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Coexistence and competition of sulfate-reducing and methanogenic populations in an anaerobic hexadecane-degrading culture
No full textBackground: Over three-fifths of the world's known crude oil cannot be recovered using state-of-the-art techniques, but microbial conversion of petroleum hydrocarbons trapped in oil reservoirs to methane is one promising path to increase the recovery of fossil fuels. The process requires cooperation between syntrophic bacteria and methanogenic archaea, which can be affected by sulfate-reducing prokaryotes (SRPs). However, the effects of sulfate on hydrocarbon degradation and methane production remain elusive, and the microbial communities involved are not well understood. Results: In this study, a methanogenic hexadecane-degrading enrichment culture was treated with six different concentrations of sulfate ranging from 0.5 to 25 mM. Methane production and maximum specific methane production rate gradually decreased to 44 and 56% with sulfate concentrations up to 25 mM, respectively. There was a significant positive linear correlation between the sulfate reduction/methane production ratio and initial sulfate concentration, which remained constant during the methane production phase. The apparent methanogenesis fractionation factor (a(app)) gradually increased during the methane production phase in each treatment, the aapp for the treatments with lower sulfate (0.5-4 mM) eventually plateaued at similar to 1.047, but that for the treatment with 10-25 mM sulfate only reached similar to 1.029. The relative abundance levels of Smithella and Methanoculleus increased almost in parallel with the increasing sulfate concentrations. Furthermore, the predominant sulfate reducer communities shifted from Desulfo-bacteraceae in the low-sulfate cultures to Desulfomonile in the high-sulfate cultures. Conclusion: The distribution of hexadecane carbon between methane-producing and sulfate-reducing populations is dependent on the initial sulfate added, and not affected during the methane production period. There was a relative increase in hydrogenotrophic methanogenesis activity over time for all sulfate treatments, whereas the total activity was inhibited by sulfate addition. Both Smithella and Methanoculleus, the key alkane degraders and methane producers, can adapt to sulfate stress. Specifically, different SRP populations were stimulated at various sulfate concentrations. These results could help to evaluate interactions between sulfate-reducing and methanogenic populations during anaerobic hydrocarbon degradation in oil reservoirs
Host species shapes the co-occurrence patterns rather than diversity of stomach bacterial communities in pikas
