固定化硝化菌群联合芽孢杆菌处理对虾养殖废水

【背景】高度集约化的对虾养殖业面临着日益严重的水污染问题,同步高效降解养殖废水中的有机物、氨氮和亚硝酸盐是对虾养殖业健康可持续发展的重要保障之一。【目的】通过分别固定化硝化菌群(Nitrifyingbacterialconsortia,NBC)和芽孢杆菌,优化菌群空间结构,提高菌群功能,实现同步高效降解对虾养殖废水中的有机物、亚硝酸盐和氨氮,保障南美白对虾养殖的可持续发展。【方法】采集养殖虾塘底泥进行硝化细菌自养富集和连续培养,利用16S rRNA基因高通量测序技术分析硝化菌群组成。从5株芽孢杆菌中筛选化学需氧量(Chemical oxygen demand,COD)降解能力最强的菌株。选用吸附和成球效果好的无毒包埋材料,通过正交实验优化固定化配方提高机械强度。选择硝化菌群和芽孢杆菌最适使用浓度进行分别固定化并联合应用于对虾养殖废水的处理。【结果】高通量分析结果显示硝化菌群中变形菌门(Proteobacteria,61.10%)占绝对优势,具有自养硝化功能的类群丰度达12.69%并呈高多样性。还包含丰度达47.44%的具有反硝化功能或者潜在反硝化功能的优势菌群和丰度达12.85%的光合细菌,是高有机负荷下硝化作用的重要补充,并可通过反硝化作用实现真正脱氮。COD降解能力最强的是解淀粉芽孢杆菌(Bacillusamyloliquefacien)YL-10,48h内COD降解率达100%。固定化最佳配方为贝壳粉5%、海藻酸钠3%、交联剂氯化钙为4%、优化后的固定化小球其机械强度可达129.68m N。固定化使硝化菌群的氨氮和亚硝酸盐降解率分别提高了128.13%和130.11%(P0.05),但对芽孢杆菌YL-10的COD降解率无明显提高。1×10~8 CFU/mL为硝化菌群和芽孢杆菌YL-10在养殖废水中最适使用浓度。在固定化硝化菌群和芽孢杆菌YL-10联合作用下,对虾养殖废水的氨氮、亚硝酸盐和COD浓度在48h内分别由初始的6.32±0.12、5.69±0.11和65.29±1.14 mg/L降至0.03±0.03、0.06±0.01和0 mg/L (P0.05),降解率分别为99.57%、99.03%和100%。【结论】通过优化固定化有效提高硝化菌群的硝化作用,联合COD降解能力强的芽孢杆菌,同步高效降解对虾养殖废水中的有机物、氨氮和亚硝酸盐,为规模化应用于南美白对虾高密度养殖提供科学依据。     

耐高温石油烃降解菌的筛选及性能研究

目的筛选耐高温石油烃降解菌并对降解条件进行优化。方法以大庆地区石油污染土壤的堆肥样品为研究材料,通过富集培养后分离得到耐高温石油降解菌株,选取降解率最高的菌株,对其降解条件进行了探讨。结果得到6株耐高温石油烃降解菌,其中WY 2最适温度52~58℃,pH值7,石油浓度0.5%,接种量2mL,最佳氮源为硫酸铵,通过16SrDNA序列分析,确定该菌株为地衣芽孢杆菌。结论确定了耐高温石油烃降解菌的最佳降解条件。     

N-甲基吡咯烷酮降解菌株的筛选鉴定及应用

N-甲基吡咯烷酮（NMP）是一种重要的化工原料，因本身结构的稳定性等使其不易被降解，造成了严重的环境及水土污染。文章在某活性污泥中通过驯化、富集培养、分离筛选和效果验证，得到一株可高效降解NMP的菌株。经16S rDNA鉴定，该菌株为地衣芽孢杆菌，20 h内对1 000 mg/L的NMP降解率达99.99%,12 h内对200 mg/L的NMP降解率达100.00%。该菌应用于芳纶纸产生的NMP废水处理中，0.1%的添加量可使256.92 mg/L含量的NMP在5 h内完全降解，达到工业处理废水的合格指标。     

