Three-reaction model for the anaerobic digestion of microalgae

Coupling an anaerobic digester to a microalgal culture has received increasing attention as an alternative process for combined bioenergy production and depollution. In this article, a dynamic model for anaerobic digestion of microalgae is developed with the aim of improving the management of such a coupled system. This model describes the dynamics of inorganic nitrogen and volatile fatty acids since both can lead to inhibition and therefore process instability. Three reactions are considered: Two hydrolysis–acidogenesis steps in parallel for sugars/lipids and for proteins, followed by a methanogenesis step. The proposed model accurately reproduces experimental data for anaerobic digestion of the freshwater microalgae Chlorella vulgaris with an organic loading rate of 1 gCOD L^?1 d^?1. In particular, the three‐reaction pathway allows to adequately represent the observed decoupling between biogas production and nitrogen release. The reduced complexity of this model makes it suitable for developing advanced, model‐based control and monitoring strategies. Biotechnol. Bioeng. 2012; 109:415–425. © 2011 Wiley Periodicals, Inc.     

Strategies to enhance cell growth and achieve high‐level oil production of a Chlorella vulgaris isolate

The autotrophic growth of an oil‐rich indigenous microalgal isolate, identified as Chlorella vulgaris C? C, was promoted by using engineering strategies to obtain the microalgal oil for biodiesel synthesis. Illumination with a light/dark cycle of 14/10 (i.e., 14 h light‐on and 10 h light‐off) resulted in a high overall oil production rate (v_oil) of 9.78 mg/L/day and a high electricity conversion efficiency (E_c) of 23.7 mg cell/kw h. When using a NaHCO₃ concentration of 1,500 mg/L as carbon source, the v_oil and E_c were maximal at 100 mg/L/day and 128 mg/kw h, respectively. A Monod type model was used to describe the microalgal growth kinetics with an estimated maximum specific growth rate (μ_max) of 0.605 day^?1 and a half saturation coefficient (K_s) of 124.9 mg/L. An optimal nitrogen source (KNO₃) concentration of 625 mg/L could further enhance the microalgal biomass and oil production, leading to a nearly 6.19 fold increase in v_oil value. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

毕赤酵母液滴微流控高通量筛选方法的建立与应用

巴斯德毕赤酵母是当前应用最为方便和广泛的外源蛋白表达系统之一,为了进一步提高其表达外源蛋白的能力,文中建立了基于液滴微流控的毕赤酵母高通量筛选方法,并以木聚糖酶融合荧光蛋白为例,筛选获得木聚糖酶表达和分泌能力提高的突变株。通过PCR扩增得到木聚糖酶xyn5基因和绿色荧光蛋白gfp基因融合片段,并克隆到毕赤酵母表达载体pPIC9K中构建出木聚糖酶融合绿色荧光蛋白的质粒pPIC9K-xyn5-gfp,电转化至毕赤酵母GS115中得到表达木聚糖酶和绿色荧光蛋白的毕赤酵母SG菌株。该菌株经过常压室温等离子体诱变后进行单细胞液滴包埋,液滴培养24h后进行微流控筛选,获得高表达木聚糖酶的突变菌株,进而用于下一轮的诱变突变库构建和筛选。以此类推,经过5轮液滴微流控筛选,获得一株高产菌株SG-m5,其木聚糖酶活为149.17U/mg,较出发菌株提升300%,分泌外源蛋白的能力较出发菌株提高160%。文中建立的毕赤酵母单细胞液滴微流控高通量筛选方法能达到每小时10万菌株的筛选通量,筛选百万级别的菌株库仅需10h,消耗荧光试剂体积100μL,对比传统的微孔板筛选方法降低试剂成本近百万倍,为高效、低成本筛选获得表达和分泌外源蛋白能力提高的毕赤酵母提供了一条新途径。     

Effects of Temperature and pH on Cell Growth of a Flocculating Microalga (Chlorella vulgaris) JSC-7