No full textThe mammalian stomach acts as an important barrier against ingested pathogens into the entire gastrointestinal tract, thereby playing a key role in host health. However, little is known regarding to the stomach microbial compositions in wild mammals and the factors that may influence the community compositions. Using high-throughput sequencing of the 16S rRNA gene, we characterized the stomach bacterial community compositions, diversity, and interactions in two common pika (Ochotona sp.) species in China, including Plateau pikas (Ochotona curzoniae) and Daurian pikas (Ochotona daurica) living in the Qinghai-Tibet Plateau and the Inner Mongolia Grassland, respectively. The bacterial communities can be divided into two distinct phylogenetic clusters. The most dominant bacteria in cluster I were unclassified bacteria. Cluster II was more diverse, predominantly consisting of Bacteroidetes, followed by unclassified bacteria, Firmicutes and Proteobacteria. Three dominant genera (Prevotella, Oscillospira, and Ruminococcus) in pika stomachs were significantly enriched in cluster II. In addition, seasons, host species, and sampling sites as well as body weight and sex had no significant impacts on the composition and diversity of pika stomach communities. Interestingly, Plateau pikas harbored a more complex bacterial network than Daurian pikas, and these two pika species showed different co-occurrence patterns. These results suggested that the pika stomach harbors a diverse but relatively stable and unique bacterial community, which is independent on host (host species, body weight, and sex) and measured environmental factors (sampling sites and seasons). Interestingly, host species shapes the microbial interactions rather than diversity of stomach bacterial communities in pikas, reflecting specific niche adaptation of stomach bacterial communities through species interactions
Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System
No full textClostridium saccharoperbutylacetonicum N1-4 is well known as a hyperbutanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR- associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk, two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P (J23119), we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii. Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum. Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production
Stereo-complementary bioreduction of saturated N-heterocyclic ketones
No full textThe asymmetric bioreduction of several saturated N-heterocyclic ketones is demonstrated in a stereo complementary fashion using the ketoreductases READH and ChKRED20 for the production of (S)- and (R)-alcohols, respectively. The reaction accepts substrates with a five-, six- or seven-membered ring, and exhibits excellent stereoselectivity when using 2-propanol as both the ultimate reducing agent and cosolvent, achieve > 99% ee in the majority of cases for both enantiomers. (C) 2017 Elsevier Ltd. All rights reserved
