高营养组分小球藻选育、代谢调控及废水资源化利用

Exploitation of Nutrient Components Riched Chlorella Strains: Breeding, Metabolic Regulation and Utilization of Organic Effluent

作者: 专业:发酵工程 导师:王武 年度:2013 学位:博士 

关键词
小球藻驯化 藻蛋白检测方法 蛋白质和油脂 NaCl 胁迫 生理响应 有机废水利用

Keywords
Taming of Chlorella, algal protein assay method, protein and lipid, NaCl stress, physiological response, utilization of organic effluent
        小球藻属绿藻门小球藻属单细胞球形微藻,营自养和异养,分布广,富含蛋白、油脂及色素等营养物质。小球藻种属差异大,导致其生长特性迥异,适应外界环境条件能力也各异,培养条件影响生长及细胞组分。开发可利用有机废水培养高营养组分小球藻,促进绿色制造和循环经济,具有积极意义。本文以此为切入点,选育利用柠檬酸废水(CAE)和城市富含N/P废水(ME)高蛋白组分小球藻,研究其生理特性及优化培养基和控制条件,并调控细胞代谢途径实现蛋白质及(或)油脂等大量积累,以期高效处理CAE及ME,同时吸收大量CO2生产高营养组分藻体。主要结果如下:建立快速检测藻蛋白方法以选育高蛋白组分藻种,优化高蛋白藻体培养工艺。利用所建立的APPD-UV法快速检测藻蛋白,选出生物量、生长速率及蛋白含量均较理想的驯化藻株C. vulgaris C9-JN2010,其生物量和比生长速率均比出发株提高了13.0%,藻蛋白含量高达54.6%,必需氨基酸组成为40.8%,氮磷利用率分别高于出发株16.6%及15.3%。并比较自养、混养及异养小球藻蛋白及氨基酸合成效率,混养时生长及蛋白质产率更佳,其比生长速率和蛋白产率分别为0.526d-1和0.097g·L-1·d-1。与纯自养模式相比,Cys、Tyr和Met含量差异显著。采用P-B、最陡爬坡及CCD试验优化后的混养培养基,藻生物量及蛋白质含量分别为1.786g·L-1和47.0%,其蛋白产率为0.210g·L-1·d-1,提高了116.2%。7.5L光反应器培养,藻的比生长速率、生物量及蛋白产率分别提高到0.630d-1、0.470和0.220g·L-1·d-1。基于氮代谢调控,辅以盐胁迫和高光照强化C. vulgaris C9-2010油脂合成流向。采用单因子、P-B、最陡爬坡及CCD响应面试验优化并确定了C. vulgaris C9-2010高效自养培养基及培养条件(接种量0.150g·L-1,pH7.0,25℃,4000lux和16h:8h)。在优化的培养条件下,藻生物量及产率分别为0.755g·L-1和0.126g·L-1·d-1,提高了6.5%和6.8%。最优条件下二步培养更易积累油脂及提高产率,低氮、2.5%NaCl胁迫和高光照强度(6000lux)组合调控油脂积累效果最佳,藻油脂含量和产率分别高达26.0%和24.2mg·L-1·d-1,为对照的2.5倍和1.75倍。7.5L光反应器与摇瓶培养结果相比,前者油脂含量及产率分别高达25.6%和27.5mg·L-1·d-1,且油脂产率提高约13.6%。研究了NaCl胁迫对C. vulgaris C9-2010生理代谢及叶绿素基因转录的影响。无NaCl时C. vulgaris C9-JN2010生长速率最佳为0.225d-1,且叶绿素和蛋白含量分别高达39.5mg·g-1和55.0%。2.5%NaCl胁迫下,藻油脂含量及产率为15.5%和16.10mg·L-1·d-1,是最低值的3.6倍。适度NaCl浓度可提高藻细胞游离Pro、MDA和GSH等抗氧化物含量和抗氧化酶活,2.5%NaCl胁迫藻的脯氨酸含量高达2.3%,为无盐的20.0倍,未见更高报道;5.5%NaCl胁迫后MDA和GSH含量分别高达70.0及9.4mg·g-1DW,分别为无NaCl的2.1和2.4倍;NaCl胁迫小球藻SOD、CAT及POD酶活均显著提高,分别高达2367.5、50.3和710.4U·g-1DW,是未胁迫的3.0、4.7及2.7倍。利用荧光定量PCR方法,首次分析了无盐和3.5%NaCl胁迫藻细胞叶绿素基因chl B、chl I、chl L及chl N转录水平,3.5%NaCl胁迫藻叶绿素基因转录水平分别为无盐的2.1、1.5、4.5及4.8倍。适当NaCl浓度能增强藻细胞叶绿素基因转录,并促进大量抗氧化活性物质及酶系合成,以抵抗胁迫产生的氧化毒害。驯养小球藻高效利用有机废水磷氮,积累蛋白质与油脂。摇瓶与光反应器中分别培养C. vulgaris C9-JN2010处理CAE和ME。首先,确定了培养该藻最适CAE浓度为20.0%,于5L光反应器获得生物量和产率分别为1.040g·L-1和0.260g·L-1·d-1,且CODCr,BOD5和TOC,氮和磷去除率均高于90.0%。其次,根据摇瓶中ME培养藻的较好结果,于7.5L光反应器分批、半连续和连续培养藻,其最低比生长速率和产率约为0.200d-1和0.106g·L-1·d-1,且NH4+-N、TN、TP、CODCr及BOD5最低去除率分别为98.3%、95.5%、90.6%、88.3%和87.4%。最后,7.5L光反应器中最佳CAE/ME混合废水(20:80)培养藻可获生物量及产率分别高达1.187g·L-1和0.237g·L-1·d-1,且废水污染因子大量被去除(>90.0%)。反应器中藻生长和处理效果均高于摇瓶,且稍高于单独处理CAE和ME,污染因子残留较低接近城镇污水厂废水排放标准(总氮≤15.0mg·L-1,总磷≤0.5mg·L-1,氨氮≤5.0mg·L-1,CODCr≤120.0mg·L-1和BOD5≤60.0mg·L-1)。并发现藻生物量总蛋白、总油脂、粗纤维、灰分和总磷含量分别为48.0%-55.0%,10.0%-30.0%,3.3%-4.0%,5.0%-5.5%和0.65%-0.80%,且必需氨基酸与多不饱和脂肪酸组成比例分别高达44.5%和73.4%。以上结果表明,利用发酵废水培养C. vulgaris C9-JN2010使废水得以资源化具有较好的前景,由于藻细胞富含必需氨基酸和不饱和脂肪酸,是一种很有潜力的优良饲料蛋白来源。
