Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log₁₀ reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.     

Oxidative assimilation treatment of a nitrogen-deficient toxic waste

Batch growth tests were performed under both replicating and nonproliferating (no nitrogen source in medium) conditions with acclimated heterogenous populations that utilized phenol as a sole source of carbon and energy. It was shown that the acclimated populations could efficiently remove the toxic waste component phenol under nonproliferating conditions by utilizing an oxidative assimilation mechanism. The phenol was assimilated and converted into nonnitrogenous storage products. During the assimilation process, the cells had a tendency to excrete some product (nonsubstrate) chemical oxygen demand (COD). Bench-scale oxidative assimilation units were operated by sequentially feeding a carbon source (phenol) and nitrogen source (ammonium sulfate) to heterogeneous populations. This demonstrated that, subsequent to the addition of the nitrogen source to the medium, the cells utilized the stored carbon for replication. Four of these units were operated at different phenol COD-to-ammonia-nitrogen ratios of 10:1, 20:1, 40:1, and 50:1. All of these units demonstrated excellent removal of phenol using an oxidative assimilation mechanism. These results suggested the feasibility of utilizing a continuous flow oxidative assimilation process for the treatment of nitrogen-deficient phenolic wastes. This process would be advantageous over conventional treatment processes in that it would realize a savings in chemical costs (ammonia as nitrogen source) and prevent leakage of excess ammonia from the system.     

Mechanism of aerobic transformation of carbon tetrachloride by poplar cells

The biochemical mechanism of carbon tetrachloride transformation by poplarcells was investigated using an axenic poplar cell culture.After one-day incubations of poplar cells under aerobic conditions, about 1.5% of dosedcarbon tetrachloride was transformed to carbon dioxide, about 0.001% to chloroform andabout 3% of the carbon was bound to insoluble poplar cellular materials. The productionof carbon dioxide increased under aerobic conditions while the formation of chloroformand cell binding of carbon tetrachloride-carbon was enhanced under anaerobic conditions.Both carbon dioxide production and cell binding were significantly inhibitedby a general inhibitor of cytochrome P-450 activity (carbon monoxide) and by specific P-450 2E1 inhibitors(chlorzoxazone, isoniazid, 4-methylpyrazole and 1-phenylimidazole). However, no inhibitory effects were observed when the cells were incubated in thepresence of lignin peroxidase inhibitors (NaVO₃ and 3-amino-1,2,4-triazole). These resultssuggest that an enzyme similar to mammalian cytochrome P450-2E1 is involved inthe metabolism of carbon tetrachloride by poplar cells. This study demonstratesan environmental biodegradative process for carbon tetrachloridethat operates under aerobic conditions.     

Molecular dynamics simulation of primary detonation process of TATB crystal under shock loading

Molecular dynamics simulations were performed to gain fundamental insights into the mechanisms for the primary detonation process of 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) under shock wave loading using self-consistent charge density-functional tight binding(SCC-DFTB) calculations combined with the multiscale shock technique (MSST). The primary process starts with shock loading and ends with the formation of dynamically stable heterocyclic clusters, which could inhibit the reactivity of TATB. The results show that the initial step of shocked TATB decomposition is the N–O bond cleavage; then carbon rings aggregate and connect by N atoms to form clusters; after the carbon rings open, heterocyclic clusters with nitrogen are formed, and persist throughout the simulation. This is a new mechanism for the primary processes of shocked TATB and this initiation mechanism is independent of the initial shock speeds.     

大尺度森林碳循环过程模拟模型综述

森林生态系统碳循环是全球陆地生态系统碳循环的重要组成部分,而碳循环模型已经成为研究森林碳循环的必要手段。森林碳循环模型可以分为统计模型和过程模型,其中过程模型以其完整的理论框架、严谨的结构分析和清晰的过程机理,逐渐占据了主导地位。从地球化学过程模型、陆面物理过程模型和生物过程模型等3个方面综述区域尺度到全球尺度(本文称为大尺度)森林碳循环过程模型研究进展,论述了各类模型的主要特征、优缺点以及应用现状,探讨了森林碳循环模拟研究中存在的问题,并讨论了森林碳循环过程模型的主流研究方向。可为不同空间尺度下森林生态系统碳循环模拟模型的选择提供参考,以及为森林碳循环研究提供借鉴。     

Bisource carbon dioxide feeding of the photosynthetic process of microalgae autotrophic cultivation

The mechanism of carbon dioxide saturation of alkaline nutrient medium for the purpose of autotrophic cultivation of microalgae is studied. Carbon dioxide desorption from aqueous solutions of sodium hydrocarbonate is investigated and the influence of chemical equilibria in the solutions on the cultivation of carbon dioxide supply is discussed. It is pointed out that these solutions can be considered to be carriers of the assimilable carbon source for the microalgae cultivation, most efficient at pH 8.5. A comparative estimation of the carbon dioxide and the hydrocarbonic ion as assimilable components of the chemical equilibria under study was done, leading to the conclusion that the more probable component in an alkaline medium autotrophic microalgae cultivation is carbon dioxide. A scheme of bisource feeding of the photosynthetic process, with carbon dioxide for the autotrophic microalgae cultivation in a alkaline nutrient medium, providing a stable and economical cultivation process, is proposed.     