纳米TiO_2的健康风险与环境毒性研究进展
No full text纳米TiO2因其特殊的理化特性以及低廉生产成本,被广泛地应用于造纸、化妆品、纺织、农业生产和环境保护等行业。随着nTiO_2被广泛使用和大量排放,其健康风险和生态毒理效应逐渐引起人们的关注。本论文综述了近些年的研究进展,探讨了nTiO_2的健康风险和生态毒理效应,总结了nTiO_2的生物毒性机制,呼吁加强相关研究和环境保护,为nTiO_2的安全使用做出贡献
Fine roots branch orders of Abies faxoniana respond differentially to warming in a subalpine coniferous forest ecosystem
No full textRoot is an important plant organ and has high heterogeneity. Global warming could change root and affect belowground ecological processes. There is little information on how fine roots branch orders responds to global change. This study examined the growth, morphological and physiological responses of fine roots of a subalpine coniferous species to warming. We investigated biomass, average diameter, specific root length (SRL), triphenyltetrazolium chloride (TTC) reducing capacity, carbon (C), total non-structural carbon (TNC) and fractions of the primal five branch order roots of Abies faxoniana in April, August, October and December. The decrease in total fine roots biomass after a growing season was significantly greater under warming treatment compared to control, suggesting that warming could accelerate the carbon input from root to soil, but the increment depended on tree species. Warming did not affect average diameter and SRL. Responses of biomass, TTC reducing capacity, C, TNC and fractions to warming significantly differed with root order and month. Significant warming effects were only observed in C and starch concentration of the first order and also TNC and soluble sugar concentration of the first three orders. The results indicated that the lower order roots (the first three orders) were more sensitive to warming, probably because they had more frequent, intense interactions with soil and low defense capability. Thus, global warming may dramatically alter root functions such as nutrients and water uptake as well as the cycle of C and nutrients at the whole subalpine coniferous forest ecosystem
硫氧化细菌的筛选及其异养生长和自养脱硫实验研究
No full text生物脱硫是一门新兴的环保技术，其应用前景良好，具有不需要催化剂、无二次污染、处理成本低等优点。该技术的核心在于硫氧化细菌。对于脱硫工程，需要选择能够耐受较高硫化物浓度、硫化物去除效率高、可较快获得高密度菌体的细菌。本研究从天然气脱硫厂污泥、污水处理厂好氧污泥、鸡粪堆肥场、硫铁矿、温泉中采样，通过富集驯化，分离复筛得到硫氧化细菌，对菌株进行了分子生物学和生理生化鉴定，考察了菌株的异养生长和自养脱硫性能，主要内容如下：（1）在驯化期间，添加500 ml含硫（S2-）为200mg/L的溶液驯化1h后硫化物去除率达到97%以上，且伴随着一定浓度的单质硫产生；对富集前后样品进行高通量测序分析发现，微生物群落结构发生了明显的变化，样品中的微生物群落多样性降低，优势菌属为盐状硫杆菌属（Halothiobacillus）、硫杆菌属（Thiobacillus）、假单胞菌属（Pseudomonas）、鞘氨醇杆菌属（Sphingobacterium）和副球菌属（Paracoccus），丰度分别达到21.03%、15.9%、4.03%、6.7%和4.8%。（2）从富集驯化液中初筛分离得到12株硫氧化细菌，并采用硫化物（S2-）浓度为650mg/L的硫化物溶液进行复筛，获得5株脱硫性能较好的菌株。5株菌在4.5h内硫化物去除率均达到95%以上，其中去除效率最高的是菌株DS-11，硫化物去除率达到97.19%。各菌脱硫液中都含有较高浓度的单质硫，其中，菌株DS-8的单质硫浓度最高，第5.5h时的单质硫浓度和转化率达到最大，分别为322.49mg/L和49.61%。菌株DS-2、DS-7、DS-8、DS-10、DS-11的脱硫产物经扫描电镜-能谱分析联用仪(SEM-EDS)分析表明，脱硫产物中硫元素为主要成分，重量百分比为47.52%~77.87%，原子量百分比为45.38%~59.48%。