微藻是可广泛用于健康食品及水产养殖的饵料,同时,微藻细胞内积累的油脂可作为可再生生物燃料,因此微藻的生长和代谢受到广泛关注。温度和pH对微藻的生物量积累有很大影响,考察不同温度和pH条件下微藻细胞的生长有助于寻找最佳的条件进行微藻的培养。自絮凝小球藻JSC-7（Chlorella vulgaris JSC-7）可实现自沉降采收,有利于降低微藻生产成本,优化其生长条件对更好地利用该微藻具有重要意义。考察了温度（22∽40℃）及pH（6.0∽10.0）对其细胞生长、叶绿素含量和油脂产量的影响。在所选取的温度及pH范围内,JSC-7细胞均可生长,显示该藻种可以适应广泛的温度和pH条件。适合细胞生长的温度依次为31℃〉28℃〉35℃〉25℃,pH依次为7.0〉8.0〉6.0。pH 8.0时生物量和油脂的积累量最多,说明该藻株在弱碱条件下更适合生长和产油。当温度为31℃、pH为7.0时,可获得最高的生长量（OD690=0.941）、叶绿素含量（19 mg/L）及油脂产量（39.07%/克干重）。     

CO2 utilization in the production of biomass and biocompounds by three different microalgae

The atmospheric CO₂ increase is considered the main cause of global warming. Microalgae are photosynthetic microorganisms that can help in CO₂ mitigation and at the same time produce value‐added compounds. In this study, Scenedesmus obliquus , Chlorella vulgaris , and Chlorella protothecoides were cultivated under 0.035 (air), 5 and 10% (v/v) of CO₂ concentrations in air to evaluate the performance of the microalgae in terms of kinetic growth parameters, theoretical CO₂ biofixation rate, and biomass composition. Among the microalgae studied, S. obliquus presented the highest values of specific growth rate (μ = 1.28 d^?1), maximum productivities (P _max = 0.28 g L^?1d^?1), and theoretical CO₂ biofixation rates (0.56 g L^?1d^?1) at 10% CO₂. The highest oil content was found at 5% CO₂, and the fatty acid profile was not influenced by the concentration of CO₂ in the inflow gas mixture and was in compliance with EN 14214, being suitable for biodiesel purposes. The impact of the CO₂ on S. obliquus cells’ viability/cell membrane integrity evaluated by the in‐line flow cytometry is quite innovative and fast, and revealed that 86.4% of the cells were damaged/permeabilized in cultures without the addition of CO₂.     

海洋经济微藻混合培养与单独培养下的生长比较

海洋微藻可以广泛应用在水产养殖、食品加工、医药保健、环境保护和生物制能等各种行业,具有非常好的开发利用前景.目前,如何突破海洋微藻培养过程中细胞生物量低下等瓶颈,提高微藻的细胞密度,以低成本、高效率开发利用海洋微藻资源成为了国内外学者关注的焦点.论文以三角褐指藻、亚心型扁藻和杜氏盐藻3 种典型经济海洋微藻为研究材料,在实验室条件下研究了它们两两混合培养与各自单独培养条件下的细胞生长情况,探讨海洋微藻混合培养在促进微藻整体细胞生长方面的可能性.结果显示,三角褐指藻和杜氏盐藻混合培养在生长前9 天的OD680 高于三角褐指藻或杜氏盐藻分别单独培养的OD680 ,而随着试验时间的延长,混合培养条件下的OD680 与单独培养三角褐指藻的OD680 相似;就三角褐指藻和亚心型扁藻混合培养的OD680而言,在整个生长时期均高于单独培养亚心型扁藻的OD680 ,但低于单独培养三角褐指藻的OD680 ;不同的是,杜氏盐藻和亚心型扁藻混合培养下的OD680 在生长第6 天后高于单独培养杜氏盐藻或单独培养亚心型扁藻的OD680 .研究结果表明,混合培养三角褐指藻和杜氏盐藻或混合培养杜氏盐藻和亚心型扁藻具有一定程度促进微藻细胞生长的潜力.研究结果将为经济海洋微藻的高密度培养及其高附加价值活性物质的开发利用提供一个崭新的研究方向.     