    Grow either in autotrophism or heterotrophism, Chlorella species, as the widespreadspherical unicellular microalgae under Chlorella genus of Chlorophyta, might be riched inprotein, lipid and pigment etc. Their growth characteristics and cellular components weresignificantly influenced by the surrounding conditions. Screening and cultivating some ofChlorella species which could utilize the pollutive elements in origanic effluent andmeanwhile accumulate high-value nutrient components, might be beneficial to promoteindustries for implementation of green manufacturing and circulation economy. In this paper,a Chlorella strain was screened and studied in details, for figuring out the relations betweencultivation models and Chlorella cellular components, the effects of NaCl concentration onthe Chlorella metabolism, and also the utilization efficiency of some organic effluent forproduction of Chlorella cell components. The main results were illustrated as follows:A novel rapid method of assaying algal protein content was established, and used toassay Chlorella protein accumulation at taming period and different culture processes. Thenewly exploited assay, so called APPD-UV method, is quite accurate and reproducible fordetermining Chlorella protein content. By this assay method, a tamed strain, named C.vulgaris C9-JN2010was screened out, which grew better in citric acid effluent andaccumulated higher level of protein. Its biomass and specific growth rate were both13.0%higher than the starter strains, and the algal protein content was54.6%, in which the essentialamino acid content was about40.8%of the total. Compared with the starter, the consumptionrates of nitrogen and phosphorus by C. vulgaris C9-JN2010increased by about16.6%and15.3%, respectively. Based on that, biosynthesis of the algal protein and amino acids werecompared under autotrophism, heterotrophism and mixotrophism forms. In the3rdprocess, C.vulgaris C9-JN2010grew better and shew higher protein productivity, with a specific growthrate of0.526d-1and the protein productivity of0.097g·L-1·d-1, respectively. Comparedamong the three processes, Cys, Tyr and Met proportions were significantly different. P-Bdesign, the steepest climbing and CCD experiments used, an algal fermentation medium ofprotein optimized that was applied to cultivate C. vulgaris C9-JN2010, in which the algalbiomass and protein content were1.786g·L-1and47.0%, respectively, with a proteinproductivity of0.210g·L-1·d-1which was116.2%higher than the control. The specific growthrate (0.630d-1), biomass productivity (0.470g·L-1·d-1) and