（3）分子生物学鉴定结果表明，菌株DS-2和DS-10分别与菌株Paracoccus denitrificans和Paracoccus.pantotrophus同源性为100%，因此鉴定这两株菌为Paracoccus denitrificans和Paracoccus.pantotrophus；菌株DS-7、DS-8、DS-11分别为鞘氨醇杆菌属（Sphingobacterium）、假单胞菌属（Pseudomonas）、副球菌属（Paracoccus），对这3株菌进行生化鉴定，其鉴定结果与分子生物学结果相结合发现，DS-7、DS-8、DS-11分别为Sphingobacterium spiritivorum、Pseudomonas monteilii、Paracoccus versutus；经生长实验表明，5株细菌都属于兼性营养型细菌。在自养培养基中生长28h后其OD值分别从0.103、0.135、0.112、0.114、0.121增加到0.213、0.239、0.199、0.213、0.193；在异养培养基中在生长28h后其OD值分别从0.118、0.123、0.105、0.101、0.099增加到1.059、1.358、1.325、1.244、1.130。5株菌在异养培养基中生长速率显著高于在无机自养培养基中的生长速率。（4）对5株菌株进行异养生长-自养脱硫实验，经过28h的异养培养后，菌体浓度OD值达到1.17以上，在随后的自养脱硫培养过程中，1h时的硫化物去除率为30.71%~38.50%；而复筛实验中自养生长(28h)-自养脱硫（1h）的脱硫效率为16.23%~34.42%。在整个异养生长-自养脱硫自过程中，最大单质硫转化率为60.74%~84.28%；而复筛实验中自养生长-自养脱硫的最大单质硫转化率为28.38%~49.61%。可见，异养生长-自养脱硫的方式能够加速脱硫的启动并提高单质硫转化率
贡嘎山东坡森林土壤好氧甲烷氧化细菌群落空间分异及其潜在驱动机制研究
No full text甲烷是仅次于CO2的第二大温室气体，森林表层土壤中由好氧甲烷氧化细菌（Aerobic methane oxidizing bacteria, MOBs）驱动的甲烷好氧氧化作用是大气甲烷重要的汇，在碳循环和减缓全球变暖方面扮演着重要的作用。研究森林土壤中好氧甲烷氧化细菌群落的空间分异及其潜在驱动机制有助于更好地理解好氧甲烷氧化细菌群落对环境变化的响应机制。在山地森林生态系统海拔梯度上，土壤理化性质、植被带谱和气候条件呈现明显的梯度分布。连续的环境、气候和植被梯度使得山地森林生态系统成为研究微生物群落对环境变化响应的理想自然平台。本研究以贡嘎山东坡海拔1800 m到4100 m区域森林土壤为研究对象，通过对甲烷氧化单加氧酶β亚基基因（pmoA）进行Miseq测序，结合生物信息学和多元生态统计学手段对贡嘎山东坡森林土壤中好氧甲烷氧化细菌群落空间分异及其潜在的驱动因素进行了研究，得出如下基本结论：（1） 为了从pmoA基因推断氨基酸水平来评估好氧甲烷氧化细菌群落结构，首先利用Python在前人的研究基础上构建了pmoA推断氨基酸序列比对和物种注释生物信息学数据库。结合现有的用于16/18S rRNA基因和ITS序列分析的生物信息工具和本研究实现的Python程序，搭建了用于功能基因扩增子测序推断氨基酸水平的生物信息学处理流程（Functional Gene Analysis Toolkit，FGAT），经测试后用于好氧甲烷氧化细菌群落结构和多样性的评估。（2）在连续的海拔和植被梯度上，好氧甲烷氧化细菌α多样性和群落结构都出现明显的空间分异；结果显示，虽然研究尺度较小，森林土壤中好氧甲烷氧化细菌群落也和土壤总细菌一样有着明显的空间分异。（3） 在连续海拔和植被梯度上，好氧甲烷氧化细菌相对丰度组成具有较大的空间分异；在低海拔1800 m到2800 m区域（常绿和落叶阔叶林与针阔叶混交林），好氧甲烷氧化细菌群落组成较为相似，而在高海拔3000 m到4100 m区域（暗针叶林和灌丛草甸），好氧甲烷氧化细菌群落组成具有一定的差异。AOB-like和AOB-rel在整个海拔梯度上都具有较高的丰度，相比高海拔区域而言，低海拔相对丰度更高。USC-α和RA21主要出现在3000 m到4100 m区域，其它好氧甲烷氧化细菌在整个研究区域都具有较低的相对丰度。Spearman秩相关分析表明，不同的好氧甲烷氧化细菌对环境变化的响应可能不同，比如Crenothrix、USC-γ、AOB-rel、AOB-like和TUSC的相对丰度与大多数环境参数呈现出正相关关系，而USC-α和RA21的相对丰度与大多数环境参数具有较高的负相关关系。（4） 好氧甲烷氧化细菌α多样性在连续的海拔梯度上呈现“单峰”分布模式；在针阔叶混交林和暗针叶林土壤中α多样性较高，而在常绿和落叶阔叶林与灌丛草甸土壤中α多样性较低。Spearman秩相关分析表明，α多样性的空间分异和多种环境参数存在相关性，其中电导率表现出较强的预测能力。进一步分析表明，好氧甲烷氧化细菌α多样性在贡嘎山东坡森林土壤中的空间变化可能是多种环境变化的综合结果，其效应表现在土壤综合指标电导率上。因此，在贡嘎山东坡1800 m到4100 m区域森林土壤中，土壤电导率可作为好氧甲烷氧化细菌α多样性变化的指示性参数。（5） 对贡嘎山东坡森林土壤好氧甲烷氧化细菌β多样性进行的研究发现，在连续海拔梯度上2200 m到4100 m区域，β呈现总体上升的趋势。在1800 m和 2000 m海拔具有较高的β多样性。暗针叶林和灌丛草甸土壤中好氧甲烷氧化细菌β多样性较高，而常绿和落叶阔叶林与针阔叶混交林土壤中β多样性相对较低。高低海拔好氧甲烷氧化细菌群落结构具有较大的差异，低海拔1800 m到2800 m好氧甲烷氧化细菌群落结构较为相似，而高海拔3000 m到4100 m区域好氧甲烷氧化细菌群落结构差异较大。基于距离的冗余分析表明，多种因素和贡嘎山东坡森林土壤好氧甲烷氧化细菌β多样性相关，其中降水、土壤pH、铵氮和温度（包括土壤温度和年均气温）相关性较高。除此之外，碳氮比、土壤总碳、土壤总氮、硝氮和土壤电导率都与好氧甲烷氧化细菌群落结构的空间分异具有显著的相关关系，表明好氧甲烷氧化细菌的群落结构空间分异可能受多种因素制约。进一步分析表明，低海拔区域好氧甲烷氧化细菌群落受到环境的束缚作用相对较大，高低海拔群落结构的差异可能和环境压力大小的差异有关。（6） Null模型分析表明，高低海拔群落构建都是确定性过程。与高海拔相比，低海拔好氧甲烷氧化细菌群落受到的确定性作用更大。变差分解分析表明，低海拔好氧甲烷氧化细菌群落构建的主要驱动力是环境过滤，而高海拔较高的β多样性可能源于扩散限制的作用。丰度差异分析也表明，和高海拔相比，低海拔贫化的OTU要比富集的OTU更多，暗示低海拔可能是由于环境过滤的作用导致部分taxa丢失或者丰度降低，从而使得群落高度相似；研究结果暗示，高低海拔好氧甲烷氧化细菌群落结构出现明显的空间分异可能源于不同的多样性维持机制