Production of Chlorella vulgaris as a source of essential fatty acids in a tubular photobioreactor continuously fed with air enriched with CO2 at different concentrations

To reduce CO₂ emissions and simultaneously produce biomass rich in essential fatty acids, Chlorella vulgaris CCAP 211 was continuously grown in a tubular photobioreactor using air alone or air enriched with CO₂ as the sole carbon source. While on one hand, nitrogen‐limited conditions strongly affected biomass growth, conversely, they almost doubled its lipid fraction. Under these conditions using air enriched with 0, 2, 4, 8, and 16% (v/v) CO₂, the maximum biomass concentration was 1.4, 5.8, 6.6, 6.8, and 6.4 g_DB L^?1 on a dry basis, the CO₂ consumption rate 62, 380, 391, 433, and 430 L^?1 day^?1, and the lipid productivity 3.7, 23.7, 24.8, 29.5, and 24.4 mg L^?1 day^?1, respectively. C. vulgaris was able to grow effectively using CO₂‐enriched air, but its chlorophyll a (3.0–3.5 g 100g_DB^?1), chlorophyll b (2.6–3.0 g 100g_DB^?1), and lipid contents (10.7–12.0 g 100g_DB^?1) were not significantly influenced by the presence of CO₂ in the air. Most of the fatty acids in C. vulgaris biomass were of the saturated series, mainly myristic, palmitic, and stearic acids, but a portion of no less than 45% consisted of unsaturated fatty acids, and about 80% of these were high added‐value essential fatty acids belonging to the ω3 and ω6 series. These results highlight that C. vulgaris biomass could be of great importance for human health when used as food additive or for functional food production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:916–922, 2014     

Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach

Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RHs show a fast apical growth, constituting a unique single cell model system for analysing cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, although high-resolution imaging is still lacking to enable an investigation of the accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) that enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area and aspect ratio of nuclei moving in growing and mature RHs. Interestingly, we observed higher aspect ratios in the nuclei of mature RHs, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.     

植物激素在小球藻异养培养中的作用

本文研究了几种植物激素对小球藻异养培养的影响。结果表明 ,IAA、IBA及 6_BA三种植物激素均不同程度地促进了小球藻的异养生长 ,培养≤ 36h时 ,IAA或IBA以 2 0mg/L的促进小球藻异养生长的效应最大 ,1 0 0mg/LIAA或IBA则抑制了藻的生长 ;>36h时 ,1 0 0mg/LIAA或IBA表现出促进小球藻生长的效应 ,并最终获最大净A540 增长量 ;6_BA以 0 1mg/L的促进作用最大。IBA与 6_BA组合同样表现出促进小球藻异养生长的效应 ,但并非IBA和 6_BA简单的加合效应 ,5mg/LIBA与 6_BA组合的效应维持 6_BA单因子的作用趋势 ,2 0mg/LIBA与 1mg/L 6_BA组合的效应大于与 0 .1mg/L 6_BA组合的 ,1 0 0mg/LIBA与 0 .1mg/L 6_BA组合的效应在≤ 36h时大于与 1mg/L 6_BA组合的 ,>36h时则相反。另外 ,高浓度IBA(≥ 2 0mg/L)与 6_BA组合抑制了前中期异养藻对葡萄糖的吸收 ,但加速了中后期葡萄的吸收。再者 ,IBA与 6_BA组合加速了异养小球藻对NO_3 的吸收。     

Overcoming shear stress of microalgae cultures in sparged photobioreactors

In the present work we identified and quantified the effect of hydrodynamic stress on two different microalgae strains, Dunaliella tertiolecta and D. salina, cultivated in bench-scale bubble columns. The cell death rate constant increased with increasing gas-entrance velocity at the sparger. Dunaliella salina was slightly more sensitive than D. tertiolecta. The critical gas-entrance velocities were approximately 50 and 30 m s(-1) for D. tertiolecta and D. salina, respectively. The effects of gas-flow rate, culture height, and nozzle diameter on the death rate constant were also studied. From these results it was concluded that bubble rising and bubble bursting are not responsible for cell death. Regarding nozzle diameter, small nozzles were more detrimental to cells. The bubble formation at the sparger was found to be the main event leading to cell death.     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Growth kinetics and mathematical modeling of Synechocystis sp. PCC 6803 under flashing light