基于稳定同位素的SPAC水碳拆分及耦合研究进展

土壤-植被-大气连续体(SPAC)是陆地水文学、生态学和全球变化领域的重要研究对象,其水碳循环过程及耦合机制是前沿性问题.稳定同位素技术示踪、整合和指示的特征有助于评估分析生态系统固碳和耗水情况.本文在简述稳定同位素应用原理和技术的基础上,重点阐释了基于稳定同位素光学技术的SPAC系统水碳交换研究进展,包括:在净碳通量中拆分光合与呼吸量,在蒸散通量中拆分蒸腾与蒸发量,以及在系统尺度上的水碳耦合研究.新兴的技术和方法实现了生态系统尺度上长期高频的同位素观测,但在测量精准度、生态系统呼吸拆分、非稳态模型适应性、尺度转换和水碳耦合机制等方面存在挑战.本文探讨了现有主要研究成果、局限性以及未来研究展望,以期对稳定同位素生态学领域的新研究和技术发展有所帮助.     

Influence of dilution rate on the acidogenic phase products distribution during two-phase lactose anaerobiosis

Acidogenic fermentation of lactose was carried out in a continuous stirred reactor with a mixed anaerobic culture. From the variation of the reactor products with pH and dilution rate two possible carbon flow schemes were proposed for the reaction. In both schemes the carbon flow from pyruvate to butyrate and lactate was assumed to occur in parallel. A change in gas composition and in product concentrations at dilution rates between 0.1 and 0.15 h(-1) for pH levels between 4.5 and 6.0 was ascribed to a shift in microbial population. To clarify the mechanism radiotracer tests were made using [U-(14)C]-butyrate, [2-(14)C]-propionate and [U-(14)C]-lactate to determine the path of carbon flow during acidogenesis of lactose using a mixed culture. At a dilution rate between 0.1 and 0.15 h(-1) and pH from 4.5 to 6.0 a rise in the lactate concentration in the product was shown to be due to a microbial population shift which disabled the conversion of lactate to other intermediary metabolites. It was also found that the flow of carbon from pyruvate to butyrate and lactate occurred by parallel pathways. Also, in the presence of hydrogen reducing methanogens, lactate was almost completely converted to acetate and not propionate. Butyrate was found to be converted to acetate at a slow rate as long as hydrogen reducing methanogens were present. The role played by propionibacteria in this lactose acidogenic eocosystem was minor. From the carbon flow model it can be concluded that lactate is the most suitable marker for optimizing an acidogenic reactor in a two-phase biomethanation process.     

Carbonization of wood and nanostructures formed from the cell wall

In this paper, formation of unique carbon nanostructures via carbonization of wood in a step-wise process is reviewed. The mechanism described for the production of carbon nanotubes improves our understanding of a historic mystery related to the production of Damascus steel.     

森林生态系统碳水关系及其影响因子研究进展

森林生态系统的碳水关系是陆地生态系统碳循环和水循环相互耦合的作用过程,对研究森林碳汇、森林生态水文过程和全球变化响应有重要意义.在全球变化背景下,森林生态系统碳水关系已成为生态水文学领域中的一个热点科学问题.本文在总结国际上森林碳汇研究的基础上,概述了森林碳水关系的过程机制,包括森林水分利用效率、不同尺度上的碳水关系、尺度推绎和碳水关系的模拟研究方面的进展;总结了影响森林碳水关系的因子和研究进展,包括水分条件、CO₂浓度升高、增温、氮沉降、臭氧浓度变化、辐射因子和海拔梯度因子对森林碳水关系的影响;最后对已有研究存在的问题进行了初步分析,并对未来研究内容和方向进行了展望.     