单室MEC处理黄水产甲烷及其微生物群落结构初步研究
No full text随着社会的不断进步，日益增大的能源消耗与大量废弃物排放造成的环境污染之间的关系正引起人们的关注。生物能源化技术通过降解废弃物而获取发展所需的能源物质，不仅有利于保护环境，还能获取其中的能源，可在一定程度上降低对化石燃料的依赖性，符合社会与经济的可持续发展理念，具有十分可观的发展前景。在有机废水的能源化途径中，生物电化学系统（bioelectrochemical systems, BESs）利用附着在电极上的电活性微生物传递电子而催化阳极上的氧化反应和（或）阴极上的还原反应，是一门环境友好型、能源集约型的处理废弃污染物并回收能源的新兴技术。其中的微生物电解池（Microbial electrolysis cell, MEC）可通过施加一定的外电压并以具有电子传递功能的微生物为催化剂，驱动反应体系中的非自发反应（ΔGθ> 0）的发生或加快自发反应的速率，在污染治理与能源回收领域具有很大的发展潜力。 黄水又名黄浆水，是酒窖内酒醅向下层渗漏而形成的黄色淋浆水，是浓香型白酒酿造过程的一种副产物。黄水pH值低，其成分复杂且含量不定，属于高浓度有机废水，是白酒酿造过程中的主要污染源之一。每生产1 000 kg大曲酒可产生300-400 kg黄水，一个大型酿酒企业每日排放的黄水可达100 t左右，污染环境的同时也造成了资源的浪费。综合利用黄水将是解决其污染问题的有效途径。目前，对黄水资源的再利用研究主要包括利用黄水作发酵培养基以及生物反硝化过程的补充碳源、培养人工窖泥、生产强化酒曲、食品防腐剂等产品等。但由于黄水产量大，上述处理并不能完全将其消纳，其资源化利用途径有待进一步拓展。 为进一步挖掘酿酒副产物黄水的资源化利用空间，本实验通过构建单室微生物电解池处理黄水并实现能源回收。实验以黄水为主要基质，以不加电压的传统厌氧消化（AD）为对照，考察了不同外加电压、黄水初始浓度、反应体系磷酸盐缓冲液（PBS）浓度以及利用生活污水作为黄水稀释液对黄水处理过程（以36h为一个处理周期）中化学需氧量（Chemical Oxygen Demand, COD）去除、有机酸降解、甲烷产生及能量平衡等的影响。结果表明，当外加0.8 V电压时，MEC中COD去除率达到94.90 ± 0.70 %，较对照组（AD）的82.00 ± 0.70%增加了12.90 ± 0.74%。同时，COD去除负荷达5.27 ± 0.51 kg m-3 d-1，是AD（3.45 ± 0.09 kg m-3 d-1）的1.53倍。对反应中甲烷产生速率和有机酸组分变化分析表明，当外加0.6 V电压时，MEC中的甲烷产生速率为1 818.54 ± 145.77 mL L-1 d-1，比AD（1 014.88 ± 121.44 mL L-1 d-1）增加了78.19%；当外加电压为0.8 V时，MEC中的乙醇去除速率（102.37 ± 14.65 mg L-1 h-1）是AD（57.31 ± 10.45 mg L-1 h-1）的1.79倍；AD的最高丙酸浓度高达1 436.10 ± 84.42 mg L-1，而外加1.0 V电压的MEC，其最高丙酸浓度较之降低了590.53 ± 7.73 mg L-1。当反应周期结束时，AD中残留的乙酸和丙酸浓度分别是MEC中（外加0.8 V电压）的93.57和5.31倍。最后，反应器能量平衡分析的结果表明，当外加电压为1.0 V时，其能量产生与净能量产生分别达到了3.93 ± 0.48、3.80 ± 0.48 kWh kg-COD-1，较AD分别增加了1.01± 0.12、0.88± 0.12kWh kg-COD-1，且MEC均获得了较AD更多的净能量。该MEC可有效促进黄水处理效率并回收甲烷且其最佳外加电压为0.8 V。 黄水初始浓度对反应器性能影响的研究结果显示，当外加0.8V电压时，反应基质的COD去除率随初始黄水浓度的升高呈先上升后下降的趋势。当初始黄水浓度为4%时，其MEC的COD去除率（94.90±0.70%）最高；当反应周期结束时，其COD残余量（426±80.73mg L-1）最低。当浓度为5%时，其甲烷产生速率最大（2493.51±124mL L-1 day-1），分别是3%和4%的2.04和1.42倍。不同PBS浓度对黄水降解过程分析以及能量平衡分析的实验结果显示，当使用纯水（PBS浓度为0mM）做稀释液时，其COD去除率达到了94.83±1.10%，甲烷产生速率（1 985.69±98.86mL L-1 day-1）也较其他更高，其净能量产生为4.24 ± 0.36 kWh kg-COD-1。直接降解未额外添加PBS溶液稀释后的黄水废水并产甲烷回收能量是可行的。 以生活污水为黄水的稀释液进行黄水降解的实际应用可行性验证实验的结果表明，MEC的COD去除率为93.21±0.9%，较AD高23.09±2.46%；MEC的COD去除负荷可达4.83±0.31kg m-3 day-1，比AD高1.19±0.23 kg m-3 day-1；MEC与AD的甲烷产生速率分别为2115.08±66.51 mL L -1day-1、1586.83±36.81 mL L -1 day-1。有机酸分析表明，MEC可显著促进乙酸、丙酸、丁酸的降解。MEC较传统厌氧发酵具有更强的底物适应性和系统稳定性。综上：以生活污水为直接稀释液的黄水底物可以被单室不锈钢MEC有效处理并回收甲烷，在实际应用方面具有可观的潜力。 本实验对各条件下运行的MEC与AD的三个不同空间位置的微生物群落结构进行了分析。结果显示，MEC阳极碳毡中可富集地杆菌属（Geobacter sp.）和脱硫弧菌属（Desulfovibrio sp.）等具有电子传递功能的微生物；反应器中的碳毡与不锈钢内壁上的产甲烷菌均是以噬氢产甲烷菌（Hydrogenotrophic methanogens）为主，其中又以甲烷粒菌属（Methanocorpusculum.sp）为主，且相对含量均高于悬浮液。MEC悬浮液中主要含有拟杆菌属（Bacteroides sp.）和链球菌属（Streptococcus sp.）等具有降解大分子物质产酸功能的菌群。AD中，各空间位置的产甲烷菌相对含量的差异不如MEC的明显，初步认为，外加电压将更加利于反应系统内菌群结构的分布与功能利用