学生食堂餐厨垃圾中淀粉的生物利用菌株筛选

目的探索地衣芽胞杆菌、枯草芽胞杆菌和蜡样芽胞杆菌分解大学生食堂厨余中淀粉的能力,以筛选和研制餐厨垃圾生物降解的使用菌种。方法将各菌种接于淀粉酶试验培养基,培养后滴加碘溶液,观察透明圈,判定产淀粉酶能力;收集大学生食堂的厨余,观察三种细菌在不同接种量（5%、10%、15%、20%、25%）、不同接种时间（24 h、48 h、72 h）及不同菌株配伍方式下发酵淀粉的能力。结果地衣芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌都能产生淀粉酶,以地衣芽胞杆菌产生的淀粉酶较多,其次为枯草芽胞杆菌,蜡样芽胞杆菌较少,三种细菌分解厨余中淀粉的最佳接种量都为15%-20%,最佳发酵时间为48 h,枯草芽胞杆菌和地衣芽胞杆菌各7.5%的接种量混合配伍发酵效果最佳。结论可采用枯草芽胞杆菌和地衣芽胞杆菌各7.5%的接种量混合配伍发酵学生食堂的厨余中的淀粉。     

副地衣芽孢杆菌FA6全基因组测序及比较基因组学分析

副地衣芽孢杆菌(Bacillus paralicheniformis)FA6是一株从草鱼肠道内分离出来的细菌, 其具有淀粉酶和纤维素酶等多种碳水化合物酶活性。为深入研究副地衣芽孢杆菌FA6可能的益生机制, 研究通过三代测序技术测定了副地衣芽孢杆菌FA6的全基因组序列, 运用生物信息学方法进行基因组组装、基因预测和功能注释。同时通过比较基因组学方法, 比较分析了副地衣芽孢杆菌FA6与4株基因组序列已经发表的芽孢杆菌基因组结构和功能的差异。结果发现副地衣芽孢杆菌FA6全基因组由1条环状染色体组成, 大小为4450579 bp, GC含量为45.9%。副地衣芽孢杆菌FA6基因组中含有128个蛋白酶基因, 32个脂肪酶基因和72个糖苷水解酶基因, 这些基因与食物降解相关; 此外, 细菌基因组中还含有7个编码羊毛硫抗生素相关的基因。比较基因组结果显示, 副地衣芽孢杆菌FA6与其他4株芽孢杆菌的基因组共线性关系较好, 但是与地衣芽孢杆菌菌株相比, 菌株FA6基因组特征更接近于副地衣芽孢杆菌菌株。副地衣芽孢杆菌FA6基因组中编码纤维素酶、半纤维素酶和淀粉酶的基因数量分别为5、7和5个, 多于其他菌株, 能够更好地降解植物多糖。研究结果表明副地衣芽孢杆菌FA6高度适应植物性成分, 反映了该菌株在草鱼肠道中的适应性进化, 该菌株可能可以作为益生菌用于水产养殖。     

氨氮降解菌的筛选、鉴定与复合菌水质调控效果研究

为寻找高效降解水体中氨氮的菌株并对其进行应用评价,研究从多种水产养殖池塘水体和底泥的混合物中筛选出2株氨氮降解菌,降解率分别达97.8%和98.5%,经鉴定均为凝结芽孢杆菌(Bacillus coagulans)。对筛选出的2菌株培养条件进行优化,2菌株pH、C/N适应范围广,并且耐高温、高盐。通过灌服试验表明所筛选菌株对养殖动物是安全的。在此基础上,将筛选菌株与本实验室前期诱变菌株B38复配后制成复合菌,通过养殖试验评价了复合菌对氨氮、亚硝酸盐及藻类数量的调控效果。与4种商品微生态制剂(光合细菌、酵母菌、强效EM和芽孢杆菌)相比,泼洒复合菌的池塘氨氮含量逐渐降低。在氨氮含量下降的同时,亚硝酸盐含量有上升的趋势,但在试验的第18天,复合菌组与酵母菌组亚硝酸盐含量有所降低。对藻类数量的影响结果显示,从第9天开始添加复合菌与芽孢杆菌组藻类数量高于其他各组,在第14天,这2组藻类数量大约为其他组的2倍。由此可见,复合菌具有明显的降氨氮特性,并能有效增加藻类数量,但对亚硝酸盐降解效果不显著。研究为复合型微生态制剂的开发提供了技术支撑。     