In photobioreactors and natural systems, microalgae are subjected to rapidly changing light intensities (LI) due to light attenuation and mixing. A controlled way to study the effect of rapidly changing LI is to subject cultures to flashing light. In this study, series of flashing-light experiments were conducted using Synechocystis sp. PCC6803 with constant overall average LI of approximately 84 μmol·m⁻²·s⁻¹ and relative times in the light and dark varied. The results were also compared with simulated results using a mathematical model including an absorbed pool of light energy, photoacclimation, and photoinhibition. With equal time in light and dark, the specific growth rate (μ) systematically decreased with increasing light duration, and µ decreased further when the ratio of light to dark was decreased. The model captured both trends with the mechanistic explanation that when the light duration was very short the changes in the pool of absorbed LI were smoothed out across the light and dark periods, whereas longer durations caused the biomass to experience discrete light and dark conditions that lead to reduced light absorption, more energy loss to nonphotochemical quenching, and more photodamage. These growth effects were accentuated as the ratio of light to dark decreased.     

A first view on the unsuspected intragenus diversity of N‐glycans in Chlorella microalgae

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health‐promoting food supplements. A mass spectrometry‐based survey of N‐glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N‐glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N‐acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan‐type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4‐O‐methyl‐N‐acetylglucosamine), and substitution of the second N‐acetylglucosamine. Analysis of barcode ITS1–5.8S–ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N‐glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella‐like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N‐glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.     

Growth and uptake kinetics of phosphate by benthic microalga Nitzschia sp. isolated from Hiroshima Bay,Japan

In the present study, we experimentally investigated the phosphate uptake kinetics of benthic microalga Nitzschia sp. isolated from Hiroshima Bay, Japan. The maximum uptake rate (ρ_max) obtained by short‐term experiments was 6.84 pmol cell^?1 h^?1 for phosphate. The half‐saturation constant for uptake (K_S) was 61.2 µmol cell^?1 h^?1. Both the ρ_max and Ks of this species were extremely high, suggesting that Nitzschia sp. is adapted to benthic environments, where nutrient concentrations are much higher than in the water column. The specific maximum growth rate (µ'_max) and minimum cell quota (Q₀) for the P‐limited condition, obtained by a semi‐continuous growth experiment, were 0.48 day^?1 and 0.045 pmol cell^?1, respectively. It is concluded that Nitzschia sp. could be a ‘storage strategist’ species, meaning it adapts so as to minimize the influence of fluctuations in phosphate conditions resulting from the change in redox conditions of sediment due to bioturbation.     

Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly diluted environments

Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. Recently, it has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a 50% higher growth rate compared to a 1 L batch cultivation [Grünberger et al. (2012) Lab Chip]. We here compare growth of C. glutamicum with glucose as substrate at different scales covering batch cultivations in the liter range down to single cell cultivations in the picoliter range. The maximum growth rate of standard batch cultures as estimated from different biomass quantification methods is ${\hat {\mu }} = 0.42\pm 0.03\,{\rm h}^{- 1} $ even for microtiter scale cultivations. In contrast, growth in a microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth rate of ${\hat {\mu }} = 0.62\pm 0.02\,{\rm h}^{- 1} $ . When in the same perfusion system cell‐free supernatant from exponentially grown shake flask cultures is used the growth rate of single cells is reduced to ${\hat {\mu }} = 0.47\pm 0.02\,{\rm h}^{- 1} $ . Likewise, when fresh medium is additionally supplied with 5 mM acetate, a growth rate of ${\hat {\mu }} = 0.51\pm 0.01\,{\rm h}^{- 1} $ is determined. These results prove that higher growth rates of C. glutamicum than known from typical batch cultivations are possible, and that growth is definitely impaired by very low concentrations of byproducts such as acetate. Biotechnol. Bioeng. 2013; 110: 220–228. © 2012 Wiley Periodicals, Inc.     

