小球藻生物量、油脂含量及脂肪酸组分进行分析。结果显示,培养8 d时,在氮浓度为1.5 g/L时,生物量达到最大,干重为3.4 g/L,而油脂含量仅为28.24%,油脂产量为0.96 g/L;在氮浓度为0 g/L时生物量最小,干重为0.49 g/L,而油脂含量最高,为44.57%,油脂产量为0.22 g/L;在氮浓度为0.75 g/L时,干重为3.2 g/L,油脂含量为40.36%,油脂产量最高为1.3 g/L,是标准氮浓度下油脂产量的1.4倍。0.75 g/L氮浓度下连续培养8 d,藻油脂肪酸组成更适于制取优质生物柴油。综合生物量、油脂含量及脂肪酸组成等指标,确定0.75 g/L氮浓度为该埃氏小球藻株系规模化培养以生产优质生物燃油的优化参数。     

产油微藻自养培养条件调控

作为新兴生物燃料的生物柴油近年来发展迅速,以微藻为代表的第二代生物能源是解决能源危机的长远之计,但如何提高其产量仍是研究的热点问题。以提高产油自养微藻生物量和油脂含量为目的,在气升式光反应器中运用均匀设计实验方法进行了条件优化试验。分别得出了氮原子浓度、通气速率、二氧化碳体积浓度和光照强度4个因素对小球藻C2生物量积累和油脂含量影响的显著回归方程和反应器优化培养条件。以生物量为指标的优化培养条件是:氮原子浓度0.178 g/L,通气速率5 L/min,二氧化碳体积浓度3%(V/V),光照强度6000 lx。该优化条件下,生物量为2.11 g/L,即生产速率为0.352 g/(L.d),比测试实验中检测到的最高生物量[1.88 g/L,即生产速率为0.313 g/(L.d)]提高了12.2%;以油脂含量为指标的优化培养条件是:进气速率0.400 L/min,二氧化碳体积浓度1.94%(V/V),得到油脂含量为22.4%,比测试实验中检测到的最高油脂量(20.7%)提高7.7%。     

乙酸钠调控小球藻生长及代谢产物

【背景】小球藻是一种单细胞绿藻,在不同培养条件下可积累高附加值的代谢产物,这些产物可用于生产生物燃料、食品、保健品、药品等。然而这些代谢产物在藻细胞中的生产率较低且很难通过经济可行的方法将其分离,这使其工业化规模生产受到限制。【目的】研究乙酸钠对小球藻生物量的影响,并分析其对小球藻代谢产物的调控作用。【方法】通过在小球藻培养液中添加不同浓度的乙酸钠(1.0、2.0、3.0、4.0、5.0 g/L),研究其调控小球藻生长和代谢的作用机理。【结果】在添加3.0 g/L乙酸钠的培养液中,小球藻的生物量是对照组的5.2倍,尽管藻细胞中蛋白质含量无明显变化,但油脂和类胡萝卜素含量是对照组的2.4倍和1.2倍,多糖和叶绿素a含量却仅为对照组的54.6%和54.4%。【结论】乙酸钠不仅会影响藻细胞的生长,还会调控其代谢过程,这为深入探索乙酸钠在调控小球藻生长及代谢过程的作用机制提供了理论基础和技术资料。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoi-dosa No.2.研究了培养基组成及培养条件对其细胞生长和油脂积累的影响.结果表明,最适培养基组成为(g/L):葡萄糖20,甘氨酸0.08,MgSO4·7H2O 0.4,K2HPO4 1.0,FeSO4·7H2O 0.004;适宜的培养温度,初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和650 Lux.在上述优化条件下培养7 d,Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L和40.15%提高到6.56 g/L和59.90%,油脂产量提高了162%.Chlorella pyrenoidosa No.2能以木糖为碳源产油脂,可望用于以木质纤维素等可再生生物质资源为原料生产油脂.气相色谱分析表明该油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达71%左右,可作为生产生物柴油的原料.     