荧光原位杂交技术对土壤石油降解菌的检测

从辽河油田样品中筛选出一株高效石油降解菌,经鉴定为地衣芽孢杆菌。针对其16S rRNA设计寡核苷酸探针。荧光原位杂交(FISH)技术利用寡核苷酸探针检测特定细胞内的互补核苷酸序列。通过对纯菌和泥浆中地衣芽孢杆菌的FISH进行优化,得到泥浆中地衣芽孢杆菌的荧光原位杂交实验条件：样品固定时间17 h,杂交温度46 ℃,杂交时间3 h,杂交液中去离子甲酰胺浓度35%,冲洗缓冲液中与去离子甲酰胺对应的NaCl的浓度88 mmol·L^-1。运用上述FISH技术监测生物泥浆反应器中地衣芽孢杆菌量的变化,并与泥浆中含油率的变化进行比较,二者的变化情况符合微生物降解石油的趋势,为监测含油污泥中微生物的变化提供了一种可行的技术。     

异养硝化细菌的筛选、鉴定及其氨氮转化特性的研究

从巢湖湖底泥床中分离筛选出一株具有氨氮转化活性的异养型硝化菌株X5.经生理生化分析、16S rDNA序列测定及系统发育学比较,菌株X5为枯草芽孢杆菌属(Bacillus sp.).菌株X5对氨氮的转化能力受温度、pH值以及接种量等条件的影响.实验结果表明,菌株X5的最适氨氮转化条件为:25～35℃,16 h,pH值7.0～8.0以及6%的接种量.将菌株X5扩大培养后接种于含蓝藻的发酵培养基中,分别测定总氮(TN)、氨氮(NH4+-N)及硝态氮(NO-3-N)的含量.结果显示,菌株X5对蓝藻中的氮素具有一定的转化作用.     

N^＋注入诱变选育改善豆粕饲用品质的菌种研究

为了选育能够提高豆粕饲用品质的高效菌株,利用能降解胰蛋白酶抑制因子(trypsin inhibitors,TI)的枯草芽孢杆菌进行N+注入诱变,挑选碱性蛋白酶高产菌株,按出发菌的优化发酵条件与出发菌分别进行豆粕固态发酵,比较其TI兀降解效果,同时采用L16(45)正交设计对诱变菌株发酵豆粕的接种量、料水比、温度、发酵时间和通气量五个因素进行优化.在诱变能量为15 keY,剂量为1.5×1015ions/cm2时,筛到一株高产碱性蛋白酶的菌株KY-103,其酶活由诱变前21.39 U/mL提高到103.07 U/mL,胰蛋白酶抑制因子的降解率由19.58%提高到36.24%,小肽含量也由4.27%提高到7.89%.综合考虑的最佳发酵条件为15%的接种量,料水比1:1,通气量为60 g/500 mL,pH自然,在40℃下发酵96 h.此条件下胰蛋白酶抑制因子的降解率达43.92%,小肽含量达8.14%.结果提示,N+注入诱变可以获得改善豆粕饲用品质的高效枯草芽孢杆菌.     

强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能

地衣芽孢杆菌2709由于易于培养、GRAS状态和完善的蛋白质分泌能力,是已经投入工业生产碱性蛋白酶的菌株。为改善该菌株的发酵生产性能,提高菌体对培养基成分的利用和碱性蛋白酶产量,对菌株的胞外分泌酶系进行完善。利用同源重组机制,在基因组复制起始位点附近引入了来源于短小芽孢杆菌的木聚糖酶基因xynA和在复制起始位点中心对称的位置引入耶氏解脂酵母来源的脂肪酶基因lipY2。整合菌株在摇瓶发酵44h时,木聚糖酶、脂肪酶酶活力分别达(58±2.07)U/mL和(207±10.62)U/mL,其分泌表达促进了地衣芽孢杆菌对发酵培养基的分解与利用,提高了培养基中还原糖、上清总氮的含量和沉淀中含氮化合物的分解;细菌生物量较地衣芽孢杆菌原始菌株提高了11.76%,同时碱性蛋白酶的发酵周期较原始菌提前了4h,碱性蛋白酶产量提高了14.41%。地衣芽孢杆菌2709分泌酶系的丰富和发酵性能的改善为在饲料行业中作为微生物制剂的地衣芽孢杆菌提供了改造的方法。     