Simulation of terrestrial carbon equilibrium state by using a detachable carbon cycle scheme

Determining the equilibrium state of terrestrial carbon is a prerequisite for scientific analysis on the carbon cycle. However, the mechanism through which the carbon cycle reaches the equilibrium state remains unclear. Moreover, the carbon cycle in most of the short–term field experiments rarely reaches the equilibrium state. In this study, a detachable carbon cycle (DCC) model was proposed to simulate the equilibrium state of each carbon pool. The model was established based on a pool–and–flux scheme and contained 14 carbon pools, or carbon flow processes, each process could be detached from the main model and evaluated as an independent component. The environmental scalar algorithms of the Integrated Terrestrial Ecosystem Carbon budget model (InTEC) and Community Atmosphere Biosphere Land Exchange (CABLE) were incorporated in the DCC model. Four situations were compared using the two environmental scalar algorithms and model structure (9 vs. 14 carbon pools). Furthermore, the size and turnover time of each carbon pool were analyzed at the equilibrium state. A sensitivity analysis was then conducted to investigate the responses of carbon density and equilibrium time to 12 key parameters of the model. Results indicated that the combination of the CABLE environmental scalar algorithm and 14 pools exhibited improved performance on carbon storage simulation than that of the other combinations, and the effect of the environmental scalar algorithm was considerably larger than that of the carbon pool number. Sensitivity analysis indicated that the carbon density of grassland and cropland was more vulnerable and sensitive to key parameters of the model than that of the other biomes. This study elucidates influencing factors and underlying control mechanisms in the carbon accumulation, and provides a framework for quantitative analysis of each component of the carbon cycle.     

Hydrolyzed molasses as an external carbon source in biological nitrogen removal

Hydrolyzed molasses was evaluated as an alternative carbon source in a biological nitrogen removal process. To increase biodegradability, molasses was acidified before thermohydrolyzation. The denitrification rate was 2.9-3.6 mg N/g VSSh with hydrolyzed molasses, in which the percentage of readily biodegradable substrate was 47.5%. To consider the hydrolysate as a carbon source, a sequencing batch reactor (SBR) was chosen to treat artificial municipal wastewater. During the 14 days (28 cycles) of operation, the SBR using hydrolyzed molasses as a carbon source showed 91.6 +/- 1.6% nitrogen removal, which was higher than that using methanol (85.3 +/- 2.0%). The results show that hydrolyzed molasses can be an economical and effective external carbon source for the nitrogen removal process.     

Crithidia fasciculata: regulation of aerobic fermentation by malic enzyme

Aerobic fermentation, described in many protozoan organisms, was investigated using the nonpathogenic protozoan Crithidia fasciculata. The major end-products of this process were organic acids, particularly succinic acid. Inhibition of malic enzyme (EC 1.1.1.40) (l-malate: NADP oxidoreductase (decarboxylating) appeared to be integral to the aerobic fermentative process; the enzyme was inhibited in a cumulative manner by oxalacetate, acetyl-coenzyme A and oxalate. Inhibition by oxalate was noncompetitive with respect to both substrate and coenzyme. The inhibition of malic enzyme by oxalacetate and acetyl-coenzyme A appeared to provide a mechanism for the diversion of carbon from oxalacetate to succinate via the fumarase, thereby avoiding recycling to pyruvate. This was corroborated by the demonstration of an inverse relationship between the concentrations of pyruvate and succinate elaborated by this organism.     

城市系统碳循环:特征、机理与理论框架

城市是地表受人类活动影响最深刻的区域,城市系统碳循环在全球和区域碳过程中具有重要的地位和作用.提出了城市“自然-社会”二元碳循环的概念,探讨了城市系统碳循环的一般特征;分析了城市系统碳循环的内部机理,主要包括:城市系统碳储量和碳输入/输出通量的主要过程和途径、城市系统碳储量、碳通量和碳流通的生命周期分析、城市系统碳输入和碳输出的类型划分等;提出了基于系统层次划分和碳流通过程的城市系统碳循环的研究框架,分析了城市自然系统和城市经济系统的主要碳流通过程和环节,构建了城市系统碳循环研究的思路和理论框架;最后提出了城市系统碳循环领域未来的研究重点.     

铁施肥促进海洋浮游植物和生物碳泵的不确定性

铁作为浮游植物所必需的微量元素,限制了全球超过三分之一海域的初级生产力,尤其是在高营养盐、低叶绿素海域(high nutrient low chlorophyll,HNLC)。长期以来海洋铁施肥被认为是一项可以降低大气二氧化碳含量的地球工程策略。然而通过13次海洋人工铁施肥(artificial ocean iron fertilization,aOIF)实验发现,铁的额外添加对海洋深层碳输出量的促进作用要显著低于预期。本文简要地总结了碳在海洋和大气中的循环过程,回顾了人工铁施肥实验对生物碳泵和碳通量等的影响,分析了从海洋铁施肥到海洋碳汇关键生物地球化学过程的影响因素。综上分析发现,科学界对生物碳泵过程及其调控机制的认识仍十分浅薄,考虑到海洋铁施肥还会对海洋生态系统带来一定的负面作用,铁施肥能否作为降低大气中CO₂的有效手段,以达到碳中和并缓解温室效应仍需进一步研究。     