裂殖壶菌Schizochytrium sp. 20888产DHA培养条件优化

【目的】为了优化裂殖壶菌产DHA的培养条件,提高油脂中DHA含量。【方法】采用单因素试验和正交设计试验方案,针对分批培养时间、培养基碳、氮源的种类和浓度以及培养温度开展试验,采用重量法测定生物量、采用索氏提取法测定油脂总量,采用气相色谱法测定油脂DHA含量,考察培养条件对细胞油脂DHA含量的影响。【结果】最适培养时间为4 d,培养温度23°C,最优碳氮源组成为(g/L):葡萄糖65、甘油80、蛋白胨6、酵母粉4和谷氨酸钠8。【结论】裂殖壶菌Schizochytrium sp.20888在葡萄糖和甘油组成的复合碳源和由蛋白胨、酵母粉和谷氨酸钠组成的复合氮源的培养基中可以得到最优的DHA产量,细胞DHA含量能达到33.68%。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoidosa No.2。研究了培养基组成及培养条件对其细胞生长和油脂积累的影响。结果表明, 最适培养基组成为(g/L)：葡萄糖 20, 甘氨酸 0.08, MgSO4·7H2O 0.4, K2HPO4 1.0, FeSO4·7H2O 0.004; 适宜的培养温度、初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和 650 Lux。在上述优化条件下培养7 d, Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L 和 40.15%提高到6.56 g/L和59.90%, 油脂产量提高了162%。Chlorella pyrenoidosa No.2能以木糖为碳源产油脂, 可望用于以木质纤维素等可再生生物质资源为原料生产油脂。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 不饱和脂肪酸含量达71％左右, 可作为生产生物柴油的原料。     

出芽短梗霉产聚苹果酸发酵条件的优化

【背景】出芽短梗霉可发酵葡萄糖生成聚苹果酸,但存在转化率和转化效率低等瓶颈,阻碍其实现商业化生产。【目的】通过优化发酵培养条件,提高出芽短梗霉的聚苹果酸产量、糖酸转化率和生产强度。【方法】采用单因素试验优化适宜出芽短梗霉BK-10菌株产生聚苹果酸的培养条件,通过Plackett-Burman法对培养基组分筛选显著性影响因素,并对其培养基中无机盐进行正交试验优化,最后进行5 L发酵罐验证。【结果】最优培养基配方和培养条件:100 g/L葡萄糖,1.5 g/L尿素,0.20 g/L KH_2PO_4,0.20 g/L ZnSO_4,0.05 g/L MgSO_4,0.75 g/L KCl,30 g/L CaCO_3,0.01%吐温-80,发酵温度26°C,250 mL摇瓶装液量50 mL。【结论】通过优化,聚苹果酸的糖酸转化率达到0.71 g/g,生产强度达到0.89 g/(L·h),较优化前分别提高了18.33%和71.15%,为发酵葡萄糖合成聚苹果酸进而生产L-苹果酸工艺的工业化生产奠定经济性基础。     

Fe~(3+)对小球藻的生长及油脂含量的影响

传统化石能源储量日益减少,生物柴油因其环保可再生性成为优质的石化柴油替代品。利用小球藻生产生物柴油速度快、油脂含量高,受到了广泛关注。为进一步提高小球藻生产生物柴油效率,分别探究了Fe3+的浓度及添加时间对自养和异养小球藻生长及产油的影响,获得最优Fe3+培养条件为:自养小球藻延滞期添加10-3 g/L Fe3+,生物量及油脂含量达2.80 g/L及30.90%;异养小球藻指数期添加10-5 g/L Fe3+,生物量及油脂含量达3.30 g/L及29.05%。经脂肪酸分析,以上条件获得的微藻油脂均可作为生物柴油生产原料。     

利用啤酒废水小球藻异养培养

摘要：【目的】利用小球藻异养培养技术处理啤酒废水,旨在为啤酒废水资源化利用和降低小球藻生产成本提供一个途径。【方法】在含有10 g/L葡萄糖的基本培养基进行异养小球藻高效藻株的筛选,并用于啤酒废水的资源化处理。【结果】从5株小球藻中得到2株适合高密度异养培养的藻株（Chlorella pyrenoidosa 15-2070 和 Chlorella vulgaris 15-2075）,在啤酒废水的资源化处理过程中这2株小球藻得到非常接近的试验结果。利用由废水配制含10 g/L葡萄糖的基本培养液培养Chlorella pyrenoidosa 15-2070获得了5.3 g/L藻细胞;并且在此过程中,啤酒废水得到有效利用,几种主要污染物最高去除率为：CODcr,92.2 %;BOD5,95.1 %;NO3--N,98.5 %;NH4+-N,92.3 %。【结论】啤酒废水中的重要环境污染物在培养小球藻的过程中可以得到有效地清除,并从中可以获得具有商业价值的小球藻细胞。     