毛枝藻对人工污水脱氮除磷能力的研究

文章探究了2株毛枝藻(Stigeoclonium sp.)SHY-370及HB1617在不同初始氨氮浓度以及不同氮磷比条件下的生长情况与氮磷去除能力。结果表明氨氮浓度对2株毛枝藻的生长及TP去除能力均有一定的影响, SHY-370可耐受最大氨氮浓度为10 mg/L, HB1617为5 mg/L;氨氮浓度为1—10 mg/L时SHY-370及HB1617对其去除率均达到97%以上,最大去除速率为3.98 mg/(L·d)。氮磷比对SHY-370的生长影响不大,但在氮磷比大于20时HB1617的生长受到抑制; SHY-370对NO_3~--N去除的最佳氮磷比为10—40, HB1617为2—10;氮磷比为2时水体中TP的含量超过了SHY-370及HB1617所能去除的最大值,去除率较低。实验结果表明SHY-370及HB1617在污水深度脱氮除磷方面具有一定的潜力,可考虑将其应用于城市生活污水二级出水(TN≤15 mg/L、TP≤0.5 mg/L、 NH_4~+-N≤5 mg/L)的深度处理。     

Antimicrobial colorants in molasses distillery wastewater and their removal technologies

Molasses is a widely used feedstock in the bioethanol distilleries, which generate the dark colored wastewater known as molasses distillery wastewater (MDWW). This type of wastewater leads to pollution problems in the local environment where it is disposed of due to the high content of pollutants, among which colorants are of great concern. The main MDWW colorants are polyphenols, melanoidin, alkaline degradation products of hexoses, and caramels whose formation, concentration and antimicrobial effects are summarized in this review. A lot of efforts have been made in the community to remove the colorants. Effective treatment methods are discussed, including biological treatment, enzymatic treatment, chemical oxidation, and coagulation. These technologies could also be applied to remove the colorants as a final treatment step after the anaerobic digestion.     

Microalgae and wastewater treatment

Organic and inorganic substances which were released into the environment as a result of domestic, agricultural and industrial water activities lead to organic and inorganic pollution. The normal primary and secondary treatment processes of these wastewaters have been introduced in a growing number of places, in order to eliminate the easily settled materials and to oxidize the organic material present in wastewater. The final result is a clear, apparently clean effluent which is discharged into natural water bodies. This secondary effluent is, however, loaded with inorganic nitrogen and phosphorus and causes eutrophication and more long-term problems because of refractory organics and heavy metals that are discharged. Microalgae culture offers an interesting step for wastewater treatments, because they provide a tertiary biotreatment coupled with the production of potentially valuable biomass, which can be used for several purposes. Microalgae cultures offer an elegant solution to tertiary and quandary treatments due to the ability of microalgae to use inorganic nitrogen and phosphorus for their growth. And also, for their capacity to remove heavy metals, as well as some toxic organic compounds, therefore, it does not lead to secondary pollution. In the current review we will highlight on the role of micro-algae in the treatment of wastewater.     

Biomass production of the mycorrhizal fungus Suillus grevillei: effect of pH and ammonium

The ability of the ectomycorrhizal fungus Suillus grevillei (Klotzsch) Singer to grow in agitated submerged culture was investigated by employing the Marx-Melin-Norkrans (MMN) medium. The operating conditions suitable for improving the biomass production were determined. Batch experimental tests were carried out in either shake flasks or a stirred tank reactor. The results showed that at least two factors strongly affected the fungal growth, namely the pH and the ammonia-nitrogen concentration in the medium. By controlling the acidity in the pH range 4-5 with a Na-citrate buffer solution and introducing the ammonia-nitrogen in a step-feed way (without exceeding a concentration of approximately 0.07 kg N/m3), the exponential growth phase continued for longer than that of the control culture (no stationary phase seemed to be reached after 17 days) and an approximately 2-fold increase of the biomass/substrate growth yield was obtained compared with the control culture.     