protein productivity (0.220g·L-1·d-1) of C. vulgaris C9-JN2010cultivated in photo-reactor were all higher than those inthe shake flask.Based on nitrogen metabolism regulation, NaCl stress plus high light intensity wassupplemented to accumulate higher level of Chlorella lipid. The autotrophic culture mediumand conditions (inoculation size0.150g·L-1, pH7.0,25℃,4000lux and light cycle16h:8h)which were successively optimized in single factor, P-B design, the steepest climbing and theCCD experiments were used for cultivating C. vulgaris C9-JN2010and biosynthesis of thealgal lipid was regulated. One-step cultivation strategy performed, the algal biomass and itsproductivity were0.755g·L-1and0.126g·L-1·d-1, increased by6.5%and6.8%, respectively. It was found that low nitrogen concentration, NaCl stress, high light intensity and long lightcycle were favorable for lipid synthesis. Moreover, under low nitrogen,2.5%NaCl stress andhigh light intensity (6000lux) together, two-step cultivation strategy was more advantageousto obtain higher biomass (0.744g·L-1) and accumulate lipid, with the higher lipid content andproductivity of26.0%and24.2mg·L-1·d-1that were2.5and1.75times as well as the algaebefore regulation, respectively. Compared with the algae inoculated in the shake flask, thealgal lipid content and productivity in7.5L photo-reactor were higher, reaching25.6%and27.5mg·L-1·d-1, respectively, and the lipid productivity increased by about13.6%.NaCl stress-inducing effects on algal physiological response mechanism and chlorophyllbiosynthesis gene expression of the tamed Chlorella strain were studied. Under free NaCl, thealgal growth was the best with a specific growth rate of0.225d-1, and its chlorophyll andprotein contents were of39.5mg·g-1and50.0%, respectively. However, the algal lipidcontent in mezzo-salinity (2.5%) was higher of around15.5%with a productivity of16.10mg·L-1·d-1which was around3.6times as well as the algae in the free NaCl. NaCl stresswould induce biosynthesis of some antioxidants and enzymes, such as free proline, GSH andMDA etc. It was the first found free proline content (2.3%) in mezzo-salinity was around20.0times as well as the lowest value. Under5.5%NaCl, MDA and GSH contents were70.0and9.4mg·g-1DW which were about2.1and2.4times as well as those in the non-salinity, andthe activities of algal cellular SOD, CAT and POD were greatly increased, with the activitiesof2367.5,50.3and710.4U·g-1DW which were3.0,4.7and2.7times as well as those in thenon-salinity, respectively. For C. vulgaris C9-JN2010individually under free NaCl and3.5%NaCl stress, the algal chlorophyll biosynthesis genes chl B, chl I, chl L and