A screening model to predict microalgae biomass growth in photobioreactors and raceway ponds

A microalgae biomass growth model was developed for screening novel strains for their potential to exhibit high biomass productivities under nutrient‐replete conditions in photobioreactors or outdoor ponds. Growth is modeled by first estimating the light attenuation by biomass according to Beer‐Lambert's Law, and then calculating the specific growth rate in discretized culture volume slices that receive declining light intensities due to attenuation. The model uses only two physical and two species‐specific biological input parameters, all of which are relatively easy to determine: incident light intensity, culture depth, as well as the biomass light absorption coefficient and the specific growth rate as a function of light intensity. Roux bottle culture experiments were performed with Nannochloropsis salina at constant temperature (23°C) at six different incident light intensities (10, 25, 50, 100, 250, and 850 µmol/m² s) to determine both the specific growth rate under non‐shading conditions and the biomass light absorption coefficient as a function of light intensity. The model was successful in predicting the biomass growth rate in these Roux bottle batch cultures during the light‐limited linear phase at different incident light intensities. Model predictions were moderately sensitive to minor variations in the values of input parameters. The model was also successful in predicting the growth performance of Chlorella sp. cultured in LED‐lighted 800 L raceway ponds operated in batch mode at constant temperature (30°C) and constant light intensity (1,650 µmol/m² s). Measurements of oxygen concentrations as a function of time demonstrated that following exposure to darkness, it takes at least 5 s for cells to initiate dark respiration. As a result, biomass loss due to dark respiration in the aphotic zone of a culture is unlikely to occur in highly mixed small‐scale photobioreactors where cells move rapidly in and out of the light. By contrast, as supported also by the growth model, biomass loss due to dark respiration occurs in the dark zones of the relatively less well‐mixed pond cultures. In addition to screening novel microalgae strains for high biomass productivities, the model can also be used for optimizing the pond design and operation. Additional research is needed to validate the biomass growth model for other microalgae species and for the more realistic case of fluctuating temperatures and light intensities observed in outdoor pond cultures. Biotechnol. Bioeng. 2013; 110: 1583–1594. © 2012 Wiley Periodicals, Inc.     

Growth kinetics of aerobic granules developed in sequencing batch reactors

AIMS: This paper attempts to develop a kinetic model to describe the growth of aerobic granules developed under different operation conditions. METHODS AND RESULTS: A series of experiments were conducted by using four-column sequencing batch reactors to study the formation of aerobic granules under different conditions, e.g. organic loading rates, hydrodynamic shear forces and substrate N/COD ratios. A simple kinetic model based on the Linear Phenomenological Equation was successfully derived to describe the growth of aerobic granules. It was found that the growth of aerobic granules in terms of equilibrium size and size-dependent growth rate were inversely related to shear force imposed to microbial community, while a high organic loading favoured the growth of aerobic granules, leading to a large size granule. The effect of substrate N/COD ratio on the growth kinetics of aerobic granules was realized through change in microbial populations, and enriched nitrifying population in aerobic granules developed at high substrate N/COD ratio resulted in a low overall growth rate of aerobic granules. CONCLUSIONS: The proposed model can provide good prediction for the growth of aerobic granules indicated by the correlation coefficient >0.95. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model proposed could offer a useful tool for studying the growth kinetics of cell-to-cell immobilization process. The study confirmed that the growth of aerobic granules and biofilms are subject to a similar kinetic pattern. This work would also be helpful for better understanding the mechanism of aerobic granulation.     

Molecular phenotyping of aging in single yeast cells using a novel microfluidic device

Budding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild-type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long-lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker - the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism.     

氧化铝气体分布器应用小球藻培养的研究

光生物反应器设计中,气体分布器对微藻生长有较大的影响,尤其是在鼓泡式光生物反应器中更为显著。实验考察了采用氧化铝烧制的多孔气体分布器的5L鼓泡式光生物反应器中通气速率、CO₂ 浓度对小球藻LICME002生物量、叶绿素含量、油脂积累的影响。对该气体分布器下的CO₂浓度和通气速率对小球藻的作用机理进行了初步的探讨。结果表明,CO₂浓度为3%时,该株微藻生物量、叶绿素、油脂积累的最佳;CO₂浓度超过6%时各项指标显著下降。通过对0.1vvm,0.4vvm,0.7vvm、1.0vvm的通气速条件下的各项指标的分析,确定最佳通气条件为0.4vvm。结论显示,在最佳通气速率和CO₂浓度下,微藻生物量能达到1.52g/L,油脂含量达到31.5%。