Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.     

越南篦齿苏铁传粉媒介的研究

利用排除法和引入昆虫的方法相结合,研究越南篦齿苏铁(Cycas elongata)的传粉媒介.结果表明,越南篦齿苏铁靠风媒传粉和虫媒传粉的雌株结实率分别55.3%和57.2%,自然传粉的结实率为63.5%,说明风和象鼻虫都是越南篦齿苏铁的有效传粉媒介.越南篦齿苏铁散粉高峰在白天,夜间散粉很少,在3.0 m以内风传花粉的密度较高,3.0 m之外的密度急剧下降.可见,风媒和虫媒都是越南篦齿苏铁的有效传粉途径,与泽米铁类植物为专一寄主性昆虫传粉的结论不一致.     

Association Between Chronic Exposure to Tobacco Smoke and Accumulation of Toxic Metals in Hair Among Pregnant Women

Tobacco smoke contains various toxic heavy metals that individuals are exposed to when they smoke. Despite the presence of heavy metals in tobacco smoke, the relationship between smoking and the accumulation of toxic metals in pregnant women after long-term exposure remains under discussion. We examined the association between long-term exposure to tobacco smoke and the accumulation of toxic metals in the hair of female participants. Our study recruited 252 women from the Shanxi and Hebei provinces of Northern China; these participants were self-reported non-active smokers, and had previously delivered healthy babies without birth defects. Scalp hair was collected and analyzed for nicotine and cotinine and five potentially toxic metals (specifically, silver, chromium, cadmium, mercury, and lead). Our results showed significant positive correlations between cotinine and four metals, including silver (r?=?0.369, p?<?0.001), cadmium (r?=?0.185, p?<?0.01), mercury (r?=?0.161, p?<?0.05), and lead (r?=?0.243, p?<?0.001). Significant positive correlations were also found between nicotine and three metals—specifically silver (r?=?0.331, p?<?0.001), cadmium (r?=?0.176, p?<?0.01), and lead (r?=?0.316, p?<?0.001). A logistic regression model showed significant associations between cotinine and potentially toxic metals including mercury, silver, and lead (with or without adjusting for potential confounders). We thus conclude that long-term passive smoking could potentially increase the exposure level of toxic metals including lead, silver, and mercury in our study, which are especially harmful for pregnant women and their unborn fetus.     

基因前定点敲入FLAG标签表达的FLAG-SND1蛋白参与胞内应激颗粒的形成

CRISPR/ Cas9 (clustered regular interspaced short palindromic repeats-Cas9 nuclease) gene editing technology is an emerging technology for gene-specific knock-out and knock-in in recent years. In this experiment, the CRISPR/ Cas9 gene editing system was used to insert 3×FLAG tags into the front of SND1 gene in HeLa cells, so that the endogenous expression of SND1 protein in cells was equipped with 3×FLAG tags, and the localization of SND1 with stress granules and processing bodies was observed. A sgRNA near the start codon ATG of the SND1 gene was designed, and a recombinant eukaryotic expression plasmid was constructed using px459 as an expression vector. A sequence containing 3×FLAG and a 150 bp homologous arm upstream and downstream of the position to be inserted was designed, and the recombinant plasmid was synthesized by the company. Two plasmids were co-transfected into HeLa cells, and positive cells were screened by puromycin. Western blotting indicated that the cells expressed the 3×FLAG-SND1 fusion protein. Genomic DNA was extracted and sequenced. Cell cycle and apoptosis were detected by flow cytometry after sequencing and the stable strain was obtained without error. It was found that there was no significant difference compared with WT cells. At the same time, the treatment with 0. 5 mmol / L sodium arsenite resulted in oxidative stress in cells, increased phosphorylation of eIF2α protein, and the presence of stress particles in the cytoplasm. There was co-localization between SND1 and stress particle marker protein TIAR, but no co-localization with processed body protein DCP1α. © 2019 The authors.     

黄麻吸附重金属Cr(Ⅵ)专用种质筛选

首次以国内外23个代表性黄麻种质为研究对象,用主茎嫩梢和一、二级分枝嫩梢制成天然重金属吸附剂,测定其对溶液中Cr(Ⅵ)离子的去除率,并对与黄麻吸附能力及产量密切相关的功能性状:生育期动态、株高、分枝习性、各级分枝嫩梢的产量进行调查、方差分析和相关性分析。结果表明,不同黄麻种质制成的生物吸附剂对Cr(Ⅵ)的吸附能力不同,去除率在85.25%~96.88%之间;不同级次分枝嫩梢的产量及对Cr(Ⅵ)的去除率不同;种质间除出苗速度外所有调查性状均存在较大差异;去除率与株高呈极显著负相关(P<0.01)。从产量和去除率方面综合考虑,J001和J011 2个种质表现优良,适宜作为吸附重金属专用品种推广种植。     

内蒙古鄂尔多斯乌兰木伦遗址MIS3阶段的植被与环境

鄂尔多斯高原拥有我国最早发现的水洞沟和萨拉乌苏旧石器遗址,是系统研究东亚现代人演化及动因的重要地区。乌兰木伦遗址位于鄂尔多斯康巴什新区乌兰木伦河岸,发掘出大量石制品、动物化石和炭屑,遗存埋藏的14C年代在41.4~33.1 cal ka BP之间,属MIS 3阶段中期。花粉和木炭化石记录显示,早期为灌丛-草原,晚期为典型草原植被,气候温凉偏干,较现今相对温暖湿润,胡颓子属和霸王属等小乔木和灌木作为先民使用的薪材。MIS 3阶段相对暖湿气候有利于人类的繁衍、扩散和交流,可能是我国北方地区旧石器时代晚期出现大量人类活动的重要原因。     

Genetic differences within and between species of deep-sea crabs (chaceon) from the North Atlantic Ocean

The deep-sea red crab Chaceon quinquedens is a commercially important crustacean on the Atlantic continental shelf and slope of North America. To assess genetic subdivision in C. quinquedens, we examined the nucleotide sequence of the mitochondrial 16S rDNA gene and the internal transcribed spacers (ITS) of the nuclear ribosomal repeat in samples from southern New England and the Gulf of Mexico. We compared those data to sequences from two congeners, a sympatric species from the Florida coast, C. fenneri, and an allopatric eastern Atlantic species, C. affinis. The 16S rDNA data consisted of 379 aligned nucleotides obtained from 37 individuals. The greatest genetic difference among geographical groups or nominal species was between C. quinquedens from southern New England and C. quinquedens from the Gulf of Mexico. Haplotypes from these two groups had a minimum of 10 differences. All 11 C. fenneri samples matched the most common haplotype found in C. quinquedens from the Gulf of Mexico, and this haplotype was not detected in C. quinquedens from southern New England. The three haplotypes of C. affinis were unique to that recognized species, but those haplotypes differed only slightly from those of C. fenneri and C. quinquedens from the Gulf of Mexico. Based on 16S rDNA and ITS data, genetic differences between C. quinquedens from southern New England and the Gulf of Mexico are large enough to conclude that these are different fishery stocks. Our results also indicate that the designation of morphological species within the commercially important genus Chaceon is not congruent with evolutionary history. The genetic similarity of C. affinis from the eastern Atlantic and C. quinquedens from the Gulf of Mexico suggests these trans-Atlantic taxa share a more recent common history than the two populations of "C. quinquedens" that we examined.