chl N werequantificationally analyzed through RT-PCR, in which the transcriptional levels of these genesfrom the sample in3.5%NaCl were2.1,1.5,4.5, and4.8times as well as those in thefree-NaCl sample, respectively. The results indicated that proper NaCl concentration wouldaccelerate synthesis of numerous antioxidants and enzymes to resist oxidation damageinduced in NaCl stress to the algal cell and promote transcription of its chlorophyllbiosynthesis genes.Nitrogen and phosphorus in the organic effluent were efficiently utilized to biosynthesizealgal protein and lipid by C. vulgaris C9-JN2010. The algae individually inoculated in shakeflask and photo-reactor would remove nutrients in CAE, ME and mixed CAE/ME effluent.First, in the optimal20.0%CAE, the algal biomass and its productivity were1.040g·L-1and0.260g·L-1·d-1in5L photo-reactor, with higher growth rate and removal rate of nutrient,respectively. Eventually, high removal efficiency of CODCr, BOD5and TOC, TN and TP inCAE were all over90.0%. Additionally, ME being in favour of the algal culture in the flask,batch, semi-continuous and continuous cultivation were performed in7.5L photo-reactors,respectively. The algal specific growth rates and biomass productivities in the three processeswere over0.200d-1and0.106g·L-1·d-1, and removals of NH4+-N, TN, TP, CODCrand BOD5were98.3,95.5,90.6,88.3and87.4%, respectively. Moreover, in the optimal (20:80) mixedeffluent, the algal biomass and specific growth rate were of1.187g·L-1and0.237d-1,respectively. Simultaneously, NH4+-N, TN, TP, TOC, BOD5and CODCrwere almost used up (above90.0%). The algal growth and removal efficiency of pollution factors in thephoto-reactor were higher than those in the shake flask, in which the former were slightlyover those in the CAE and ME separately treated. The above effluents were effectively treatedso that low level residual was close to urban sewage plant wastewater discharge standards(TN≤15.0mg·L-1, TP≤0.5mg·L-1, NH4+-N≤5.0mg·L-1, CODCr≤120.0mg·L-1and BOD5≤60.0mg·L-1). Ultimately, the algal biomass gathered in these effluents was analyzed, and itscrude protein, crude fat, crude fiber, ash and total phosphorus contents were48.0%-55.0%,10.0%-30.0%,3.3%-4.0%,5.0%-5.5%and0.65%-0.80%, respectively, and the essentialamino acid was44.5%of the total protein. Moreover, polyunsaturated fatty acids were about73.4%of the total fatty acids.Above results indicated the promising potencial of utilizing Chlorella to makefermentation waste profitable, especially on supplying the high nutrient feed from Chlorellawith riched essential amino acids and unsaturized fatty acids.
        

高营养组分小球藻选育、代谢调控及废水资源化利用

摘要3-5