Quantitative analyses of ammonia-oxidizing Archaea and bacteria in the sediments of four nitrogen-rich wetlands in China

With the rapid development of ammonia-synthesizing industry, the ammonia-nitrogen pollution in wetlands acting as the sink of point and diffuse pollution has been increased dramatically. Most of ammonia-nitrogen is oxidized at least once by ammonia-oxidizing prokaryotes to complete the nitrogen cycle. Current research findings have expanded the known ammonia-oxidizing prokaryotes from the domain Bacteria to Archaea. However, in the complex wetlands environment, it remains unclear whether ammonia oxidation is exclusively or predominantly linked to Archaea or Bacteria as implied by specific high abundance. In this research, the abundance and composition of Archaea and Bacteria in sediments of four kinds of wetlands with different nitrogen concentration were investigated by using quantitative real-time polymerase chain reaction, cloning, and sequencing approaches based on amoA genes. The results indicated that AOA distributed widely in wetland sediments, and the phylogenetic tree revealed that archaeal amoA functional gene sequences from wetlands sediments cluster as two major evolutionary branches: soil/sediment and sediment/water. The bacteria functionally dominated microbial ammonia oxidation in different wetlands sediments on the basis of molecule analysis, potential nitrification rate, and soil chemistry. Moreover, the factors influencing AOA and AOB abundances with environmental indicator were also analyzed, and the results addressed the copy numbers of archaeal and bacterial amoA functional gene having the higher correlation with pH and ammonia concentration. The pH had relatively great negative impact on the abundance of AOA and AOB, while ammonia concentration showed positive impact on AOB abundance only. These findings could be fundamental to improve understanding of the importance of AOB and AOA in nitrogen and other nutrients cycle in wetland ecosystems.     

LINKING BIG: THE CONTINUING PROMISE OF EVOLUTIONARY SYNTHESIS

Synthetic science promises an unparalleled ability to find new meaning in old data, extant results, or previously unconnected methods and concepts, but pursuing synthesis can be a difficult and risky endeavor. Our experience as biologists, informaticians, and educators at the National Evolutionary Synthesis Center has affirmed that synthesis can yield major insights, but also revealed that technological hurdles, prevailing academic culture, and general confusion about the nature of synthesis can hamper its progress. By presenting our view of what synthesis is, why it will continue to drive progress in evolutionary biology, and how to remove barriers to its progress, we provide a map to a future in which all scientists can engage productively in synthetic research.     

古DNA提取技术对比及概述

从古代原始材料中提取古DNA的方法多种多样,但是古DNA的研究受限于降解严重,内源性古DNA含量低,微生物和现生人群DNA污染严重等因素的影响。能否从古代人类遗骸中成功获取可靠且足量的内源性古DNA,一直是古DNA研究领域面临的实际困难和挑战。控制污染最直接且简便的策略就是在古DNA提取阶段的有效排除,本文整理了古DNA提取常用的去除污染的方法,对比分析了每种方法表现出来的优缺点。介绍了通常使用的骨粉裂解时间,并研究了在常温环境下,不同的裂解时间对古DNA回收效率的影响,提出了常温裂解过程中最佳孵育时间。同时对常用的古DNA纯化方法及其原理和在实际应用中的表现进行了概述与讨论。本文对古DNA提取技术的概述和实践经验,为古DNA相关领域的研究提供借鉴与参考。     

Antibody purification from CHO cell supernatant using new multimodal membranes

This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG₁ following a size‐exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two‐step MMM chromatography process achieved high selectivity for capturing hIgG₁ from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG₁ could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658–665, 2017     

A modified IMAC method for the capture of target protein from mammalian cell culture harvest containing metal chelating species

Although immobilized metal affinity chromatography (IMAC) offers high capacity and protein selectivity it is not typically used commercially for the capture of native proteins from mammalian cell culture harvest. This is due mainly to the potential for low target recovery due to the presence of strong metal ion chelating species in the harvest that compete for the metal immobilized on the resin. To address this issue a buffer exchange step, such as tangential flow filtration (TFF), is added after harvest clarification and prior to IMAC to remove the interfering harvest components. The addition of a TFF step adds process time and cost and reduces target protein recovery. The elimination of the TFF might make IMAC competitive with other orthogonal methods of protein capture. In this study, we developed a modified IMAC method to allow the direct loading of clarified mammalian harvest without prior buffer exchange (direct IMAC). Although the target enzyme recovery was lower than that from standard IMAC the elimination of the buffer exchange step resulted in a 19% increase in overall enzyme recovery. The target enzyme capacity in direct IMAC was higher, in our experience, than the capacity of hydrophobic interaction (HIC) and ion-exchange (IEX) for protein capture. An economic evaluation of using direct IMAC as a capture step in manufacturing is also discussed.     

Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography

Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.