不同碳源条件下广叶绣球菌转录组分析

【背景】广叶绣球菌(Sparassis latifolia)是一种名贵的食用菌,其木质纤维素降解的分子机制尚不明确。【目的】了解广叶绣球菌在不同碳源条件下木质纤维素降解相关基因表达动态。【方法】通过转录组测序技术对分别以葡萄糖、纤维素+木质素、纤维素及松木屑为碳源的广叶绣球菌基因表达谱进行分析。以葡萄糖为碳源的样本为对照,分别对不同碳源下广叶绣球菌显著差异表达的基因进行功能分析。【结果】Geneontology(Go)富集分析表明,以葡萄糖为碳源的样本为对照,差异表达基因主要富集在碳水化合物利用的过程,如多糖催化过程、碳水化合物催化过程、碳水化合物代谢过程及多糖代谢过程等。碳水化合物活性酶(Carbohydrate-activeenzymes,CAZymes)功能注释表明,碳源种类主要影响了半纤维素和纤维素降解相关糖苷水解酶家族基因的表达,其中涉及半纤维素降解的相关酶基因上调幅度最大。同时,在纤维素+木质素、松木屑为碳源的处理组中一些转录因子基因上调表达显著。【结论】不同碳源显著影响了广叶绣球菌基因表达谱,这种对碳源的适应也可能反映了广叶绣球菌攻击植物细胞壁的机制,研究结果为深入了解广叶绣球菌木质纤维素降解的分子机理和相关功能基因提供了一些参考。     

Carbon source recovery from waste activated sludge by alkaline hydrolysis and gamma-ray irradiation for biological denitrification

The recovery of an organic carbon source from a waste activated sludge by using alkaline hydrolysis and radiation treatment was studied, and the feasibility of the solubilized sludge carbon source for a biological denitrification was also investigated. The effects of an alkaline treatment and gamma-ray irradiation on a biodegradability enhancement of the sludge were also studied. A modified continuous bioreactor for a denitrification (MLE reactor) was operated by using a synthetic wastewater for 47 days. Alkaline treatment of pH 10 and gamma-ray irradiation of 20 kGy were found to be the optimum carbon source recovery conditions. COD removal of 84% and T-N removal of 51% could be obtained by using the solubilized sludge carbon source through the MLE denitrification process. It can be concluded that the carbon source recovered from the waste activated sludge was successfully employed as an alternative carbon source for a biological denitrification.     

土壤非保护性有机碳对荒漠草原沙漠化的响应

沙化草地的恢复与重建是干旱半干旱区生态建设的重要内容,分析荒漠草原沙漠化过程中土壤非保护性有机碳分配比例的变异规律对于探讨沙化草地恢复机制具有重要的理论价值。以干旱、半干旱地区荒漠草原不同沙化阶段的土壤为研究对象,分析土壤粗颗粒有机碳、细颗粒有机碳、轻组有机碳含量和分配比例的分布特征、土壤非保护性有机碳转化为保护性有机碳的速率。结果表明:荒漠草原发生逆向演替后,土壤细颗粒有碳含量和分配比例表现为固定沙地荒漠草地半固定沙地流动沙地;粗颗粒有机碳含量表现为荒漠草地半固定沙地流动沙地固定沙地,粗颗粒有机碳分配比例表现为流动沙地半固定沙地荒漠草地固定沙地;轻组有机碳含量和分配比例递减。颗粒有机碳、轻组有机碳、土壤有机碳对草地沙漠化的敏感性不同,颗粒有机碳较轻组有机碳和土壤有机碳敏感性强,其中细颗粒有机碳较粗颗粒有机碳敏感性强。随着草地沙漠化程度的增强土壤非保护性有机碳分配比例呈下降趋势,表明草地沙漠化降低土壤质量。荒漠草地退化至流动沙地土壤非保护性有机碳转化为保护性有机碳的速率整体呈上升趋势。     

Cobalt hydroxide nanoparticles modified glassy carbon electrode as a biosensor for electrooxidation and determination of some amino acids

The electrochemical behavior of some amino acids was investigated on cobalt hydroxide nanoparticles modified glassy carbon (CHM-GC) electrode in alkaline solution. The process of oxidation and its kinetics were established by using cyclic voltammetry, chronoamperometry techniques, and steady-state polarization measurements. The results revealed that cobalt hydroxide promotes the rate of oxidation by increasing the peak current, so these bimolecular reactions are oxidized at lower potentials. Cyclic voltammograms and chronoamperometry indicate a catalytic EC′ mechanism to be operative with electrogeneration of Co(IV) as the electrochemical process. Also, the process is diffusion controlled and the current-time responses follow Cottrellian behavior. This result was confirmed by steady-state measurements. The rate constants of the catalytic oxidation of amino acids and the electron transfer coefficients are reported.