Abstract5-7
第一章 绪论12-20
    1.1 小球藻概述12
    1.2 小球藻营养组分及应用12-13
        1.2.1 小球藻应用于食品与饲料12-13
        1.2.2 小球藻应用于化妆品、医药及保健制品13
    1.3 小球藻培养技术研究进展13-16
        1.3.1 小球藻培养条件13-15
        1.3.2 小球藻的培养模式与系统15-16
    1.4 小球藻降污研究进展16-17
        1.4.1 小球藻减少温室气体排放16
        1.4.2 小球藻处理废水16-17
    1.5 小球藻叶绿体基因研究17
    1.6 选题依据和主要研究内容17-20
        1.6.1 立题背景17-18
        1.6.2 研究意义18
        1.6.3 本课题的主要研究内容18-20
第二章 利用废水 N/P 且富含蛋白质的藻种选育与优化培养20-46
    2.1 前言20
    2.2 材料与方法20-27
        2.2.1 藻种20
        2.2.2 培养基20-21
        2.2.3 仪器与试剂21
        2.2.4 培养方法21-22
        2.2.5 改良 APPD-UV 法测定藻蛋白22-23
        2.2.6 藻蛋白质提取及蛋白检测方法23
        2.2.7 柠檬酸废水驯化选育小球藻23
        2.2.8 筛选高效处理柠檬酸废水小球藻23
        2.2.9 藻蛋白自养、混养与异养发酵23
        2.2.10 高效混养培养基的优化23-24
        2.2.11 7.5 L 光反应器培养藻细胞生产蛋白24-25
        2.2.12 发酵参数检测及分析方法25-27
        2.2.13 数据分析27
    2.3 结果与讨论27-44
        2.3.1 建立快捷定量检测小球藻蛋白质的新方法27-31
        2.3.2 高蛋白组分及高效利用废水 N/P 藻种的筛选31-35
        2.3.3 优化藻细胞生长及蛋白质合成代谢调控策略35-38
        2.3.4 高蛋白混养条件优化及 7.5 L 光反应器放大38-44
    2.4 本章小结44-46
第三章 基于氮代谢调控增强藻油合成流向的研究46-68
    3.1 前言46
    3.2 材料与方法46-49
        3.2.1 藻种46
        3.2.2 培养基46
        3.2.3 仪器与试剂46
        3.2.4 高效生长培养基的优化46-48
        3.2.5 优化小球藻自养培养条件48
        3.2.6 高效自养培养基稳定性检验48
        3.2.7 单步培养调控油脂积累试验48
        3.2.8 二步培养调控油脂积累试验48
        3.2.9 7.5 L 光反应器培养藻积累油脂48-49
        3.2.10 叶绿素含量测定49
    3.3 结果与讨论49-67
        3.3.1 优化藻细胞高效生长自养培养基49-59
        3.3.2 高效自养生长调控策略优化59-63
        3.3.3 自养小球藻油脂合成代谢调控策略优化63-66
        3.3.4 7.5 升光反应器放大及藻细胞组分比较66-67
    3.4 本章小结67-68
第四章 NaCl 胁迫影响小球藻生理代谢及叶绿素基因转录差异68-82
    4.1 前言68
    4.2 材料与方法68-70
        4.2.1 藻种68
        4.2.2 培养基68
        4.2.3 仪器与试剂68
        4.2.4 NaCl 胁迫的培养条件68-69
        4.2.5 藻细胞叶绿素基因表达检测方法69-70
        4.2.6 细胞活性物质检测70
        4.2.7 其余发酵参数检测70
    4.3 结果与讨论70-81
        4.3.1 NaCl 胁迫影响藻细胞的生理代谢70-74
        4.3.2 NaCl 压胁迫增强藻细胞抗氧化性74-77
        4.3.3 NaCl 胁迫促进叶绿素相关基因转录而降低叶绿素含量77-81
    4.4 本章小结81-82
第五章 小球藻高效利用两种废水积累高营养组分的研究82-98
    5.1 前言82-83
    5.2 材料与方法83-84
        5.2.1 试验藻种及材料83
        5.2.2 摇瓶优化培养藻细胞的 CAE 浓度83
        5.2.3 5L 光反应器中 20.0%CAE 分批培养83
        5.2.4 市政生活废水摇瓶培养83
        5.2.5 分批、半连续及连续处理市政生活废水83
        5.2.6 摇瓶优化 CAE/ME 混合废水比例83
        5.2.7 7.5 L 光反应器中最佳 CAE/ME 混合废水放大83
        5.2.8 培养参数与细胞营养组分分析方法83-84
    5.3 结果与讨论84-96
        5.3.1 高效利用 CAE 转化为高营养组分84-87
        5.3.2 培养模式调控藻细胞转化 ME 为高营养组分87-91
        5.3.3 CAE 与 ME 互济强化培养效果及细胞高营养组分91-94
        5.3.4 分析废水培养藻细胞的营养组分94-96
    5.4 本章小结96-98
结论与展望98-101
    1 结论98-100
    2 展望100
    3 创新点100-101
致谢101-102
参考文献102-112
附录Ⅰ:作者攻读博士学位期间取得的研究成果112-113
附录Ⅱ:实验照片及样品检测图谱113-114


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