无机碳源对小球藻自养产油脂的影响

旨在研究小球藻利用无机碳自养产油脂,考察了3种无机碳源 (Na2CO3、NaHCO3和CO2) 及其初始浓度对小球藻产油特性的影响。结果表明,小球藻能利用Na2CO3、NaHCO3和CO2产油;经Na2CO3、NaHCO3和CO2培养10 d后,随着每种无机碳源浓度的增加,小球藻产量均先增加后减少。小球藻经3种无机碳源培养后,其培养液pH值上升。最适宜的Na2CO3和NaHCO3添加量均为40 mmol/L,其生物量分别达到0.52 g/L和0.67 g/L,产油量分别达到0.19 g/L和0.22 g/L。在3种无机碳源中,CO2是最佳无机碳源,当CO2浓度为6％时,小球藻生长最快,生物量达2.42 g/L,产油量最高达0.72 g/L;当CO2浓度过低时,无机碳供应不足,油脂产量低;当CO2浓度过高时,培养液pH偏低,小球藻油脂积累受到抑制。Na2CO3和NaHCO3较CO2更有利于小球藻积累不饱和脂肪酸。     

培养条件对产油微生物生长的影响

为了筛选出高产油菌株, 首先采用细胞形态学方法与细胞化学方法(苏丹III染色法)对4株高产油脂菌株进行初筛, 并通过索氏提取法对初筛菌株油脂含量进行分析, 确定M2菌株为实验菌株, 其油脂含量达53.09%。为了增加产油微生物油脂产量, 本试验考察了不同发酵条件对其细胞生长和油脂积累的影响。优化工艺参数为: 10° Bx玉米皮渣水解液为培养基质, 0.2% NaNO3为氮源, pH 6.0、28oC下发酵培养6 d, 微生物油脂含量75.21%, 菌体生物量30.40 g/L, 油脂产量22.86 g/L。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 主要含有16碳和18碳系脂肪酸, 可作为生物柴油的原料, 不饱和脂肪酸含量达68%, 可应用于医药化工领域。     

高产莪术细胞悬浮系建立及产物前体对挥发油合成的调控研究

研究了高产莪术细胞悬浮系培养的条件及前体物质添加对挥发油合成的调控。结果表明：淡黄色颗粒状愈伤组织是建立高产细胞悬浮系的最佳供试愈伤组织;最佳培养基成分是MS培养基添加葡萄糖与蔗糖各15—30g／L(1：1),氮源为NH4^ 和NO3^-,比例为1：3,总量为80mmol／L;激素组合为6-BA3．0—5．0mg／L、2,4-D1．0mg／L;光下培养10—15天再转入优化条件下的暗培养,可形成稳定的高产细胞悬浮系;其细胞周期中的最大细胞生长量及挥发油含量分别是248g／L和2．28％;前体物质泛酸钙、乙酸铵、乙酸钾的添加均可有效提高培养细胞合成挥发油的百分含量,其中乙酸铵最有效,在指数生长中期添加0．5mmol／L乙酸铵,挥发油的最高含量可达3．11％,产量为8．27g/L,分别是添加前的1.25倍及1.2倍。     

拉曼被孢霉F_5发酵生产γ—亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ—亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨。最适发酵培养基组成为 (g/L) :葡萄糖 1 0 0 ,酵母浸出粉 4 ,蛋白胨 1 ,K2 HPO4 1 ,CaCl2 1× 1 0 - 2 ,MgSO4 5× 1 0 - 2 ,FeSO4 1× 1 0 - 2 ,ZnSO4 7.5× 1 0 - 3,CuSO4 0 .5× 1 0 - 3,MnSO4 2× 1 0 - 3,pH 6.0。培养温度为 2 5℃ ,1 4 0r/min振荡培养 1 0天 ,培养 8天后 (即收获前 2天 )补加 5 %葡萄糖。发酵结果为 :DC 2 4 .5 9g/L ,TL 1 0 .84g/L ,TL/DC 4 4.0 9% ,GLA/TL 1 0 .67% ,GLA产量为 1 1 5 6.63mg/L。GLA产量较初始结果提高 1 5 6.1 5 %。该菌株已达到工业化生产菌株要求     

吸水链霉菌ATCC 29253产Hygrocin A发酵条件的优化

【背景】Hygrocins是一种萘安莎抗生素,具有良好的新药开发潜能。但在常见培养基及发酵条件下菌体内Hygrocin A含量一般很低,甚至难以直接进行准确检测。【目的】提高吸水链霉菌ATCC 29253发酵物中Hygrocin A的产量。【方法】采用单因素与正交试验设计优化相结合的方法系统考察碳源、氮源、磷酸盐、MgCl_2浓度、NaCl浓度、种子菌龄等因素对吸水链霉菌ATCC 29253产Hygrocin A能力的影响。【结果】最佳发酵条件为(g/L):葡萄糖4.0,黄豆饼粉8.0,麦芽提取物10.0,K_2HPO_4 1.5,KH_2PO_4 1.5,NaCl 1.5,Mg Cl2 1.0;种子最佳活化时间为48 h;培养参数:摇床转速200 r/min,初始pH为6.8-7.0,瓶装量50 m L/250 m L,接种量5%,30°C培养10 d。在优化条件下,Hygrocin A产量与其原始培养基M10相比提高了500%,Rapamycin产量同时下降了95%。【结论】通过培养基优化,可显著提高吸水链霉菌ATCC 29253中Hygrocin A产量,为Hygrocin A合成应用研究奠定基础,同时可使Rapamycin产量明显下降。这说明可通过选择培养条件有目的地调节两种抗生素的代谢通量,进而开展多种抗生素同时表达的代谢调控研究。     

Application of high-salinity stress for enhancing the lipid productivity of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> HS1 in a two-phase process

Increased lipid accumulation of algal cells as a response to environmental stress factors attracted much attention of researchers to incorporate this stress response into industrial algal cultivation process with the aim of enhancing algal lipid productivity. This study applies high-salinity stress condition to a two-phase process in which microalgal cells are initially grown in freshwater medium until late exponential phase and subsequently subjected to high-salinity condition that induces excessive lipid accumulation. Our initial experiment revealed that the concentrated culture of Chlorella sorokiniana HS1 exhibited the intense fluorescence of Nile red at the NaCl concentration of 60 g/L along with 1 g/L of supplemental bicarbonate after 48 h of induction period without significantly compromising cultural integrity. These conditions were further verified with the algal culture grown for 7 days in a 1 L bottle reactor that reached late exponential phase; a 12% increment in the lipid content of harvested biomass was observed upon inducing high lipid accumulation in the concentrated algal culture at the density of 5.0 g DW/L. Although an increase in the sum of carbohydrate and lipid contents of harvested biomass indicated that the external carbon source supplemented during the induction period increased overall carbon assimilation, a decrease in carbohydrate content suggested the potential reallocation of cellular carbon that promoted lipid droplet formation under high-salinity stress. These results thus emphasize that the two-phase process can be successfully implemented to enhance algal lipid productivity by incorporating high-salinity stress conditions into the pre-concentrated sedimentation ponds of industrial algal production system.     

Waste molasses alone displaces glucose-based medium for microalgal fermentation towards cost-saving biodiesel production

The by-product of sugar refinery—waste molasses was explored as alternative to glucose-based medium of Chlorella protothecoides in this study. Enzymatic hydrolysis is required for waste molasses suitable for algal growth. Waste molasses hydrolysate was confirmed as a sole source of full nutrients to totally replace glucose-based medium in support of rapid growth and high oil yield from algae. Under optimized conditions, the maximum algal cell density, oil content, and oil yield were respectively 70.9 g/L, 57.6%, and 40.8 g/L. The scalability of the waste molasses-fed algal system was confirmed from 0.5 L flasks to 5 L fermenters. The quality of biodiesel from waste molasses-fed algae was probably comparable to that from glucose-fed ones. Economic analysis indicated the cost of oil production from waste molasses-fed algae reduced by 50%. Significant cost reduction of algal biodiesel production through fermentation engineering based on the approach is expected.     

The effect of processing of Chlorellavulgaris: K-5 on in vitro and in vivo digestibility in rats

Two experiments were done to clarify whether or not cell rupture is necessary to improve the digestibility of major components of Chlorella vulgaris: K-5. Chlorella was treated with or without high pressure homogenization (1 × 10⁸ N/m² at less than −20°C) after a heating process (100-120°C). Chlorella (air-dry matter) contained 934 g dry matter and 244 g essential amino acids (total)/kg. Chemical composition was hardly altered irrespective of the treatment. In the first experiment, pepsin digestibility of chlorella protein was determined in vitro. The cell rupture by high pressure homogenization caused a small but significant improvement in pepsin digestibility of chlorella protein compared with the control. In the second experiment, total tract apparent digestibilities of chlorella were determined in the rat. Digestibility of chlorella protein was significantly enhanced by high pressure homogenization, but the difference (88.6% vs. 87.4%, P < 0.01) due to treatment was small and similar to that observed in the in vitro experiment. These results suggested that Chlorella strain vulgaris: K-5 may be an efficient protein source even without cell rupture.     

High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture

The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea. In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source. As a result, a maximum cell dry weight concentration of 48 g/L was achieved. This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions. In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed-batch mode to establish a nitrogen-limited culture. Such a modification resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the N-sufficient culture. The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This N-limited fed-batch culture was successfully scaled up from 3.7 L to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L). The maintenance of the culture at a higher temperature (i.e., 32 degrees C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed-batch culture (30 L) without such a treatment. The enhancement of lutein production resulted from the combination of nitrogen limitation and high-temperature stress.